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Am.J.Respir.Cell Mol.Biol. 01/2002; 26:371-379.
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K Chisaki,
T Nakajima, K Iwasawa,
H Iida,
A Matsumoto,
M Tada,
Y Komatsu,
K Hirose,
K Miyamoto,
Y Okuda,
Y Shiratori,
A Goto,
Y Hirata,
R Nagai,
M Omata
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ABSTRACT: The purpose of this study was to clarify whether physiological concentrations of bile acids could affect endothelial nitric oxide production. We investigated the relationships between clinical concentrations of individual bile acids observed in patients with hepatobiliary diseases and endothelial nitric oxide production induced by each bile acid. Fifteen serum bile acids were measured using high-performance liquid chromatography combined with enzymatic fluorometry in 8 patients with liver cirrhosis, obstructive jaundice, and 8 healthy subjects. The effects of individual bile acids on nitric oxide production were examined in human umbilical endothelial cells by measuring the concentration of NO2- in the cultured medium. NO release in the blood was also determined by measuring the NO2-/NO3- concentration in these patients. In patients with hepatobiliary diseases, the plasma concentrations of chenodeoxycholic acid, ursodeoxycholic acid and cholic acid (free acid, taurine and glycine conjugates) were markedly elevated. Incubation of cells with chenodeoxycholic acid and deoxycholic acid (free acid, taurine and glycine conjugates) enhanced NO2- production in a concentration-dependent manner, while cholic acid (free and its conjugates) did not. The effects of individual bile acids on nitric oxide production were additive. Patients with liver cirrhosis and obstructive jaundice had higher plasma levels of NO2-/NO3- levels than the control subjects. These results suggest that increased plasma concentrations of chenodeoxycholic acid (free, taurine and glycine conjugates) in patients with hepatobiliary diseases may induce endothelial nitric oxide production. Thus, nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in patients with liver diseases.
Japanese Heart Journal 06/2001; 42(3):339-53. · 0.40 Impact Factor
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ABSTRACT: The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.
Journal of Biological Chemistry 10/2000; 275(37):28739-49. · 4.77 Impact Factor
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ABSTRACT: The effects of endothelin (ET)-1 on cultured human bronchial smooth muscle cells (HBSMC) were investigated and compared with those of histamine, using the patch clamp techniques and measurements of intracellular Ca(2+) ([Ca(2+)](i)). Both ET-1 and histamine caused an initial transient elevation of [Ca(2+)](i) by Ca(2+) mobilization, followed by a sustained rise due to Ca(2+) entry. Nicardipine inhibited the sustained phase, but La(3+) abolished it. With low ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and K(+) internal solutions, both ET-1 and histamine induced a sustained depolarization from approximately -40 to -20 mV. Under voltage clamp conditions, both drugs transiently activated an outward K(+) current at a holding potential of 0 mV. Additionally, with a Cs(+) internal solution, they elicited another transient inward current, frequently followed by current oscillations. These transient currents were blocked by high EGTA or heparin. With high EGTA and Cs(+) internal solutions, both drugs activated a long-lasting inward current. The reversal potential of these agonist-induced currents was approximately 0 mV and was not altered by the replacement of internal or external concentration of Cl(-), suggesting that the inward current was a nonselective cation current (I(cat)). The half-maximal effective concentration to activate I(cat) was 12 nM for ET-1 and 11 microM for histamine. La(3+) and Cd(2+) abolished these agonist-induced I(cat). The effects of ET-1 on [Ca(2+)](i) and I(cat) could be blocked by combined pretreatment with BQ-123 and BQ-788. Sarafotoxin S6c also increased [Ca(2+)](i) and activated I(cat). By polymerase chain reaction of reverse transcribed RNA, we detected both ET-A and ET-B receptor messenger RNA. These results provide the first evidence that ET-1 is a potent activator of I(cat) in HBSMC via ET-A and ET-B receptors, and the activation of I(cat) plays an important role in ET-1-induced Ca(2+) entry in human airways.
American Journal of Respiratory Cell and Molecular Biology 09/2000; 23(2):213-21. · 5.13 Impact Factor
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T Nakajima,
Y Okuda,
K Chisaki,
W S Shin, K Iwasawa,
T Morita,
A Matsumoto,
J I Suzuki,
S Suzuki,
N Yamada,
T Toyo-Oka,
R Nagai,
M Omata
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ABSTRACT: The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.
British Journal of Pharmacology 09/2000; 130(7):1457-67. · 4.41 Impact Factor
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ABSTRACT: 1. The effects of troglitazone and pioglitazone on agonist-induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura-2 AM and incorporation of [3H]-thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed. 2. Vasopressin and platelet-derived growth factor-BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it. 3. Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i. 4. Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage-dependent L-type Ca2+ current (ICa.L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration-dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa.L and Icat were 4.6 and 5.7 microM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat. 5. Vasopressin and PDGF increased incorporation of [3H]-thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration-dependently. 6. These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)-induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa.L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.
British Journal of Pharmacology 11/1999; 128(3):673-83. · 4.41 Impact Factor
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M Asano,
T Nakajima, K Iwasawa,
Y Asakura,
T Morita,
F Nakamura,
T Tomaru,
Y Wang,
A Goto,
T Toyo-oka,
M Soma,
S Suzuki,
Y Okuda
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ABSTRACT: The purpose of this study was to clarify how eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, modulates the vascular action of vasopressin in rat aortic smooth muscle cell lines. The effects of EPA on Ca2+ mobilization and DNA synthesis elicited by vasopressin were investigated and compared to those of Ca2+ channel blocking agents, by means of Ca2+ measurements and the incorporation of [3H]thymidine. Patch-clamp techniques were also employed. Vasopressin (100 nM) elicited an initial peak of intracellular Ca2+ ([Ca2+]i), followed by a sustained phase due to Ca2+ entry. Nifedipine or nicardipine (1 microM), a potent L-type Ca2+ channel blocker, partly inhibited the sustained phase, but La3+ completely abolished it. EPA (10 microM) also inhibited it even in the presence of nicardipine. Under voltage-clamp conditions with CsCl-internal solution, depolarizing pulses positive to -30 mV from a holding potential of -40 mV elicited a slow inward current. The inward current was blocked by La3+, nicardipine, and nifedipine (1 microM), suggesting that the inward current mainly consisted of the voltage-dependent L-type Ca2+ channel (ICa.L). EPA (1-30 microM) also inhibited ICa.L in a concentration-dependent manner. The inhibitory effect of EPA was observed at concentrations higher than 1 microM, and its half-maximal inhibitory concentration (IC50) was 7.6 microM. Vasopressin induced a long-lasting inward current at a holding potential of -40 mV. The vasopressin-induced current was considered as a non-selective cation current (Icat) with a reversal potential of approximately +0 mV. Both nifedipine and nicardipine (10 microM) failed to inhibit it significantly, but La3+ completely abolished Icat. EPA also inhibited vasopressin-induced Icat in a concentration-dependent manner; its IC50 value was 5.9 microM. Vasopressin (100 nM) stimulated [3H]thymidine incorporation. Exclusion of extracellular Ca2+ with EGTA or La3+ markedly inhibited it. EPA (3-30 microM) also inhibited the incorporation induced by vasopressin, while nifedipine and nicardipine (1 microM) only partly inhibited it. These results suggested that EPA, unlike nifedipine and nicardipine, inhibited vasopressin-induced Ca2+-entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of EPA on Icat as well as ICa.L might be involved. Thus, EPA would exert hypotensive and antiatherosclerotic effects.
European Journal of Pharmacology 09/1999; 379(2-3):199-209. · 2.52 Impact Factor
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T Nakajima, K Iwasawa,
H Oonuma,
H Imuta,
H Hazama,
M Asano,
T Morita,
F Nakamura,
J Suzuki,
S Suzuki,
Y Kawakami,
M Omata,
Y Okuda
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ABSTRACT: It has been suggested that intracellular Ca2+ overload in cardiac myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has been shown to prevent diabetes-induced myocardial changes. To elucidate the underlying mechanism of troglitazone action on cardiac myocytes, the effects of troglitazone on voltage-dependent Ca2+ currents were examined and compared with classic Ca2+ antagonists (verapamil and nifedipine).
Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T) and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude of ICa,L in a concentration-dependent manner. Troglitazone also suppressed ICa,T, but the effect of troglitazone on ICa,T was less potent than that on ICa,L. The current-voltage relationships for ICa,L and the reversal potential for ICa,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on ICa,L measured at a holding potential of -40 mV was 6.3 micromol/L, and 30 micromol/L troglitazone almost completely inhibited ICa,L. Troglitazone 10 micromol/L did not affect the time courses for inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion, without shifting the voltage-dependency of inactivation. This effect was different from those of verapamil and nifedipine. Troglitazone also reduced isoproterenol- or cAMP-enhanced ICa,L.
These results demonstrate that troglitazone inhibits voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the effects of isoproterenol in cardiac myocytes, thus possibly playing a role in preventing diabetes-induced intracellular Ca2+ overload and subsequent myocardial changes.
Circulation 07/1999; 99(22):2942-50. · 14.74 Impact Factor
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ABSTRACT: A 59-year-old man receiving hemodialysis was hospitalized due to severe hypoglycemic attack. The patient had been treated with disopyramide (50 mg/day) because of paroxysmal atrial fibrillation. Hypoglycemia occurred after taking clarithromycin (CAM, 600 mg/day), a macrolide antibiotic. The serum disopyramide concentration reached 8.0 micrograms/ml (23.6 microM) in the presence of CAM, while it was 1.5 micrograms/ml before the addition of CAM. A 75 g oral glucose tolerance test and daily profiles of blood glucose value showed that blood glucose levels were significantly lower in the presence of CAM and disopyramide compared to that in the absence of these drugs. The Turner index in the presence of CAM and disopyramide was significantly higher than that in the absence of these drugs, suggesting that a toxic concentration of disopyramide enhanced insulin secretion, resulting in the induction of hypoglycemic attacks, in which the inhibitory effects of CAM on the hepatic chytochrome P-450 might be involved. QT and QTc intervals were prolonged in the presence of CAM and disopyramide, but torsades de points were not observed in this patient receiving nicorandil (15 mg/day). Thus, it should be taken into account that life-threatening hypoglycemia may result from the interaction between clarithromycin and disopyramide.
Japanese Heart Journal 02/1999; 40(1):91-6. · 0.40 Impact Factor
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H Hazama,
T Nakajima,
M Asano, K Iwasawa,
T Morita,
K Igarashi,
T Nagata,
T Horiuchi,
J Suzuki,
M Soma,
Y Okuda
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ABSTRACT: Omega-3 polyunsaturated fatty acids have been reported to be associated with favorable changes in the respiratory system. To determine one of the mechanisms for this effect, membrane currents were recorded in guinea-pig tracheal myocytes by using the whole-cell voltage clamp technique. Without EGTA in the patch pipette containing the Cs-internal solution, command voltage pulses positive to +0 mV from a holding potential of -60 mV elicited a voltage-dependent L-type Ca2+ current (I(Ca x L)) and a subsequent outward current. Upon repolarization, slowly decaying inward tail currents were recorded. The outward currents and the inward tail current were enhanced by methyl-1,4,-dihydro-2,6-dimethyl-3-nitro-4-(2-trigluromethylphenyl )-pyridine-5-carboxylate, and blocked by Cd2+ or nifedipine. Inclusion of EGTA (5 mM) in the patch pipette also abolished these currents, indicating that they were Ca2+-dependent. When [Cl-]o or [Cl-]i was changed, the reversal potential of these currents shifted, thus behaving like a Cl(-)-sensitive ion channel. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid. a Cl- channel blocker, inhibited the currents. The omega-3 polyunsaturated fatty acids eicosapentaenoic acid (3-30 microM) and docosahexaenoic acid (30 microM) suppressed I(Ca x L) and then inhibited I(Ca x Cl) in a reversible manner. Similar inhibitory effects of eicosapentaenoic acid on I(Ca x L) were observed with 5 mM EGTA in the patch pipette. Neurokinin A (1 microM) and caffeine (10 mM) also transiently activated I(Cl x Ca), probably due to Ca2+ release from Ca2+ storage sites. Pretreatment of the cells with eicosapentaenoic acid markedly suppressed the activation of I(Cl x Ca) by neurokinin A or caffeine. These results suggest that omega-3 polyunsaturated fatty acids inhibit voltage-dependent L-type Ca2+ currents and also Ca2+-activated Cl- currents in tracheal smooth muscle cells from the guinea-pig, which may play a role in modulation of tracheal smooth muscle tone.
European Journal of Pharmacology 09/1998; 355(2-3):257-66. · 2.52 Impact Factor
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M Asano,
T Nakajima,
H Hazama, K Iwasawa,
T Tomaru,
M Omata,
M Soma,
Y Asakura,
M Mizutani,
S Suzuki,
K Yamashita,
Y Okuda
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ABSTRACT: Long-term treatment with n-3 eicosapentaenoic acid (EPA) has been shown to exert hypotensive effects and have beneficial effects on atherosclerosis. To elucidate one of the underlying mechanisms of these effects, intracellular calcium concentration [Ca2+]i, and resting membrane potential were measured in rat vascular smooth muscle cells (A7r5 cell) treated with EPA, using Ca2+-sensitive dye fura-2 AM and the patch clamp technique. The alterations in fatty acid compositions of phospholipids and cell migration after treatment with EPA (30 microM) for 6 h-7 days were also examined. After treating cells with EPA, the EPA and DPA (docosapentaenoic acid) content of the phospholipid fraction (mol.%) increased in a time-dependent manner. Alternatively, arachidonic acid (AA) decreased, and then the ratio of EPA and AA (EPA/AA) increased significantly. The resting [Ca2+]i decreased from 170 +/- 46 nM (n = 16) in control cells to 123 +/- 29 nM (n = 16) in cells treated with EPA (30 microM) for 7 days. Vasopressin (100 nM), endothelin-1 (100 nM) and platelet-derived growth factor (PDGF 5 ng/ml) evoked an initial peak of [Ca2+]i, followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular Ca2+. In EPA-treated cells, both the peak and the sustained rise of [Ca2+]i induced by these agonists decreased in comparison to the control cells. EPA treatment also decreased the transient [Ca2+]i rise evoked by these agonists in the absence of extracellular Ca2+. Under the current clamp condition, resting membrane potential was significantly higher in EPA-treated cells (-49.8 +/- 10.4 mV, n = 41) than in control cells (-44.6 +/- 7.4 mV, n = 41, P < 0.05), and the input resistance of the cell was lower in EPA-treated cells, while cell size and capacitance were not statistically different. In addition, long-term treatment with EPA for 7 days significantly inhibited PDGF-induced cell migration. These results suggest that cellular incorporation of n-3 eicosapentaenoic acid attenuates intracellular mechanisms related to changes of [Ca2+]i and affects membrane potential, thereby inhibiting migration of vascular smooth muscle cells. These actions of EPA may contribute to its vasorelaxant and antiatherosclerotic effects.
Atherosclerosis 05/1998; 138(1):117-27. · 3.79 Impact Factor
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ABSTRACT: Electrophysiological effects of pirmenol hydrochloride (pirmenol) were investigated in single atrial myocytes obtained from rabbit and guinea-pig hearts by using a whole-cell clamp technique. Under current clamp conditions, pirmenol (2-30 microM) prolonged action potential duration in a concentration-dependent manner without affecting resting membrane potential in rabbit atrial myocytes. However, in the presence of 4-aminopyridine (4 mM), pirmenol (10 microM) failed to prolong the action potential duration further. Pirmenol also suppressed acetylcholine-induced hyperpolarization and action potential duration shortening, resulting in a significant prolongation of the action potential duration in the presence of acetylcholine. Under voltage clamp conditions, pirmenol (1-1000 microM) inhibited transient outward current (I(to)) in a concentration-dependent manner. The concentration for half-maximal inhibition (IC50) of pirmenol on I(to) was about 18 microM. Pirmenol did not show the use and frequency dependent inhibition of I(to). The voltage dependence of the steady-state inactivation of I(to) and the recovery from inactivation were not significantly affected by pirmenol. Pirmenol accelerated the inactivation of I(to) and blocked I(to) as an exponential function of time, consistent with a time-dependent open channel blockade. Pirmenol (30 microM) did not affect the inwardly rectifying K+ current significantly, but it decreased the voltage-dependent L-type Ca2+ current by about 20%. In guinea-pig atrial myocytes, both acetylcholine and adenosine induced a specific K+ current activated by GTP-binding proteins. Pirmenol suppressed both the acetylcholine- and adenosine-induced K+ current effectively. The IC50 of pirmenol for acetylcholine- and adenosine-induced current was about 1 and 8 microM, respectively. The present results suggest that pirmenol prolongs the action potential duration by primarily inhibiting the transient outward current in atrial myocytes. In addition, since pirmenol inhibits acetylcholine- and adenosine-induced K+ current, pirmenol may effectively prolong the action potential duration in atrial myocytes under various physiological conditions as in the whole heart or ischemia.
European Journal of Pharmacology 04/1998; 344(2-3):287-97. · 2.52 Impact Factor
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ABSTRACT: 1. The effects of extracellular pH (pHo) on receptor (vasopressin or endothelin-1)-mediated Ca2- entry and Ca(2+)-permeable channels were investigated in aortic smooth muscle cells (A7r5) from rat embryonic thoracic aorta. Intracellular Ca2+ ([Ca2+]i) was measured using fura-2 AM and whole-cell voltage clamp techniques were employed. 2. Vasopressin and endothelin-1 (100 nM) in the presence of nicardipine (10 microM) evoked a sustained rise in [Ca2+]i due to calcium entry. Extracellular acidosis decreased receptor (vasopressin or endothelin-1)-mediated Ca2+ entry, while extracellular alkalosis potentiated it. 3. Depletion of intracellular Ca2+ stores with thapsigargin (1 microM) also evoked Ca2+ entry activated by emptying of intracellular Ca2+ stores (capacitative Ca2+ entry). Extracellular acidosis decreased this capacitative Ca2+ entry, while extracellular alkalosis potentiated it. 4. Under voltage-clamp conditions with Ca+ internal solution, vasopressin and endothelin-1 activated non-selective cation currents (ICAT). Ba2+ or Ca2+ were also charge carriers of ICAT. Reducing the pHo inhibited ICAT, while increasing pHo potentiated it in a reversible manner. 5. Intracellular pH (pHi) changes did not cause the same marked effects as pHo changes, and a high concentration of Hepes (50 mM) in the patch pipette did not inhibit the effects of pHo on ICAT. 6. Similar results were obtained when ICAT was activated by GTP gamma S (1 mM) applied through the patch pipette, even in the absence of agonists, probably because of direct activation of GTP-binding proteins coupled to the receptors. 7. In cells treated with thapsigargin, addition of Ca2+ to the bath solution induced Ca(2+)-dependent K+ currents activated by capacitative Ca2+ entry. However, no measurable ionic currents activated by capacitative Ca2+ entry (ICRAC) were observed under conditions with Cs+ internal solution and EGTA (5 mM), although vasopressin still activated ICAT. 8. These results suggest that the contractile agonists vasopressin and endothelin-1 evoked Ca2+ entry through two different types of Ca(2+)-permeable channel (ICAT and ICRAC) and pHo affects these channels, which may modulate receptor-mediated Ca2+ influx in A7r5 cells. Thus, pH-induced changes of these channels may play a pathophysiological role in the control of receptor-mediated contractions.
The Journal of Physiology 10/1997; 503 ( Pt 2):237-51. · 4.72 Impact Factor
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ABSTRACT: Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (I(K.Ca)) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of I(K.Ca) induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion.
Biochimica et Biophysica Acta 05/1997; 1356(2):198-206. · 4.66 Impact Factor
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ABSTRACT: 1. The effects of omega-3 polyunsaturated fatty acids on receptor-mediated non-selective cation current (Icat) and K+ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole-cell voltage clamp technique was employed. 2. With a K(+)-containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 microM) produced an outward current at a holding potential of -40 mV. This response was inhibited by tetraethylammonium (20 mM) or Cs+ in the patch pipette solution, and the reversal potential of the EPA-induced current followed the K+ equilibrium potential in a near Nernstian manner. 3. Under conditions with a Cs(+)-containing pipette solution, both vasopressin and endothelin-1 (100 nM) induced a long-lasting inward current at a holding potential of -60 mV. The reversal potential of these agonist-induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl+ concentration, suggesting that the induced current was a cation-selective current (Icat). 4. La3+ and Cd2+ (1 mM) completely abolished these agonist-induced Icat, but nifedipine (10 microM) failed to inhibit it significantly. 5. omega-3 polyunsaturated fatty acids (3-100 microM), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist-induced Icat in a concentration-dependent manner. The potency of the inhibitory effect was EPA > DHA > DPA, and the half maximal inhibitory concentration (IC50) of EPA was about 7 microM. 6. Arachidonic and linoleic acids (10, 30 microM) showed a smaller inhibitory effect compared to omega-3 fatty acids. Also, oleic and stearic acids (30 microM) did not show a significant inhibitory effect on Icat. 7. A similar inhibitory action of EPA was observed when Icat was activated by intracellularly applied GTP gamma S in the absence of agonists, suggesting that the site of action of omega-3 fatty acids is not located on the receptor. 8. These results demonstrate that omega-3 polyunsaturated fatty acids can activate a K+ current and also effectively inhibit receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that omega-3 fatty acids may play an important role in the regulation of vascular tone.
British Journal of Pharmacology 05/1997; 120(7):1367-75. · 4.41 Impact Factor
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ABSTRACT: The effects of extracellular Mg2+ on receptor-mediated Ca(2+)-permeable non-selective cation currents were investigated in a cultured aortic smooth muscle cell line (A7r5) from rat thoracic aorta, using the whole-cell voltage-clamp technique. Under the Cs(+)-containing internal solution, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current with a high noise level. The reversal potential of these agonists-induced current was approximately +0 mV, and was not significantly altered by the replacement of [Cl-]i or [Cl-]o, suggesting that the inward current was a cation-selective channel. La3+ and Cd2+ (1 mM) almost completely abolished the vasopressin or endothelin-induced non-selective cation current; however, nifedipine (10 microM) failed to inhibit it significantly. Extracellular Mg2+ (3-20 mM) also markedly inhibited the vasopressin- or endothelin-induced non-selective cation current in a concentration-dependent manner. When a non-hydrolysable GTP-analogue, GTP gamma S (1 mM), was applied from the patch pipette, the non-selective cation current was gradually activated even in the absence of agonist (vasopressin or endothelin-1), probably due to the direct activation of GTP-binding proteins coupled to the receptors. Extracellular Mg2+ (3-20 mM) also suppressed the activation of non-selective cation current induced by GTP gamma S, suggesting that the inhibitory sites of Mg2+ are not located on the receptors. These results suggest that extracellular Mg2+ inhibits receptor-mediated non-selective cation current, which may contribute to the relaxation effects of Mg2+ in vascular smooth muscle cells.
European Journal of Pharmacology 03/1997; 320(1):81-6. · 2.52 Impact Factor
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ABSTRACT: To determine whether isoproterenol could reverse enhancement of electrical defibrillation effectiveness by class III antiarrhythmic agents, we measured the internal defibrillation threshold (DFT) in 12 anesthetized dogs during infusion of (a) saline (baseline), (b) isoproterenol, (c) isoproterenol + E4031 (a new class III antiarrhythmic agent), and (d) E4031 alone. The isoproterenol infusion was adjusted so that heart rate (HR) was at least 30 beats/min greater than baseline. E4031 was given as a 40-micrograms/kg bolus at the beginning of the third stage of the study, followed by constant infusion at 2 micrograms/kg/min. Eight dogs completed the study. Although the energy-based DFT was not affected by isoproterenol (from 6.1 +/- 1.5 to 6.0 +/- 1.7 J), it was decreased to 3.7 +/- 1.6 J in the third stage by infusion of E4031 and isoproterenol (p < 0.01 vs. baseline and vs. isoproterenol). After the discontinuation of isoproterenol in the fourth stage, i.e., during infusion of E4031 alone, DFT was 3.4 +/- 1.6 J (p < 0.01 vs. baseline and vs. isoproterenol). Therefore, isoproterenol did not antagonize the effect of E4031 on the DFT, suggesting the possible clinical usefulness of class III agents for facilitating defibrillation even in the presence of augmented sympathetic activity.
Journal of Cardiovascular Pharmacology 03/1995; 25(3):393-6. · 2.29 Impact Factor
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ABSTRACT: Shock-induced refractory period extension (RPE) has been suggested as a mechanism of electrical defibrillation. We measured RPE caused by localized field stimulation measured before and during infusion of disopyramide (n = 5), flecainide (n = 5), or E-4031 (n = 5) in anesthetized dogs and determined the effect of the drugs on the internal defibrillation threshold (DFT). In the baseline state (n = 15), 16 V/cm S2 field stimulation prolonged the effective RP by 36 +/- 15 ms (22 +/- 12% of RP without S2), whereas 4 and 8 V/cm S2 stimuli did not cause marked RPE. The RPE normalized by the RP without S2 was not significantly influenced by any drug (16 V/cm: disopyramide 30 +/- 11 vs. 27 +/- 11, flecainide 25 +/- 5 vs. 19 +/- 12, and E-4031 18 +/- 13 vs. 22 +/- 14%). Disopyramide did not alter the defibrillation threshold (4.2 +/- 0.6-4.4 +/- 0.6 J). In 2 dogs given flecainide, ventricular fibrillation became refractory to defibrillation. In contrast, E-4031 lowered the threshold from 4.5 +/- 2.4 to 2.2 +/- 1.2 J (p < 0.01). The results suggest that flecainide and E-4031 do not modulate defibrillation efficiency through their effects on RPE.
Journal of Cardiovascular Pharmacology 06/1994; 23(5):822-5. · 2.29 Impact Factor
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ABSTRACT: The anticholinergic effects of cibenzoline were examined and compared with those of disopyramide in atrial myocytes isolated from guinea pig heart. The tight-seal whole-cell voltage clamp technique was performed with a patch pipette filled with guanosine-5'-triphosphate (GTP) or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). In GTP-loaded cells, both acetylcholine (ACh) and adenosine (Ado) induced a specific K channel current through GTP-binding proteins by binding to the muscarinic and Ado receptors, respectively. Both cibenzoline and disopyramide suppressed the ACh-induced K current effectively in a concentration-dependent manner. The concentrations for half-maximal inhibition of the current (EC50) caused by cibenzoline and disopyramide were 8 and 3 microM, respectively. In GTP gamma S-loaded cells, the K current was irreversibly activated because GTP binding proteins were directly elicited by GTP gamma S. Cibenzoline effectively caused a decrease in the GTP gamma S-induced K current, whereas the extent of disopyramide action on the GTP gamma S-induced K current was much less. Cibenzoline also caused significant inhibition of Ado-induced K current in GTP-loaded cells. However, the action of disopyramide was less effective in inhibiting Ado-induced K current. These results indicate that cibenzoline has less potent anticholinergic effects than disopyramide in atrial myocytes. In addition, cibenzoline effectively inhibits the muscarinic K channel itself and/or GTP-binding proteins coupled to the channel, whereas the effect of disopyramide is attributed mainly to blockade of muscarinic receptors. These findings provide novel understanding of the molecular mechanism of anticholinergic action of cibenzoline.
Journal of Cardiovascular Pharmacology 05/1994; 23(4):618-23. · 2.29 Impact Factor
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American Heart Journal 10/1993; 126(3 Pt 1):719-22. · 4.65 Impact Factor
