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 3ʹ5ʹ-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. @*Conclusions@#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.

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.

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.

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 ([Ca2+]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 [Ca2+]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 / 대한마취과학회지

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 ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]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 [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca2+]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.

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 Ca2+ 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 beta-actin, alpha-enolase and Charcot-Leyden crystal protein. In particular, the expression of alpha-enolase was significantly greater in TC compared with normal tissue, but decreased in comparison to UC, implying that alpha-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.

Mechanism of vasodilation by propofol in the rabbit renal artery

BACKGROUND: Propofol directly inhibits vascular reactivity. However, available information regarding the underlying mechanisms of propofol is poor. Therefore, mechanisms of the underlying relaxant action of propofol were investigated using rabbit renal arteries. METHODS: Propofol-induced relaxation of rabbit renal arteries was studied in contracted preparations with 50 mM KCl or 10microM histamine. Vessel tension was recorded with a pen recorder. We were interested in determining whether propofol-induced vasodilation is affected by endothelium-denudation, L-NG-nitroarginine methyl ester (L-NAME), tetraethylammonium (TEA), iberiotoxin, glibenclamide, 4-aminopyridine, 7-ethoxyresorufin, caffeic acid, baiclalein, ryanodine, and thapsigargin. RESULTS: Propofol-induced concentration-dependent vasodilation was not affected either by endothelium denudation or by L-NAME during histamine-induced contraction. The relaxing effect of propofol on histamine-induced contraction was inhibited by either TEA, a K+ channel inhibitor, or iberiotoxin (100 nM), a selective blocker of the large conductance Ca(2+)-activated K+ channel (BKCa channel). In contrast, the relaxing effect of propofol was unaffected by 10microM glibenclamide, an ATP-sensitive K+ channel blocker, by 5 mM 4-aminopyridine, a blocker of delayed rectifier, by 7-ethoxyresorufin, a cytochrome P450 inhibitor, by 10microM caffeic acid and 10microM baiclalein, lipooxygenase inhibitors, or by 10microM ryanodine and thapsigargin, Ca2+store inhibitors. CONCLUSIONS: These results suggest that the relaxant effect of propofol may result from activation of BKCa channels by inhibiting voltage-gated Ca2+ influx in a prolonged manner.

Myoplasmic Ca2+, 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 [Ca2+], 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 22degrees C and 37degrees C: myoplasmic [Ca2+], MRLC phosphorylation, 1/half-time, and contractile stress increased transiently and then all decreased to intermediate values. Carbachol (CCh)-induced contractions exhibited latch at 37degrees C: stress was maintained at high levels despite decreasing myoplasmic [Ca2+], MRLC phosphorylation, and 1/half-time. At 22degrees C CCh induced sustained elevations in all parameters. 1/half-time depended on both myoplasmic [Ca2+] and MRLC phosphorylation. The steady-state dependence of stress on MRLC phosphorylation was very steep at 37degrees C in the CCh- or K(+)-depolarized tissue and reduced temperature flattend the dependence of stress on MRLC phosphorylation compared to 37degrees C. These data suggest that phasic smooth muscle also exhibits latch behavior and latch is less prominent at lower temperature.

C-Jun NH2-Terminal Kinase Contributes to Dexmedetomidine-Induced Contraction in Isolated Rat Aortic Smooth Muscle

PURPOSE: Dexmedetomidine, a full agonist of alpha2B-adrenoceptors, is used for analgesia and sedation in the intensive care units. Dexmedetomidine produces an initial transient hypertension due to the activation of post-junctional alpha2B-adrenoceptors on vascular smooth muscle cells (SMCs). The aims of this in vitro study were to identify mitogen-activated protein kinase (MAPK) isoforms that are primarily involved in full, alpha2B-adrenoceptor agonist, dexmedetomidine-induced contraction of isolated rat aortic SMCs. MATERIALS AND METHODS: Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dexmedetomidine (10(-9) to 10(-6) M) dose-response curves were generated in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD 98059, p38 MAPK inhibitor SB 203580, c-Jun NH2-terminal kinase (JNK) inhibitor SP 600125, L-type calcium channel blocker (verapamil and nifedipine), and alpha2-adrenoceptor inhibitor atipamezole. Dexmedetomidine-induced phosphorylation of ERK, JNK, and p38 MAPK in rat aortic SMCs was detected using Western blotting. RESULTS: SP 600125 (10(-6) to 10(-5) M) attenuated dexmedetomidine-evoked contraction in a concentration-dependent manner, whereas PD 98059 had no effect on dexmedetomidine-induced contraction. SB 203580 (10(-5) M) attenuated dexmedetomidine-induced contraction. Dexmedetomidine-evoked contractions were both abolished by atipamezole and attenuated by verapamil and nifedipine. Dexmedetomidine induced phosphorylation of JNK and p38 MAPK in rat aortic SMCs, but did not induce phosphorylation of ERK. CONCLUSION: Dexmedetomidine-induced contraction involves a JNK- and p38 MAPK-mediated pathway downstream of alpha2-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca2+ level ([Ca2+]i), and Ca2+ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 micrometer) inhibited high K+ (25 mM and 40 mM)- or histamine (3 micrometer)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K+-evoked contractions. Spontaneous changes in [Ca2+]i and contractions were inhibited by forskolin (1 micrometer) without changing the resting [Ca2+]i. Forskoln (10 micrometer) inhibited muscle tension more strongly than [Ca2+]i stimulated by high K+, and thus shifted the [Ca2+]i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 micrometer) inhibited both [Ca2+]i and muscle tension without changing the [Ca2+]i-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 micrometer) inhibited the 0.3 micrometer Ca2+-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca2+-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca2+ sensitivity of contractile elements in high K+-stimulated muscle and a decrease in [Ca2+]i in histamine-stimulated muscle.

Effects of Polyamines on Contractility of Guinea-Pig Gastric Smooth Muscle

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current (I(Ba)) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16+/-6.4% of the control (n=9), and significantly inhibited I(Ba) in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 micrometer)-induced phasic contraction to 5+/-6.4% of the control. Inhibitory effect of spermine on I(Ba) was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of I(Ba) was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited I(Ba) to 51+/-5.7% and 81+/-5.3% of the control, respectively. And putrescine (1 mM) inhibited I(Ba) at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+ -induced contraction to 53+/-7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.

Sodium-activated Potassium Current in Guinea pig Gastric Myocytes

This study was designed to identify and characterize Na+ -activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+] degree). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]degree (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+ - and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+ -selective, since reversal potentials (E(rev)) of various concentrations of [K+]degree-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microgram), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes.

Lysophosphatidylcholine Attenuates Endothelium-dependent Relaxation Responses through Inhibition of ACh-induced Endothelial Ca2+i Increase

Lysophosphatidylcholine (LPC), which accumulates in atherosclerotic arteries, has been reported to inhibit endothelium-dependent relaxation (EDR) in many different species. However, the underlying mechanism of LPC-induced inhibition of EDR is still uncertain. In the present study, we measured simultaneously both isometric tension and cytosolic free Ca2+ ([Ca2+]i) in rabbit carotid strips, and examined the effect of LPC on tension and [Ca2+]i. In carotid strips with intact-endothelium, high K+ (70 mM) increased both tension and [Ca2+]i, and cumulative addition of acetylcholine (ACh) from 0.1 to 10microM induced dose dependent increase of [Ca2+]i with concomitant relaxation. In the presence of L-NAME (0.1 mM), ACh increased [Ca2+]i without affecting the amplitude of high K+-induced tension. These ACh-induced change of [Ca2+]i and tension was abolished by removal of endothelium or 10 nM 4-DAMP (muscarinic receptor antagonist) pretreatment. Pretreatment of LPC (10microM) inhibited ACh (10microM)-induced change of tension and [Ca2+]i in endothelium-intact carotid artery. On the other hand, LPC had no effect on ACh-induced change of tension and [Ca2+]i in endothelium denuded artery. In Ca2+-free external solution, ACh transiently increased [Ca2+]i, and pretreatment of LPC significantly inhibited ACh-induced transient [Ca2+]i change. Based on the above results, it may be concluded that LPC inhibits the ACh-induced [Ca2+]i change through inhibition of Ca2+ mobilization in vascular endothelial cells, resulting in decreased production of NO and concomitant inhibition of endothelium-dependent vascular relaxation.

Mechanism of Acetylcholine-induced Endothelium-dependent Relaxation in the Rabbit Carotid Artery by M3-receptor Activation

The present study were designed to characterize the action mechanisms of acetylcholine (ACh) -induced endothelium-dependent relaxation in arteries precontracted with high K (70 mM). For this, we simultaneously measured both muscle tension and cytosolic free Ca2 concentration ([Ca2 ]i), using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high K increased both [Ca2 ]i and muscle tension whereas ACh (10microM) significantly relaxed the muscle and increased [Ca2 ]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca2 ]i without relaxing the muscle. In the artery without endothelium, high K increased both [Ca2 ]i and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine (0.1microM) abolished ACh-induced increase in [Ca2 ]i and relaxation. The increase of [Ca2 ]i and vasorelaxation by ACh was siginificantly reduced by either 3microM gadolinium, 10microM lanthanum, or by 10microM SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM K -induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the M3 subtype, and activation of the M3 subtype is suggested to stimulate nonselective cation channels, leading to increase of [Ca2 ]i in endothelial cells.

Testosterone Causes Simultaneous Decrease of Ca2+i and Tension in Rabbit Coronary Arteries: by Opening Voltage Dependent Potassium Channels

The relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.

The effect of NO donor on contraction, cytosolic Ca2+ level and ionic currents in guinea-pig ileal smooth muscle

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on contraction, cytosolic Ca2+ level ((Ca2+)i), and ionic currents in guinea-pig ileum. SIN-1 (0.01~100 micrometer) inhibited 25 mM KCl- or histamine (10 micrometer)-induced contraction in a concentration-dependent manner. SIN-1 reduced both the 25 mM KCl- and the histamine-stimulated increases in muscle tension in parallel with decreased (Ca2+)i. Using the patch clamp technique with a holding potential of -60 mV, SIN-1 (10 micrometer) decreased peak Ba currents (IBa) by 30.9+/-5.4% (n=6) when voltage was stepped from -60 mV to +10 mV and this effect was blocked by ODQ (1 micrometer), a soluble guanylyl cyclase inhibitor. Cu/Zn SOD (100 U/ml), the free radical scavenger, had little effect on basal IBa, and SIN-1 (10 micrometer) inhibited peak IBa by 32.4+/-5.8% (n=5) in the presence of Cu/Zn SOD. In a cell clamped at a holding-potential of -40 mV, application of 10 micrometer histamine induced an inward current. The histamine-induced inward current was markedly and reversibly inhibited by 10 micrometer SIN-1, and this effect was abolished by ODQ (1 micrometer). In addition, SIN-1 markedly increased the depolarization-activated outward K+ currents in the all potential ranges. We concluded that SIN-1 inhibits smooth muscle contraction mainly by decreasing (Ca2+)i resulted from the inhibition of L-type Ca2+ channels and the inhibition of nonselective cation currents and/or by the activation of K+ currents via a cGMP-dependent pathway.

Mechanism of L-NAME-resistant endothelium-dependent relaxation induced by acetylcholine in rabbit renal artery

In the rabbit renal artery, acetylcholine (ACh, 1 nM ~ 10 micrometer) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 micrometer) in a dose-dependent manner. NG-nitro-L-arginine (L-NAME, 0.1 mM), an inhibitor of NO synthase, or ODQ (1 micrometer), a soluble guanylate cyclase inhibitor, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was abolished in the presence of 25 mM KCl and L-NAME. The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10 micrometer), miconazole (10 micrometer), or 17-octadecynoic acid (17-ODYA, 10 micrometer), failed to inhibit the ACh-induced relaxation in the presence of L-NAME. 11,12-epoxyeicosatrienoic acid (11,12-EET, 10 micrometer) had no relaxant effect. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.3 micrometer) and apamin (1 micrometer), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 micrometer), in a dose-dependent manner. Furthermore, 2-methylthio-ATP (2MeSATP), a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue. In conclusion, in renal arteries isolated from rabbit, ACh produced non-NO relaxation that is mediated by an EDHF. The results also suggest that ACh may activate the release of ATP from endothelial cells, which in turn activates P2Y receptor on the endothelial cells. Activation of endothelial P2Y receptors induces a release of EDHF resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. The results further suggest that EDHF does not appear to be a cytochrome P450 metabolite.

The effect of carbon monoxide on contraction, cytosolic Ca2+ level and ionic currents in guinea pig ileal smooth muscle

The aim of this study was to clarify the mechanism of the inhibitory action of carbon monoxide (CO) on contraction, by measuring cytosolic Ca2+ level ((Ca2+)i) and ionic currents in guinea-pig ileum. CO (10%) inhibited 40 mM KCl-induced contraction and this effect was blocked by ODQ (1 micrometer), a soluble guanylyl cyclase (sGC) inhibitor. CO inhibited the 40 mM KCl-induced contraction without changing (Ca2+)i. Cumulative addition of KCl induced a graded increase in (Ca2+)i and muscle tension. In the presence of CO, cumulative addition of KCl induced smaller contraction than in the absence of CO. On the other hand, the increase in (Ca2+)i induced by cumulative addition of KCl was only slightly decreased in the presence of CO, and the (Ca2+)i-tension relationship shifted downwards. Using the patch clamp technique with a holding potential of -60 mV, we found that CO had little effect on the peak Ba currents (IBa) when voltage was stepped from -60 mV to 0 mV. In addition, CO showed no effect on the depolarization-activated outward K+ currents in the all potential ranges. We conclude that CO inhibits smooth muscle contraction mainly by decreasing the Ca2+ sensitivity of contractile elements via a cGMP-dependent pathway, not by involving L-type Ca2+ and outward-potassium currents in guinea-pig ileum.

The involvement of K+ channels and the possible pathway of EDHF in the rabbit femoral artery

Experiments were designed to characterize the cellular mechanisms of action of endothelium-derived vasodilator substances in the rabbit femoral artery. Acetylcholine (ACh, 10(-8)-10(-5) M) induced a concentration-dependent relaxation of isolated endothelium-intact arterial rings precontracted with norepinephrine (NE, 10(-6) M). The ACh-induced response was abolished by the removal of endothelium. NG-nitro-L-arginine (L-NAME, 10(-4) M), an inhibitor of NO synthase, partially inhibited ACh-induced endothelium-dependent relaxation, whereas indomethacin (10(-5) M) showed no effect on ACh-induced relaxation. 25 mM KCl partially inhibited ACh-induced relaxation by shifting the concentration-response curve and abolished the response when combined with L-NAME and NE. In the presence of L-NAME, ACh-induced relaxation was unaffected by glibenclamide (10(-5) M) but significantly reduced by apamin (10(-6) M), and almost completely blocked by tetraethylammonium (TEA, 10(-3) M), iberiotoxin (10(-7) M) and 4-aminopyridine (4-AP, 5 x 10(-3) M). The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10(-5) M) and miconazole (10(-5) M) also significantly inhibited ACh-induced relaxation. Ouabain (10(-6) M), an inhibitor of Na+, K(+)-ATPase, or K(+)-free solution, also significantly inhibited ACh-induced relaxation. ACh-induced relaxation was not significantly inhibited by 18-alpha-glycyrrhetinic acid (18 alpha-GA, 10(-4) M). These results of this study indicate that ACh-induced endothelium-dependent relaxation of the rabbit femoral artery occurs via a mechanism that involves activation of Na+, K(+)-ATPase and/or activation of both the voltage-gated K+ channel (Kv) and the large-conductance, Ca(2+)-activated K+ channel (BKCa). The results further suggest that EDHF released by ACh may be a cytochrome P450 product.