462 results
Search Results
2. Cortical excitability measured with transcranial magnetic stimulation in children with epilepsy before and after antiepileptic drugs.
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Andreasson, Ann‐Charlotte, Sigurdsson, Gudmundur V, Pegenius, Göran, Thordstein, Magnus, Hallböök, Tove, and Andreasson, Ann-Charlotte
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TRANSCRANIAL magnetic stimulation ,CHILDHOOD epilepsy ,ANTICONVULSANTS ,VALPROIC acid ,PARTIAL epilepsy ,VAGUS nerve ,RESEARCH ,ELECTROENCEPHALOGRAPHY ,EPILEPSY ,RESEARCH methodology ,EVALUATION research ,MEDICAL cooperation ,COMPARATIVE studies ,RESEARCH funding ,PHARMACODYNAMICS - Abstract
Aim: To evaluate cortical excitability with transcranial magnetic stimulation (TMS) in children with new-onset epilepsy before and after antiepileptic drugs (AEDs).Method: Fifty-five drug-naïve patients (29 females, 26 males; 3-18y), with new-onset epilepsy were recruited from 1st May 2014 to 31st October 2017 at the Child Neurology Department, Queen Silvia's Children's Hospital, Gothenburg, Sweden. We performed TMS in 48 children (23 females, 25 males; mean [SD] age 10y [3y], range 4-15y) with epilepsy (27 generalized and 21 focal) before and after the introduction of AEDs. We used single- and paired-pulse TMS. We used single-pulse TMS to record resting motor thresholds (RMTs), stimulus-response curves, and cortical silent periods (CSPs). We used paired-pulse TMS to record intracortical inhibition and facilitation at short, long, and intermediate intervals.Results: There were no differences in cortical excitability between children with generalized and focal epilepsy at baseline. After AED treatment, RMTs increased (p=0.001), especially in children receiving sodium valproate (p=0.005). CSPs decreased after sodium valproate was administered (p=0.050). As in previous studies, we noted a negative correlation between RMT and age in our study cohort. Paired-pulse TMS could not be performed in most children because high RMTs made suprathreshold stimulation impossible.Interpretation: Cortical excitability as measured with RMT decreased after the introduction of AEDs. This was seen in children with both generalized and focal epilepsy who were treated with sodium valproate, although it was most prominent in children with generalized epilepsy. We suggest that TMS might be used as a prognostic tool to predict AED efficacy.What This Paper Adds: Resting motor threshold (RMT) correlated negatively with age in children with epilepsy. No differences in cortical excitability were noted between patients with generalized and focal epilepsy. Treatment with antiepileptic drugs decreased cortical excitability as measured with transcranial magnetic stimulation (TMS). Decreased cortical excitability with increased RMT was recorded, especially after sodium valproate treatment. Paired-pulse TMS was difficult to perform because of high RMTs in children. [ABSTRACT FROM AUTHOR]- Published
- 2020
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3. Response to clobazam in continuous spike-wave during sleep.
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Vega, Clemente, Sánchez Fernández, Ivan, Peters, Jurriaan, Thome‐Souza, Maria S., Jackson, Michele, Takeoka, Masanori, Wilkening, Greta N., Pearl, Phillip L., Chapman, Kevin, Loddenkemper, Tobias, and Thome-Souza, Maria S
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DRUG efficacy ,SLEEP physiology ,NEUROPSYCHOLOGICAL tests ,COGNITION ,ELECTROENCEPHALOGRAPHY ,BENZODIAZEPINES ,BRAIN physiology ,CLINICAL trials ,COMPARATIVE studies ,EPILEPSY ,LONGITUDINAL method ,RESEARCH methodology ,MEDICAL cooperation ,RESEARCH ,RESEARCH funding ,SLEEP ,EVALUATION research ,TRANQUILIZING drugs ,PHARMACODYNAMICS - Abstract
Copyright of Developmental Medicine & Child Neurology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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- 2018
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4. Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase.
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Tymvios, C, Moore, C, Jones, S, Solomon, A, Sanz-Rosa, D, and Emerson, M
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BLOOD platelet aggregation ,NITRIC oxide ,ENDOTHELIUM ,THROMBIN ,NITRIC-oxide synthases ,LABORATORY mice ,ANIMAL experimentation ,ARGININE ,COMPARATIVE studies ,ENZYME inhibitors ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,OXIDOREDUCTASES ,RESEARCH ,RESEARCH funding ,EVALUATION research ,PLATELET function tests ,PHARMACODYNAMICS - Abstract
Background and Purpose: Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.Experimental Approach: We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.Key Results: Acute NOS inhibition by N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3(-/-) mice but responses in NOS-3(-/-) mice were enhanced by L-NAME.Conclusions and Implications: Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3. [ABSTRACT FROM AUTHOR]- Published
- 2009
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5. Submandibular duct ligation after botulinum neurotoxin A treatment of drooling in children with cerebral palsy.
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Bekkers, Stijn, Pruijn, Ineke M J, Van Hulst, Karen, Delsing, Corinne P, Erasmus, Corrie E, Scheffer, Arthur R T, and Van Den Hoogen, Frank J A
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DROOLING , *CHILDREN with cerebral palsy , *BOTULINUM toxin , *CEREBRAL palsy , *PAROTID gland surgery , *RESEARCH , *MUSCLE relaxants , *SUBMANDIBULAR gland , *RESEARCH methodology , *RETROSPECTIVE studies , *EVALUATION research , *MEDICAL cooperation , *COMPARATIVE studies , *PHARMACODYNAMICS , *DISEASE complications - Abstract
Aim: To assess: (1) the effect on drooling of bilateral submandibular duct ligation as surgical therapy after the administration of submandibular botulinum neurotoxin A (BoNT-A) for excessive drooling and (2) the predictive value of treatment success with BoNT-A on treatment success after bilateral submandibular duct ligation.Method: This was a within-participant retrospective observational study in which 29 children with severe drooling (15 males, 14 females) received BoNT-A treatment at a mean age of 9 years 6 months (SD 2y 5mo), followed by bilateral submandibular duct ligation at a mean age of 10 years 11 months (SD 2y 4mo). Fifteen children were diagnosed with cerebral palsy (CP), with 12 children classified in Gross Motor Function Classification System levels IV and V. The 14 children without CP had non-progressive developmental disorders. The primary drooling severity outcomes were the Visual Analogue Scale (VAS; subjective assessment) and drooling quotient (objective assessment). Measurements were taken before each intervention and again at 8 and 32 weeks.Results: The VAS was significantly lower after bilateral submandibular duct ligation at follow-up compared to BoNT-A treatment (mean difference -33, p≤0.001; 95% confidence interval [CI]=-43.3 to -22.9). The mean drooling quotient did not significantly differ between BoNT-A treatment and bilateral submandibular duct ligation at follow-up (3.3, p=0.457; 95% CI=-4.35 to 9.62) or between 8 and 32 weeks (4.7, p=0.188; 95% CI=-2.31 to 11.65).Interpretation: BoNT-A treatment and bilateral submandibular duct ligation are both effective treatment modalities for drooling. At 32-week follow-up, subjective drooling severity after bilateral submandibular duct ligation was significantly lower compared to previous BoNT-A injections in participants. However, treatment success with BoNT-A is no precursor to achieving success with bilateral submandibular duct ligation.What This Paper Adds: Bilateral submandibular duct ligation is an effective therapy for drooling after treatment with botulinum neurotoxin A (BoNT-A). Treatment success with BoNT-A is not a predictor of successful therapy with bilateral submandibular duct ligation. [ABSTRACT FROM AUTHOR]- Published
- 2020
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6. Ca2+/calcineurin regulation of cloned vascular K ATP channels: crosstalk with the protein kinase A pathway.
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Orie, N. N., Thomas, A. M., Perrino, B. A., Tinker, A., and Clapp, L. H.
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PROTEIN kinases ,PHARMACOLOGY ,HYPOGLYCEMIC agents ,CHEMICAL reactions ,CARDIOVASCULAR agents ,VASODILATION ,SMOOTH muscle physiology ,BIOLOGICAL models ,BIOLOGICAL transport ,CALCIUM ,CELL lines ,COMPARATIVE studies ,CYTOLOGICAL techniques ,GENETIC techniques ,HYDROLASES ,IMMUNOSUPPRESSIVE agents ,ISOENZYMES ,RESEARCH methodology ,MEDICAL cooperation ,PHOSPHORYLATION ,POTASSIUM ,RESEARCH ,RESEARCH funding ,SMOOTH muscle ,TRANSFERASES ,EVALUATION research ,POTASSIUM antagonists ,CHEMICAL inhibitors ,PHARMACODYNAMICS ,PHYSIOLOGY - Abstract
Background and purpose: Vascular ATP-sensitive potassium (K
ATP ) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca2+ -dependent phosphatase, inhibits KATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular KATP channel (KIR 6.1/SUR2B). The effect of intracellular Ca2+ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aα but not Aβ significantly attenuated KATP currents activated by low intracellular Ca2+ , whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal KATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca2+ (equivalent to increasing calcineurin activity), almost completely inhibited KATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and implications: Calcineurin Aα regulates KIR 6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the KATP channel. British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009 This article is commented on by Tammaro, pp. 551–553 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]- Published
- 2009
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7. Dual effect of nitric oxide on uterine prostaglandin synthesis in a murine model of preterm labour.
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Cella, M, Farina, MG, Dominguez Rubio, AP, Di Girolamo, G, Ribeiro, ML, Franchi, AM, Farina, M G, Dominguez Rubio, A P, Ribeiro, M L, and Franchi, A M
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PROSTAGLANDIN synthesis ,NITRIC oxide ,ANIMAL models in research ,PREMATURE labor ,ENDOTOXINS ,HEALTH outcome assessment ,RESORPTION (Physiology) ,ANIMAL experimentation ,BIOLOGICAL models ,COMPARATIVE studies ,ENZYME inhibitors ,INFLAMMATION ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NONSTEROIDAL anti-inflammatory agents ,ORGANIC compounds ,OXIDOREDUCTASES ,PROSTAGLANDINS ,RADIOIMMUNOASSAY ,RESEARCH ,SULFUR compounds ,THIAZOLES ,UTERUS ,WESTERN immunoblotting ,CYCLOOXYGENASE 2 ,EVALUATION research ,LIPOPOLYSACCHARIDES ,DINOPROSTONE ,PHARMACODYNAMICS - Abstract
Background and Purpose: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs).Experimental Approach: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO.Key Results: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them.Conclusions and Implications: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes. [ABSTRACT FROM AUTHOR]- Published
- 2010
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8. 5-Lipoxygenase inhibitors induce potent anti-proliferative and cytotoxic effects in human tumour cells independently of suppression of 5-lipoxygenase activity.
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Fischer, AS, Metzner, J, Steinbrink, SD, Ulrich, S, Angioni, C, Geisslinger, G, Steinhilber, D, Maier, TJ, Fischer, A S, Steinbrink, S D, and Maier, T J
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LIPOXYGENASES ,ENZYME inhibitors ,CELL-mediated cytotoxicity ,CANCER cells ,ANTINEOPLASTIC agents ,CARCINOGENS ,GENE expression ,RESEARCH ,WESTERN immunoblotting ,RESEARCH methodology ,CELL physiology ,MEDICAL cooperation ,EVALUATION research ,COMPARATIVE studies ,TRANSFERASES ,GENES ,OXIDOREDUCTASES ,TUMORS ,CELL lines ,CELL death ,PHARMACODYNAMICS - Abstract
Background and Purpose: Certain 5-lipoxygenase (5-LO) inhibitors exhibit anti-carcinogenic activities against 5-LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5-LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5-LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti-proliferative and cytotoxic potency of a number of 5-LO inhibitors with different inhibitory mechanisms in 5-LO-positive and 5-LO-negative tumour cells.Experimental Approach: Cell viability was measured by the WST-1 assay; cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay. Cell death was analysed by annexin V staining, Western blot analysis of PARP (poly ADP-ribose polymerase) cleavage and a cytotoxicity assay. 5-LO product formation was quantified by a 5-LO activity assay.Key Results: The common 5-LO inhibitors AA-861, Rev-5901 and MK-886 induced cytotoxic and anti-proliferative effects in 5-LO-positive Capan-2 pancreatic cancer cells; BWA4C and CJ-13,610 only caused anti-proliferative effects, while zileuton failed to impair cell viability. Moreover, the concentrations of the 5-LO inhibitors required to induce anti-proliferation and cytotoxicity highly exceeded those for suppression of 5-LO. Supplementation with mitogenic 5-LO products failed to protect Capan-2 cells from the effects of 5-LO inhibitors. Finally, the cytotoxic and anti-proliferative 5-LO inhibitors also potently reduced the viability of 5-LO-deficient tumour cell lines (HeLa, Panc-1 and U937).Conclusions and Implications: Certain 5-LO inhibitors cause cytotoxic and anti-proliferative effects independently of suppression of 5-LO activity. Thus, the role of 5-LO overexpression in tumour cell viability remains unclear and requires further elucidation. [ABSTRACT FROM AUTHOR]- Published
- 2010
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9. Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions.
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Oliveira, C, Navarro-Xavier, RA, Anjos-Vallota, EA, Martins, JO, Silveira, VLF, Gonçalves, LRC, Araújo, MS, Motta, G, Sannomiya, P, Oliva, MLV, Navarro-Xavier, R A, Anjos-Vallota, E A, Martins, J O, Silveira, V L F, Gonçalves, L R C, Araújo, M S, and Oliva, M L V
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LEUCOCYTE elastase ,CELL migration ,CELL adhesion ,CYTOKINES ,INFLAMMATION ,METHIONINE ,BRADYKININ ,POLYSACCHARIDES ,BIOLOGICAL models ,RESEARCH ,ANTI-inflammatory agents ,LEUCOCYTES ,ANIMAL experimentation ,MICROSCOPY ,RESEARCH methodology ,PROTEOLYTIC enzymes ,CELL physiology ,MEDICAL cooperation ,EVALUATION research ,PLANT proteins ,RATS ,CELL motility ,PLANTS ,COMPARATIVE studies ,SEEDS ,ENZYME-linked immunosorbent assay ,EDEMA ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process.Experimental Approach: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Key Results: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1β or tumour necrosis factor-α, however, did not change.Conclusions and Implications: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles. [ABSTRACT FROM AUTHOR]- Published
- 2010
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10. Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin-dependent control of epithelial Na+ absorption.
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Mansley, Morag K and Wilson, Stuart M
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PROTEIN kinases ,KIDNEY tubules ,EPITHELIAL cells ,EDEMA ,HYPERTENSION ,PEOPLE with diabetes ,PHOSPHORYLATION ,SODIUM ions ,CELLULAR signal transduction ,PROTEIN metabolism ,SODIUM metabolism ,AMINES ,ANIMAL experimentation ,ANTI-infective agents ,BIOLOGICAL transport ,CELL culture ,COMPARATIVE studies ,HETEROCYCLIC compounds ,INSULIN ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,PHOSPHOTRANSFERASES ,PROTEINS ,PYRIDINE ,RESEARCH ,STEROIDS ,SULFONAMIDES ,TRANSFERASES ,EVALUATION research ,ABSORPTION ,CHEMICAL inhibitors ,PHARMACODYNAMICS ,PHYSIOLOGY ,CELL physiology - Abstract
Background and Purpose: Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention.Experimental Approach: We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins.Key Results: Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases.Conclusion and Implications: While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1. [ABSTRACT FROM AUTHOR]- Published
- 2010
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11. Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway.
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Li, Feng, Rajendran, Peramaiyan, and Sethi, Gautam
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QUINONE ,CELL proliferation ,APOPTOSIS ,MULTIPLE myeloma ,CELLULAR signal transduction ,TRANSCRIPTION factors ,PHOSPHORYLATION ,BLACK cumin ,NEOVASCULARIZATION ,PROTEIN metabolism ,ANTINEOPLASTIC agents ,BIOCHEMISTRY ,BORON compounds ,CARRIER proteins ,CELL lines ,CELL physiology ,COMPARATIVE studies ,HETEROCYCLIC compounds ,INTERLEUKINS ,PHENOMENOLOGY ,RESEARCH methodology ,MEDICAL cooperation ,PHOSPHATASES ,RESEARCH ,THALIDOMIDE ,VANADATES ,EVALUATION research ,BENZOQUINONES ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) pathway is frequently encountered in several human cancers including multiple myeloma (MM). Thus, agents that suppress STAT3 phosphorylation have a potential for treatment of MM. In the present report, we investigated whether thymoquinone (TQ), the main component isolated from the medicinal plant Nigella sativa, modulated the STAT3 signalling pathway in MM cells.Experimental Approach: The effect of TQ on both constitutive and IL-6-induced STAT3 activation, associated protein kinases, STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation and apoptosis in MM cells, was investigated.Key Results: We found that TQ inhibited both constitutive and IL-6-inducible STAT3 phosphorylation which correlated with the inhibition of c-Src and JAK2 activation. Vanadate reversed the TQ-induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that TQ can induce the expression of Src homology-2 phosphatase 2 that correlated with suppression of STAT3 activation. TQ also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, TQ induced the accumulation of cells in sub-G1 phase, inhibited proliferation and induced apoptosis, as indicated by poly ADP ribose polymerase cleavage. TQ also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells.Conclusions and Implications: Our study has identified STAT3 signalling as a target of TQ and has thus raised its potential application in the prevention and treatment of MM and other cancers. [ABSTRACT FROM AUTHOR]- Published
- 2010
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12. Reduced signal transduction by 5-HT4 receptors after long-term venlafaxine treatment in rats.
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Vidal, R, Valdizan, EM, Vilaró, MT, Pazos, A, Castro, E, Valdizan, E M, and Vilaró, M T
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CELLULAR signal transduction ,VENLAFAXINE ,LABORATORY rats ,PROTEINS ,DRUG dosage ,NEURAL transmission ,AUTORADIOGRAPHY ,ADENYLATE cyclase ,ELECTROPHYSIOLOGY ,ADRENERGIC uptake inhibitors ,GENE expression ,HIPPOCAMPUS physiology ,NEURAL physiology ,BRAIN metabolism ,ACTION potentials ,ALCOHOLS (Chemical class) ,ANIMAL experimentation ,BENZAMIDE ,BRAIN ,CELL receptors ,COMPARATIVE studies ,DRUG interactions ,HETEROCYCLIC compounds ,HIPPOCAMPUS (Brain) ,RESEARCH methodology ,MEDICAL cooperation ,RADIOISOTOPES in medical diagnosis ,RATS ,RESEARCH ,EVALUATION research ,SECOND-generation antidepressants ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: The 5-HT(4) receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT(4) receptor-mediated signalling events.Experimental Approach: The effects of 21 days treatment (p.o.) with high (40 mg·kg(-1)) and low (10 mg·kg(-1)) doses of venlafaxine, were evaluated at different levels of 5-HT(4) receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg(-1), 21 days) was also evaluated on 5-HT(4) receptor density.Key Results: Treatment with a high dose (40 mg·kg(-1)) of venlafaxine did not alter 5-HT(4) mRNA expression, but decreased the density of 5-HT(4) receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg(-1)) while it was attenuated in rats treated with 40 mg·kg(-1) of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT(4) receptor density.Conclusions and Implications: Our data indicate a functional desensitization of 5-HT(4) receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake. [ABSTRACT FROM AUTHOR]- Published
- 2010
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13. Neuroprotective effects of andrographolide in a rat model of permanent cerebral ischaemia.
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Chan, Su Jing, Wong, WS Fred, Wong, Peter TH, Bian, Jin-Song, Wong, W S Fred, and Wong, Peter T H
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NEUROPROTECTIVE agents ,DITERPENES ,LABORATORY rats ,CEREBRAL ischemia ,ANTI-inflammatory agents ,ARTERIAL occlusions ,MICROGLIA ,HISTOLOGY ,TRANSCRIPTION factors ,NF-kappa B ,IMMUNOASSAY ,TUMOR necrosis factors ,PROSTAGLANDINS ,INTERLEUKINS ,CELL metabolism ,BIOLOGICAL models ,BRAIN ,RESEARCH ,INFARCTION ,ANIMAL experimentation ,DINOPROSTONE ,RESEARCH methodology ,INTERLEUKIN-1 ,MEDICAL cooperation ,EVALUATION research ,HYDROCARBONS ,RATS ,COMPARATIVE studies ,DNA-binding proteins ,PHARMACODYNAMICS - Abstract
Background and Purpose: Andrographolide is a diterpenoid lactone isolated from a traditional medicinal herb, Andrographis paniculata. It possesses potent anti-inflammatory activity. The present study examined potential therapeutic effects of andrographolide on cerebral ischaemia using a rat model with permanent middle cerebral artery occlusion (pMCAO).Experimental Approach: The MCA in rats was permanently occluded (by cautery), and 24 h later neurological effects were assessed with behavioural scores. Infarct volume and microglial activation were determined histologically. The p65 form of the transcription factor, nuclear factor-κB (NF-κB), was measured by Western blot, and cytokines by immunoassay of brain extracts.Key Results: Andrographolide, given i.p. 1 h after pMCAO, reduced infarct volume with a maximum reduction of approximately 50% obtained at 0.1 mg·kg(-1). Neurological deficits were also reduced by andrographolide, reflecting a correlation between infarct volume and neurological deficits. pMCAO was found to induce activation of microglia and elevate tumour necrosis factor (TNF)-α, interleukin (IL)-1β and prostaglandin (PG)E(2) in the ischaemic brain areas. Andrographolide (0.1 mg·kg(-1)) significantly attenuated or abolished these effects. In addition, andrographolide suppressed the translocation of p65 from cytosol to nucleus, indicating reduced NF-κB activation.Conclusions and Implications: Andrographolide exhibited neuroprotective effects, with accompanying suppression of NF-κB and microglial activation, and reduction in the production of cytokines including TNF-α and IL-1β, and pro-inflammatory factors such as PGE(2). Our findings suggest that andrographolide may have therapeutic value in the treatment of stroke. [ABSTRACT FROM AUTHOR]- Published
- 2010
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14. Differential effects of Kv11.1 activators on Kv11.1a, Kv11.1b and Kv11.1a/Kv11.1b channels.
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Larsen, AP, Bentzen, BH, Grunnet, M, Larsen, A P, and Bentzen, B H
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POTASSIUM channels ,TISSUES ,BRAIN ,HEART ,SMOOTH muscle ,XENOPUS laevis ,ELECTRIC potential ,ELECTRODES ,ION channels ,OVUM physiology ,ANIMAL experimentation ,BIOLOGICAL transport ,CARRIER proteins ,COMPARATIVE studies ,CYTOLOGICAL techniques ,GENETIC techniques ,RESEARCH methodology ,MEDICAL cooperation ,OVUM ,PHENOLS ,PIPERIDINE ,PROTEINS ,QUINOLINE ,RESEARCH ,UREA ,VERTEBRATES ,EVALUATION research ,PHARMACODYNAMICS ,PHYSIOLOGY - Abstract
Background and Purpose: K(v)11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K(v)11.1a and K(v)11.1b, with different kinetics, are expressed. K(v)11.1 activators are potential therapeutic agents, but their effects have only been tested on the K(v)11.1a isoform. In this study, the effects of two different K(v)11.1 activators, NS1643 and RPR260243, were characterized on K(v)11.1a and K(v)11.1b channels.Experimental Approach: K(v)11.1a and K(v)11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243.Key Results: NS1643 increased steady-state currents through Kv11.1b several fold more than through K(v)11.1a channels, without affecting EC(50) values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for K(v)11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on K(v)11.1b than on K(v)11.1a. Effects of both compounds on heteromeric K(v)11.1a/K(v)11.1b channels were similar to those on K(v)11.1a.Conclusions and Implications: Both NS1643 and RPR260243 displayed differential effects on K(v)11.1a and K(v)11.1b channels, the effects being relatively more pronounced on K(v)11.1b channels. This affirms the importance of testing the effect of K(v)11.1 activators on different channel isoforms. [ABSTRACT FROM AUTHOR]- Published
- 2010
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15. A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents.
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Cluny, NL, Vemuri, VK, Chambers, AP, Limebeer, CL, Bedard, H, Wood, JT, Lutz, B, Zimmer, A, Parker, LA, Makriyannis, A, Sharkey, KA, Cluny, N L, Vemuri, V K, Chambers, A P, Limebeer, C L, Wood, J T, Parker, L A, and Sharkey, K A
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CANNABINOIDS ,CELL receptors ,INGESTION ,BODY weight ,LABORATORY rodents ,CENTRAL nervous system ,OBESITY treatment ,BRAIN metabolism ,ANIMAL experimentation ,BRAIN ,COMPARATIVE studies ,CONDITIONED response ,CYCLIC adenylic acid ,DOSE-effect relationship in pharmacology ,EPITHELIAL cells ,GASTROINTESTINAL motility ,HETEROCYCLIC compounds ,HYDROCARBONS ,LEARNING ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,PIPERIDINE ,RATS ,RESEARCH ,EVALUATION research ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects.Experimental Approach: Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated.Key Results: AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice.Conclusions and Implications: Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications. [ABSTRACT FROM AUTHOR]- Published
- 2010
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16. Resistance to endotoxic shock in mice lacking natriuretic peptide receptor-A.
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Panayiotou, Catherine M, Baliga, Reshma, Stidwill, Raymond, Taylor, Valerie, Singer, Mervyn, and Hobbs, Adrian J
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SEPTIC shock ,ATRIAL natriuretic peptides ,NITRIC-oxide synthases ,MULTIPLE organ failure ,HYPOTENSION ,ENDOTOXINS ,LABORATORY mice ,ANIMAL experimentation ,BIOLOGICAL models ,BLOOD pressure ,VASODILATION ,CELL receptors ,COMPARATIVE studies ,CYTOKINES ,DOSE-effect relationship in pharmacology ,HEMODYNAMICS ,INFLAMMATORY mediators ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NITRIC oxide ,NUCLEOTIDES ,OXIDOREDUCTASES ,RESEARCH ,RESEARCH funding ,TIME ,VASOCONSTRICTORS ,VASODILATORS ,EVALUATION research ,VASOCONSTRICTION ,LIPOPOLYSACCHARIDES ,THORACIC aorta ,PHARMACODYNAMICS ,PREVENTION - Abstract
Background and Purpose: Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.Experimental Approach: Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.Key Results: LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO(x)] and cyclic GMP).Conclusions and Implications: These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition. [ABSTRACT FROM AUTHOR]- Published
- 2010
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17. Preferential in vivo action of F15599, a novel 5-HT(1A) receptor agonist, at postsynaptic 5-HT(1A) receptors.
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Lladó-Pelfort, L, Assié, M-B, Newman-Tancredi, A, Artigas, F, Celada, P, Lladó-Pelfort, L, and Assié, M-B
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ANTIDEPRESSANTS ,DRUG efficacy ,SEROTONIN ,MICRODIALYSIS ,COMPARATIVE studies ,PREFRONTAL cortex ,NEURONS ,BRAIN metabolism ,BRAIN ,FRONTAL lobe ,RESEARCH ,HIPPOCAMPUS (Brain) ,INJECTIONS ,NERVOUS system ,HETEROCYCLIC compounds ,TIME ,ANIMAL experimentation ,SEROTONIN antagonists ,RESEARCH methodology ,CELL receptors ,MEDICAL cooperation ,EVALUATION research ,PIPERIDINE ,HYDROCARBONS ,DOPAMINE ,RATS ,SEROTONIN agonists ,DOSE-effect relationship in pharmacology ,ACTION potentials ,HEMODIALYSIS ,BRAIN stem ,DOPAMINE antagonists ,ANTIPSYCHOTIC agents ,PHARMACODYNAMICS - Abstract
Background and Purpose: F15599, a novel 5-hydroxytryptamine (5-HT)(1A) receptor agonist with 1000-fold selectivity for 5-HT compared with other monoamine receptors, shows antidepressant and procognitive activity at very low doses in animal models. We examined the in vivo activity of F15599 at somatodendritic autoreceptors and postsynaptic 5-HT(1A) heteroreceptors.Experimental Approach: In vivo single unit and local field potential recordings and microdialysis in the rat.Key Results: F15599 increased the discharge rate of pyramidal neurones in medial prefrontal cortex (mPFC) from 0.2 microg x kg(-1) i.v and reduced that of dorsal raphe 5-hydroxytryptaminergic neurones at doses >10-fold higher (minimal effective dose 8.2 microg x kg(-1) i.v.). Both effects were reversed by the 5-HT(1A) antagonist (+/-)WAY100635. F15599 did not alter low frequency oscillations (approximately 1 Hz) in mPFC. In microdialysis studies, F15599 increased dopamine output in mPFC (an effect dependent on the activation of postsynaptic 5-HT(1A) receptors) with an ED(50) of 30 microg x kg(-1) i.p., whereas it reduced hippocampal 5-HT release (an effect dependent exclusively on 5-HT(1A) autoreceptor activation) with an ED(50) of 240 microg x kg(-1) i.p. Likewise, application of F15599 by reverse dialysis in mPFC increased dopamine output in a concentration-dependent manner. All neurochemical responses to F15599 were prevented by administration of (+/-)WAY100635.Conclusions and Implications: These results indicate that systemic administration of F15599 preferentially activates postsynaptic 5-HT(1A) receptors in PFC rather than somatodendritic 5-HT(1A) autoreceptors. This regional selectivity distinguishes F15599 from previously developed 5-HT(1A) receptor agonists, which preferentially activate somatodendritic 5-HT(1A) autoreceptors, suggesting that F15599 may be particularly useful in the treatment of depression and of cognitive deficits in schizophrenia. [ABSTRACT FROM AUTHOR]- Published
- 2010
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18. Symptomatic and neuroprotective effects following activation of nigral group III metabotropic glutamate receptors in rodent models of Parkinson's disease.
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Austin, PJ, Betts, MJ, Broadstock, M, O'Neill, MJ, Mitchell, SN, Duty, S, Austin, P J, Betts, M J, O'Neill, M J, and Mitchell, S N
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NEUROPROTECTIVE agents ,GLUTAMIC acid ,PARKINSON'S disease treatment ,SUBSTANTIA nigra ,MOVEMENT disorders ,MICRODIALYSIS ,LABORATORY rodents ,ASPARTIC acid metabolism ,DRUG therapy for Parkinson's disease ,GLUTAMIC acid metabolism ,AMINOBUTYRIC acid ,ANIMAL experimentation ,BIOLOGICAL models ,BRAIN stem ,CELL receptors ,COMPARATIVE studies ,HEMODIALYSIS ,IMMUNOHISTOCHEMISTRY ,RESEARCH methodology ,MEDICAL cooperation ,MOTOR ability ,PARKINSON'S disease ,RATS ,RESEARCH ,EVALUATION research ,SERINE ,EXCITATORY amino acid agonists ,PHARMACODYNAMICS ,THERAPEUTICS - Abstract
Background and Purpose: Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinson's disease (PD). This study aimed to establish whether activation of pre-synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD.Experimental Approach: Broad-spectrum group III mGlu receptor agonists, O-phospho-l-serine (l-SOP) and l-2-amino-4-phosphonobutyrate (l-AP4), were assessed for their ability to inhibit KCl-evoked [(3)H]-d-aspartate release in rat nigral prisms or inhibit KCl-evoked endogenous glutamate release in the SNpr in vivo using microdialysis. Reversal of akinesia in reserpine-treated rats was assessed following intranigral injection of l-SOP and l-AP4. Finally, the neuroprotective effect of 7 days' supra-nigral treatment with l-AP4 was examined in 6-hydroxydopamine (6-OHDA)-lesioned rats.Key Results: l-SOP and l-AP4 inhibited [(3)H]-d-aspartate release by 33 and 44% respectively. These effects were blocked by the selective group III mGlu antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). l-SOP also reduced glutamate release in the SNpr in vivo by 48%. Injection of l-SOP and l-AP4 into the SNpr reversed reserpine-induced akinesia. Following administration above the SNpc, l-AP4 provided neurochemical, histological and functional protection against 6-OHDA lesion of the nigrostriatal tract. Pretreatment with CPPG inhibited these effects.Conclusions and Implications: These findings highlight group III mGlu receptors in the SN as potential targets for providing both symptomatic and neuroprotective relief in PD, and indicate that inhibition of glutamate release in the SN may underlie these effects. [ABSTRACT FROM AUTHOR]- Published
- 2010
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19. Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation.
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Schwalm, Stephanie, Pfeilschifter, Josef, and Huwiler, Andrea
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SPHINGOSINE ,PROTEIN kinases ,NITRIC oxide ,ENDOTHELIUM ,CELL migration ,CELL proliferation ,CELL differentiation ,NUCLEOTIDE metabolism ,REVERSE transcriptase polymerase chain reaction ,BIOCHEMISTRY ,RESEARCH ,CELL culture ,NEOVASCULARIZATION ,WESTERN immunoblotting ,TIME ,RESEARCH methodology ,ORGANIC compounds ,RNA ,MEDICAL cooperation ,EVALUATION research ,CELL motility ,CARDIOVASCULAR agents ,PHENOMENOLOGY ,COMPARATIVE studies ,TRANSFERASES ,DOSE-effect relationship in pharmacology ,EPITHELIAL cells ,GENETIC techniques ,POLYMERASE chain reaction ,OXIDOREDUCTASES ,CELL lines ,PHARMACODYNAMICS - Abstract
Background and Purpose: Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.Experimental Approach: We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.Key Results: We showed that exposure of EA.hy926 cells to Deta-NO (125-1000 microM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 microM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.Conclusions and Implications: These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis. [ABSTRACT FROM AUTHOR]- Published
- 2010
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20. Depression-resistant endophenotype in mice overexpressing cannabinoid CB(2) receptors.
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García-Gutiérrez, MS, Pérez-Ortiz, JM, Gutiérrez-Adán, A, Manzanares, J, García-Gutiérrez, M S, Pérez-Ortiz, J M, and Gutiérrez-Adán, A
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MENTAL depression ,GENE expression ,CANNABINOIDS ,DRUG resistance ,PHENOTYPES ,LABORATORY mice ,ANTIDEPRESSANTS ,ANIMAL behavior ,ANIMAL experimentation ,ANXIETY ,BIOLOGICAL models ,CELL receptors ,COMPARATIVE studies ,FOOD habits ,HIPPOCAMPUS (Brain) ,IMMUNOHISTOCHEMISTRY ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NERVE tissue proteins ,RESEARCH ,RESTRAINT of patients ,PSYCHOLOGICAL stress ,SWIMMING ,EVALUATION research ,INDOLE compounds ,PHARMACODYNAMICS ,THERAPEUTICS - Abstract
Background and Purpose: The present study evaluated the role of CB(2) receptors in the regulation of depressive-like behaviours. Transgenic mice overexpressing the CB(2) receptor (CB2xP) were challenged with different types of acute and chronic experimental paradigms to evaluate their response in terms of depressive-like behaviours.Experimental Approach: Tail suspension test (TST), novelty-suppressed feeding test (NSFT) and unpredictable chronic mild stress tests (CMS) were carried out in CB2xP mice. Furthermore, acute and chronic antidepressant-like effects of the CB(2) receptor-antagonist AM630 were evaluated by means of the forced swimming test (FST) and CMS, respectively, in wild-type (WT) and CB2xP mice. CB(2) gene expression, brain-derived neurotrophic factor (BDNF) gene and protein expressions were studied in mice exposed to CMS by real-time PCR and immunohistochemistry, respectively.Key Results: Overexpression of CB(2) receptors resulted in decreased depressive-like behaviours in the TST and NSFT. CMS failed to alter the TST and sucrose consumption in CB2xP mice. In addition, no changes in BDNF gene and protein expression were observed in stressed CB2xP mice. Interestingly, acute administration of AM630 (1 and 3 mg x kg(-1), i.p.) exerted antidepressant-like effects on the FST in WT, but not in CB2xP mice. Chronic administration of AM630 for 4 weeks (1 mg x kg(-1); twice daily, i.p.) blocked the effects of CMS on TST, sucrose intake, CB(2) receptor gene, BDNF gene and protein expression in WT mice.Conclusion and Implications: Taken together, these results suggest that increased CB(2) receptor expression significantly reduced depressive-related behaviours and that the CB(2) receptor could be a new potential therapeutic target for depressive-related disorders. [ABSTRACT FROM AUTHOR]- Published
- 2010
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21. Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta.
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AL-Zobaidy, Mohammed J, Craig, John, and Martin, William
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ACETYLCHOLINE ,NITRIC oxide ,SENSES ,ARGININE ,ETHANES ,LABORATORY rats ,AORTA ,ANIMAL experimentation ,COMPARATIVE studies ,DOSE-effect relationship in pharmacology ,ENDOTHELIUM ,RESEARCH methodology ,MEDICAL cooperation ,RATS ,RESEARCH ,EVALUATION research ,PHENYLEPHRINE ,PHARMACODYNAMICS - Abstract
Background and Purpose: Previous work has shown that N(G)-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N(G), N(G)-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.Experimental Approach: The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.Key Results: ADMA (100 microM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300-1000 microM) had no greater effect. Although ADMA (100 microM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 microM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 microM), protected ACh-induced relaxation against blockade by l-NAME (30 microM).Conclusions and Implications: In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA. [ABSTRACT FROM AUTHOR]- Published
- 2010
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22. Block and allosteric modulation of GABAergic currents by oenanthotoxin in rat cultured hippocampal neurons.
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Wyrembek, Paulina, Lebida, Katarzyna, Mercik, Katarzyna, Szczuraszek, Katarzyna, Szczot, Marcin, Pollastro, Federica, Appendino, Giovanni, and Mozrzymas, Jerzy W
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ALLOSTERIC regulation ,GABA ,LABORATORY rats ,HIPPOCAMPUS (Brain) ,NEURONS ,POLYACETYLENES ,PHARMACOLOGY ,ALCOHOLS (Chemical class) ,ALKENES ,ANIMAL experimentation ,BIOCHEMISTRY ,BIOLOGICAL transport ,CELL culture ,CELL receptors ,COMPARATIVE studies ,CYTOLOGICAL techniques ,GABA antagonists ,PHENOMENOLOGY ,RESEARCH methodology ,MEDICAL cooperation ,NEURAL transmission ,PLANTS ,RATS ,RESEARCH ,EVALUATION research ,PHARMACODYNAMICS - Abstract
Background and Purpose: Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABA(A) receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX.Experimental Approach: Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches.Key Results: OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABA(A) receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs.Conclusions and Implications: OETX shows a complex blocking mechanism of GABA(A) receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX. [ABSTRACT FROM AUTHOR]- Published
- 2010
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23. The phospholipase C inhibitor U-73122 inhibits Ca(2+) release from the intracellular sarcoplasmic reticulum Ca(2+) store by inhibiting Ca(2+) pumps in smooth muscle.
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MacMillan, D, McCarron, JG, and McCarron, J G
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PHOSPHOLIPASE C ,ENZYME inhibitors ,CALCIUM ions ,SARCOPLASMIC reticulum ,SMOOTH muscle ,ION pumps ,PHOTOCHEMISTRY ,CALCIUM metabolism ,PROTEIN metabolism ,AMINES ,ANIMAL experimentation ,CAFFEINE ,CALCIUM ,CARRIER proteins ,CELLS ,COLON (Anatomy) ,COMPARATIVE studies ,CYTOLOGICAL techniques ,CYTOPLASM ,ESTERASES ,GUINEA pigs ,HETEROCYCLIC compounds ,RESEARCH methodology ,MEDICAL cooperation ,PARASYMPATHOMIMETIC agents ,PROTEINS ,RESEARCH ,RESEARCH funding ,STEROIDS ,EVALUATION research ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.Experimental Approach: Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine).Key Results: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release.Conclusions and Implications: U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC. [ABSTRACT FROM AUTHOR]- Published
- 2010
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24. Molecular determinants of state-dependent block of voltage-gated sodium channels by pilsicainide.
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Desaphy, J-F, Dipalma, A, Costanza, T, Bruno, C, Lentini, G, Franchini, C, George, AL, and Conte Camerino, D
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EPITOPES ,SODIUM channels ,MYOCARDIAL depressants ,BIOLOGY experiments ,GENE expression ,BRAIN metabolism ,MUSCLE protein metabolism ,BRAIN ,CELL lines ,COMPARATIVE studies ,CYTOLOGICAL techniques ,FLECAINIDE ,HEART ,LIDOCAINE ,RESEARCH methodology ,MEDICAL cooperation ,MEMBRANE proteins ,MEXILETINE ,MUSCLE proteins ,NERVE tissue proteins ,RESEARCH ,RESEARCH funding ,EVALUATION research ,SODIUM channel blockers ,SKELETAL muscle ,MEMBRANE transport proteins ,PHARMACODYNAMICS - Abstract
Background and Purpose: Pilsicainide, an anti-arrhythmic drug used in Japan, is described as a pure sodium channel blocker. We examined the mechanisms by which it is able to block open channels, because these properties may be especially useful to reduce hyperexcitability in pathologies characterized by abnormal sodium channel opening.Experimental Approach: The effects of pilsicainide on various heterologously expressed human sodium channel subtypes and mutants were investigated using the patch clamp technique.Key Results: Pilsicainide exhibited tonic and use-dependent effects comparable to those of mexiletine and flecainide on hNav1.4 channels. These use-dependent effects were abolished in the mutations F1586C and Y1593C within segment 6 of domain IV, suggesting that the interaction of pilsicainide with these residues is critical for its local anaesthetic action. Its affinity constants for closed channels (K(R)) and channels inactivated from the closed state (K(I)) were high, suggesting that its use-dependent block (UDB) requires the channel to be open for it to reach a high-affinity blocking site. Accordingly, basic pH, which slightly increased the proportion of neutral drug, dramatically decreased K(R) and K(I) values. Effects of pilsicainide were similar on skeletal muscle hNav1.4, brain hNav1.1 and heart hNav1.5 channels. The myotonic R1448C and G1306E hNav1.4 mutants were more and less sensitive to pilsicainide, respectively, due to mutation-induced gating modifications.Conclusions and Implications: Although therapeutic concentrations of pilsicainide may have little effect on resting and closed-state inactivated channels, it induces a strong UDB due to channel opening, rendering the drug ideally suited for inhibition of high-frequency action potential firing. [ABSTRACT FROM AUTHOR]- Published
- 2010
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25. Co-administration of ibuprofen and nitric oxide is an effective experimental therapy for muscular dystrophy, with immediate applicability to humans.
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Sciorati, Clara, Buono, Roberta, Azzoni, Emanuele, Casati, Silvana, Ciuffreda, Pierangela, D'Angelo, Grazia, Cattaneo, Dario, Brunelli, Silvia, and Clementi, Emilio
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IBUPROFEN ,NITRIC oxide ,BIOLOGY experiments ,MUSCULAR dystrophy treatment ,ADRENOCORTICAL hormones ,ANTI-inflammatory agents ,STEM cells ,ANIMAL experimentation ,ANIMAL diseases ,CARDIOVASCULAR agents ,COMPARATIVE studies ,CYTOSKELETAL proteins ,DRUG synergism ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NONSTEROIDAL anti-inflammatory agents ,RESEARCH ,RESEARCH funding ,EVALUATION research ,ISOSORBIDE dinitrate (Drug) ,PHARMACODYNAMICS - Abstract
Background and Purpose: Current therapies for muscular dystrophy are based on corticosteroids. Significant side effects associated with these therapies have prompted several studies aimed at identifying possible alternative strategies. As inflammation and defects of nitric oxide (NO) generation are key pathogenic events in muscular dystrophies, we have studied the effects of combining the NO donor isosorbide dinitrate (ISDN) and the non-steroidal anti-inflammatory drug ibuprofen.Experimental Approach: alpha-Sarcoglycan-null mice were treated for up to 8 months with ISDN (30 mg.kg(-1)) plus ibuprofen (50 mg.kg(-1)) administered daily in the diet. Effects of ISDN and ibuprofen alone were assessed in parallel. Drug effects on animal motility and muscle function, muscle damage, inflammatory infiltrates and cytokine levels, as well as muscle regeneration including assessment of endogenous stem cell pool, were measured at selected time points.Key Results: Combination of ibuprofen and ISDN stimulated regeneration capacity, of myogenic precursor cells, reduced muscle necrotic damage and inflammation. Muscle function in terms of free voluntary movement and resistance to exercise was maintained throughout the time window analysed. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination.Conclusions and Implications: Co-administration of NO and ibuprofen provided synergistic beneficial effects in a mouse model of muscular dystrophy, leading to an effective therapy. Our results open the possibility of immediate clinical testing of a combination of ISDN and ibuprofen in dystrophic patients, as both components are approved for use in humans, with a good safety profile. [ABSTRACT FROM AUTHOR]- Published
- 2010
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26. The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels.
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Singh, A, Hildebrand, ME, Garcia, E, Snutch, TP, Hildebrand, M E, and Snutch, T P
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TRP channels ,CALCIUM antagonists ,CALCIUM channels ,PURKINJE cells ,GENE expression ,BIOLOGY experiments ,MOLECULAR genetics ,CALCIUM metabolism ,ANIMAL experimentation ,CALCIUM ,CELL lines ,COMPARATIVE studies ,CYTOLOGICAL techniques ,IMIDAZOLES ,KIDNEYS ,RESEARCH methodology ,MEDICAL cooperation ,NEURONS ,RATS ,RESEARCH ,RESEARCH funding ,EVALUATION research ,PHARMACODYNAMICS - Abstract
Background and Purpose: SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents.Experimental Approach: Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed.Key Results: SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca(V)1, Ca(V)2 and Ca(V)3) at concentrations typically utilized to assay TRPC function (10 microM). Particularly, human Ca(V)3.1 T-type Ca channels were more potently inhibited by SKF (IC(50) approximately 560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca(V)3.1 T-type currents in a rat cerebellar PC slice preparation.Conclusions and Implications: SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues. [ABSTRACT FROM AUTHOR]- Published
- 2010
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27. AE9C90CB: a novel, bladder-selective muscarinic receptor antagonist for the treatment of overactive bladder.
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Sinha, S, Gupta, S, Malhotra, S, Krishna, NS, Meru, AV, Babu, V, Bansal, V, Garg, M, Kumar, N, Chugh, A, Ray, A, Krishna, N S, and Meru, A V
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OVERACTIVE bladder ,MUSCARINIC receptors ,ACETAMIDE ,SALIVARY glands ,DRUG antagonism ,OXYBUTYNIN (Drug) ,CLINICAL drug trials ,THERAPEUTICS ,BRAIN metabolism ,ANIMAL experimentation ,CELLS ,COMPARATIVE studies ,DOSE-effect relationship in pharmacology ,HAMSTERS ,INTRAVENOUS injections ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,PARASYMPATHOMIMETIC agents ,RABBITS ,RATS ,RESEARCH ,RODENTS ,EVALUATION research ,MUSCARINIC antagonists ,PHARMACODYNAMICS - Abstract
Background and Purpose: AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).Experimental Approach: Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.Key Results: In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M(3) muscarinic receptors with pKi of 9.90 +/- 0.11 and was 20-fold more selective for M(3) than for M(2) muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK(B), 9.13 +/- 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.Conclusions and Implications: We have identified AE9C90CB, a compound exhibiting moderate selectivity for M(3) over M(2) receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs. [ABSTRACT FROM AUTHOR]- Published
- 2010
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28. Enhancement of mesenteric artery contraction to 5-HT depends on Rho kinase and Src kinase pathways in the ob/ob mouse model of type 2 diabetes.
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Matsumoto, Takayuki, Kobayashi, Tsuneo, Ishida, Keiko, Taguchi, Kumiko, and Kamata, Katsuo
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MESENTERIC artery ,MUSCLE contraction ,SEROTONIN ,PROTEIN kinases ,LABORATORY mice ,TYPE 2 diabetes ,GENE expression ,CYCLOOXYGENASES ,IN vitro studies ,RESEARCH ,PHOSPHOTRANSFERASES ,ANIMAL experimentation ,RESEARCH methodology ,DIABETES ,MEDICAL cooperation ,EVALUATION research ,CELLULAR signal transduction ,COMPARATIVE studies ,TRANSFERASES ,DOSE-effect relationship in pharmacology ,OXIDOREDUCTASES ,MICE ,ENZYME inhibitors ,CARRIER proteins ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identified the molecular mechanisms involved.Experimental Approach: Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay.Key Results: Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups.Conclusions and Implications: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase. [ABSTRACT FROM AUTHOR]- Published
- 2010
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29. Cell context-dependent activities of parthenolide in primary and metastatic melanoma cells.
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Czyz, M, Lesiak-Mieczkowska, K, Koprowska, K, Szulawska-Mroczek, A, and Wozniak, M
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SESQUITERPENE lactones ,METASTASIS ,MELANOMA ,CISPLATIN ,CELL lines ,FLUORESCENCE microscopy ,APOPTOSIS ,PROTEIN metabolism ,BIOCHEMISTRY ,RESEARCH ,CLINICAL drug trials ,RESEARCH methodology ,ANTINEOPLASTIC agents ,CELL physiology ,MEDICAL cooperation ,EVALUATION research ,HYDROCARBONS ,CELL cycle ,PHENOMENOLOGY ,CELL motility ,COMPARATIVE studies ,DNA-binding proteins ,DOSE-effect relationship in pharmacology ,PHARMACODYNAMICS - Abstract
Background and Purpose: Growing evidence implicates NF-kappaB as an important contributor to metastasis and increased chemoresistance of melanoma. Here, we report the effects of parthenolide on either untreated, cisplatin- or TNFalpha-treated melanoma cell lines A375, 1205Lu and WM793, exhibiting different levels of constitutive NF-kappaB activity.Experimental Approach: Electrophoretic mobility shift assay was used to assess changes in NF-kappaB activity, and real-time PCR to evaluate expression of NF-kappaB-regulated genes. Cell cycle arrest and apoptosis were assessed by flow cytometry. Cell death was also visualized by fluorescence microscopy. Migration was determined by scratch assay and invasiveness by Matrigel assay.Key Results: Parthenolide suppressed both constitutive and induced NF-kappaB activity in melanoma cells. This was accompanied by down-regulation of cancer-related genes, with NF-kappaB-binding sites in their promoters, including: Bcl-X(L), survivin, cyclin D1, interleukin 8 and matrix metalloproteinase 9. When the various effects of 6 microM parthenolide were compared, apoptosis associated with loss of mitochondrial membrane potential was most efficiently induced in 1205Lu cells, cell cycle arrest in G(0)/G(1) phase was observed in WM793 cells, and high metastatic potential was markedly reduced in A375 cells. These findings not only reflected differences between melanoma cell lines in basal expression of NF-kappaB-regulated genes, but also suggested other parthenolide targets involved in cell cycle progression, migration, invasiveness and survival.Conclusions: Inhibition of constitutive and therapeutically induced NF-kappaB pathway by parthenolide might be useful in the treatment of melanoma, although the diversity of changes induced in melanoma cells with different genetic backgrounds indicate context-dependent poly-pharmacological properties of this compound. [ABSTRACT FROM AUTHOR]- Published
- 2010
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30. Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase Calpha-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1.
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Hwang, Yong P and Jeong, Hye G
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METFORMIN ,CANCER cells ,CELL migration ,METALLOPROTEINASES ,PROTEIN kinases ,PHOSPHORYLATION ,MITOGEN-activated protein kinases ,CALCIUM metabolism ,PROTEINS ,RESEARCH ,CARCINOGENS ,CLINICAL drug trials ,CANCER invasiveness ,RESEARCH methodology ,PROTEOLYTIC enzymes ,ANTINEOPLASTIC agents ,MEDICAL cooperation ,EVALUATION research ,CELLULAR signal transduction ,CELL motility ,COMPARATIVE studies ,TRANSFERASES ,DOSE-effect relationship in pharmacology ,CELL lines ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: Population studies have revealed that treatment with the anti-diabetic drug metformin is significantly associated with reduced cancer risk, but the underlying mode of action has not been elucidated. The aim of our study was to determine the effect of metformin on tumour invasion and migration, and the possible mechanisms, using human fibrosarcoma HT-1080 cells.Experimental Approach: We employed invasion, migration and gelatin zymography assays to characterize the effect of metformin on HT-1080 cells. Transient transfection assays were performed to gene promoter activities, and immunoblot analysis to study its molecular mechanisms of action.Key Results: Metformin inhibited migration and invasion by HT-1080 cells at sub-toxic concentrations. In these cells, metformin also suppressed phorbol-12-myristate-13-acetate (PMA)-enhanced levels of matrix metalloproteinases-9 (MMP-9) protein, mRNA and transcription activity through suppression of activator protein-1 (AP-1) activation. In addition, metformin strongly repressed the PMA-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and protein kinase C(PKC)alpha, whereas the phosphorylation of p38 mitogen-activated protein kinase was not affected by metformin. Metformin decreased the PMA-induced Ca(2+) influx. Furthermore, treatment with an intracellular Ca(2+) chelator (BAPTA-AM) or a selective calmodulin antagonist (W7) markedly decreased PMA-induced MMP-9 secretion and cell migration, as well as activation of ERK and JNK/AP-1.Conclusions and Implications: Metformin inhibited PMA-induced invasion and migration of human fibrosarcoma cells via Ca(2+)-dependent PKCalpha/ERK and JNK/AP-1-signalling pathways. Metformin therefore has the potential to be a potent anti-cancer drug in therapeutic strategies for fibrosarcoma metastasis. [ABSTRACT FROM AUTHOR]- Published
- 2010
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31. Effects of cytochrome P450 3A (CYP3A) and the drug transporters P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) on the pharmacokinetics of lopinavir.
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Van Waterschoot, RAB, Ter Heine, R, Wagenaar, E, Van Der Kruijssen, CMM, Rooswinkel, RW, Huitema, ADR, Beijnen, JH, Schinkel, AH, van Waterschoot, R A B, van der Kruijssen, C M M, Rooswinkel, R W, Huitema, A D R, Beijnen, J H, and Schinkel, A H
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CYTOCHROME P-450 ,P-glycoprotein ,PHARMACOKINETICS ,LOPINAVIR-ritonavir ,BIOAVAILABILITY ,BIOLOGY experiments ,ANIMAL experimentation ,CARRIER proteins ,COMPARATIVE studies ,DOGS ,DRUG interactions ,GLYCOPROTEINS ,HETEROCYCLIC compounds ,INTESTINES ,LIVER ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,OXIDOREDUCTASES ,RESEARCH ,EVALUATION research ,HIV protease inhibitors ,RITONAVIR ,PHARMACODYNAMICS - Abstract
Background and Purpose: Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2.Experimental Approach: Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/- ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine.Key Results: Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration - time curve (AUC)(oral) was increased in Abcb1a/b(-/-) mice (approximately ninefold vs. wild-type) but not in Abcc2(-/-) mice. Increased lopinavir AUC(oral) (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a(-/-)) mice compared with wild-type mice. No difference in AUC(oral) between Cyp3a(-/-) and Cyp3a/Abcb1a/b/Abcc2(-/-) mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC(oral) (>100-fold), compared with Cyp3a(-/-) mice. Ritonavir markedly increased lopinavir AUC(oral) in all CYP3A-containing mouse strains.Conclusions and Implications: CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity. [ABSTRACT FROM AUTHOR]- Published
- 2010
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32. Mechanisms mediating the ability of caffeine to influence MDMA ('Ecstasy')-induced hyperthermia in rats.
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Vanattou-Saïfoudine, N, McNamara, R, Harkin, A, and Vanattou-Saïfoudine, N
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CAFFEINE ,FEVER ,ECSTASY (Drug) ,DRUG administration ,CATECHOLAMINES ,DOPAMINE ,KETANSERIN ,LABORATORY rats ,FRONTAL lobe ,ADRENERGIC alpha blockers ,RESEARCH ,NEURONS ,TIME ,ANIMAL experimentation ,RESEARCH methodology ,SEROTONIN ,NEUROTRANSMITTERS ,MEDICAL cooperation ,EVALUATION research ,ADRENERGIC uptake inhibitors ,RATS ,COMPARATIVE studies ,DRUG interactions ,DRUGS ,HYPOTHALAMUS ,BODY temperature regulation ,DOPAMINE agents ,PHOSPHODIESTERASE inhibitors ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: Caffeine exacerbates the hyperthermia associated with an acute exposure to 3,4 methylenedioxymethamphetamine (MDMA, 'Ecstasy') in rats. The present study investigated the mechanisms mediating this interaction.Experimental Approach: Adult male Sprague-Dawley rats were treated with caffeine (10 mg x kg(-1); i.p.) and MDMA (15 mg x kg(-1); i.p.) alone and in combination. Core body temperatures were monitored before and after drug administration.Key Results: Central catecholamine depletion blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Caffeine provoked a hyperthermic response when the catecholamine releaser d-amphetamine (1 mg x kg(-1)) was combined with the 5-HT releaser D-fenfluramine (5 mg x kg(-1)) or the non-selective dopamine receptor agonist apomorphine (1 mg x kg(-1)) was combined with the 5-HT(2) receptor agonist DOI (2 mg x kg(-1)) but not following either agents alone. Pretreatment with the dopamine D(1) receptor antagonist Schering (SCH) 23390 (1 mg x kg(-1)), the 5-HT(2) receptor antagonist ketanserin (5 mg x kg(-1)) or alpha(1)-adreno- receptor antagonist prazosin (0.2 mg x kg(-1)) blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Co-administration of a combination of MDMA with the PDE-4 inhibitor rolipram (0.025 mg x kg(-1)) and the adenosine A(1/2) receptor antagonist 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-C]quinazolin-5-amine 15943 (10 mg x kg(-1)) or the A(2A) receptor antagonist SCH 58261 (2 mg x kg(-1)) but not the A(1) receptor antagonist DPCPX (10 mg x kg(-1)) exacerbated MDMA-induced hyperthermia.Conclusions and Implications: A mechanism comprising 5-HT and catecholamines is proposed to mediate MDMA-induced hyperthermia. A combination of adenosine A(2A) receptor antagonism and PDE inhibition can account for the exacerbation of MDMA-induced hyperthermia by caffeine. [ABSTRACT FROM AUTHOR]- Published
- 2010
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33. Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety.
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Samer, CF, Daali, Y, Wagner, M, Hopfgartner, G, Eap, CB, Rebsamen, MC, Rossier, MF, Hochstrasser, D, Dayer, P, Desmeules, JA, Samer, C F, Eap, C B, Rebsamen, M C, Rossier, M F, and Desmeules, J A
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GENETIC polymorphisms ,DRUG interactions ,OXYCODONE ,DRUG efficacy ,PHARMACODYNAMICS ,KETOCONAZOLE ,ORAL drug administration ,CYTOCHROME P-450 ,THERAPEUTIC use of narcotics ,ANALGESICS ,BIOTRANSFORMATION (Metabolism) ,CLINICAL trials ,COMPARATIVE studies ,CROSSOVER trials ,ENZYME inhibitors ,RESEARCH methodology ,MEDICAL cooperation ,PSYCHOLOGY of movement ,NARCOTICS ,OXIDOREDUCTASES ,REFLEXES ,RESEARCH ,PHENOTYPES ,EVALUATION research ,RANDOMIZED controlled trials ,BLIND experiment ,PAIN threshold ,GENOTYPES ,CHEMICAL inhibitors ,THERAPEUTICS - Abstract
Background and Purpose: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored.Experimental Approach: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed.Key Results: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers.Conclusions and Implications: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism. [ABSTRACT FROM AUTHOR]- Published
- 2010
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34. The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase.
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Chung, Hui-Hsuan, Dai, Zen-Kong, Wu, Bin-Nan, Yeh, Jwu-Lai, Chai, Chee-Yin, Chu, Koung-Shing, Liu, Chung-Pin, and Chen, Ing-Jun
- Subjects
PULMONARY hypertension ,XANTHINE ,ENZYME inhibitors ,NITRIC-oxide synthases ,GENE expression ,PHOSPHORYLATION ,LABORATORY rats ,PULMONARY hypertension prevention ,CELL metabolism ,NUCLEOTIDE metabolism ,ANTIHYPERTENSIVE agents ,IN vitro studies ,RESEARCH ,CELL culture ,ENDOTHELIUM ,PHOSPHOTRANSFERASES ,LUNGS ,ANIMAL experimentation ,RIGHT ventricular hypertrophy ,RESEARCH methodology ,PULMONARY artery ,MEDICAL cooperation ,EVALUATION research ,PIPERIDINE ,RATS ,CELLULAR signal transduction ,NUCLEOTIDES ,VASODILATION ,COMPARATIVE studies ,TRANSFERASES ,CELLS ,OXIDOREDUCTASES ,ESTERASES ,CARRIER proteins ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats.Experimental Approach: We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats.Key Results: In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 microM) or the thromboxane A(2)-mimetic U46619 (0.5 microM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 microg x kg(-1) x min(-1), 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca(2+) efflux and angiotensin II-induced Ca(2+) influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression.Conclusion and Implications: KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca(2+) desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH. [ABSTRACT FROM AUTHOR]- Published
- 2010
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35. The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone.
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Samer, CF, Daali, Y, Wagner, M, Hopfgartner, G, Eap, CB, Rebsamen, MC, Rossier, MF, Hochstrasser, D, Dayer, P, Desmeules, JA, Samer, C F, Eap, C B, Rebsamen, M C, Rossier, M F, and Desmeules, J A
- Subjects
CYTOCHROME P-450 ,PHARMACOKINETICS ,GENETIC polymorphisms ,OXYCODONE ,OXIDATION ,QUINIDINE ,ENZYME inhibitors ,KETOCONAZOLE ,ANALGESICS ,BIOTRANSFORMATION (Metabolism) ,CELL receptors ,CLINICAL trials ,COMPARATIVE studies ,CROSSOVER trials ,DRUG interactions ,RESEARCH methodology ,MEDICAL cooperation ,NARCOTICS ,OXIDOREDUCTASES ,RESEARCH ,PHENOTYPES ,EVALUATION research ,RANDOMIZED controlled trials ,BLIND experiment ,GENOTYPES ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug-drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.Experimental Approach: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg x kg(-1) and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session.Key Results: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C(max) (-0.71 < Spearman correlation coefficient rhos < -0.92). Oxymorphone C(max) was 62% and 75% lower in PM than EM and UM. Noroxymorphone C(max) reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C(max) by 40% and 80%, and increased noroxycodone AUC(infinity) by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC(infinity) and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition.Conclusions and Implications: Drug-drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype. [ABSTRACT FROM AUTHOR]- Published
- 2010
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36. GPR55 ligands promote receptor coupling to multiple signalling pathways.
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Henstridge, Christopher M, Balenga, Nariman AB, Schröder, Ralf, Kargl, Julia K, Platzer, Wolfgang, Martini, Lene, Arthur, Simon, Penman, June, Whistler, Jennifer L, Kostenis, Evi, Waldhoer, Maria, Irving, Andrew J, and Schröder, Ralf
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LIGANDS (Biochemistry) ,DRUG receptors ,CELLULAR signal transduction ,LYSOPHOSPHOLIPIDS ,CANNABINOIDS ,ENZYME inhibitors ,BIOLOGICAL assay ,CELL receptors ,CELLS ,COMPARATIVE studies ,ENDOCYTOSIS ,HETEROCYCLIC compounds ,HYDROCARBONS ,RESEARCH methodology ,MEDICAL cooperation ,PHOSPHOLIPIDS ,PIPERIDINE ,RESEARCH ,RESEARCH funding ,EVALUATION research ,PHARMACODYNAMICS - Abstract
Background and Purpose: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alpha-lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55.Experimental Approach: We evaluated Ca(2+) signalling, stimulation of extracellular signal regulated kinase (ERK1/2) mitogen activated kinase MAP-kinases, induction of transcriptional regulators that are downstream of GPR55, including nuclear factor of activated T cells (NFAT), nuclear factor-kappaB (NF-kappaB) and cAMP response element binding protein (CREB), as well as receptor endocytosis. In addition, we assessed the suitability of a novel, label-free assay for GPR55 ligands that involves optical measurement of dynamic mass redistribution following receptor activation.Key Results: GPR55 linked to a range of downstream signalling events and that the activity of GPR55 ligands was influenced by the functional assay employed, with differences in potency and efficacy observed.Conclusions and Implications: Our data help to resolve some of the issues surrounding the pharmacology of cannabinoid ligands at GPR55 and highlight some differences in effector coupling associated with distinct GPR55 ligands. [ABSTRACT FROM AUTHOR]- Published
- 2010
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37. Effects of COX-2 inhibition on spinal nociception: the role of endocannabinoids.
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Staniaszek, LE, Norris, LM, Kendall, DA, Barrett, DA, Chapman, V, Staniaszek, L E, Norris, L M, Kendall, D A, and Barrett, D A
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CYCLOOXYGENASE 2 inhibitors ,NOCICEPTORS ,CANNABINOIDS ,DRUG receptors ,ANTI-inflammatory agents ,ENZYME inhibitors ,FATTY acids ,HYDROLASES ,DRUG metabolism ,NEURAL physiology ,SPINAL cord physiology ,ANIMAL experimentation ,CELL receptors ,COMPARATIVE studies ,DRUGS ,DRUG administration ,DOSE-effect relationship in pharmacology ,EVOKED potentials (Electrophysiology) ,HETEROCYCLIC compounds ,RESEARCH methodology ,MEDICAL cooperation ,NEUROTRANSMITTERS ,OXIDOREDUCTASES ,PAIN ,PIPERIDINE ,RATS ,RESEARCH ,SPINAL cord ,SULFONAMIDES ,EVALUATION research ,PHARMACODYNAMICS ,CELL physiology - Abstract
Background and Purpose: Recent studies suggest that the effects of cyclooxygenase-2 (COX-2) inhibition are mediated by cannabinoid receptor activation. However, some non-steroidal anti-inflammatory drugs inhibit the enzyme fatty acid amide hydrolase, which regulates levels of some endocannabinoids. Whether COX-2 directly regulates levels of endocannabinoids in vivo is unclear. Here, the effect of the COX-2 inhibitor nimesulide, which does not inhibit fatty acid amide hydrolase, on spinal nociceptive processing was determined. Effects of nimesulide on tissue levels of endocannabinoids and related compounds were measured and the role of cannabinoid 1 (CB(1)) receptors was determined.Experimental Approach: Effects of spinal and peripheral administration of nimesulide (1-100 microg per 50 microL) on mechanically evoked responses of rat dorsal horn neurones were measured, and the contribution of the CB(1) receptor was determined with the antagonist AM251 (N-(piperidin-1-yl)-5-(-4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), in anaesthetized rats. Effects of nimesulide on spinal levels of endocannabinoids and related compounds were quantified using liquid chromatography-tandem mass spectrometry.Key Results: Spinal, but not peripheral, injection of nimesulide (1-100 microg per 50 microL) significantly reduced mechanically evoked responses of dorsal horn neurones. Inhibitory effects of spinal nimesulide were blocked by the CB(1) receptor antagonist AM251 (1 microg per 50 microL), but spinal levels of endocannabinoids were not elevated. Indeed, both anandamide and N-oleoylethanolamide (OEA) were significantly decreased by nimesulide.Conclusions and Implications: Although the inhibitory effects of COX-2 blockade on spinal neuronal responses by nimesulide were dependent on CB(1) receptors, we did not detect a concomitant elevation in anandamide or 2-AG. Further understanding of the complexities of endocannabinoid catabolism by multiple enzymes is essential to understand their contribution to COX-2-mediated analgesia. [ABSTRACT FROM AUTHOR]- Published
- 2010
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38. Vasorelaxation to N-oleoylethanolamine in rat isolated arteries: mechanisms of action and modulation via cyclooxygenase activity.
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Wheal, AJ, Alexander, SPH, Randall, MD, Wheal, A J, Alexander, S P H, and Randall, M D
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BIOCHEMICAL mechanism of action ,ETHANOLAMINES ,LABORATORY rats ,CYCLOOXYGENASES ,SMALL intestine ,DRUG receptors ,SENSORY neurons ,INTRACELLULAR calcium ,ENDOTHELIUM physiology ,MESENTERIC artery physiology ,AMIDES ,ANIMAL experimentation ,ARACHIDONIC acid ,VASODILATION ,CAFFEINE ,CAPSAICIN ,CELL receptors ,COMPARATIVE studies ,DRUGS ,ENDOTHELIUM ,HETEROCYCLIC compounds ,INDOMETHACIN ,RESEARCH methodology ,MEDICAL cooperation ,MEMBRANE proteins ,MESENTERIC artery ,NEUROTRANSMITTERS ,NITRIC oxide ,OXIDOREDUCTASES ,PIPERIDINE ,RATS ,RESEARCH ,RESEARCH funding ,SENSORY receptors ,UNSATURATED fatty acids ,EVALUATION research ,POTASSIUM antagonists ,IN vitro studies ,THORACIC aorta ,CHEMICAL inhibitors ,PHARMACODYNAMICS ,PHYSIOLOGY - Abstract
Background and Purpose: The endocannabinoid-like molecule N-oleoylethanolamine (OEA) is found in the small intestine and regulates food intake and promotes weight loss. The principal aim of the present study was to evaluate the vascular effects of OEA.Experimental Approach: Perfused isolated mesenteric arterial beds were pre-contracted with methoxamine or high potassium buffers and concentration-response curves to OEA were constructed. Combinations of inhibitors to block nitric oxide production, sensory nerve activity, cyclooxygenase activity, potassium channels, chloride channels and gap junctions, and a cannabinoid CB(1) receptor antagonist, were used during these experiments. The effects of OEA on caffeine-induced contractions in calcium-free buffer were also assessed. Isolated thoracic aortic rings were used as a comparison.Key Results: OEA caused concentration-dependent vasorelaxation in rat isolated mesenteric arterial beds and thoracic aortic rings, with a greater maximal response in mesenteric vessels. This relaxation was sensitive to inhibition of sensory nerve activity and endothelial removal in both preparations. The cyclooxygenase inhibitor indomethacin reversed the effects of capsaicin pre-treatment in perfused mesenteric arterial beds and indomethacin alone enhanced vasorelaxation to OEA. The OEA-induced vasorelaxation was inhibited by a CB(1) receptor antagonist only in aortic rings. In mesenteric arteries, OEA suppressed caffeine-induced contractions in calcium-free buffer.Conclusions and Implications: The vasorelaxant effects of OEA are partly dependent on sensory nerve activity and a functional endothelium in the vasculature. In addition, vasorelaxation to OEA is enhanced following cyclooxygenase inhibition. OEA may also interfere with the release of intracellular calcium in arterial preparations. [ABSTRACT FROM AUTHOR]- Published
- 2010
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39. Selective alpha7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis.
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Snoek, Susanne A, Verstege, Marleen I, Van Der Zanden, Esmerij P, Deeks, Nigel, Bulmer, David C, Skynner, Michael, Lee, Kevin, Te Velde, Anje A, Boeckxstaens, Guy E, and De Jonge, Wouter J
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CHOLINERGIC receptors ,COLITIS ,VAGUS nerve ,INFLAMMATION ,DRUG dosage ,CYTOKINES ,TARGETED drug delivery ,LABORATORY mice ,ANIMAL experimentation ,BIOLOGICAL models ,CELL culture ,COMPARATIVE studies ,DOSE-effect relationship in pharmacology ,HYDROCARBONS ,INJECTIONS ,MACROPHAGES ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NICOTINE ,RESEARCH ,DNA-binding proteins ,EVALUATION research ,SEVERITY of illness index ,NICOTINIC agonists ,PHARMACODYNAMICS - Abstract
Background and Purpose: In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis.Experimental Approach: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored.Key Results: Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis.Conclusions and Implications: Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis. [ABSTRACT FROM AUTHOR]- Published
- 2010
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40. Structurally diverse amphiphiles exhibit biphasic modulation of GABAA receptors: similarities and differences with neurosteroid actions.
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Chisari, M, Shu, H-J, Taylor, A, Steinbach, JH, Zorumski, CF, Mennerick, S, Steinbach, J H, Zorumski, C F, and Mennerick, Steven
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AMPHIPHILES ,GABA receptors ,STEROID drugs ,NEUROLOGICAL disorders ,THERAPEUTICS ,DRUG synergism ,DRUG antagonism ,PHARMACOLOGY ,PHARMACEUTICAL chemistry ,OVUM physiology ,ANIMAL experimentation ,BIOCHEMISTRY ,CAPSAICIN ,CELL receptors ,COMPARATIVE studies ,DRUG interactions ,DRUGS ,GLYCOSIDES ,PHENOMENOLOGY ,RESEARCH methodology ,MEDICAL cooperation ,GENETIC mutation ,NEUROTRANSMITTERS ,OVUM ,RESEARCH ,RESEARCH funding ,SURFACE active agents ,VERTEBRATES ,DOCOSAHEXAENOIC acid ,EVALUATION research ,GABA modulators ,PHARMACODYNAMICS ,CELL physiology - Abstract
Background and Purpose: Some neurosteroids, notably 3alpha-hydroxysteroids, positively modulate GABA(A) receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions.Experimental Approach: Xenopus laevis oocytes were challenged with Triton X-100, octyl-beta-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations.Key Results: These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 microM) promoted potentiation and a high concentration (20 microM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (alpha1V256S) and positive (alpha1Q241L or alpha1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in alpha1V256S-mutated receptors, but potentiation was unaffected. In alpha1Q241L- or alpha1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact.Conclusions and Implications: Structurally diverse amphiphiles have antagonist actions at GABA(A) receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA(A) receptors does not have a clear pharmacophore requirement. [ABSTRACT FROM AUTHOR]- Published
- 2010
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41. Beta-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA.
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Stuenæs, Jorid T, Bolling, Astrid, Ingvaldsen, Ada, Rommundstad, Camilla, Sudar, Emina, Lin, Fang-Chin, Lai, Yu-Chiang, Jensen, Jørgen, Stuenaes, Jorid T, and Jensen, Jørgen
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ADRENERGIC receptors ,INSULIN ,PHOSPHORYLATION ,LABORATORY rats ,HEART cells ,GENETICS ,SKELETAL muscle ,CELLULAR signal transduction ,CELL metabolism ,ADRENERGIC beta agonists ,BIOCHEMISTRY ,RESEARCH ,CYCLIC adenylic acid ,DOBUTAMINE ,ANIMAL experimentation ,ISOPROTERENOL ,RESEARCH methodology ,BETA adrenoceptors ,MEDICAL cooperation ,EVALUATION research ,RATS ,PHENOMENOLOGY ,COMPARATIVE studies ,TRANSFERASES ,CELLS ,DRUG synergism ,CALCIUM-binding proteins ,PHARMACODYNAMICS - Abstract
Background and Purpose: Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and beta-adrenergic stimulation in regulation of PKB phosphorylation.Experimental Approach: Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies.Key Results: Isoprenaline increased insulin-stimulated PKB Ser(473) and Thr(308) phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3beta Ser(9) phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (beta(1)-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (beta(2)-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser(16) phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N(6)-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N(6)-benzoyl-cAMP, but not 007, increased phospholamban Ser(16) phosphorylation.Conclusions and Implications: beta-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during beta-adrenergic receptors stimulation. [ABSTRACT FROM AUTHOR]- Published
- 2010
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42. The melanocortin MC(1) receptor agonist BMS-470539 inhibits leucocyte trafficking in the inflamed vasculature.
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Leoni, G, Voisin, M-B, Carlson, K, Getting, SJ, Nourshargh, S, and Perretti, M
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MELANOCORTIN receptors ,LEUCOCYTES ,BLOOD vessels ,PHARMACEUTICAL research ,ANTI-inflammatory agents ,DRUG development ,MICROCIRCULATION ,LABORATORY mice ,ANIMAL experimentation ,CELL physiology ,CELL receptors ,COMPARATIVE studies ,CYTOKINES ,IMIDAZOLES ,INFLAMMATORY mediators ,RESEARCH methodology ,MEDICAL cooperation ,MESENTERIC blood vessels ,MICE ,PHOSPHOLIPIDS ,RESEARCH ,VASCULITIS ,EVALUATION research ,PHARMACODYNAMICS - Abstract
Background and Purpose: Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC(1)) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC(1) receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds.Experimental Approach: Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC(1) receptors (the recessive yellow e/e colony).Key Results: BMS-470539, given before an ischaemia-reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC(1) receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC(1) receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor.Conclusions and Implications: Activation of MC(1) receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC(1) receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions. [ABSTRACT FROM AUTHOR]- Published
- 2010
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43. Amitriptyline does not block the action of ATP at human P2X4 receptor.
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Sim, JA, North, RA, Sim, J A, and North, R A
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AMITRIPTYLINE ,ADENOSINE triphosphate ,NEUROPATHY ,PAIN management ,MICROGLIA ,LABORATORY rats ,RNA ,ANALGESIA ,ANALGESICS ,ANIMAL experimentation ,ANTIDEPRESSANTS ,CELL lines ,CELL receptors ,COMPARATIVE studies ,CYTOLOGICAL techniques ,DRUGS ,IMMUNITY ,RESEARCH methodology ,MEDICAL cooperation ,MICE ,NEUROTRANSMITTERS ,RATS ,RESEARCH ,EVALUATION research ,CHEMICAL inhibitors ,PHARMACODYNAMICS - Abstract
Background and Purpose: Amitriptyline is a tricyclic antidepressant that is also widely used to treat neuropathic pain in humans, but the mechanism of this anti-hyperalgesic effect is unknown. Microglia in the mouse spinal cord become activated in neuropathic pain, and expression of P2X4 receptors by these microglia is increased. Antisense RNA targeting P2X4 receptors suppresses the development of tactile allodynia in rats. This suggests that blockade of P2X4 receptors might be the mechanism by which amitriptyline relieves neuropathic pain.Experimental Approach: We expressed human, rat and mouse P2X receptors (P2X2, P2X4, P2X7) in human embryonic kidney cells and evoked inward currents by applying ATP. We compared the action of ATP on control cells and cells treated with amitriptyline.Key Results: Amitriptyline (10 microM), either applied acutely or by pre-incubation for 2-6 h, had no effect on inward currents evoked by ATP (0.3-100 microM) at human P2X4 receptors. At rat and mouse receptors, amitriptyline (10 microM) caused a modest reduction in the maximum responses to ATP, without changes in EC(50) values, but it had no effect at 1 microM. Amitriptyline also had no effects on currents evoked by ATP at rat P2X2 receptors, or at rat or human P2X7 receptors.Conclusion and Implications: The results do not support the view that amitriptyline owes its pain-relieving actions in man to the direct blockade of P2X4 receptors. [ABSTRACT FROM AUTHOR]- Published
- 2010
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44. Impaired transactivation of the human CYP2J2 arachidonic acid epoxygenase gene in HepG2 cells subjected to nitrative stress.
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Cui, Pei H, Lee, Andy C, Zhou, Fanfan, and Murray, Michael
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CYTOCHROME P-450 ,ARACHIDONIC acid ,ENZYMES ,CANCER cell proliferation ,LIVER cancer ,PHYSIOLOGICAL stress ,SODIUM nitroferricyanide ,APOPTOSIS ,BIOCHEMISTRY ,RESEARCH ,ELECTROPHORESIS ,RESEARCH methodology ,NITRATES ,MEDICAL cooperation ,EVALUATION research ,NUCLEOTIDES ,PHENOMENOLOGY ,DNA probes ,COMPARATIVE studies ,GENES ,TRANSFERASES ,POLYMERASE chain reaction ,CELL lines ,HEMOPROTEINS ,PHARMACODYNAMICS - Abstract
Background and Purpose: Human cytochrome P450 2J2 (CYP2J2) generates epoxyfatty acids that modulate cellular apoptosis and proliferation. CYP2J2 regulation has not been intensively studied but induction of the activator protein-1 (AP-1) subunit c-fos mediates CYP2J2 down-regulation in hypoxia, a component of ischaemic injury. Decreased CYP2J2 expression may contribute to tissue injury.Experimental Approach: HepG2 cells were treated with sodium nitroprusside (SNP) to induce nitrative stress, which has been associated with inflammation and infection in liver and other tissues. CYP2J2 protein and mRNA expression were evaluated by immunoblotting and real-time PCR respectively. The role of mitogen-activated protein (MAP) kinases in CYP2J2 dysregulation was assessed using specific inhibitors and dominant negative MAP kinase expression plasmids. CYP2J2-luciferase reporter constructs and electromobility shift assays (EMSAs) were used to identify SNP-regulated regions in the CYP2J2 gene.Key Results: Cytochrome P450 2J2 was down-regulated by SNP while the AP-1 proteins c-jun and c-fos were up-regulated; inhibition of p38 and ERK MAP kinases normalized their expression. The gene elements at -105/-95 and -56/-63 were required for the down-regulation of CYP2J2 induced by nitrative stress.Conclusions and Implications: p38 and ERK MAP kinases transduce stress stimuli that down-regulate CYP2J2. Targeting these kinases may prevent the loss of CYP2J2 and epoxy-fatty acids that protect cells against deleterious stresses. [ABSTRACT FROM AUTHOR]- Published
- 2010
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45. The anti-protozoal drug pentamidine blocks KIR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel.
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De Boer, TP, Nalos, L, Stary, A, Kok, B, Houtman, MJC, Antoons, G, Van Veen, TAB, Beekman, JDM, De Groot, BL, Opthof, T, Rook, MB, Vos, MA, Van Der Heyden, MAG, de Boer, T P, Houtman, M J C, van Veen, T A B, Beekman, J D M, de Groot, B L, Rook, M B, and Vos, M A
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ANTIPROTOZOAL agents ,POTASSIUM channels ,CYTOPLASM ,PROTOZOAN disease treatment ,CARDIAC arrest ,ARRHYTHMIA ,HEART cells ,PATCH-clamp techniques (Electrophysiology) ,RESEARCH ,GENETIC mutation ,WESTERN immunoblotting ,ANIMAL experimentation ,RESEARCH methodology ,POTASSIUM ,MEDICAL cooperation ,EVALUATION research ,PENTAMIDINE ,COMPARATIVE studies ,CELL lines ,CYTOLOGY ,DOGS ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (I(K1)), we examined the action and mechanism of pentamidine-mediated I(K1) block.Experimental Approach: Patch clamp measurements of I(K1) were made on cultured adult canine ventricular cardiomyocytes, K(IR)2.1-HEK293 cells and K(IR)2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of K(IR)2.1 channels was studied by molecular modelling.Key Results: Pentamidine application (24 h) decreased I(K1) in cultured canine cardiomyocytes and K(IR)2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited I(K1) in K(IR)2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of I(K1) was acute (IC(50)= 0.17 microM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of K(IR)2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of I(K1). Block was independent of the presence of spermine. K(IR)2.2, and K(IR)2.3 based I(K1) was also sensitive to pentamidine blockade.Conclusions and Implications: Pentamidine inhibits cardiac I(K1) by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific I(K1) blocking compounds. [ABSTRACT FROM AUTHOR]- Published
- 2010
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46. Inhalational anaesthetics and n-alcohols share a site of action in the neuronal Shaw2 Kv channel.
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Bhattacharji, Aditya, Klett, Nathan, Go, Ramon Christopher V, and Covarrubias, Manuel
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ANESTHETICS ,INHALATION anesthesia ,ALCOHOLS (Chemical class) ,POTASSIUM channels ,BINDING sites ,HALOTHANE ,MOLECULAR models ,VOLTAGE-clamp techniques (Electrophysiology) ,INHALATION anesthetics ,RESEARCH ,NEURONS ,GENETIC mutation ,VERTEBRATES ,ANIMAL experimentation ,RESEARCH methodology ,EVALUATION research ,MEDICAL cooperation ,COMPARATIVE studies ,QUESTIONNAIRES ,RESEARCH funding ,PHARMACODYNAMICS - Abstract
Background and Purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K(+) (K(v)) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel.Experimental Approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant K(v) channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system.Key Results: Shaw2 K(v) channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K(0.5)= 400 microM; n(H)= 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and K(v)3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity.Conclusions and Implications: These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 K(v) channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in K(v) channel activation. [ABSTRACT FROM AUTHOR]- Published
- 2010
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47. Statins suppress interleukin-6-induced monocyte chemo-attractant protein-1 by inhibiting Janus kinase/signal transducers and activators of transcription pathways in human vascular endothelial cells.
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Jougasaki, Michihisa, Ichiki, Tomoko, Takenoshita, Yoko, and Setoguchi, Manabu
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STATINS (Cardiovascular agents) ,INTERLEUKIN-6 ,MONOCYTES ,CHEMOTAXIS ,PROTEIN-tyrosine kinases ,ENZYME inhibitors ,CELLULAR signal transduction ,GENETIC transcription ,VASCULAR endothelium ,ANTI-inflammatory agents ,INTERLEUKINS ,RESEARCH ,ENDOTHELIUM ,ANTILIPEMIC agents ,CELL culture ,IMMUNOHISTOCHEMISTRY ,RESEARCH methodology ,CELL physiology ,MEDICAL cooperation ,EVALUATION research ,JANUS kinases ,COMPARATIVE studies ,GENES ,INFLAMMATORY mediators ,EPITHELIAL cells ,AORTA ,PHOSPHORYLATION ,PHARMACODYNAMICS - Abstract
Background and Purpose: The mechanisms of anti-inflammatory actions of statins, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors, remain unclear. We investigated the effects of statins on interleukin (IL)-6-induced monocyte chemo-attractant protein (MCP)-1 expression and monocyte chemotaxis.Experimental Approach: Cultures of human aortic endothelial cells (HAECs) were stimulated with IL-6 in the absence and presence of statins. Gene expression and protein secretion of MCP-1, phosphorylation of Janus kinase (JAK) and the signal transducers and activators of transcription (STAT) pathway, and human monocyte migration were examined.Key Results: IL-6 plus its soluble receptor sIL-6R (IL-6/sIL-6R) promoted THP-1 monocyte migration, and increased gene expression and protein secretion of MCP-1, more than IL-6 alone or sIL-6R alone. Various statins inhibited IL-6/sIL-6R-promoted monocyte migration and MCP-1 expression in HAECs. Co-incubation of mevalonate and geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, reversed the inhibitory effects of statins on MCP-1 expression. Geranylgeranyl transferase inhibitor, but not farnesyl transferase inhibitor, suppressed IL-6/sIL-6R-stimulated MCP-1 expression. IL-6/sIL-6R rapidly phosphorylated JAK1, JAK2, TYK2, STAT1 and STAT3, which were inhibited by statins. Transfection of STAT3 small interfering RNA (siRNA), but not STAT1 siRNA, attenuated the ability of IL-6/sIL-6R to enhance THP-1 monocyte migration. In addition, statins blocked IL-6/sIL-6R-induced translocation of STAT3 to the nucleus.Conclusions and Implications: Statins suppressed IL-6/sIL-6R-induced monocyte chemotaxis and MCP-1 expression in HAECs by inhibiting JAK/STAT signalling cascades, explaining why statins have anti-inflammatory properties beyond cholesterol reduction. [ABSTRACT FROM AUTHOR]- Published
- 2010
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48. SAR150640, a selective beta3-adrenoceptor agonist, prevents human myometrial remodelling and activation of matrix metalloproteinase in an in vitro model of chorioamnionitis.
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Lirussi, F, O'Brien, M, Wendremaire, M, Goirand, F, Sagot, P, Dumas, M, Morrison, JJ, Bardou, M, and Morrison, J J
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METALLOPROTEINASES ,BETA adrenoceptors ,MYOMETRIUM ,INFLAMMATION ,FETAL membranes ,PATHOLOGICAL physiology ,ENDOTOXINS ,PHYSIOLOGY ,ADRENERGIC beta agonists ,LIPOPOLYSACCHARIDES ,BIOLOGICAL models ,IN vitro studies ,RESEARCH ,PROPRANOLOL ,WESTERN immunoblotting ,RESEARCH methodology ,ANTI-infective agents ,MEDICAL cooperation ,EVALUATION research ,FETAL diseases ,MATRIX metalloproteinases ,ADRENERGIC beta blockers ,COMPARATIVE studies ,DOSE-effect relationship in pharmacology ,SULFONAMIDES ,PHARMACODYNAMICS - Abstract
Background and Purpose: The uterine pathophysiology underlying inflammatory conditions such as chorioamnionitis remains largely unclear. As we have shown that beta(3)-adrenoceptors act as regulators of myometrial inflammation, we wanted to investigate the potential role of beta(3)-adrenoceptors in preventing uterine remodelling induced by inflammation.Experimental Approach: The consequences of human chorioamnionitis on myometrial remodelling were characterized by Sirius Red staining and metalloproteinase (MMP) expression, and compared with the effects of incubating human myometrial samples with Escherichia coli lipopolysaccharide (LPS) in vitro. We also assessed the effect of SAR150640, a selective beta(3)-adrenoceptor agonist, on the production and activity of MMPs.Key Results: Chorioamnionitis was associated with a 46% decrease in total collagen, as well as over-expression of MMP2 (+61%) and MMP9 (+84%); both effects were reproduced by incubation with LPS (10 microg x mL(-1), 48 h). LPS-induced over-expression of MMP2 and MMP9 in normal human myometrium was paralleled by an overactivity of the proteins. Both over-expression and overactivity were prevented by the beta(3)-adrenoceptor agonist SAR150640 in a concentration-dependent manner. SAR150640, by itself, did not exhibit any effect on MMP production in control tissues.Conclusions and Implications: This study shows that inflammation was associated with an intense remodelling of human myometrium, a process likely to be explained by MMP activation. Our study emphasizes the potential therapeutic relevance of beta(3)-adrenoceptor agonists to the treatment of preterm labour and other uterine inflammatory conditions. [ABSTRACT FROM AUTHOR]- Published
- 2010
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49. Mechanisms involved in the antinociception induced by systemic administration of guanosine in mice.
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Schmidt, AP, Böhmer, AE, Schallenberger, C, Antunes, C, Tavares, RG, Wofchuk, ST, Elisabetsky, E, Souza, DO, Schmidt, A P, Böhmer, A E, Tavares, R G, Wofchuk, S T, and Souza, D O
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NOCICEPTORS ,DRUG administration ,ADENINE nucleotides ,PAIN ,CEREBROSPINAL fluid ,DRUG toxicity ,METHYL aspartate ,LABORATORY mice ,SPINAL cord ,ANIMAL behavior ,BIOLOGICAL models ,RESEARCH ,INJECTIONS ,HIGH performance liquid chromatography ,ANALGESICS ,NUCLEOSIDES ,ANIMAL experimentation ,ORAL drug administration ,RESEARCH methodology ,GLYCOSIDES ,MEDICAL cooperation ,EVALUATION research ,PAIN threshold ,COMPARATIVE studies ,DOSE-effect relationship in pharmacology ,TOXICITY testing ,MOTOR ability ,EDEMA ,MICE ,CEREBRAL cortex ,PHARMACODYNAMICS - Abstract
Background and Purpose: It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines on pain transmission. The aim of this study was to investigate the effects of intraperitoneal (i.p.) and oral (p.o.) administration of guanosine on mice pain models. Additionally, investigation into the mechanisms of action of guanosine, its potential toxicity and cerebrospinal fluid (CSF) purine levels were also assessed.Experimental Approach: Mice received an i.p. or p.o. administration of vehicle (0.1 mM NaOH) or guanosine (up to 240 mg x kg(-1)) and were evaluated in several pain models.Key Results: Guanosine produced dose-dependent antinociceptive effects in the hot-plate, glutamate, capsaicin, formalin and acetic acid models, but it was ineffective in the tail-flick test. Additionally, guanosine produced a significant inhibition of biting behaviour induced by i.t. injection of glutamate, AMPA, kainate and trans-ACPD, but not against NMDA, substance P or capsaicin. The antinociceptive effects of guanosine were prevented by selective and non-selective adenosine receptor antagonists. Systemic administration of guanosine (120 mg x kg(-1)) induced an approximately sevenfold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by intraplantar capsaicin.Conclusions and Implications: This study provides new evidence on the mechanism of action of the antinociceptive effects after systemic administration of guanosine. These effects seem to be related to the modulation of adenosine A(1) and A(2A) receptors and non-NMDA glutamate receptors. [ABSTRACT FROM AUTHOR]- Published
- 2010
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50. Nicotinic acetylcholine receptors expressed in the ventralposterolateral thalamic nucleus play an important role in anti-allodynic effects.
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Ueda, M, Iida, Y, Tominaga, A, Yoneyama, T, Ogawa, M, Magata, Y, Nishimura, H, Kuge, Y, and Saji, H
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CHOLINERGIC receptors ,NICOTINIC receptors ,THALAMUS ,NOCICEPTORS ,PYRIDINE ,ALLODYNIA ,LABORATORY rats ,NICOTINIC agonists ,BIOLOGICAL models ,RESEARCH ,PAIN ,HETEROCYCLIC compounds ,ANIMAL experimentation ,RESEARCH methodology ,RADIOGRAPHY ,MEDICAL cooperation ,EVALUATION research ,SCIATICA ,PAIN threshold ,RATS ,IODINE radioisotopes ,COMPARATIVE studies ,PHARMACODYNAMICS - Abstract
Background and Purpose: Much interest is currently being focused on the anti-nociceptive effects mediated by nicotinic acetylcholine (nACh) receptors, including their location and mechanism of action. The purpose of this study was to elucidate these issues using 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5IA), a nACh receptor agonist, and [(125)I]5IA.Experimental Approach: We partially ligated the sciatic nerve of Sprague-Dawley rat to induce neuropathic pain [Seltzer's partial sciatic nerve ligation (PSL) model]. We then examined the changes in nACh receptor density in the CNS using [(125)I]5IA autoradiography and the involvement of nACh receptors in anti-nociceptive effects in the region where changes occurred.Key Results: Autoradiographic studies showed that the accumulation of [(125)I]5IA and the number of nACh receptors in the thalamus of PSL rats were increased about twofold compared with those in the sham-operated rats. No change was observed in other brain regions. Rats injected in the ventral posterolateral thalamic nucleus (VPL) with 5IA demonstrated a significant and dose-dependent anti-allodynic effect and this effect was completely antagonized by mecamylamine, injected with 5IA, into the VPL. The blockade of nACh receptors in the VPL by mecamylamine decreased by 70% the anti-allodynic effect of 5IA, given i.c.v. Moreover, mecamylamine given intra-VPL by itself, induced significant hyperalgesia.Conclusions and Implications: Our findings suggest that the nACh receptors expressed in the VPL play an important role in the anti-allodynic effects produced by exogenous and endogenous agonists. [ABSTRACT FROM AUTHOR]- Published
- 2010
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