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The synthesis of a 6-CF 3 -substituted 2-amino-dihydro-1,3-thiazine via N, N-diethylaminosulfur trifluoride (DAST)-mediated cyclization of N-hydroxypropyl thiourea 6 is described. This reaction gave 6-CF 3 -1,3-thiazine 7 with high chemical yield and chemoselectivity, suppressing the common byproduct of oxazine 8. This new protocol enabled access to 6-CF 3 -substituted 1,3-thiazine β-secretase inhibitor 2.

Objectives: The aim of this article was to use copaiba oil (C.O) to improve skin permeability and topical anti-inflammatory activity of celecoxib (Cxb)., Methods: Formulations containing C.O (1-50%) were associated with Cxb (2%). In vitro skin permeability studies were conducted using porcine ear skin. Histological analysis of the hairless mice skin samples after application of formulations was achieved with the routine haematoxylin/eosin technique. The anti-inflammatory activity was assessed using the AA-induced ear oedema mice model., Key Findings: The formulation containing 25% C.O promoted the highest levels of in vitro Cxb permeation through pig ear skin, retention in the stratum corneum (SC) and epidermis/dermis of pig ear skin in vitro (~5-fold) and hairless mice skin in vivo (~2.0-fold), as compared with the control formulation. At 25%, C.O caused SC disorganization and increased cell infiltration and induced angiogenesis without clear signs of skin irritation. The formulation added to 25% C.O as adjuvant inhibited ear oedema and protein extravasation by 77.51 and 89.7%, respectively, and that it was, respectively, 2.0- and 3.4-fold more efficient than the commercial diethylammonium diclofenac cream gel to suppress these inflammatory parameters., Conclusions: 25% C.O is a potential penetration enhancer for lipophilic drugs like Cxb that can improve cutaneous drug penetration and its anti-inflammatory activity., (© 2018 Royal Pharmaceutical Society.)

Introduction: Thyroid hormones are essential for proper development of many systems and organs, including circulatory system. Thyroid deficiency during pregnancy may affect the cardiovascular function in children early on and later in adulthood. However, long-term effects of early thyroid deficiency are poorly understood. We hypothesized that antenatal/early postnatal hypothyroidism will influence anticontractile effect of NO in coronary arteries of adult rats., Design and Methods: To model antenatal/early postnatal hypothyroidism dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%, w/v) from the first day of pregnancy till 2 weeks after delivery. Control females were supplied with pure water. Their male offspring was grown up till the age of 10-12 weeks. Systolic blood pressure was measured using tail cuff method. Septal coronary arteries were isolated and studied in wire myograph. Blood serum thyroid hormones concentrations (ELISA) and NO metabolites level (Griess method) were evaluated., Results: At the age of 10-12 weeks thyroid hormones, TSH concentrations, NO metabolites and systolic blood pressure level didn't differ between groups. Arterial responses to acetylcholine and exogenous NO-donor DEA/NO were similar in control and PTU groups. Along with that, in control rats endothelium denudation strongly potentiated basal tone of arteries and their contractile responses to thromboxane A2 receptor agonist U46619. The effects of endothelium denudation were absent in PTU rats indicating that anticontractile effect of endothelium is abolished in their arteries. Further, NO-synthase inhibitor L-NNA (100 μM) caused significant elevation of basal tone and increased U46619-induced contraction of endothelium-intact arteries only in control rats, while had no effect in PTU group., Conclusions: Our data demonstrate that NO-mediated anticontractile effect of endothelium is eliminated in coronary arteries of adult rats, which suffered from antenatal/early postnatal hypothyroidism. Therefore, maternal thyroid hormones deficiency may have detrimental consequences in adult offspring including coronary circulation pathologies, despite normal blood levels of thyroid hormones., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The effects of the Pseudomonas aeruginosa virulence factor pyocyanin (PCN) on the contractile function of porcine coronary arteries was investigated in vitro. Artery rings (5 mm) were suspended in organ baths containing Krebs' solution for the measurement of isometric tension. The effect of PCN on resting and precontracted coronary arteries was initially investigated with various agents. Arteries were precontracted with prostaglandin (PG) F2α or potassium chloride and endothelium-dependent relaxations were induced by various agents in the presence of PCN. Pyocyanin (0.1-10 μmol/L) evoked small-amplitude, dose-dependent contractions in resting porcine coronary arteries. In addition, PCN amplified the contractile response to PGF2α , but did not alter responses to carbachol. Pyocyanin (0.1-10 μmol/L) significantly inhibited endothelium-dependent relaxations evoked by neurokinin A. Pyocyanin also inhibited relaxations evoked by diethylamine nitric oxide (a nitric oxide donor), forskolin (an adenylate cyclase activator), dibuytyryl-cAMP (a cAMP analogue), 8-bromo-cGMP (a cGMP analogue) and P1075 (a KATP channel activator), but not isoprenaline (β-adrenoceceptor agonist). These results indicate that physiological concentrations of PCN interfere with multiple intracellular processes involved in vascular smooth muscle relaxation, in particular pathways downstream of nitric oxide release. Thus, PCN may alter normal vascular function in patients infected with P. aeruginosa., (© 2014 Wiley Publishing Asia Pty Ltd.)

Adaptation of the horizontal vestibulo-ocular reflex (HVOR) provides an experimental model for cerebellum-dependent motor learning. We developed an eye movement measuring system and a paradigm for induction of HVOR adaptation for the common marmoset. The HVOR gain in dark measured by 10° (peak-to-peak amplitude) and 0.11-0.5Hz turntable oscillation was around unity. The gain-up and gain-down HVOR adaptation was induced by 1h of sustained out-of-phase and in-phase 10°-0.33Hz combined turntable-screen oscillation in the light, respectively. To examine the role of long-term depression (LTD) of parallel fiber-Purkinje cell synapses, we intraperitonially applied T-588 or nimesulide, which block the induction of LTD in vitro or in vivo preparations, 1h before the test of HVOR adaptation. T-588 (3 and 5mg/kg body weight) did not affect nonadapted HVOR gains, and impaired both gain-up and gain-down HVOR adaptation. Nimesulide (3 and 6mg/kg) did not affect nonadapted HVOR gains, and impaired gain-up HVOR adaptation dose-dependently; however, it very little affected gain-down HVOR adaptation. These findings are consistent with the results of our study of nimesulide on the adaptation of horizontal optokinetic response in mice (Le et al., 2010), and support the view that LTD underlies HVOR adaptation., (Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 receptor, specifically for substituted benzamides (SBAs) having polar 4- (para) and/or 5- (meta) benzamide ring substituents. D4-T7.39A was fully functional, and the mutation did not alter the sodium-mediated positive and negative allostery observed with SBAs and agonists, respectively. With the exception of the non-SBA ligand (+)-butaclamol, which, in contrast to certain SBAs, had decreased affinity for the D4-T7.39A mutant, the interactions of numerous other ligands were unaffected by this mutation. SBAs were docked into D4 models in the same mode as observed for eticlopride in the D3 crystal structure. In this mode, interactions with TM5 and TM6 residues constrain the SBA ring position that produces distal steric crowding between pyrrolidinyl/diethylamine moieties and D4-Thr7.39. Ligand-residue interaction energy profiles suggest this crowding is mitigated by substitution with a smaller alanine. The profiles indicate sites that contribute to the SBA binding interaction and site-specific energy changes imparted by the D4-T7.39A mutation. Substantial interaction energy changes are observed at only a few positions, some of which are not conserved among the dopamine receptor subtypes and thus seem to account for this D4 subtype-specific structure-activity relationship.

Background: Rubbing a topical NSAID (non steroidal anti-inflammatory drug) on the skin may increase local drug permeation, affecting its distribution to the site of pain and inflammation. The present study evaluates this hypothesis, by assessing in vitro the effect on skin permeation of applying diclofenac-dieythylamine 1.16% gel with or without rubbing., Methods: A single dose of 5 mg/cm2 diclofenac-diethylamine 1.16% gel was applied on excised human skin mounted in Franz-type diffusion cells without or with rubbing for 45 s. Drug penetration into the skin layers was determined after 1 h using the tape stripping technique. In vitro cutaneous permeation into the receptor fluid of the diffusion chamber was measured up to 24 h. Skin electrical resistance was also recorded., Results: Application of diclofenac-diethylamine 1.16% gel with rubbing resulted to a 5-fold higher flux of diclofenac through the skin than when applied without rubbing at 8 h (P = 0.04). Skin rubbing for 45 s decreased by 2-fold skin electrical resistance when compared to the standard application. Application of diclofenac-diethylamine 1.16% gel with rubbing tended to result in higher accumulation in the stripped skin vs. the superficial skin layers when applied without rubbing (P = 0.2)., Conclusion: These results suggest that rubbing may alter the superficial skin layer resulting in a transient faster initial diffusion of topically applied diclofenac through the stratum corneum into the deeper skin layer of the dermis to the tissue target.

Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(•-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(•)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(•-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (•)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nitroxyl (HNO) displays distinct pharmacology to its redox congener nitric oxide (NO(•)) with therapeutic potential in the treatment of heart failure. It remains unknown if HNO donors are resistant to tolerance development following chronic in vivo administration. Wistar-Kyoto rats received a 3-day subcutaneous infusion of one of the NO(•) donors, glyceryl trinitrate (GTN) or diethylamine/NONOate (DEA/NO), or the HNO donor Angeli's salt (AS). GTN infusion (10 μg/kg/min) resulted in significantly blunted depressor responses to intravenous bolus doses of GTN, demonstrating tolerance development. By contrast, infusion with AS (20 μg/kg/min) or DEA/NO (2 μg/kg/min) did not alter their subsequent depressor responses. Similarly, ex vivo vasorelaxation responses in isolated aortae revealed that GTN infusion elicited a significant 6-fold decrease in the sensitivity to GTN and reduction in the maximum response to acetylcholine (ACh). Chronic infusion of AS or DEA/NO had no effect on subsequent vasorelaxation responses to themselves or to ACh. No functional cross-tolerance between nitrovasodilators was evident, either in vivo or ex vivo, although an impaired ability of a nitrovasodilator to increase tissue cGMP content was not necessarily indicative of a reduced functional response. In conclusion, HNO donors may represent novel therapies for cardiovascular disease with therapeutic potential over clinically used organic nitrates.

Aims: The present study aims to investigate the interaction between nitric oxide (NO) and hydrogen sulfide (H(2)S), the two important gaseous mediators in rat hearts., Methods and Results: Intracellular calcium in isolated cardiomyocytes was measured with a spectrofluorometric method using Fura-2. Myocyte contractility was measured with a video edge system. NaHS (50 µM, an H(2)S donor) had no significant effect on the resting calcium level, electrically induced (EI) calcium transients, and cell contractility in ventricular myocytes. Stimulating endogenous NO production with l-arginine or exogenous application of NO donors [sodium nitroprusside (SNP) and 2-(N,N-diethylamino)-diazenolate-2-oxide] decreased myocyte twitch amplitudes accompanied by slower velocities of both cell contraction and relaxation. Surprisingly, NaHS reversed the negative inotropic and lusitropic effects of the above three NO-increasing agents. In addition, the mixture of SNP + NaHS increased, whereas SNP alone decreased, the resting calcium level and the amplitudes of EI calcium transients. Angeli's salt, a nitroxyl anion (HNO) donor, mimicked the effect of SNP + NaHS on calcium handling and myocyte contractility. Three thiols, N-acetyl-cysteine, l-cysteine, and glutathione, abolished the effects of HNO and SNP + NaHS on myocyte contraction. Neither Rp-cAMP [a protein kinase A (PKA) inhibitor] nor Rp-cGMP [a protein kinase G (PKG) inhibitor] affected the effects of SNP + NaHS, suggesting a cAMP/PKA- or cGMP/PKG-independent mechanism., Conclusion: H(2)S may interact with NO to form a thiol sensitive molecule (probably HNO) which produces positive inotropic and lusitropic effects. Our findings may shed light on the interaction of NO and H(2)S and provide new clues to treat cardiovascular diseases.

Objectives: To investigative the effects of oxidative stress simultaneously on Glucose-6-phosphate dehydrogenase (G6PD) activity and RBC fragility under normal and G6PD defective conditions., Methods: The effects of several nitric oxide (NO) generating compounds and sulfhydryl blocking agents were simultaneously tested in vitro on hemolysate G6PD activities and RBC fragility. These effects were compared between normal subjects and patients with G6PD deficiency., Results: The NO donor compounds nitrosocysteine, nitrosoarginine and diethylamine caused strong inhibition on normal and defective G6PD activities, while a similar inhibition was observed only at higher concentrations of the sulfhydryl blocking agents: 2-mercaptoethanol , cysteine and reduced glutathione. All these oxidative compounds promoted RBC hemolysis in parallel to their inhibition extents on G6PD activities. The protection of RBC from this hemolysis was achieved by preincubation with NADPH or SNP but not NAD(+) compound., Conclusion: A concomitant response of G6PD activities and RBC fragility towards the oxidative stress was established., (Copyright 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Traditional herbal formulations, such as Juzen-taiho-to (TJ-48), are used extensively in medical practice in Asia even though their mechanism of action remains elusive. This study tested a hypothesis that TJ-48 is protective against hepatocarcinogenesis by impeding Kupffer cell-induced oxidative stress. Forty-eight patients were randomly assigned to receive TJ-48 (n = 10), or no supplementation (n = 38) for up to 6 years after surgical treatment for hepatocellular carcinoma (HCC). In addition, to investigate the mechanism of protective action of TJ-48, diethylnitrosamine-containing water was administered for 22 weeks to male mice that were fed regular chow or TJ-48-containing diet. Liver tumor incidence, cell proliferation, number of 8-hydroxy-2'-deoxyguanosine- or F4/80-positive cells, and cytokine expression were evaluated. Although most of the patients experienced recurrence of HCC, a significantly longer intrahepatic recurrence-free survival was observed in the TJ-48 group. In mice, TJ-48 inhibited the development of liver tumors, reduced oxidative DNA damage, inflammatory cell infiltration and cytokine expression. Administration of TJ-48 improves intrahepatic recurrence-free survival after surgical treatment of hepatocellular carcinoma. On the basis of animal experiments, we reason that the protective mechanism of TJ-48 involves inhibition of Kupffer cells. This leads to lower levels of pro-inflammatory cytokines and oxidants in liver which may slow down the process of hepatocarcinogenesis and improves hepatic recurrence-free survival in patients with HCC., ((c) 2008 Wiley-Liss, Inc.)

Store-operated Ca(2+) entry (SOCE) plays an important role in the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs). The aim of this study was to characterise the pharmacological properties of the SOCE pathway in freshly isolated PASMCs from rat lung and to determine whether this Ca(2+) entry pathway is sensitive to nitric oxide donor drugs. Following depletion of Ca(2+) from the sarcoplasmic reticulum, by treating cells with thapsigargin, re-addition of Ca(2+) produced an increase in cytosolic fluo-4 fluorescence that was sustained for the period that extracellular Ca(2+) was present. Thapsigargin also increased the rate of quench of fura-2 fluorescence, confirming that SOCE was activated. The SOCE pathway was not affected by nifedipine or verapamil; however, it was inhibited by the divalent cations Ni(2+) (10 microM) and Cd(2+) (10 microM) by 47+/-5% and 49+/-5% respectively. SOCE was also inhibited 42+/-5% by 2-aminoethoxydiphenyl borate (2-APB; 75 microM) and 58+/-4% by Gd(3+) (10 microM), although La(3+) (100 microM) had little effect. None of the NO donors examined, including sodium nitroprusside, glyceryl trinitrate, and 2-(N,N-diethylamino)-diazenolate-2-oxide had any effect on SOCE. Thus, the pulmonary vasorelaxation produced by NO does not involve direct inhibition of SOCE in PASMCs. Western blot and immunocytochemistry using antibodies directed against specific TRPC subunits detected the presence of TRPC1, 3, and 6 in pulmonary artery and the pharmacological profile of SOCE in PASMCs favours a role for TRPC1 in mediating the underlying channels that are activated by store depletion.

Two new alkaloids, hypserpanines A and B (1, 11), together with eleven known compounds, phenolbetain (2), acutumine (3), acutumidine (4), dechloroacutumine (5), dauricumine (6), dauricumidine (7), pronuciferine (8), glaziovine (9), S-reticuline (10), magnoflorine (12) and laurifoline(13), were isolated from Hypserpa nitida Miers. (Menispermaceae) and chemically elucidated through spectral analyses. All the isolated alkaloids were evaluated for their anti-HBV activities in vitro using the HBV transfected Hep G2.2.15 cell line. The most active compound, dauricumidine (7), exhibited an IC(50) value of 0.450 mM (SI=4.13) on hepatitis B virus (HBV) surface antigen (HBsAg) secretion of the Hep G2.2.15 cell line.

Diabetes is associated with vascular dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. We investigated whether the angiotensin II antagonist telmisartan improves vascular dysfunction and reduces platelet activation in diabetic rats. Therefore, male Wistar rats were injected with streptozotocin (50 mg kg(-1) i.v.) to induce insulin-deficient diabetes. Treatment with telmisartan (10 mg kg(-1)day(-1)) or vehicle was initiated 2 weeks after injection of streptozotocin and continued for 2 weeks. At week 4, platelet activation was assessed in fresh whole blood and vascular function was characterized in isolated aortic segments in organ bath chambers. Diabetic rats displayed severe impairment of endothelium-dependent relaxation induced by acetylcholine as well as endothelium-independent relaxation evoked by a nitric oxide donor, which were improved by treatment with telmisartan. Treatment with telmisartan also improved endogenous platelet vasodilator-stimulated phosphoprotein phosphorylation, which was reduced in platelets from diabetic rats indicating augmented intraluminal vascular nitric oxide bioavailability. Platelets from diabetic rats had increased surface-bound fibrinogen, which was attenuated by telmisartan. Telmisartan normalizes vascular dysfunction and reduces platelet activation in diabetic rats. These effects may contribute to the reduction of cardiovascular events by angiotensin II receptor blockers in diabetic patients.

Objectives/hypothesis: We have previously shown that gene therapy using Insulin-like growth factor (IGF)-I, glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), or a combination of these trophic factors, is a treatment option for recurrent laryngeal nerve (RLN) palsy. However, there remain some difficulties preventing this option from becoming a common clinical therapy for RLN injury. Thus, we need to develop novel treatment option that overcomes the problems of gene therapy.R(-)-1-(benzothiophen-5-yl)-2-[2-N,N-diethylamino]ethoxy]ethanol hydrochloride (T-588), a synthetic compound, is known to have neuroprotective effects on neural cells. In the present study, the possibility of new drug treatments using T-588 for RLN injury was assessed using rat models., Study Design: Animal study., Methods: Animals were administered T-588 for 4 weeks. The neuroprotective effects of T-588 administration after vagal nerve avulsion and neurofunctional recovery after recurrent laryngeal nerve crush were studied using motoneuron cell counting, evaluation of choline acetyltransferase immunoreactivity, the electrophysiologic examination, and the re-mobilization of the vocal fold., Results: T-588 administration successfully prevented motoneuron loss and ameliorated the choline acetyltransferase immunoreactivity in the ipsilateral nucleus ambiguus after vagal nerve avulsion. Significant improvements of motor nerve conduction velocity of the RLN and vocal fold movement were observed in the treatment group when compared to controls., Conclusion: These results indicate that oral administration of T-588 might be a promising therapeutic option in treating peripheral nerve injury.

The nucleus tractus solitarii (NTS) is a key central link in control of multiple homeostatic reflexes. A number of studies have demonstrated that exogenous and endogenous nitric oxide (NO) within NTS regulates visceral function, but further understanding of the role of NO in the NTS is hampered by the lack of information about its intracellular actions. We studied effects of NO in acute rat brainstem slices. Aqueous NO solution (NO(aq)) potentiated electrically evoked excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in different neuronal subpopulations and, in some neurones, caused a depolarization. Similar effects were observed using the NO donor diethylamine NONOate (DEA/NO). The threshold NO concentration as determined using an NO electrochemical sensor was estimated as approximately 0.4 nm (EC(50) approximately 0.9 nm) for potentiating glutamatergic EPSPs but approximately 3 nm for monosynaptic GABAergic IPSPs. Bath application of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) abolished NO(aq)- and DEA/NO-induced potentiation of evoked EPSPs, IPSPs and depolarization. All NO actions were mimicked by the non-NO-dependent guanylate cyclase activator Bay 41-2272. The effects of NO on EPSPs and IPSPs persisted in cells where postsynaptic sGC was blocked by ODQ and therefore were presynaptic, owing to a direct modulation of transmitter release combined with depolarization of presynaptic neurones. Therefore, while lower concentrations of NO may be important for fine tuning of glutamatergic transmission, higher concentrations are required to directly engage GABAergic inhibition. This differential sensitivity of excitatory and inhibitory connections to NO may be important for determining the specificity of the effects of this freely diffusible gaseous messenger.

Nitric oxide (NO) functions as an intercellular messenger throughout the brain. For this role to be performed efficiently, there must be a mechanism for neutralizing NO, but whether an active biological process exists, or whether NO is lost mainly through diffusion is unclear. To investigate this issue, rat cerebellar slices were exposed to constant levels of NO and the cGMP generated within the slice used as an indicator of NO concentrations therein. NO was about 1000-fold less potent in slices (EC50, 1 microM) than in separated cells from the same tissue (EC50, 1.6 nM), consistent with access of NO to the slice interior being greatly hindered by inactivation. Supporting this interpretation, immunohistochemical analysis indicated a marked concentration gradient of cGMP across the thickness of slices exposed to subsaturating NO concentrations, signifying a marked NO gradient. Several known NO-degrading processes, including reaction with lipid peroxyl radicals, erythrocytes and superoxide ions, were eliminated as contributing factors, indicating a novel mechanism. A diffusion-inactivation model was used to estimate the kinetics of NO consumption by the slices. The best fits to experimental data indicated a Michaelis-Menten-type reaction having a Vmax of 1-2 microM s-1 and a Km of around 10 nM. The rates predict that inactivation would impose a very short half-life (<10 ms) on NO in physiological concentrations (up to 10 nM) and that it would play an important role in shaping the NO concentration profiles when it is synthesized by multiple nearby sites.

The purpose of this study was to examine the levels of norepinephrine (NE) turnover in skin tissues and to determine the effect of nitric oxide (NO) on NE production in acupuncture points (acupoints) and meridians. The rats were pretreated with alpha-methyl-tyrosine methyl ester and intravenously infused with L-(2,3,5,6-(3)H)-tyrosine. Blood was withdrawn and skin tissues were excised from the low skin resistance points, non-acupoint, and non-meridian areas located on leg, arm, or trunk. The results showed that the skin NE concentration and (3)H-NE release in acupoints were significantly higher than those in non-acupoints and non-meridian controls. (3)H-NE releases in the acupoints were increased by intravenous infusion of 2-N,N-diethylamino-diazenolate-2-oxide, an NO donor, but lowered by N(G)-Propyl-L-arginine, an inhibitor of neuronal NO synthesis. NE turnover rates in the acupoints were lower in the NO donor treated group while the inhibitor of NO synthesis reversed the trend. In contrast, NE turnover rates were not altered by NO donor and inhibitor of NO synthesis in non-acupoint and non-meridian control tissues. This is the first evidence that NE turnover was consistently decreased in acupoints and enhanced NE synthesis/release in acupoints were facilitated by presence of an NO donor and inhibited by an inhibitor of NO synthesis. The data suggest that skin NE synthesis/release in acupoints/meridians is increased in skin acupoints, which is modulated by L-arginine-derived NO synthesis in sympathetic nervous system.

Critical roles for nitric oxide (NO) in regulating cell and tissue physiology are broadly appreciated, but aspects remain to be explored. In the mollusk Pleurobranchaea, NO synthase activity is high in CNS ganglia containing motor networks for feeding and locomotion, where a cAMP-gated cation current (I(Na,cAMP)) is also prominent in many neurons. We examined effects of NO on I(Na,cAMP) using voltage-clamp methods developed to analyze cAMP signaling in the live neuron, focusing on the identified metacerebral giant neuron of the feeding network. NO donors enhanced the I(Na,cAMP) response to injected cAMP by an averaged 85%. In dose-response measures, NO increased the current stimulated by cAMP injection without altering either apparent cAMP binding affinity or cooperativity of current activation. NO did not detectably alter levels of native cAMP or synthesis or degradation rates as observable in both current saturation and decay rate of I(Na,cAMP) responses to cAMP injection. NO actions were not exerted by cGMP signaling, as they were not mimicked by cGMP analogue nor blocked by inhibitors of guanylate cyclase and protein kinase G. NO potentiation of I(Na,cAMP) was broadly distributed among many other neurons of the feeding motor network in the buccal ganglion. However, NO did not affect a second type of I(Na,cAMP) found in locomotor neurons of the pedal ganglia. These results suggest that NO acts through a novel mechanism to regulate the gain of cAMP-dependent neuromodulatory pathways that activate I(Na,cAMP) and may thereby affect the set points of feeding network excitability and reactivity to exogenous input.

Our aim was to study whether nitric oxide (NO) donor-induced inhibition of pregnant rat myometrium contractility correlates with the release of NO. Uterine rings from mid-pregnant and late pregnant Sprague-Dawley rats were used for isometric tension recording. Concentration-response relationships to sodium nitroprusside (SNP), nitroglycerine (NTG) and diethylamine (DEA)/NO were assessed. The time course of NO release after addition to the organ chambers of the 3 NO-donors was assessed by the detection of NO products NOx (NO3+NO2) using the microdialysis probe by a HPLC-NO detector system. DEA/ NO induced greater inhibition of the spontaneous contractile activity of uterine rings from mid-pregnant rats than SNP or NTG. In uterine rings from late pregnant rats, however, the maximal inhibition of the contractility by all 3 NO-donors were significantly less. The NOx levels measured in the uterine ring walls from either mid-pregnant or late pregnant rats significantly increased after DEA/ NO as compared to the basal levels or the levels after NTG or SNP. The decrease of NO-donor-induced inhibition of rat myometrium contractility, with unchanged formation of NOx, at term, suggests that the changes in NO signaling are responsible for gestational age-dependent attenuation of the inhibitory effect.

Systemic delivery of (1R-1-benzo thiophen-5-yl-2[2-diethylamino)-ethoxy] ethanol hydrochloride (T-588) prevented long-term depression (LTD) of the parallel fiber (PF)-Purkinje cell (PC) synapse induced by conjunctive climbing fiber and PF stimulation in vivo. However, similar concentrations of T-588 in the brains of behaving mice and rats affected neither motor learning in the rotorod test nor the learning of motor timing during classical conditioning of the eyeblink reflex. Rats given doses of T-588 that prevented PF-PC LTD were as proficient as controls in learning to adapt the timing of their conditioned eyeblink response to a 150- or 350-ms change in the timing of the paradigm. The experiment indicates that PF-PC LTD under control of the climbing fibers is not required for general motor adaptation or the learning of response timing in two common models of motor learning for which the cerebellum has been implicated. Alternative mechanisms for motor timing and possible functions for LTD in protection from excitotoxicity are discussed.

Long-term depression (LTD) of the parallel-fiber (PF) Purkinje synapse induced by four different experimental paradigms could be prevented in rat cerebellar slices by T-588, a neuroprotective compound. The paradigms consisted of pairing PF activation with climbing-fiber activation, direct depolarization, glutamic iontophoretic depolarization, or caffeine. In all cases, LTD was determined by patch-clamp recording of PF excitatory postsynaptic currents at the Purkinje cell somata. T-588 at 1 muM prevented the triggering of LTD reversibly and did not generate LTD on its own. Two-photon calcium-sensitive dye imaging demonstrated that T-588 reduces intracellular calcium concentration ([Ca(2+)](i)) increase by blocking calcium release from intracellular stores. Because [Ca(2+)](i) increase has been widely shown to trigger LTD and glutamate excitotoxicity, we propose that LTD may act as a neuroprotective mechanism. As such, LTD would serve to decrease glutamatergic-receptor sensitivity to limit deleterious [Ca(2+)](i) increase rather than to act as a mechanism for cerebellar learning.

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.

S-Nitrosating nitric oxide (NO) donors like S-nitrosoglutathione (GSNO) induce a persistent inhibition of vascular tone, through the formation of releasable NO stores. In this study, we investigate whether GSNO also induces NO stores-related effects in vessels exhibiting tolerance to glyceryl-trinitrate. Rat aortic rings treated with glyceryl-trinitrate (100 microM for 1 h) exhibited increased level of superoxide and a decrease in NO elevation and relaxation induced by subsequent addition of glyceryl-trinitrate. In glyceryl-trinitrate-treated rings as in controls, pre-exposure to GSNO (1 microM for 30 min) induced a persistent hyporesponsiveness to noradrenaline and a relaxant response to N-acetylcysteine (a low molecular weight thiol which can displace NO from NO stores), both of which being inhibited by guanylyl-cyclase or cyclic GMP-dependent protein kinase inhibitors. These data indicate that GSNO can promote the formation of releasable NO stores in arteries exhibiting increased superoxide level and tolerance to glyceryl-trinitrate. Formation of releasable NO stores is of potential interest to restore the protective effect of NO in organic nitrate-tolerant blood vessels.

Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.

The effects of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a cognitive enhancer, on sodium nitroprusside (SNP)-induced cytotoxicity were examined in cultured rat astrocytes. Treatment with 100 microM SNP for 72 h decreased cell viability and mitochondrial function assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenil tetrazolium bromide (MTT) reduction activity, mitochondrial transmembrane potential, and intracellular ATP level. T-588 at 100 microM prevented SNP-induced mitochondrial dysfunction and cell injury. Furthermore, T-588 increased MTT reduction activity without affecting cell proliferation in astrocytes. These results suggest that T-588 has a protective effect against SNP-mediated toxicity via improvement of mitochondrial dysfunction in astrocytes.

Our objective was to evaluate the role of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in rat uterine and cervical contractility at mid- and late gestation. Rings of uterus and cervix from Sprague Dawley rats on day 14 of pregnancy (mid-) and day 21 of pregnancy (late) were equilibrated at 2 g passive tension in organ chambers filled with Krebs-Henseleit solution and bubbled with 5% CO2 in air (37 degrees C, pH approximately 7.4). Rings were treated with an inhibitor of outward potassium current, tetraethylammonium, to activate phasic contractions, and the concentration-response relationships to diethylamine/NO and 8-bromo-cGMP (8-br-cGMP) were assessed. Baseline spontaneous activity was significantly higher at term gestation in both uterine and cervical rings compared with mid-gestation. Spontaneous contractile activity was not apparent in cervical rings from rats in mid-gestation, but was persistent after treatment with tetraethylammonium. Oxyhemoglobin did not affect NO-induced inhibition of activation by tetraethylammonium contractile activity in either cervical or uterine tissues in mid- or late gestation. The 8-br-cGMP concentration-dependently inhibited tetraethylammonium-activated contractions that were more pronounced in uterine tissues compared with cervical tissues in both mid- and late gestation. We concluded that activation of the NO-cGMP pathway inhibits both uterine and cervical smooth muscle contractility. Both tissues demonstrated refractoriness to NO at term.

Nitric oxide (NO) signal transduction occurs through guanylyl cyclase-coupled receptors, which exist in both cytosolic and membranous locations. It has recently been reported from experiments using heart tissue that the membrane-associated receptor has enhanced sensitivity to NO. Owing to its potential importance, we tested this finding using a method of applying NO in known, constant concentrations. The results showed that the concentration-response curves for receptor activation in cytosolic and membrane preparations of two different tissues (cerebellum and platelets) were indistinguishable. In all cases, half-maximal activation required about 1 nM NO and the curves had Hill coefficients of close to 1. The differential sensitivity reported for the heart is attributed to NO being scavenged by myoglobin in the cytosol, but not in the membrane fraction.

R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy] ethanol hydrochloride) (T-588) enhances acetylcholine release. This compound slows the motor deterioration of wobbler mouse motor neuron disease and enhances neurite outgrowth and choline acetyltransferase activity in cultured rat spinal motor neurons. We examined the ability of T-588 on axotomized spinal motor neuron death in the rat spinal cord. After the postnatal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of T-588 for 14 consecutive days rescued spinal motor neuron death. Our results showing in vivo neurotrophic activity of T-588 for motor neurons support the applicability of T-588 for the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis and motor neuropathies.

We have previously shown that the cognition enhancer (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588) protects astrocytes against hydrogen peroxide (H2O2) injury via activation of extracellular signal-regulated kinase (ERK) pathway. The present study examines whether the effect of T-588 on astrocytes contributes to neuroprotection in neuronal injury models. Astrocyte-conditioned medium (ACM) protected against neuronal injury induced by amyloid-beta protein (A beta) in cultured cortical neurons. The effect of ACM on A beta-induced injury was blocked by the ERK kinase inhibitor 2'-amino-3'-methoxyflavone. ACM stimulated ERK phosphorylation in cultured neurons. ACM derived from astrocytes exposed to H2O2 lost the activities to stimulate ERK phosphorylation and protect against neuronal injury. T-588 blocked the H2O2-induced loss of the activities of ACM. These results suggest that ACM protects against neuronal injury by an ERK-dependent mechanism, and the effect of T-588 on astrocytic injury results in neuroprotection.

To examine the possible neuroprotective effect of T-588 against glutamate-induced neurotoxicity, we analyzed the pharmacological utility of T-588 in a postnatal organotypic culture model of motor neuron degeneration. Treatment with 10(-5) M of glutamate resulted a motor neuron loss and decreased activity of choline acetyltransferase (ChAT). Cotreatment of 10(-5) M of glutamate and T-588 revealed a protective effect against motor neuron death and decreased ChAT activity. We concluded that T-588 may play important roles in the survival and maintenance of spinal motor neurons in its neuroprotection against glutamate-induced neurotoxicity. Our data may provide a rationale for designing a therapeutic strategy for protection against pathologically induced motor neuron damage or cell death such as amyotrophic lateral sclerosis and motor neuropathy.

In vivo studies show a dynamic cycle in which alpha-nitrosylated hemoglobin is mainly in the relaxed state in arterial blood of rats treated with 2-(N,N-diethylamino)-diazenolate-2-oxide, but converts mainly to the tense state during the arterial-venous transit. A detailed analysis shows that different electron paramagnetic resonance spectra recorded for alpha-nitrosyl hemoglobin in arterial and venous blood at 77 K originate only from a different ratio between 5- and 6-coordinate heme without any change in the concentration of nitrosyl hemoglobin. In venous blood, the five- and six-coordination equilibrium of the alpha-nitrosyl heme is shifted in favor of the 5-coordinate state (58% venous vs. 20% arterial). These results are not consistent with the recently proposed exchange of nitrosyl heme with the beta-93 nitrosothiol group of hemoglobin during the arterial-venous cycle.

Administration of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a cognitive enhancer, like the acetylcholine esterase inhibitors physostigmine and tacrine, stimulated phosphorylation of extracellular signal-regulated kinases (ERK) in the mouse hippocampus. The effect of T-588 on ERK phosphorylation was persistent from 2 to 6 h after injection. Immunohistochemical study showed that T-588 stimulated neuronal ERK phosphorylation in rat hippocampal CA1 pyramidal subfield. These findings suggest that systemic T-588 stimulates the ERK kinase pathway in the hippocampal neurons.

Cortical spreading depression (CSD) is a transient disruption of local ionic homeostasis that may promote migraine attacks and the progression of stroke lesions. We reported previously that the local inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) delayed markedly the initiation of the recovery of ionic homeostasis from CSD. Here we describe a novel method for selective, controlled generation of exogenous NO in a functioning brain region. It is based on microdialysis perfusion of the NO donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO). As DEA/NO does not generate NO at alkaline pH, and as the brain has a strong acid-base buffering capacity, DEA/NO was perfused in a medium adjusted at alkaline (but unbuffered) pH. Without DEA/NO, such a microdialysis perfusion medium did not alter CSD. DEA/NO (1, 10 and 100 microM) had little effect on CSD by itself, but it reversed in a concentration-dependent manner the effects of NOS inhibition by 1 mM L-NAME. These data demonstrate that increased formation of endogenous NO associated with CSD is critical for subsequent, rapid recovery of cellular ionic homeostasis. In this case, the molecular targets for NO may be located either on brain cells to suppress mechanisms directly involved in CSD genesis, or on local blood vessels to couple flow to the increased energy demand associated with CSD.

The present study examines the effect of the cognition enhancer (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588) on neuronal injury induced by serum deprivation or amyloid-beta protein (A beta). T-588 protected partially against neuronal injury induced by serum deprivation or A beta in cultured cortical neurons. T-588 did not affect the phosphorylation of extracellular signal-regulated kinase (ERK) in cortical neurons and SH-SY5Y cells. These results suggest that T-588 has a protective effect in neuronal injury models and the effect is not mediated by an ERK signal pathway.

Objective: R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxylethanol hydrochloride (T-588), a synthetic compound, has been shown to have neuroprotective potentials for neuronal cells. We investigated whether orally administered T-588 can rescue injured motoneurons after facial nerve avulsion in adult rats., Methods: The right facial nerves of adult Fischer 344 male rats were avulsed and the animals were freely administered solution of 0.05% (w/v) T-588 or received T-588 (3-30 mg/kg/day) through an oral tube for 1-4 weeks. Facial motoneurons on both sides of the facial nuclei were counted in Nissl-stained sections, and choline acetyltransferase (ChAT) immunoreactivity in injured motoneurons and ChAT enzyme activities in the ventral brain stem tissue containing the facial nuclei were examined., Results: Both free oral administration of 0.05% T-588 solution and oral tube administration of T-588 (30mg/kg/day) improved the survival of facial motoneurons at 3 or 4 weeks after avulsion. These treatments ameliorated ChAT immunoreactivity in injured motoneurons and the tissue ChAT enzyme activities at 1-week postoperation examined., Conclusion: These results indicate that oral administration of T-588 ameliorates the survival of injured motoneurons and supports their neuronal function after facial nerve avulsion in adult rats. T-588 may have therapeutic potential in motoneuron injury or motor neuron diseases in humans.

Anticarcinogenic activity of d-limonene has been well documented within last few years. We have also reported the anticarcinogenic activity of d-limonene in N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis. The involvement of oncogenes which adds to the mechanisms of d-limonene mediated chemprevention were also suggested in the same model system. The overexpression of c-myc oncoprotein in different durations of NDEA induced hepatrocarcinogenesis is observed which is inhibited completely when d-limonene was treated prior to and along with NDEA. To work further in this direction, an attempt has been made here to know the role of YY1 (Yin Yang 1) transcription factor in N-nitrodiethylamine (NDEA) induced hepatocarcinogenesis and its chemoprevention by d-limonene. Electrophoretic mobility shift assay results have clearly indicated the binding of YY1 in control liver tissue. But this binding is blocked in 60 days and 150 days NDEA treated liver tumors. Thus, it is assumed that there is deregulation of YY1 transcription factor in NDEA induced hepatocarcinogenesis. A similar type of binding to that of control liver tissue has also observed when limonene was given prior to NDEA administration. Western blot analysis has shown inhibition of YY1 protein in NDEA induced liver tumor samples in comparison to normal and both NDEA and limonene treated samples. On the otherhand RT-PCR analysis does not indicate any correlation between YY1 mRNA level and inhibition of YY1 protein. However, along with our earlier information about c-myc with the present study, clearly indicated the involvement of YY1 in NDEA induced hepatocarcinogenesis and d-limonene mediated chemoprevention which might be regulated by c-myc oncoprotein.

Purpose: We investigated whether T-588 can attenuate retinal ganglion cell (RGC) death induced by elevated intraocular pressure (IOP)., Methods: IOP elevation was induced unilaterally by argon laser irradiation of the rat trabecular meshwork 4 days after an intracameral injection of India ink. We orally administered either the vehicle, or 10, 30, or 100 mg/kg body weight (BW) of T-588 24 hours before the laser application. Five days after the laser application, 1.5 microL of 3% Fast Blue was injected into the superior colliculi bilaterally. Three days after the Fast Blue injection, the eye was enucleated and the retinal whole flatmount was prepared. Labeled ganglion cells were counted by fluorescence microscope with an ultraviolet filter., Results: Laser treatment significantly increased the IOP. The percentages of labeled RGCs in the lasered eyes as compared with the nonlasered contralateral eyes were 78.0 +/- 11.6% in the control group, 78.7 +/- 12.9% in the 10 mg/kg BW group, 79.1 +/- 13.0% in the 30 mg/kg BW group, and 91.0 +/- 9.0% in the 100 mg/kg BW T-588-treated group. The survival rate of RGCs was significantly higher in the 100 mg/kg BW T-588-treated group than in the control group., Conclusion: T-588 appears to have a neuroprotective effect on retinal ganglion cells in this ocular hypertensive model.

1. Nitric oxide (NO) is a key modulator of cellular Ca(2+) signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca(2+) entry (CCE) into HEK293 cells by impairment of mitochondrial Ca(2+) handling. 2. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 micro M, respectively. 3. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 micro M), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 micro M) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 micro M) failed to antagonize the inhibitory action of NO on CCE. 4. DEANO (1-10 micro M) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca(2+) levels, respectively, indicated that DEANO (10 micro M) depolarized mitochondria and suppressed mitochondrial Ca(2+) sequestration. The inhibitory effect of DEANO on Ca(2+) uptake into mitochondria was confirmed by recording mitochondrial Ca(2+) during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5. DEANO (10 micro M) failed to inhibit Ba(2+) entry into TG-stimulated cells when extracellular Ca(2+) was buffered below 1 micro M, while clear inhibition of Ba(2+) entry into store depleted cells was observed when extracellular Ca(2+) levels were above 10 micro M. Moreover, buffering of intracellular Ca(2+) by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca(2+) dependent-regulatory process. 6. Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca(2+) handling by mitochondria as a key mechanism of NO induced suppression of CCE.

Nitric oxide (NO) signal transduction may involve at least two targets: the guanylyl cyclase-coupled NO receptor (NO(GC)R), which catalyzes cGMP formation, and cytochrome c oxidase, which is responsible for mitochondrial O(2) consumption and which is inhibited by NO in competition with O(2). Current evidence indicates that the two targets may be similarly sensitive to NO, but quantitative comparison has been difficult because of an inability to administer NO in known, constant concentrations. We addressed this deficiency and found that purified NO(GC)R was about 100-fold more sensitive to NO than reported previously, 50% of maximal activity requiring only 4 nm NO. Conversely, at physiological O(2) concentrations (20-30 microM), mitochondrial respiration was 2-10-fold less sensitive to NO than estimated beforehand. The two concentration-response curves showed minimal overlap. Accordingly, an NO concentration maximally active on the NO(GC)R (20 nm) inhibited respiration only when the O(2) concentration was pathologically low (50% inhibition at 5 microM O(2)). Studies on brain slices under conditions of maximal stimulation of endogenous NO synthesis suggested that the local NO concentration did not rise above 4 nm. It is concluded that under physiological conditions, at least in brain, NO is constrained to target the NO(GC)R without inhibiting mitochondrial respiration.

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.

The effects of nitric oxide (NO) donors on the L-type Ca(2+) current (I(Ca,L)) and the muscarinic activated K(+) current (I(K,ACh)) were studied in isolated rat cardiac myocytes. The nitrosothiol S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 pM-1 microM) strongly potentiated the stimulation of the I(Ca,L) elicited by subthreshold concentrations of isoprenaline (Iso, 0.1-0.5 nM) in ventricular myocytes. The effect of SNAP was mimicked by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO, 1 pM-1 nM), a NONOate that spontaneously releases NO in a pH-controlled manner, and was blunted by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM), a NO trap. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (10 microM), a guanylyl cyclase inhibitor, did not alter the effect of SNAP. SNAP (1 pM-1 microM) did not modify the effect of L858051 (0.1-0.3 microM), a forskolin analogue that activates adenylyl cyclase, on I(Ca,L) and did not enhance the basal I(Ca,L) in the presence of rolipram (1 microM), a phosphodiesterase type 4 inhibitor. Superfusion with Rp-CPT-cAMPS (500 microM), or internal dialysis with cAMP-dependent protein kinase (cA-PK) inhibitory peptide (PKI; 20 microM), inhibitors of the cA-PK, blunted the effect of SNAP (1 nM and 1 microM) on the Iso-stimulated (1-100 pM) I(Ca,L). SNAP (1 nM and 1 microM) potentiated the threshold stimulation of I(Ca,L) elicited by internal GTP-gammaS (10 microM), a non-hydrolysable analogue of GTP. SNAP (1 pM-1 microM) and DEANO (1 microM) potentiated the stimulation of I(K,ACh) elicited by low concentrations of ACh (1-2 nM) in rat atrial myocytes. The threshold stimulation of I(K,ACh) elicited by internal 5'-guanylylimidodiphosphate (10 microM) was also potentiated by NO donors. SNAP (1 microM) did not modify I(K,ACh) reconstituted in human embryonic kidney 293 cells, in the absence or in the presence of ACh (1 or 10 nM). Taken together, these data suggest that NO is a cGMP-independent modulator of G-protein-coupled muscarinic and beta-adrenergic receptor actions on cardiac ion channels. Although this action of NO seemed to occur at the level of G proteins, it appeared to require a component distinct from receptors, G proteins or their effectors.

T-588(R(-)-1-(benzo(b)thiophen-5yl)-2-[2(N,N-diethylamino)ethoxy]ethanol hydrochloride) is a novel compound which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. This compound can slow the motor deterioration of wobbler mouse motor neuron disease. However, it is not known whether this compound has a trophic effect on spinal motor neurons. We have studied the effect of T-588 on neurite outgrowth and choline acetyltransferase(ChAT) activity in primary explant cultures of ventral spinal cord of fetal rats(VSCC). Cultures were treated with T-588 from day 1 to 1 week. T-588 treated VSCC, compared with control VSCC, had a significant neurite promoting effect at ranged between 10(-8) molar(M) and 10(-5) M, with 2.3 to 5.3 fold increased over that of control VSCC. In T-588 treated VSCC, ChAT activity was increased 1.5 times over that of control at 10(-6), and 10(-5) M respectively. Our data showing T-588 has neurotrophic action on VSCC and suggests a potential use of T-588 in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and motor neuron disease.

This study was undertaken to determine the concentration of malondialdehyde, an end product of lipoperoxidation, in lens and retinal tissue postvitrectomy associated with oral administration of antioxidant flavonoids cyaninosides chloride and chromocarb diethylamine salt or N -acetylcysteine. Fifty adult pigmented rabbits were divided into five groups: (1) controls (normal eyes, malondialdehyde concentration in lens and retina); (2) vitrectomy with BSS Plus (malondialdehyde level measured 2hr after vitrectomy); (3) vitrectomy with BSS Plus and pretreatment with oral cyaninosides chloride 100mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery); (4) vitrectomy with BSS Plus and pretreatment with oral chromocarb diethylamine salt 100 mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery); and (5) vitrectomy with BSS Plus and pretreatment with oral N -acetylcysteine 200 mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery). Lens and retina samples were used to determine malondialdehyde levels using ion-pairing high performance liquid chromatography. Statistical analysis was done using analysis of variance (P<0.05). The content of malondialdehyde in the normal lens was 0.036 +/- 0.017 microg g(-1); in the vitrectomized groups the malondialdehyde concentrations were as follows: (2) 0.027 +/- 0.013 microg g(-1); (3) under detection limit (detection limit=1.75x e-3 microg g(-1)); (4) under detection limit; and (5) 0.020 +/- 0.006 microg g(-1). The results showed that the malondialdehyde concentration in the normal retina was 1.160 +/- 0.361 microg g(-1), while in the vitrectomized groups with or without pretreatment (cyaninosides chloride, chromocarb diethylamine salt, and N -acetylcysteine) the malondialdehyde levels were 2.091 +/- 0.982 microg g(-1), 0.069 +/- 0.024 microg g(-1), 0.082 +/- 0.027 microg g(-1), and 0.215 +/- 0.134 microg/g(-1), respectively, all significantly different from the normal eyes (P<0.05). Vitrectomy induced increased malondialdehyde levels in the retina. Oral flavonoids are an effective protective therapy for surgically induced lipoperoxidation, especially in the retina., (Copyright 2002 Elsevier Science Ltd.)

The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.

Pulmonary vascular responses to the novel diazeniumdiolate nitric oxide (NO) donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt, were investigated and compared in the intact-chest cat. Under conditions of controlled blood flow, when tone in the pulmonary vascular bed had been raised to a high steady level, intralobar injections of diethylamine/NO (0.3-10 microg), diethylenetriamine/NO (10-30 microg), spermine/NO (10-30 microg), sulfite/NO (10-30 microg), and angeli's salt (10-30 microg) caused dose-related decreases in lobar arterial pressure without changing left atrial pressure. In terms of relative vasodilator activity in the pulmonary vascular bed, the dose of the compounds that decreased lobar arterial pressure 4 mm Hg (ED(4) mm Hg) was significantly lower for diethylamine/NO compared to S-nitroso-N-acetylpenicillamine which was significantly less than diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt. The half-life of the vasodilator responses, as measured by 50% response recovery time, to diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt was similar for doses with similar magnitudes of vasodilation, while the half-life to S-nitroso-N-acetylpenicillamine was significantly less than the diazeniumdiolate NO donors. The present data demonstrate that the diazeniumdiolate NO donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt have potent but relatively short-lasting vasodilator activity in the pulmonary vascular bed of the cat.

Exercise generates free radicals only when it is exhaustive. Free radicals are involved in tissue damage caused by exercise. Antioxidant vitamins (vitamin C and E) and other antioxidants such as coenzyme Q, and N-acetyl cysteine prevent muscle damage and decrease muscle fatigue. The main aim of this paper was to test the possible protective effect of two new antioxidants, cyanoside chloride and chromocarbe diethylamine, on the oxidative stress generated by exhaustive exercise. The antioxidants were given to rats daily (50 mg/kg) in drinking water for 30 days. Blood oxidized glutathione/ reduced glutathione ratio, and plasma malondialdehyde levels were determined as indexes of oxidative stress. Plasma creatine kinase, alanine-aminotransferase and lactate dehydrogenase activities were used as markers of muscle damage. Both cyanoside chloride and chromocarbe diethylamine were more effective than vitamin C in the prevention of glutathione oxidation in blood. Furthermore, cyanoside chloride and chromocarbe diethylamine partially prevented muscle damage. Chromocarbe diethylamine was the most effective compound in the prevention of exercise-induced lipid peroxidation (malondialdehyde) in plasma.