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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.

1. The effects of oestrogens on action potential and membrane currents were examined in single guinea-pig atrial myocytes. 2. 17Beta-estradiol (3-10 microM) shortened the action potential duration without significant changes in the resting membrane potential. E-4031 (1 microM) markedly prolonged the action potential duration and induced early afterdepolarization, and 17beta-estradiol (10 microM) abolished it. 3. When cells were perfused in isoproterenol-containing solution, action potentials due to abnormal automaticity caused by membrane depolarization developed, and were also inhibited by 17beta-estradiol. 4. Under voltage clamp conditions, the voltage-dependent Ca2+ currents consisted of both T-(I(Ca,T)) and L-type (I(Ca,L)). 17Beta-estradiol reduced I(Ca,L) concentration-dependently, while it (10 microM) suppressed I(Ca,T) only by approximately 10%. 17Beta-estradiol did not affect time courses of I(Ca,L) inactivation, but it shifted the steady-state inactivation curve to more negative potentials. 5. 17Beta-estradiol (10 microM) did not affect the time-dependent K+ current (I(K)), referred to as I(Kr) and I(Ks) and inwardly rectifying K+ current. However, 17beta-estradiol (30 microM) or diethylstilbestrol (10 microM) inhibited K+ currents. 6. DES and ethinylestradiol (EES) also suppressed I(Ca,L), but testosterone and progesterone failed to inhibit I(Ca,L) The potency of the inhibitory effect on I(Ca,L) was DES> EES> 17beta-estradiol. 7. 17Beta-estradiol and DES also inhibited the cyclic AMP-enhanced I(Ca,L), but cyclic GMP in the pipette or pretreatment of L-NAME could not block the effects of oestrogen on I(Ca,L). 8 These results suggest that oestrogen specifically has antiarrhythmic effects, possibly by acting the L-type Ca2+ channels. The antiarrhythmic effects of oestrogens may contribute to the cardioprotective actions of oestrogens.

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.

Monocrotaline (MCT)-induced pulmonary hypertension (PH) is a useful model for the investigation of this disorder in humans. The role of thrombocytes in the genesis of PH has already been addressed; however, the exact mechanism by which they induce PH remains to be elucidated. We investigated the effects of a thromboxane A2 (TXA2) synthase inhibitor (OKY-046) and a TXA2/prostaglandin H2 (PGH2) receptor antagonist (ONO-8809) on the development of MCT-induced PH. A single dose of MCT (60 mg/kg bodyweight; BW) was injected subcutaneously in Wistar rats 24 h after the administration of OKY-046 or ONO-8809. The TXA2 inhibitors were administered by gavage daily for 3 weeks. Urinary excretion of eicosanoids was determined by radioimmunoassay. At the end of the treatment period, the lungs, heart and kidneys were morphologically examined. The per cent medial thickness of the muscular pulmonary arteries (%MT) and the ratio of the right to the left ventricular mass including the septum (RV/LV + S) increased significantly in MCT-treated rats compared with the control rats. The %MT was attenuated by the administration of ONO-8809. Either OKY-046 or ONO-8809 attenuated the increase in RV/LV + S. In addition, both TXA2 inhibitors reduced urinary excretion of 11-dehydro-TXB2, particularly during the early phase of PH, suggesting that platelet aggregation was reduced. These findings suggest that the inhibition of TXA2 by synthase inhibition or receptor antagonism reduces or delays the development of MCT-induced PH in rats, probably by inhibiting platelet aggregation.

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.

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.

Molecular mechanisms related to sodium retention have been implicated in the pathogenesis of hypertension. It is unclear how sodium retention leads to a rise in blood pressure, but ouabainlike compound may act as a final common pathway in sodium-induced hypertension. In ectopic corticotropin syndrome, hypertension has been attributed to cortisol inactivation overload, giving rise to mineralocorticoid-type hypertension. We sequentially measured plasma and urinary levels of ouabainlike compound over 2 months to evaluate its role in the hypertensive mechanisms in a 64-year-old man with this syndrome caused by lung cancer. His data included hypokalemia and increased cortisol concentrations, corticotropin levels, and urinary 17-hydroxycorticosteroid excretion. Plasma renin activity was suppressed. Plasma and urinary levels of ouabainlike compound were markedly increased concomitantly with high blood pressure. The maximum plasma level was 40-fold the normal range of the subject. After chemotherapy, ouabainlike compound levels gradually decreased in parallel with the decline in blood pressure and rise in potassium concentration. A correlation was observed between plasma and urinary levels of ouabainlike compound ( P P P 3 H]ouabain binding to human erythrocytes. These findings suggest that ouabainlike compound with biological activity could partly account for hypertension in ectopic corticotropin syndrome.

The effects of vasopressin and endothelin-1 on cultured aortic smooth muscle cell lines (A7r5) were investigated by measurements of intracellular calcium [Ca2+]i and the patch-clamp techniques. Vasopressin and endothelin-1 (100 nM) evoked an initial peak followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular calcium [Ca2+]o. In the absence of [Ca2+]o, only the initial peak of [Ca2+]i was observed. Therefore, the initial peak of [Ca2+]i was mainly due to calcium release from the storage sites, whereas the later sustained rise of [Ca2+]i was due to the calcium entry from outside. The sustained rise of [Ca2+]i was unaffected by nifedipine (10 microM) significantly, but was completely abolished by La3+ (1 mM). Under current clamp conditions with K(+)-internal solution, vasopressin and endothelin-1 (100 nM) produced hyperpolarization, then followed by depolarization. Under voltage clamp conditions at a holding potential of -40 mV, both vasopressin and endothelin-1 first activated the outward current, then followed by a long-lasting inward current with a high noise level. The first outward current was abolished by charybdotoxin (100 nM), Cs+ in the patch pipette and high EGTA (10 mM) in the pipette, suggesting that it was a Ca(2+)-sensitive K+ current (IK.Ca). The inward current was still elicited with the patch pipette containing Cs(+)-internal solution, and reversed at about 0 mV. The reversal potential was not significantly altered by the replacement of [Cl-]i or [Cl-]o, proposing that the inward current is a cation selective channel (IN.S.). The inward current was also observed even when extracellular cations are Ca2+. La3+ (1 mM), Cd2+ (1 mM) completely abolished the vasopressin-induced (IN.S.), however, nifedipine (10 microM) failed to inhibit it significantly. Single channel activities were recorded in the cell-attached configurations when vasopressin or endothelin-1 was applied to the bathing solution. The unitary conductance of the channels was approximately 20 pS with 140 mM Na+, Cs+, or K+ in the pipette, but was 15 pS with 110 mM Ca2+ in the pipette. Permeabilities sequence calculated from the reversal potentials was Na+ not equal to Cs+ not equal to K+ > Ca+. These results provide evidence that calcium entry and membrane depolarization elicited by vasopressin or endothelin-1 are mediated by a receptor-mediated Ca(2+)-permeable non-selective cation channel in aortic smooth muscle cells.

To elucidate the mechanisms of estrogens-induced relaxation effects on vascular smooth muscle cells, the effects of estrogens and the related hormones were examined in cultured rat thoracic aortic smooth muscle cell lines (A7r5), using the whole-cell voltage clamp technique. The patch pipette was filled with 140 mM CsCl- or KCl-containing internal solution. With CsCl-internal solution, 17beta-estradiol and synthetic estrogens, ethynylestradiol and diethylstilbestrol (0.1-30 mu M) inhibited the Ba2+ inward current (IBa) through the voltage-dependent L-type Ca2+ channel in a concentration-dependent and reversible manner. The potency of the inhibitory effects on IBa was 17beta-estradiol < ethynylestradiol < diethylstilbestrol. 17beta-Estradiol (10 mu M) appeared to reduce the maximal conductance of IBa with only a slight shift of voltage-dependency of inactivation and to affect IBa in a use-independent fashion. On the other hand, testosterone and progesterone (30 mu M) failed to affect IBa. At a holding potential of -40 mV, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current. After endothelin-1 (100 nM) activated the current, the additional application of vasopressin (100 nM) could not induce it furthermore, suggesting that each agonist activates the same population of the channels. The reversal potential of the current was about 0 mV and was not significantly altered by replacement of [Cl-]i or [Cl-]0 and the inward current was also observed even when extracellular cations are Ca2+, proposing that it was a Ca2+-permeable non-selective cation channel (IN.S.). La3+ or Cd2+ (1 nM) completely abolished IN.S., however, nifedipine (10 mu M) failed to inhibit it at all. Diethylstilbestrol (1-30 mu M) suppressed the IN.S. evoked by both endothelin-1 and vasopressin in a concentration-dependent manner, while 17beta-estradiol, ethynylestradiol, progesterone and testosterone (30 mu M) failed to inhibit it significantly. In addition, at a holding potential of +0 mV, 17beta-estradiol by itself did not affect the holding currents, and did not inhibit K+ currents evoked by endothelin-1 or vasopressin, possibly due to the Ca2+ release from the storage sites. These results suggest that 17beta-estradiol may play a role in regulating vascular tone, selectively by inhibiting the voltage-dependent L-type Ca2+ current in vascular smooth muscle cells.

Background Recent experimental and clinical studies on atrial flutter have demonstrated that the crista terminalis (CT) plays an important role in the genesis of atrial reentry. To elucidate the underlying mechanism of its role, we characterized the electrophysiological repolarization properties of CT cells by comparing them with those of the pectinate muscles (PM). Methods and Results After action potential properties of both regions were compared by conventional microelectrode technique in multicellular atrial tissues, the whole-cell clamp experiments were applied in atrial cells isolated from both regions. Action potential duration (APD) was more prolonged in CT than in PM in multicellular preparations (APD 90 77±5 ms versus 52±8 ms at 1 Hz, P 90 63±7 ms versus 41±6 ms at 0.1 Hz, P 2+ current and the inwardly rectifying K + current between these cells but revealed a significant reduction of the density of the 4-aminopyridine–sensitive transient outward current (I to ) in CT cells compared with that in PM cells (6.3±0.7 pA/pF versus 10.3±0.8 pA/pF at +20 mV, P to were observed between the cells. The time course of recovery from inactivation of I to was also similar in both types of cells. Conclusions These results suggest that the preferential reduction in the density of I to in the CT cells could contribute to prolong their APD, which may be related to the genesis of atrial reentry.

Neurokinin A (NKA) caused single tracheal smooth muscle cells (TSMCs) to contract. The effects of NKA on the electrical activity of guinea-pig TSMCs were examined using the tight-seal whole-cell patch-clamp technique. Under current-clamp conditions at rest, the membrane potential of TSMCs spontaneously oscillated at about -40 mV and NKA rapidly depolarized the membrane potential to nearly 0 mV, which then gradually repolarized to about -20 mV in the presence of NKA. The oscillations in potential disappeared transiently during the rapid phase of depolarization in response to NKA and reappeared during the sustained phase of depolarization. Under voltage-clamp conditions, NKA evoked an inward current which faded quickly. Subsequently, the cell conductance in the presence of NKA at potentials greater than -40 mV decreased gradually. The reversal potential of the NKA-induced inward current was about 0 mV, and shifted with changes in the Cl- equilibrium potential. The Cl- current was not elicited by NKA when using a pipette solution containing 10 mM ethylenebis(oxonitrilo)tetraacetic acid (EGTA). During the sustained phase, K+ currents evoked by depolarizing voltage steps were inhibited by NKA. The present results indicate that NKA causes rapid and sustained depolarization of TSMCs by two distinct mechanisms: (1) initial transient activation of the Ca(2+)-dependent Cl- current, and (2) sustained inhibition of K+ currents.

OBJECTIVE Nitric oxide (NO) production is enhanced in patients with liver cirrhosis (LC). Although most patients with mild LC have neither dyspnea nor platypnea, they might have mild oxygenation abnormalities due to intrapulmonary vasodilatation caused by increased NO. We investigated whether oxygenation abnormalities, such as hypoxemia and orthodeoxia, are present in patients with mild LC. METHODS We investigated 148 consecutive patients with biopsy-proven chronic liver diseases such as CH (noncirrhotic chronic hepatitis) (n=46), LC(A), LC(B), and LC(C) (LC Child's A, B, and C) (n=18, 51, 33, respectively). The oxygen saturation by pulse oximetry (SpO2) in the supine and upright positions was determined in patients and controls (normal subjects, n=29). The change in SpO2 on standing was defined as deltaSpO2. NO output in exhaled air was measured in 16 patients. RESULTS Four patients [two LC(B) and two LC(C)] had hypoxemia (supine SpO2< or =94% and/or upright SpO2< or =94%). Although there was no intergroup difference in the supine SpO2 or the upright SpO2, the deltaSpO2 decreased [control, +0.2+/-0.6%; CH, +0.1+/-0.9%; LC(A), -0.3+/-0.8%; LC(B), -0.2+/-0.9%; LC(C), -0.5+/-1.1%; mean+/-SD; p=0.005] with worsening liver disease, and the prevalence of desaturation on standing (deltaSpO2< or =-1%) increased [control, 7%; CH, 20%; LC(A), 33%; LC(B), 35%; LC(C), 42%; p=0.01]. The NO output was inversely correlated with deltaSpO2 (r=-0.66, p=0.006). CONCLUSIONS Desaturation on standing is present in one-third of normoxemic patients with mild LC of Child's A, and is associated with the severity of liver disease. This postural desaturation is correlated with the exhaled NO, which suggests that intrapulmonary vasodilatation may play some role in this phenomenon.

A woman with a long history of chronic bronchitis and chronic sinusitis, i.e., sinobronchial syndrome, was admitted with a fever. Radiologically, there were areas of longstanding consolidation in both lungs, with areas of active inflammation demonstrated by gallium-67 scintigraphy. Antineutrophil cytoplasmic antibody specific for myeloperoxidase was highly positive. Pulmonary hemorrhage and hematuria occurred 2 weeks after admission and responded to steroid therapy. However, the patient died of pneumonia. An autopsy revealed systemic necrotizing vasculitis affecting multiple organs, consistent with microscopic polyangiitis. The vasculitis might have been caused by the chronic inflammation in the lungs associated with sinobronchial syndrome.

Background —It has been suggested that intracellular Ca 2+ 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 Ca 2+ currents were examined and compared with classic Ca 2+ antagonists (verapamil and nifedipine). Methods and Results —Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca 2+ currents consisted of both T-type ( I Ca,T ) and L-type ( I Ca,L ) Ca 2+ currents. Troglitazone effectively reduced the amplitude of I Ca,L in a concentration-dependent manner. Troglitazone also suppressed I Ca,T , but the effect of troglitazone on I Ca,T was less potent than that on I Ca,L . The current-voltage relationships for I Ca,L and the reversal potential for I Ca,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on I Ca,L measured at a holding potential of −40 mV was 6.3 μmol/L, and 30 μmol/L troglitazone almost completely inhibited I Ca,L . Troglitazone 10 μmol/L did not affect the time courses for inactivation of I Ca,L and inhibited I Ca,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 I Ca,L . Conclusions —These results demonstrate that troglitazone inhibits voltage-dependent Ca 2+ 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 Ca 2+ overload and subsequent myocardial changes.

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 μ M) 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 μ M) 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 μ M) inhibited transient outward current ( I to ) in a concentration-dependent manner. The concentration for half-maximal inhibition (IC 50 ) of pirmenol on I to was about 18 μ M. 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 μ M) did not affect the inwardly rectifying K + current significantly, but it decreased the voltage-dependent L-type Ca 2+ 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 IC 50 of pirmenol for acetylcholine- and adenosine-induced current was about 1 and 8 μ M, 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.

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 (IK.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 IK.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. © 1997 Elsevier Science B.V. All rights reserved.

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.

1. Membrane currents were recorded by a patch clamp technique in guinea-pig tracheal myocytes, using the whole cell mode with Cs(+) internal solution. 2. Both neurokinin A (NKA, 1 mu M) and caffeine (10 mM) evoked Ca(2+)-activated Cl- currents (I[Cl(Ca)]) transiently. In Ca(2+)-free bathing solution, the first application of NKA or caffeine elicited I[Cl(Ca)] but the second application of these substances failed to activate it. In addition, pretreatment with ryanodine in the presence of caffeine abolished the response to both NKA and caffeine whilst heparin (200 mu g ml(-1)) only blocked the NKA-induced response. I[Cl(Ca)] was also elicited by inositol 1,4,5-trisphosphate (IP(3)). 3. Command voltage pulses positive to 0 mV from a holding potential of -60 mV activated the voltage-dependent L-type Ca2+ current (I(Ca,L)) and late outward current. Upon repolarization to the holding potential, slowly decaying inward tail currents were recorded. The outward current during the depolarizing pulses and the inward tail current were enhanced by Bay K 8644, but completely blocked by Cd2+ or nifedipine. Replacement of external Ca2+ with Ba2+, removal of Ca2+ from the bath solution, or inclusion of EGTA (5 mM) in the patch pipette, also led to abolition of these currents, indicating that they were Ca2+ dependent, and that Ca2+ influx due to I(Ca,L) activated the currents. 4. When [Cl(-)](O) or [Cl(-)](i) was changed, the reversal potential (E(rev)) of the Ca2+-activated currents shifted, thus behaving like a Cl(-)-selective ion channel as predicted by the Nernst equation. DIDS (1 mM) completely abolished the currents, also suggesting that they were I[Cl(Ca)]. 5. NKA (1 mu M) and caffeine (30 mM) transiently activated I[Cl(Ca)], and after that both agents markedly reduced I[Cl(Ca)] induced by I(Ca,L). This is probably due to sarcoplasmic reticulum (SR) Ca2+ release induced by NKA or caffeine, followed by inhibition of the Ca(2+)-induced Ca2+ release from the SR. 6. The present results indicate that I[Cl(Ca)] can be activated by SR Ca2+ release due to NKA or caffeine (through IP(3) or ryanodine receptors) as well as by Ca2+ influx due to I(Ca,L). It also suggests that activation of I[Cl(Ca)] by NKA may be mediated by the production of IP(3), which releases Ca2+ from the SR.

Azelastine [4-( p -chlorobenzyl)-2-(hexahydro-1-methyl -1 H-azepin-4-yl)-1-(2 H )-phthalazinone hydrochloride], an anti-allergic agent, inhibited the high K + -induced contraction in tracheal smooth muscle cells isolated from the guinea-pig. In order to investigate the ionic mechanisms, we examined the effects of azelastine on membrane currents, using the tight-seal whole cell voltage clamp technique. Azelastine (1–100 μM) caused an inhibition of the Ba 2+ inward current (I Ba ) through the voltage-dependent L-type Ca 2+ channel in a concentration-dependent manner. The inhibitory effect of azelastine on I Ba was fully reversible. The IC 50 value for azelastine-induced inhibition of I Ba was approximately 8 μM, and 100 μM azelastine completely suppressed I Ba . Azelastine exerted mainly a tonic block of I Ba but did not show use dependence. Azelastine (10 μM) shifted the quasi-steady-state inactivation curve of I Ba to more negative membrane potentials by approximately −20 mV, suggesting that the inhibitory effect of azelastine on I Ba was voltage-dependent. In addition, azelastine produced inhibitory actions on other membrane currents (i.e. the voltage-dependent transient outward K + current and the Ca 2+ -activated oscillatory K + current) at doses higher than 10 μM. These results suggest that azelastine inhibits the voltage-dependent L-type Ca 2+ current in single tracheal smooth muscle cells, which may contribute to the anti-allergic actions of azelastine in airways.

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.