Protective effects of sigma 1 receptor agonist PRE084 on 2,4,6-trinitrobenzene sulfonic acid-induced experimental colitis in mice.

Purpose: We aimed to investigate the protective effect of sigma 1 receptor agonist and antagonist, PRE084 and BD1047, respectively, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Methods: Thirty male ICR mice were randomly divided into 5 groups: control, 50% ethanol, colitis, PRE084 + colitis, and combined (PRE084 + BD1047 + colitis). Colitis was induced by intrarectal administration of TNBS. PRE084 and BD1047 were injected daily, starting 3 days before colitis induction. Distal colon tissue was excised for histopathological evaluation, and levels of glutathione (GSH), superoxide dismutase (SOD), myeloperoxidase (MPO), and lipid peroxidation were determined. Results: Colitis caused weight loss, mucosal damage, upregulation of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, MPO, and thiobarbituric acid reactive substance activities, and downregulation of GSH and SOD activities. These changes caused by TNBS-induced colitis were significantly ameliorated by PRE084 pretreatment. However, the combined pretreatment with BD1047 significantly attenuated the protective effect of PRE084, thereby reverting to the colitis-induced state. Conclusion: We conclude that the sigma 1 receptor agonist PRE084 exhibits significant protective effects against TNBS-induced colitis, which appears to be at least partly mediated by the inhibition of inflammation and oxidative stress, and enhancement of antioxidant properties. Collectively, these results suggest that PRE084 might be an effective drug for the treatment of ulcerative colitis.

Dexmedetomidine-Induced Aortic Contraction Involves Transactivation of the Epidermal Growth Factor Receptor in Rats.

In this study, we examined whether aortic contraction, induced by the alpha-2 adrenoceptor agonist dexmedetomidine, is involved in the transactivation of the epidermal growth factor receptor (EGFR) in isolated endothelium-denuded rat aortas. Additionally, we aimed to elucidate the associated underlying cellular mechanisms. The effects of the alpha-2 adrenoceptor inhibitor rauwolscine, EGFR tyrosine kinase inhibitor AG1478, Src kinase inhibitors PP1 and PP2, and matrix metalloproteinase inhibitor GM6001 on EGFR tyrosine phosphorylation and c-Jun NH2-terminal kinase (JNK) phosphorylation induced by dexmedetomidine in rat aortic smooth muscles were examined. In addition, the effects of these inhibitors on dexmedetomidine-induced contraction in isolated endothelium-denuded rat aorta were examined. Dexmedetomidine-induced contraction was inhibited by the alpha-1 adrenoceptor inhibitor prazosin, rauwolscine, AG1478, PP1, PP2, and GM6001 alone or by a combined treatment with prazosin and AG1478. AG1478 (3 × 10-6 M) inhibited dexmedetomidine-induced contraction in isolated endothelium-denuded rat aortas pretreated with rauwolscine. Dexmedetomidine-induced EGFR tyrosine and JNK phosphorylation were inhibited by rauwolscine, PP1, PP2, GM6001, and AG1478. Furthermore, dexmedetomidine-induced JNK phosphorylation reduced upon EGFR siRNA treatment. Therefore, these results suggested that the transactivation of EGFR associated with dexmedetomidine-induced contraction, mediated by the alpha-2 adrenoceptor, Src kinase, and matrix metalloproteinase, caused JNK phosphorylation and increased calcium levels.

Gastroprotective effects of irsogladine maleate on ethanol/hydrochloric acid induced gastric ulcers in mice.

BACKGROUND/AIMS: This study was conducted to investigate the inhibitory effect of irsogladine maleate (IM) on gastric ulcers induced by ethanol and hydrochloric acid (HCl). METHODS: Mice were pretreated with IM for 1 hours before ulcer induction. Gastric ulcers were induced by oral administration of an ethanol/HCl mixture. To clarify the action mechanism of IM, the roles of 3'5'-cyclic adenosine monophosphate (cAMP), nitric oxide (NO), adenosine triphosphate-sensitive potassium (KATP) channels, prostaglandins and transient receptor potential cation channel subfamily V member 1 (TRPV1) were investigated, and lipid peroxidation in the stomach of IM-treated and -untreated animals was also measured. RESULTS: IM significantly reduced the extent of ethanol/HCl mixture-induced gastric ulceration. It exhibited dose-related gastroprotection against the ethanol/ HCl-induced lesions, while pretreatment with glibenclamide but not N(ω)-nitro- L-arginine methyl ester, reversed this action. While pretreatment with the TRPV1 antagonist capsazepine failed to effectively block the gastroprotective effect of IM, the non-selective cyclooxygenase inhibitor indomethacin almost abolished it. IM also decreased the level of thiobarbituric acid reactive substances. CONCLUSION: We concluded that IM exhibited significant gastroprotective effects in an ethanol/HCl-induced ulcer model, which appear to be mediated, at least in part, by NO, cAMP, endogenous prostaglandins, KATP channel opening, activation of TRPV1 channels, and antioxidant properties.

Linolenic acid enhances contraction induced by phenylephrine in isolated rat aorta.

This study examined the effect of linolenic acid on the contraction of isolated endothelium-intact and -denuded rat aorta induced by phenylephrine and its underlying mechanism. This was conducted in the presence or absence of NW-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, and calmidazolium. The effects of linolenic acid on contraction induced by calcium chloride in calcium-free Krebs solution containing 60 mM potassium chloride were also examined. Moreover, the effect of linolenic acid on the association between intracellular calcium level ([Ca2+]i) and tension induced by phenylephrine was examined. Finally, we examined the effects of linolenic acid on cGMP formation and endothelial nitric oxide synthase (eNOS) phosphorylation induced by phenylephrine. Linolenic acid (5 × 10-5 M) increased phenylephrine-induced contraction in endothelium-intact aorta (standardized mean difference [SMD] of log ED50: 2.23), whereas it decreased this contraction in endothelium-denuded aorta (SMD: 1.96). L-NAME, ODQ, methylene blue, and calmidazolium increased phenylephrine-induced contraction in endothelium-intact aorta. Linolenic acid decreased contraction induced by calcium chloride in calcium-free Krebs solution containing 60 mM potassium chloride in endothelium-denuded aorta. Linolenic acid caused an increase in [Ca2+]i (SMD at 3 × 10-7 M phenylephrine: 1.63) and calcium sensitivity induced by phenylephrine in endothelium-intact aorta. Conversely, linolenic acid decreased [Ca2+]i (SMD: 0.99) induced by phenylephrine in endothelium-denuded aorta. Linolenic acid decreased cGMP formation and eNOS phosphorylation induced by phenylephrine. These results suggest that linolenic acid increases phenylephrine-induced contraction, which is attributed to linolenic acid inhibition of endothelial NO release rather than its decrease of [Ca2+]i in vascular smooth muscle.

Levobupivacaine-induced vasoconstriction involves caldesmon phosphorylation mediated by tyrosine kinase-induced ERK phosphorylation.

The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase.

Linoleic Acid Attenuates the Toxic Dose of Bupivacaine-Mediated Reduction of Vasodilation Evoked by the Activation of Adenosine Triphosphate-Sensitive Potassium Channels.

The goal of this study was to investigate the effect of lipid emulsion on a toxic dose of local anesthetic-mediated reduction of vasodilation evoked by the ATP-sensitive potassium (KATP) channel agonist levcromakalim. The effect of lipid emulsion (LE) and linoleic acid on the local anesthetic-mediated reduction of vasodilation and membrane hyperpolarization evoked by levcromakalim was assessed in isolated endothelium-denuded vessels (rat aorta and mesenteric artery) and aortic vascular smooth muscle cells. The effect of LE and linoleic acid on KATP channel activity in transfected HEK-293 cells was investigated, as was the effect of LE on bupivacaine concentration. The efficacy of LE in attenuating the local anesthetic-mediated reduction of vasodilation evoked by levcromakalim was correlated with the lipid solubility of the local anesthetic. Linoleic acid attenuated the bupivacaine-mediated reduction of vasodilation evoked by levcromakalim. LE decreased the bupivacaine-mediated reduction of membrane hyperpolarization evoked by levcromakalim but did not significantly alter the mepivacaine-mediated reduction. LE and linoleic acid both reversed the bupivacaine-mediated decrease of KATP activity and enhanced KATP activity. LE decreased the bupivacaine concentration. Linoleic acid may be the major contributor to LE-induced attenuation of bupivacaine-mediated reduction of vasodilation evoked by levcromakalim via the direct activation of KATP channels and indirect effects.

A Lipid Emulsion Reverses Toxic-Dose Bupivacaine-Induced Vasodilation during Tyrosine Phosphorylation-Evoked Contraction in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of a lipid emulsion on toxic-dose bupivacaine-induced vasodilation in a model of tyrosine phosphatase inhibitor sodium orthovanadate-induced contraction in endothelium-denuded rat aortae and to elucidate the associated cellular mechanism. The effect of a lipid emulsion on vasodilation induced by a toxic dose of a local anesthetic during sodium orthovanadate-induced contraction was examined. In addition, the effects of various inhibitors, either bupivacaine alone or a lipid emulsion plus bupivacaine, on protein kinase phosphorylation induced by sodium orthovanadate in rat aortic vascular smooth muscle cells was examined. A lipid emulsion reversed the vasodilation induced by bupivacaine during sodium orthovanadate-induced contraction. The lipid emulsion attenuated the bupivacaine-mediated inhibition of the sodium orthovanadate-induced phosphorylation of protein tyrosine, c-Jun NH2-terminal kinase (JNK), myosin phosphatase target subunit 1 (MYPT1), phospholipase C (PLC) γ-1 and extracellular signal-regulated kinase (ERK). These results suggest that a lipid emulsion reverses toxic-dose bupivacaine-induced vasodilation during sodium orthovanadate-induced contraction via the activation of a pathway involving either tyrosine kinase, JNK, Rho-kinase and MYPT1 or tyrosine kinase, PLC γ-1 and ERK, and this reversal is associated with the lipid solubility of the local anesthetic and the induction of calcium sensitization.

Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine.

BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca2+]i) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca2+]i. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.

Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas.

Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca2+]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.

Lipid Emulsion Inhibits Vasodilation Induced by a Toxic Dose of Bupivacaine via Attenuated Dephosphorylation of Myosin Phosphatase Target Subunit 1 in Isolated Rat Aorta.

Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca(2+)]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca(2+)]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF.

Dexmedetomidine-Induced Contraction in the Isolated Endothelium-Denuded Rat Aorta Involves PKC-δ-mediated JNK Phosphorylation.

Vasoconstriction mediated by the highly selective alpha-2 adrenoceptor agonist dexmedetomidine leads to transiently increased blood pressure and severe hypertension. The dexmedetomidine-induced contraction involves the protein kinase C (PKC)-mediated pathway. However, the main PKC isoform involved in the dexmedetomidine-induced contraction remains unknown. The goal of this in vitro study was to examine the specific PKC isoform that contributes to the dexmedetomidine-induced contraction in the isolated rat aorta. The endothelium-denuded rat aorta was suspended for isometric tension recording. Dexmedetomidine dose-response curves were generated in the presence or absence of the following inhibitors: the pan-PKC inhibitor, chelerythrine; the PKC-α and -ß inhibitor, Go6976; the PKC-α inhibitor, safingol; the PKC-ß inhibitor, ruboxistaurin; the PKC-δ inhibitor, rottlerin; the c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125; and the myosin light chain kinase inhibitor, ML-7 hydrochloride. Western blot analysis was used to examine the effect of rottlerin on dexmedetomidine-induced PKC-δ expression and JNK phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) and to investigate the effect of dexmedetomidine on PKC-δ expression in VSMCs transfected with PKC-δ small interfering RNA (siRNA) or control siRNA. Chelerythrine as well as SP600125 and ML-7 hydrochloride attenuated the dexmedetomidine-induced contraction. Go6976, safingol, and ruboxistaurin had no effect on the dexmedetomidine-induced contraction, whereas rottlerin inhibited the dexmedetomidine-induced contraction. Dexmedetomidine induced PKC-δ expression, whereas rottlerin and PKC-δ siRNA transfection inhibited dexmedetomidine-induced PKC-δ expression. Dexmedetomidine also induced JNK phosphorylation, which was inhibited by rottlerin. Taken together, these results suggest that the dexmedetomidine-induced contraction involves PKC-δ-dependent JNK phosphorylation in the isolated rat aorta.

Carvacrol inhibits atherosclerotic neointima formation by downregulating reactive oxygen species production in vascular smooth muscle cells.

OBJECTIVE: Carvacrol (2-methyl-5-(1-methylethyl) phenol), a cyclic monoterpene, exerts protective activities in a variety of pathological states including tumor growth, inflammation, and oxidative stress. However, it is unknown whether carvacrol affects events in vascular cells during the development of atherosclerotic neointima. We investigated the effects of carvacrol on the migration and proliferation of rat aortic smooth muscle cells (RASMCs) and on vascular neointima formation. METHODS AND RESULTS: Carvacrol significantly inhibited platelet-derived growth factor (PDGF)-BB-stimulated RASMC migration and proliferation in a concentration-dependent manner. Cell viability was not affected by treatment with carvacrol. Carvacrol attenuated the expression of NADPH oxidase (NOX) 1 and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 in response to PDGF-BB. Moreover, carvacrol suppressed the PDGF-BB-stimulated generation of H2O2 and inhibited the activity of NOX in RASMCs. Treatment with carvacrol inhibited PDGF-BB-induced aortic sprout outgrowth, balloon injury-evoked vascular neointima formation, and expression of proliferating cell nuclear antigen in the neointima. CONCLUSION: These findings indicate that carvacrol inhibits migration and proliferation of RASMCs by suppressing the reactive oxygen species-mediated MAPK signaling pathway in these cells, thereby attenuating vascular neointimal formation. Carvacrol may be a promising agent for preventing vascular restenosis or atherosclerosis.

Bupivacaine-induced Vasodilation Is Mediated by Decreased Calcium Sensitization in Isolated Endothelium-denuded Rat Aortas Precontracted with Phenylephrine.

BACKGROUND: A toxic dose of bupivacaine produces vasodilation in isolated aortas. The goal of this in vitro study was to investigate the cellular mechanism associated with bupivacaine-induced vasodilation in isolated endotheliumdenuded rat aortas precontracted with phenylephrine. METHODS: Isolated endothelium-denuded rat aortas were suspended for isometric tension recordings. The effects of nifedipine, verapamil, iberiotoxin, 4-aminopyridine, barium chloride, and glibenclamide on bupivacaine concentration-response curves were assessed in endothelium-denuded aortas precontracted with phenylephrine. The effect of phenylephrine and KCl used for precontraction on bupivacaine-induced concentration-response curves was assessed. The effects of verapamil on phenylephrine concentration-response curves were assessed. The effects of bupivacaine on the intracellular calcium concentration ([Ca(2+)]i) and tension in aortas precontracted with phenylephrine were measured simultaneously with the acetoxymethyl ester of a fura-2-loaded aortic strip. RESULTS: Pretreatment with potassium channel inhibitors had no effect on bupivacaine-induced relaxation in the endothelium-denuded aortas precontracted with phenylephrine, whereas verapamil or nifedipine attenuated bupivacaine-induced relaxation. The magnitude of the bupivacaine-induced relaxation was enhanced in the 100 mM KCl-induced precontracted aortas compared with the phenylephrine-induced precontracted aortas. Verapamil attenuated the phenylephrine-induced contraction. The magnitude of the bupivacaine-induced relaxation was higher than that of the bupivacaine-induced [Ca(2+)]i decrease in the aortas precontracted with phenylephrine. CONCLUSIONS: Taken together, these results suggest that toxic-dose bupivacaine-induced vasodilation appears to be mediated by decreased calcium sensitization in endothelium-denuded aortas precontracted with phenylephrine. In addition, potassium channel inhibitors had no effect on bupivacaine-induced relaxation. Toxic-dose bupivacaine- induced vasodilation may be partially associated with the inhibitory effect of voltage-operated calcium channels.

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.

BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca(2+)]i). However, the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca(2+)]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd(3+)), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca(2+)]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca(2+)]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca(2+)]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca(2+)]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca(2+)]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca(2+)]i increase. Gd(3+) had no effect on either vasoconstriction or the [Ca(2+)]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca(2+)]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta.

Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca(2+)]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca(2+)]i-tension curve was higher than that of the KCl-induced [Ca(2+)]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca(2+)]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways.

Ropivacaine-induced contraction is attenuated by both endothelial nitric oxide and voltage-dependent potassium channels in isolated rat aortae.

This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS) inhibitor N (ω) -nitro-L-arginine methyl ester (L-NAME), the neuronal NOS inhibitor N (ω) -propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC) inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP), the calcium-activated potassium channel inhibitor tetraethylammonium (TEA), the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, N (ω) -propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

Mechanisms of motility change on trinitrobenzenesulfonic Acid-induced colonic inflammation in mice.

Ulcerative colitis is an inflammatory bowel disease (IBD) characterized by recurrent episodes of colonic inflammation and tissue degeneration in human or animal models. The contractile force generated by the smooth muscle is significantly attenuated, resulting in altered motility leading to diarrhea or constipation in IBD. The aim of this study is to clarify the altered contractility of circular and longitudinal smooth muscle layers in proximal colon of trinitrobenzen sulfonic acid (TNBS)-induced colitis mouse. Colitis was induced by direct injection of TNBS (120 mg/kg, 50% ethanol) in proximal colon of ICR mouse using a 30 G needle anesthetized with ketamin (50 mg/kg), whereas animals in the control group were injected of 50% ethanol alone. In TNBS-induced colitis, the wall of the proximal colon is diffusely thickened with loss of haustration, and showed mucosal and mucular edema with inflammatory infiltration. The colonic inflammation is significantly induced the reduction of colonic contractile activity including spontaneous contractile activity, depolarization-induced contractility, and muscarinic acetylcholine receptor-mediated contractile response in circular muscle layer compared to the longitudinal muscle layer. The inward rectification of currents, especially, important to Ca(2+) and Na(+) influx-induced depolarization and contraction, was markedly reduced in the TNBS-induced colitis compared to the control. The muscarinic acetylcholine-mediated contractile responses were significantly attenuated in the circular and longitudinal smooth muscle strips induced by the reduction of membrane expression of canonical transient receptor potential (TRPC) channel isoforms from the proximal colon of the TNBS-induced colitis mouse than the control.

Proteomic analysis of colonic mucosal tissue from tuberculous and ulcerative colitis patients.

Changes in the expression profiles of specific proteins leads to serious human diseases, including colitis. The proteomic changes related to colitis and the differential expression between tuberculous (TC) and ulcerative colitis (UC) in colon tissue from colitis patients has not been defined. We therefore performed a proteomic analysis of human TC and UC mucosal tissue. Total protein was obtained from the colon mucosal tissue of normal, TC, and UC patients, and resolved by 2-dimensional electrophoresis (2-DE). The results were analyzed with PDQuest using silver staining. We used matrix-assisted laser desorption ionization time-of-flight/time-of-flight spectrometry (MALDI TOF/TOF) to identify proteins differentially expressed in TC and UC. Of the over 1,000 proteins isolated, three in TC tissue and two in UC tissue displayed altered expression when compared to normal tissue. Moreover, two proteins were differentially expressed in a comparative analysis between TC and UC. These were identified as mutant ß-actin, α-enolase and Charcot-Leyden crystal protein. In particular, the expression of α-enolase was significantly greater in TC compared with normal tissue, but decreased in comparison to UC, implying that α-enolase may represent a biomarker for differential diagnosis of TC and UC. This study therefore provides a valuable resource for the molecular and diagnostic analysis of human colitis.

Protein kinases participate in the contraction in response to levobupivacaine in the rat aorta.

Levobupivacaine is a long-acting amide local anesthetic that intrinsically produces vasoconstriction both in vivo and in vitro. Levobupivacaine increases intracellular calcium concentrations ([Ca(2+)](i)) in vascular smooth muscle cells. The goals of this in vitro study were to investigate whether levobupivacaine-induced contraction is associated with increased Ca(2+) sensitivity and to identify the protein kinases involved in mediating contraction in response to levobupivacaine in isolated rat aortic smooth muscle. The effect of levobupivacaine and potassium chloride (KCl) on the [Ca(2+)](i) and tension was measured simultaneously with acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative levobupivacaine concentration-response curves were generated in the presence or absence of the following antagonists: GF 109203X; Y-27632; genistein; SP600125; PD 98059; and SB 203580. Levobupivacaine-induced protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) phosphorylation and Rho-kinase (ROCK-2) membrane translocation were detected in rat aortic vascular smooth muscle cells using Western blotting. The slope of the [Ca(2+)](i)-tension curve for levobupivacaine was higher than that for KCl. Y-27632, GF 109203X, and SP600125 attenuated levobupivacaine-induced contraction in a concentration-dependent manner. Genistein, PD 98059, and SB 203580 attenuated levobupivacaine-induced contraction. Pretreatment with GF 109203X and Y-27632 inhibited levobupivacaine-induced PKC phosphorylation and Rho-kinase (ROCK-2) membrane translocation, respectively. Pretreatment with SP600125 or PD 98059 attenuated the levobupivacaine-induced phosphorylation of JNK and ERK, respectively. These results indicate that levobupivacaine-induced contraction involving an increase in myofilament Ca(2+) sensitivity involves the primary activation of Rho-kinase-, PKC-, and JNK-mediated pathways of rat aortic smooth muscle.

Myoplasmic [ca], crossbridge phosphorylation and latch in rabbit bladder smooth muscle.

TONIC SMOOTH MUSCLE EXHIBIT THE LATCH PHENOMENON: high force at low myosin regulatory light chains (MRLC) phosphorylation, shortening velocity (Vo), and energy consumption. However, the kinetics of MRLC phosphorylation and cellular activation in phasic smooth muscle are unknown. The present study was to determine whether Ca(2+)-stimulated MRLC phosphorylation could suffice to explain the agonist- or high K(+)-induced contraction in a fast, phasic smooth muscle. We measured myoplasmic [Ca(2+)], MRLC phosphorylation, half-time after step-shortening (a measure of Vo) and contractile stress in rabbit urinary bladder strips. High K(+)-induced contractions were phasic at both 22℃ and 37℃: myoplasmic [Ca(2+)], MRLC phosphorylation, 1/half-time, and contractile stress increased transiently and then all decreased to intermediate values. Carbachol (CCh)-induced contractions exhibited latch at 37℃: stress was maintained at high levels despite decreasing myoplasmic [Ca(2+)], MRLC phosphorylation, and 1/half-time. At 22℃ CCh induced sustained elevations in all parameters. 1/half-time depended on both myoplasmic [Ca(2+)] and MRLC phosphorylation. The steady-state dependence of stress on MRLC phosphorylation was very steep at 37℃ in the CCh- or K(+)-depolarized tissue and reduced temperature flattend the dependence of stress on MRLC phosphorylation compared to 37℃. These data suggest that phasic smooth muscle also exhibits latch behavior and latch is less prominent at lower temperature.