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Na + -K + -2Cl - cotransporter (NKCC) is expressed at exceptionally high levels in gastric parietal cells. Bumetanide, a potent loop diuretic that blocks NKCC, usually causes a decrease in gastric acid secretion. Endotoxaemia causes hypochlorhydria in vivo, in which lipopolysaccharide (LPS) plays an important role. This study aimed to investigate the effect of NKCC2 on gastric acid secretion and its alteration in LPS-treated mice. The scanning ion-selective electrode technique and real-time pH titration combined with RNA interference were used to determine the effects of bumetanide on gastric acid secretion. Immunochemistry and Western blotting were performed to investigate the changes in NKCC2 expression in LPS-treated mice. Immunoreactivity of NKCC1 and NKCC2 was mainly observed near the basolateral and apical membranes of parietal cells, respectively. Pretreatment with bumetanide reduced the histamine-stimulated H + flux in the mouse gastric mucosa. The apical, but not the basolateral, addition of bumetanide inhibited forskolin- or histamine/3-isobutyl-1-methylxanthine(IBMX)-induced gastric acid secretion. In vivo treatment with NKCC2 siRNA inhibited forskolin-induced acid secretion. Upon histamine stimulation, the majority of NKCC2 was targeted to the apical membrane in the gastric mucosa and in primary cultured parietal cells. The expression of NKCC2 and vesicle-associated membrane protein-2 (VAMP2), but not that of H + /K + -ATPase, was decreased in the gastric mucosa of LPS-treated mice. Blocking apical NKCC2, but not basolateral NKCC1, by bumetanide inhibited secretagogue-induced gastric acid secretion, during which the membrane trafficking of NKCC2 may be necessary. The downregulation of NKCC2 and VAMP2 may be related to the reduced gastric acid secretion induced by LPS., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Airway remodeling in asthma is difficult to treat because of its complex pathophysiology that involves proinflammatory cytokines, as well as the arachidonic acid cytochrome P-450 (CYP) pathway; however, it has received little attention. In this study, we assessed the efficacy of a soluble epoxide hydrolase (sEH) on airway remodeling in a mouse model of chronic asthma. The expression of sEH and CYP2J2 and the level of 14,15-epoxyeicosatrienoic acid (14,15-EET), airway remodeling and hyperresponsiveness (AHR) were analyzed to determine the level of sEH inhibition. AUDA, a sEH inhibitor, was given daily for 9 weeks orally, which significantly increased the level of 14,15-EET by inhibiting the expression of sEH and increasing the expression of CYP2J2 in lung tissues. The inhibition of sEH reduced the expression of remodeling-related molecular markers, such as interleukin (IL)-13, IL-17, matrix metalloproteinase 9, N-cadherin, α-smooth muscle actin (α-SMA), S100A4, Twist, epithelial goblet cell metaplasia, and collagen deposition in bronchoalveolar lavage fluid (BAL fluid) and lung tissues. Moreover, remodeling-related eosinophil accumulation in the BAL fluid and infiltration into the lung tissue were improved by AUDA. Finally, AUDA alleviated AHR, which is a functional indicator of airway remodeling. The effect of AUDA on airway remodeling was related to the downregulation of extracellular-regulated protein kinases (Erk1/2), c-Jun N-terminal kinases (JNK) and signal transducer and activator of transcription 3 (STAT3). To our knowledge, this is the first report to demonstrate that inhibition of sEH exerts significant protective effects on airway remodeling in asthma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ferulic acid (FA), a naturally derived phenolic compound, has antioxidant and antidepressant-like effects. It is still a challenge to study its mechanism due to the complexity of the pathophysiology of depression. In this study, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to perform metabolomics studies based on biochemical changes in differentiated rat pheochromocytoma (PC12) cells treated with corticosterone-induced neurological damage after FA treatment. A total of 31 metabolites were identified as potential biomarkers for corticosterone-induced PC12 cells injury. Among them, 24 metabolites were regulated after FA treatment. Pathway analysis revealed that these metabolites were mainly involved in the amino acid metabolism, energy metabolism and glycerophospholipid metabolism. In addition, based on the results of metabolomics, three cell signaling pathways related to glutamate were discovered. To further study the interactions between FA and major targets in three signaling pathways, a molecular docking method was employed. The results showed that FA had the strongest binding power with protein kinase B (AKT). Furthermore, the result of mRNA changes analyzed by quantitative real time RT-PCR indicated that AKT and protein kinase A (PKA) in the signaling pathway were up regulated after treatment with FA compared with model group. This study shows that strategies based on cell metabolomics associated with molecular docking and molecular biology is a helpful tool to elucidate the neuroprotective mechanism of FA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mosapride, a gastrointestinal prokinetic drug, is an agonist of 5-hydroxytryptamine (5-HT) receptor 4 that also reduces blood glucose. Whether 5-HT4 receptor is distributed in pancreatic islets and whether mosapride can directly stimulate insulin secretion is unclear. In the present study, the protein expression and cellular location of 5-HT4 receptor in pancreas was detected through western blotting and immunofluorescence. The acute effects of 5-HT4 receptor agonists, mosapride and prucalopride, on insulin secretion were investigated in vivo and in vitro in normal and alloxan-induced diabetes rats. The results indicated that 5-HT4 receptor immunoreactivity was co-existed in the islets insulin-immunoreactive cells of rat, mouse, pig and human. However the immunoreactive cells of insulin and 5-HT4 receptor and the protein expression of 5-HT4 receptor were significantly decreased in the pancreas of alloxan-induced diabetes rats. In normal rats, mosapride and prucalopride decreased blood glucose and increased insulin secretion during glucose tolerance test, in association with an increase in glucose-stimulated insulin secretion, which was abolished by the 5-HT4 receptor antagonist GR113808. In diabetes rats, mosapride and prucalopride failed to improve blood glucose and insulin levels in the group of 180mg/kg alloxan, but increased glucose-stimulated insulin secretion in the group of 120mg/kg alloxan in vitro. We conclude that 5-HT4 receptor is distributed in the islet β cell. Activation of 5-HT4 receptor is able to stimulate insulin secretion directly, thereby reduce blood glucose. The study provides important experimental evidences for the 5-HT4 receptor regulating insulin secretion and acting as a potential drug target in diabetes treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ambroxol, a metabolite of bromhexine, is shown to exert several pharmacological activities, including secretolytic, anti-inflammatory and antioxidant actions. Oral and intravenous administration of ambroxol is useful for the airway inflammatory diseases. However, little is known about its potential in inhalation therapy for lipopolysaccharide (LPS)-induced mucous hypersecretion and inflammatory response. In the present study, we compared the pharmacological effects of ambroxol by inhalation with intravenous administration and preliminarily explored its mechanism of action. Our results demonstrated that ambroxol administered by inhalation inhibited MUC5AC expression, reduced glycosaminoglycan levels, enhanced the function of mucociliary clearance and promoted sputum excretion, suggesting that ambroxol increases expectoration of sputum by reducing its viscosity. Moreover, ambroxol significantly alleviated LPS-induced the influx of inflammatory cells and the extracellular signal-regulated kinase 1/2 (Erk 1/2) expression in lung tissues, and inhibited increases in the mRNA expression of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, CCL-2 (monocyte chemotactic protein-1), KC (keratinocyte cell protein) and interleukin (IL)-1β in lung tissues. The secretolytic and anti-inflammatory effects of inhaled ambroxol at a dose of 7.5 mg/ml was comparable to that of ambroxol at 20 mg/ml i.v. and dexamethasone at 0.5 mg/kg i.p. In addition, we found that ambroxol dose-dependently inhibited LPS-induced increases in the mRNA expression of MUC5AC, TNF-α, and IL-1β in human bronchial epithelial cell (NCI-H292) by inhibiting the Erk signaling pathway. These results demonstrate the beneficial effects of ambroxol in inhalation therapy for the airway inflammatory diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Inhaled corticosteroid (ICS) therapy in combination with long-acting β-adrenergic agonists (LABA) is the most important treatment for allergic asthma, although the mechanism still remains unclear. However, mast cells play a central role in the pathogenesis of asthma. In this study, we explored the sole or synergetic effects of des-ciclesonide (ICS) and formoterol (LABA) on the cytokines IL-4 and IL-13 and on histamine release from mast cells (RBL-2H3 cells). We found that des-ciclesonide (0.1, 1 and 10nM) and formoterol (0.1, 1 and 10μM) alone attenuated DNP-BSA-induced IL-4 and IL-13 production, respectively, in a concentration-dependent manner in DNP-IgE-sensitized mast cells. Des-ciclesonide (0.2nM) and formoterol (1μM) alone also reduced histamine production. However, the combination of des-ciclesonide (0.2nM) and formoterol (1μM) had a synergistic inhibition effect on IL-4 mRNA expression and protein production but not IL-13 and histamine release. The JNK inhibitor SP600125 (10μM) inhibited antigen-induced mRNA expression and protein production of IL-4. Des-ciclesonide and formoterol alone inhibited the activation of JNK in a concentration-dependent manner, and the combination of des-ciclesonide (0.2nM) and formoterol (1μM) exhibited greater inhibition effect compared with des-ciclesonide (0.2nM) or formoterol (1μM) alone. Taken together, these synergistic effects on mast cells might provide the rationale for the development of the most recent ICS/LABA combination approved for asthma therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Central neuropathic pain can arise from injury of the spinal cord and can become chronic. Treatment is difficult and, because complete pain relief is currently very hard to achieve, there is a need for new, more effective treatment options. In this study we used an animal model of spinal cord injury to evaluate the potency of a bioactive fragment of substance P (SP), i.e. SP1-7, in alleviating signs of allodynia and acute pain. SP1-7 is known from earlier studies to possess antinociceptive properties. We also studied the effects of intraperitoneal injection of an amidated analog of this heptapeptide and of its truncated analogs, all of which had high affinity to the SP1-7 binding site, to evaluate the importance of the removed amino acids for the biodistribution and stability of the peptides. Most of the examined compounds alleviated mechanical allodynia without any signs of sedation or motor impairment in the rats. In contrast, the response threshold to acute nociceptive stimulation was not affected by any of the compounds tested. Most of the amino acids in the heptapeptide structure were essential for retaining the biological effect after peripheral injection. These observations suggest that the heptapeptide and its N-terminal truncated hexa- and pentapeptide analogs could be of interest for further development of analgesics in the management of mechanical allodynia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Oxidized low-density lipoprotein (Ox-LDL) is associated with atherosclerotic events through the modulation of arachidonic acid (AA) metabolism and activation of inflammatory signaling. Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) mitigate inflammation through nuclear factor-κB (NF-κB). In this study, we explored the effects and mechanisms of exogenous EETs on the ox-LDL-induced inflammation of pulmonary artery endothelial cells (PAECs), which were cultured from rat pulmonary arteries. We determined that pre-treatment with 11,12-EET or 14,15-EET attenuated the ox-LDL-induced expression and release of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1) in a concentration-dependent manner. In addition, the ox-LDL-induced expression of CYP2J4 was upregulated by 11,12-EET and 14,15-EET (1μM). Furthermore, the endothelial receptor of lectin-like oxidized low-density lipoprotein (LOX-1) was downregulated in PAECs treated with EETs. The inflammatory responses evoked by ox-LDL (100μg/mL) were blocked by pharmacological inhibitors of Erk1/2 mitogen-activated protein kinase (MAPK) (U0126), p38 MAPK (SB203580), and NF-κB (PDTC). In addition, we confirmed that 11,12-EET suppresses phosphorylation of p38, degradation of IκBα, and activation of NF-κB (p65), whereas 14,15-EET can significantly suppress the phosphorylation of p38 and Erk1/2. Our results indicate that EETs exert beneficial effects on ox-LDL-induced inflammation primarily through the inhibition of LOX-1 receptor upregulation, MAPK phosphorylation, and NF-κB activation and through the upregulation of CYP2J4 expression. This study helps focus the current understanding of the contribution of EETs to the regulation of the inflammation of pulmonary vascular endothelial cells. Furthermore, the therapeutic potential of targeting the EET pathway in pulmonary vascular disease will be highlighted., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Genistein, an isoflavone naturally found in soy products, displays estrogenic properties. Our previous study clearly demonstrated that genistein can activate the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in human breast cancer MCF-7 cells. The present study aims to test the hypothesis that the IGF-I receptor signaling pathway is involved in the neuroprotective effects of genistein in neuroblastoma SK-N-SH cells. Our results revealed that pretreatment with genistein resulted in an enhancement in the survival of human neuroblastoma SK-N-SH cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. 6-OHDA arrested the cells at G(0)G(1) phase and prevented S phase entry. Genistein pretreatment could reverse the cytostatic effect of 6-OHDA on cell cycle. The decreased mitochondrial membrane potential induced by 6-OHDA could be also reversed by genistein pretreatment. These effects could be completely blocked by co-treatment with JB-1, which is the specific antagonist of the IGF-I receptor. Furthermore, genistein pretreatment restored the 6-OHDA-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expression. Genistein treatment alone could significantly increase the phosphorylation level of MEK and induce ERE luciferase activity. Co-treatment with IGF-I could enhance the effect of genistein on cell proliferation and MEK phosphorylation. This study provides the first evidence that genistein has neuroprotective effects against 6-OHDA-induced neurotoxicity in SK-N-SH cells and activation of the IGF-I receptor signaling pathway might be involved in actions of genistein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have demonstrated that the activation of 5-hydroxytryptamine (5-HT) receptor 3 in the submucosal plexus suppresses 5-HT-induced colonic ion secretion by increasing submucosal somatostatin release. A number of psychological and physical stresses have impacts on the intestinal mucosal functions, including secretion and the epithelial barrier. Whether the 5-HT(3) receptor-mediated somatostatin-dependent secretoinhibitory pathway in the rat distal colon is involved in the stress process is still unknown. The present study aims to investigate the effect of the water-immersion restraint stress on this inhibitory pathway and its underlying mechanisms. Mucosa/submucosa preparations from the rat distal colon were mounted in the Ussing chambers for the measurement of short-circuit current (I(SC)). Real-time PCR and western blot were performed to study the expression of the 5-HT(3) receptor, 5-HT(4) receptor, and somatostatin receptor 2. Radioimmunoassay was used to measure somatostatin release. After 2h of water-immersion restraint stress, the membrane resistance (Rte) of rat mucosa/submucosa preparations was significantly decreased, but the baseline I(SC) and 5-HT-induced I(SC) responses were significantly increased. The protein expression of the submucosal 5-HT(3) receptors and mucosal somatostatin receptor 2 were down-regulated, and the 5-HT-induced somatostatin release from the mucosa/submucosa preparations was significantly reduced in the stress group. Taken together, these results suggest that the 5-HT(3) receptor-mediated somatostatin-dependent secretoinhibitory pathway is suppressed in the water-immersion restraint stressed rats, which may contribute to the acute stress-induced increase in colonic secretion., (Copyright © 2011 Elsevier B.V. All rights reserved.)

M(3) muscarinic receptors are localized on inflammatory cells, airway smooth muscle, and submucosal glands, known to mediate bronchoconstriction, mucus secretion, and airway remodeling. It is hypothesized bencycloquidium bromide (BCQB), a novel M(3) receptor antagonist, might have potential effects on airway hyperresponsiveness, inflammation and airway remodeling in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was examined to determine the total and differential cell counts, and cytokine levels. Lung tissues were evaluated for cell infiltration, mucus hypersecretion, airway remodeling, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Inhalation administration of BCQB significantly not only reduced ovalbumin-induced airway hyperresponsiveness comparing to methacholine, and prevented the ovalbumin-induced increase in total cell counts and eosinophil counts. Reverse transcriptase polymerase chain reaction analysis of whole lung lysates revealed that BCQB markedly suppressed ovalbumin-induced mRNA expression of eotaxin, IL-5, IL-4 and MMP-9, and increased mRNA expression of IFN-γ and TIMP-1 in a dose-dependent manner. Substantial IFN-γ/IL-4 (Th1/Th2) levels were recovered in bronchoalveolar lavage fluid after BCQB treatment. In addition, histological studies showed that BCQB dramatically inhibited ovalbumin-induced lung tissue eosinophil infiltration, airway mucus production and collagen deposition in lung tissues. Results reported in current paper suggest that M(3) receptors antagonist may provide a novel therapeutic approach to treat airway inflammation, hyperresponsiveness and remodeling., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

In this study we have investigated the antagonist affinity, efficacy and duration of action of bencycloquidium bromide (BCQB), a selective muscarinic M(3) receptor antagonist, as a possible clinical bronchodilator for the treatment of chronic obstructive pulmonary disease (COPD) and asthma. In competition studies, BCQB showed high affinity toward the M(3) receptor in Chinese hamster ovary (CHO) cells (M(3) pKi=8.21, M(2) pKi=7.21, and M(1) pKi=7.86); pA(2)=8.85, 8.71 and 8.57 in methacholine-induced contraction of trachea, ileum and urinary bladder, 8.19 in methacholine-induced bradycardia of right atrium in vitro, respectively. In function studies, duration of inhibition of carbachol-induced tonic contraction, BCQB and ipratropium had a very similar onset and offset of action, but onset faster and offset slower than that of tiotropium. After treatment with intratracheally instilled or the inhalation route, BCQB protects against methacholine or antigen-induced bronchoconstriction in a dose-dependent manner in the normal and sensitized guinea pigs in vivo. BCQB and ipratropium-induced inhibitory activity was short lasting, as it declined quickly when compared to tiotropium. These results suggest that BCQB bind muscarinic M(3) receptors with high affinity. On this basis we speculate that a putative BCQB-based therapy for COPD might require more than once-a-day administration to be as effective as the currently employed once-daily therapy with tiotropium. Nevertheless, Inhalable M(3)-selective compounds may spare M(2)-cardiac receptors and reduce the risks of cardiovascular events associated with the long-term treatment of these agents., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

5-hydroxytryptamine (5-HT)-evoked intestinal secretion can be divided into neural and non-neural pathway. Recently, 5-HT(3) receptor in neural pathway received much attention as a possible target in bowel diseases. The present study aims to investigate the effects of 5-HT(3) receptor in different enteric neural plexus (myenteric plexus and submucosal plexus) on rat colonic ion transport by using rat intact colon and mucosa/submucosa preparations. Ussing chamber and real-time PCR techniques were performed in our present study. Surprisingly, we found that in mucosa/submucosa preparations, 5-HT-induced DeltaI(SC) (change in short-circuit current) was not inhibited, but further increased by pretreatment with 5-HT(3) receptor antagonists, MDL72222 and Tropanyl-3, 5-dimethylbenzoate. And this response was significantly attenuated in the presence of tetrodotoxin (TTX). Conversely, in rat intact colon, 5-HT(3) receptor antagonists significantly inhibited 5-HT-induced DeltaI(SC). The results from real-time PCR proved the existence of 5-HT(3) receptor in muscularis externa and submucosa. Taken together, 5-HT(3) receptors possess a role of dual regulation on electrolyte secretion in rat distal colon, the neural stimulatory effect of 5-HT(3) receptor in myenteric plexus and the inhibitory effect of 5-HT(3) receptor in submucosal plexus.

Freshly isolated rat colonic mucosa close to anus (the late distal colonic mucosa) was used to investigate the effect of dopamine on the rat late distal colonic ion transport by means of short-circuit current (I(SC)) recording and reverse transcription PCR (RT-PCR) analysis. The results showed that the basolateral addition of dopamine (0.1-1000 micromol/l) produced a concentration-dependent downward deflection in I(SC) with an apparent EC(50) of 20.06 micromol/l in the late distal colon. The dopamine-induced I(SC) decrease was abolished by bilateral removal of Cl(-) or HCO(3)(-), apical Cl(-) replacement and apical pretreatment with non-specific Cl(-) channel blocker/transporter inhibitor, DPC (1 mmol/l) or glibenclamide (1 mmol/l), and reversed by subsequent addition of glibenclamide. Removal of basolateral Na(+) or reducing basolateral HCO(3)(-) (3 mmol/l) as well as basolateral pretreatment with DIDS (4,4'-didsothio- cyanostilbene-2, 2'-disulfonic acid) (250 micromol/l), an inhibitor of NBC or AE, could also inhibit the dopamine-induced I(SC) response. However, apical pretreatment with epithelial Na(+) channel blocker, amiloride (10 micromol/l), Ca(2+)-dependent Cl(-) channel blocker/anion exchanger, DIDS (100 micromol/l), or putative K(+) blockers such as Ba(2+) (5 mmol/l), TEA (tetraethylammonium) (5 mmol/l) or 293B (trans-6-cyano-4- (N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chromane) (10 micromol/l) did not significantly affect the dopamine-induced I(SC) response. RT-PCR results showed the expression of anion exchanger, SLC26A3, but not SLC26A6, in rat late distal colon. In conclusion, the present results suggest that dopamine may promote rat late distal colonic epithelial Cl(-) absorption coupled with HCO(3)(-) secretion, which may be mediated by apical electrogenic anion exchanger, SLC26A3, and require basolateral entry of HCO(3)(-) through Na(+)-HCO(3)(-) cotransporter. The present findings reveal a previously unreported dopamine-regulated anion transport process in rat late distal colon, which may have implication in Parkinson's disease.

Artemisinin and its derivatives exhibit potent immunosuppressive activity. The aim of this study was to investigate the suppressive effects of SM905, a new water-soluble artemisinin derivative, on T lymphocytes both in vitro and in vivo, and explore its potential mode of action. The results showed that SM905 had a high inhibitory activity in Concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction, and a relatively low cytotoxicity in vitro. In ovalbumin-immunized mice, oral administration of SM905 dose-dependently suppressed T cell proliferative response to ovalbumin, and inhibited anti-ovalbumin interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by T cells. Further studies showed that SM905 inhibited TCR (T cell receptor)/CD3 plus CD28-mediated primary T cell proliferation and cytokine production (IL-2 and IFN-gamma), and exerted an inhibitory action on the phosphorylation of mitogen-activated protein (MAP) kinases including extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK), and the activation of Ras. The results of this study provided experimental evidence that the new artemisinin derivative SM905 had immunosuppressive effects both in vitro and in vivo. SM905 suppressed T cell activation, which was associated with the inhibition of MAP kinases and Ras activation. Our results suggested a potential of SM905 to be developed as a new type agent for treating T cell-mediated immune disorder.

The effect of systemic administration of lacosamide, a newly developed anti-epileptic, on neuropathic pain-like behaviors was examined in rats after ischemic injury to the infraorbital nerve or spinal cord using a photochemical method. In rats with infraorbital nerve injury, lacosamide reduced mechanical hypersensitivity and the effect was markedly stronger in female than in male rats. In spinal cord injured female rats 10-20 mg/kg lacosamide dose-dependently alleviated the mechanical and cold allodynia-like behaviors without causing motor impairments or marked sedation. Administration of lacosamide twice daily at 20 mg/kg for 7 days totally alleviated the allodynia-like state in spinally-injured rats with no tolerance. Following treatment cessation the cold and the static allodynia reappeared but the effect on dynamic mechanical allodynia (brushing) was maintained until day 11. Lacosamide also produced hypothermia at antinociceptive doses in rats. It is suggested that this novel compound may be useful as an analgesic for treating central and trigeminal neuropathic pain. Furthermore, there may be a gender difference to the effect of lacosamide with female rats being more responsive to the treatments.

We have previously demonstrated that tetramethylpyrazine (TMP) could stimulate colonic and pancreatic anion secretion. The present study investigated the signaling pathways and cellular mechanisms underlying the effect of TMP using human colonic Caco-2 cells, with permeabilized apical or basolateral membranes, in conjunction with Ussing chamber technique, intracellular cAMP and Ca2+ measurements as well as competitive RT-PCR for mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+)-dependent Cl- channels (CACC). Basolateral addition of TMP induced a short circuit current (I(SC)) response, which could be mimicked by forskolin and 3-isobutyl-1-methylxanthine (IBMX). Adenylate cyclase inhibitor, MDL12330A, and intracellular Ca2+ chelator, BAPTA-AM, significantly inhibited the TMP-induced I(SC). In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an I(SC) response, which was sensitive to MDL-12330A, H89, and specific channel blocker CFTR(inh-172), but insensitive to apical application of 4-4'-didsothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and basolateral pretreatment with BAPTA-AM. In apical membrane-permeabilized cells, TMP, similar to forskolin and IBMX, produced a very small current increase, which was sensitive to K+ channel blockers, BaCl2 and tetraethylammonium (TEA), but not Chromanol 293B and charybdotoxin (ChTX), alone or combined. However, in intact Caco-2 monolayers, the TMP-induced I(SC) could be partially inhibited by ChTX. TMP (5 mM) could stimulate intracellular cAMP production. Intracellular Ca2+ was also increased by TMP (5 mM) in both Ca(2+)-containing and Ca(2+)-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca(2+)-activated Cl- channel (CACC). In conclusion, TMP stimulates Cl- secretion by activating cAMP and [Ca2+]i signaling pathways leading to subsequent activation of apical CFTR and basolateral K+ channels.

We have found in the present study that incubation of neuroblastma N2a with calyculin A, an inhibitor of protein phosphatase-2A (PP-2A) and protein phosphatase-1 (PP-1), reduces cell viability in a dose-dependent manner, and leads to tau hyperphosphorylation at tau-1 (Ser198/199/202) and PHF-1 (Ser396/404) epitopes. In addition to inhibit PP-2A, calyculin A treatment also results in significant activation of glycogen synthase kinase-3 (GSK-3). Calyculin A induces oxidative stress manifested by elevated level of malondialdehyde and decreased activity of superoxide dismutase. When the cells were incubated simultaneously with calyculin A and melatonin (25 microM or 50 microM), the calyculin A-induced decrease in cell viability, tau hyperphosphorylation, PP-2A/GSK-3 imbalance and oxidative stress were attenuated accordingly. These results suggest (i) that calyculin A induces tau hyperphosphorylation not only by inhibition of PP-2A, but also by activation of GSK-3 in N2a cells; (ii) that melatonin efficiently attenuates the calyculin A-induced damages through not only its antioxidant effect but also its modulation to the phosphorylation system.

The selective, high-efficacy 5-HT(1A) receptor agonist, (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl]piperidin-1-yl]-methanone (F 13640) has been reported to produce long-term analgesia in rodent models of chronic nociceptive and neuropathic pain; it also preempts allodynia following spinal cord injury. Here, rats underwent spinal cord injury, fully developed allodynia, and were infused with saline or 0.63 mg/day of F 13640 for 56 days. Infusion was then discontinued, and further assessments of allodynia (vocalization threshold to von Frey filament stimulation, responses to brush and cold) were conducted for another 70 days. F 13640-induced analgesia persisted during this post-treatment period. The data offer initial evidence that high-efficacy 5-HT(1A) receptor activation produces an unprecedented curative-like action on pathological pain.

We have examined the effect of systemically administered galnon, a novel low-molecular weight agonist of galanin receptors, on neuropathic pain-like behaviors in rats after photochemically induced partial nerve injury. Galnon is a galanin receptor ligand with moderate affinity to spinal cord membranes (K(D) of 6+/-0.6 microM). While intraperitoneally applied galnon produced no significant effect on mechanical or cold hypersensitivity, it dose-dependently prolonged heat withdrawal latency in nerve-injured rats. The effect of galnon was more potent on the injured side which has significantly shorter latency than the contralateral side. The anti-hyperalgesic effect of galanon was prevented by intrathecal M35, a galanin receptor antagonist. No side effects, such as sedation or motor impairment, were seen following systemic galnon treatment at the doses used. It is concluded that systemic galnon alleviated heat-hyperalgesic response in rats with partial sciatic nerve injury. This effect was likely to be mediated by activation of spinal galanin receptors.

Central neuropathic pain after spinal cord injury (SCI) presents a challenging clinical problem with limited treatment options. [(3-chloro-4-fluoro-phenyl)-[4-fluoro-4-([(5-methyl-pyridin-2-ylmethyl)-amino]-methyl)piperidin-1-yl]]-methadone (F 13640) is a recently discovered very-high-efficacy, selective 5-HT1A receptor agonist that produces a remarkably powerful, central analgesia through unprecedented neuroadaptive mechanisms. In a rat model of spinal cord injury pain, we previously found that chronic infusion of F 13640 alleviated pain-like behaviors. Here, we report that infusion of 0.63 mg/day of F 13640 for 8 weeks starting 24 h before the induction of injury significantly attenuates the development of chronic allodynia-like behavior in rats sustaining a photochemically-induced, ischaemic injury of the dorsal laminae of the L3-L5 segments of the spinal cord. Importantly, the preemptive effect of F 13640 persisted for 2 months after treatment was discontinued. The data warrant the study of the possible effects of the early administration of F 13640 in patients sustaining spinal cord injury.

The pyranocoumarins, (+/-)-3'-angeloyl-4'-acetoxy-cis-khellactone, were isolated from Radix Peucedani, the dry root of Peucedanum praeruptorum Dunn, through bioassay-guided fractionation. The chemical structure of pyranocoumarins was determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography showed that there are eight molecules (i.e. two each of four conformers) in each unit cell with their optical activities equally cancelled out. The four conformers are 3'(R)-angeloyl-4'(R)-acetoxy-khellactone in two conformational forms, and 3'(S)-angeloyl-4'(S)-acetoxy-khellactone in two conformational forms. Pyranocoumarins caused apoptotic cell death with IC50 of 41.9+/-2.8 and 17.3+/-8.2 microM for drug-sensitive KB-3-1 and multidrug resistant (MDR) KB-V1, respectively. The two- to threefold sensitivity difference between the two cell lines is interesting considering that the same ratio for doxorubicin is 50-300. Strong synergistic interactions were demonstrated when pyranocoumarins were combined with common anti-tumor drugs including doxorubicin, paclitaxel, puromycin or vincristine in MDR KB-V1 cell line, but not in drug-sensitive KB-3-1 cells. Pyranocoumarins increased doxorubicin accumulation in KB-V1 cells by about 25% after 6 h of incubation. Pyranocoumarins treatment for 24 h down-regulated the expression of P-glycoprotein in KB-V1 cells at both protein and mRNA levels. Pyranocoumarins also transiently reduced the cellular ATP contents in KB-V1 cells in a dose-dependent manner. Our results suggest that pyranocoumarins could be a potential MDR reversing agent.

The pain-relieving effects of various voltage-activated Na(+) channel blockers have been evaluated in two rat models of neuropathic pain; the photochemically induced nerve injury model (Gazelius) and spared nerve injury model. Lidocaine (up to 40 mg/kg, i.p.) and lamotrigine (up to 60 mg/kg, i.p.) had no effect on mechanical or cold allodynia in either model. However, lamotrigine (10, 30 and 60 mg/kg) significantly attenuated mechanical hyperalgesia in the spared nerve injury model, while mexiletine (25 and 37.5 mg/kg, i.p.) attenuated mechanical allodynia in the Gazelius model. Tocainide (50, 75 and 100 mg/kg, i.p.) significantly reduced all types of pain behaviour measured. The present results show that these voltage-activated Na(+) channel blockers have broadly similar antinociceptive effects in these two models of neuropathic pain. They also show that these drugs can have markedly different effects on distinct neuropathic pain-related behaviours within models.

We have studied and compared the antinociceptive and anti-hyperalgesic effect of the partial opioid receptor agonist buprenorphine in normal and neuropathic rats. In normal rats, systemic buprenorphine produced dose-dependent antinociception on the hot plate test. In rats with peripheral nerve or spinal cord injury, buprenorphine markedly alleviated neuropathic pain-related behaviors, including mechanical and cold allodynia/hyperalgesia at doses comparable to that producing antinociception. The results suggest that buprenorphine may be a useful analgesic for treating neuropathic pain and thus is an atypical opioid since morphine tends to be less potent after nerve injury.