Inhibition Of ICa Research Articles

Alpha-adrenergic modulation of ionic currents in cultured parasympathetic neurons from rat intracardiac ganglia

1. Modulation of ionic conductances by alpha-adrenergic agonists was investigated in cultured parasympathetic neurons from rat intracardiac ganglia. Application of norepinephrine (NE, 25-100 microM) to the soma of isolated neurons reversibly reduced both the amplitude and duration of the Ca(2+)-dependent action potential evoked by injection of depolarizing current when Na+ and K+ currents were blocked pharmacologically. 2. In the whole-cell voltage-clamp mode, application of NE reversibly reduced the amplitude and rate of activation of Ca2+ current (ICa). The amplitude inhibition was greater at the peak of the current (55%) than at the end of a 700-ms pulse (20%). Maximal doses of NE produced only approximately 60% inhibition of peak ICa amplitude. 3. Inactivation of ICa was best fit by the sum of two exponential functions in the absence of NE, but was described by a single exponential function in the presence of NE. These results suggest that NE preferentially inhibited a fast inactivating component of the Ca2+ current in these parasympathetic neurons. 4. NE reversibly reduced the amplitude of Ba2+ tail currents through open Ca channels at all voltages from -40 to +150 mV with a slight shift in the activation curve determined from the current-voltage (I-V) relationship for the tail currents. NE did not change the voltage dependence of the steady-state inactivation of the calcium channels. 5. NE inhibited Ca2+ current either in the absence or presence of nifedipine but to a lesser extent in the presence of omega-conotoxin (omega-CGTX), suggesting that the Ca channels inhibited by NE are predominantly omega-CGTX sensitive. 6. The inhibition of ICa by NE was mimicked by the alpha 1-adrenergic agonists methoxamine and phenylephrine and potentiated in the presence of the alpha 2-adrenoceptor antagonist yohimbine (10 microM). NE inhibition of ICa was antagonized by bath application of the alpha-adrenergic antagonist phentolamine (1 microM), but not by prazosin (1-10 microM), yohimbine, or the beta-adrenergic antagonist propranolol (1 microM). Taken together, these results suggest that NE inhibition of Ca2+ current in rat parasympathetic cardiac neurons is mediated by an alpha-adrenergic receptor with properties that may differ from alpha 1- and alpha 2-adrenoceptors. 7. In approximately 35% of neurons studied, NE not only reduced depolarization-activated inward Ca2+ current but also increased an outward current, with a shift of the I-V curve and reversal potential to more negative voltages.(ABSTRACT TRUNCATED AT 400 WORDS)

Fendiline inhibits L‐type calcium channels in guinea‐pig ventricular myocytes: a whole‐cell patch‐clamp study

1. Fendiline, a diphenylalkylamine type of antianginal drug, was examined for its effects on L-type calcium channels in guinea-pig ventricular myocytes by the whole-cell patch-clamp technique. 2. Fendiline (0.3-100 microM) applied extracellularly inhibited the calcium channel current (ICa) in a concentration- and time-dependent manner. The IC50 of fendiline was 17.0 +/- 2.43 microM and the Hill slope was 1.39 +/- 0.23. 3. Inhibition of ICa by fendiline appeared with an onset of less than 3 s. 4. Fendiline inhibited ICa at all the membrane potentials tested and shifted the current-voltage curve upwards. The overall calcium channel conductance (gCa) of the cell was reduced and conductance-voltage curve was shifted to the left in the presence of fendiline. 5. Isoprenaline (0.5-1 microM), a beta-adrenoceptor agonist, partially reversed the inhibitory effect of fendiline on ICa. 6. It is suggested that fendiline applied extracellularly blocks L-type calcium channels and reduces calcium channel conductance of the cell. The calcium channels thus inhibited are, nevertheless, still available for beta-adrenoceptor stimulation.

Inhibitory effects of histamine and bradykinin on calcium current in smooth muscle cells isolated from guinea-pig ileum.

1. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum and within 10 h Ca(2+)-currents (ICa) were recorded using the whole-cell patch clamp technique. 2. Histamine (10 microMs) and bradykinin (BK, 1 microM) suppressed ICa; the effect had two phases: a rapid and transient suppression of ICa followed by a sustained suppression. Acetylcholine and substance P appeared to have similar effects but these were not investigated in detail. 3. The effects of histamine and BK on ICa were established by high intracellular concentrations of the Ca2+ buffer EGTA (30 mM) or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (5 mM) in the absence of Ca2+ added to the pipette solution. When [Ca2+]i was strongly buffered to 125 or 190 nM by BAPTA-Ca2+ mixtures in the pipette the transient suppression of ICa was blocked but the sustained effect still occurred. This indicated that the transient effect was caused by a rise in [Ca2+]i. The sustained effect, in contrast, did not seem to be caused by a rise in [Ca2+]i but did show Ca2+ dependence because it did not occur if [Ca2+]i was abnormally low. 4. Application of caffeine (10 mM) to deplete stored Ca2+ or intracellular heparin (1 mM) to block the action of D-myo-inositol 1,4,5-trisphosphate (IP3) to release stored Ca2+ prevented the transient but not the sustained suppression of ICa. Heparin also blocked the transient Ca(2+)-activated K+ current in response to histamine or BK. Both transient and sustained suppressions of Ca2+ channel activity were observed in the absence of extracellular Ca2+ when current was carried mostly by Na+ ions. 5. Intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 10 or 100 microM) induced a gradual decline of ICa upon which transient decreases of current were superimposed. Histamine caused a larger than normal inhibition of ICa and no recovery occurred on wash-out. Intracellular guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 1 mM) abolished the effects of histamine and BK on ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Agonist‐independent effects of muscarinic antagonists on Ca2+ and K+ currents in frog and rat cardiac cells.

1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of the Inhibitory Action of Semotiadil Fumarate, a Novel Ca Antagonist, on the Voltage-Dependent Ca Current in Smooth Muscle Cells of the Rabbit Portal Vein

Effects of semotiadil on the voltage-dependent Ca current (ICa) were investigated in dispersed smooth muscle cells of the rabbit portal vein. At a holding potential of -100 mV, semotiadil (> or = 0.1 microM; dissolved in dimethylsulphoxide, DMSO) inhibited the ICa in a concentration-dependent manner (IC50 = 2.0 microM, Hill's coefficient = 1.0). At a holding potential of -80 mV or -60 mV, the concentration-inhibition curve observed in the presence of semotiadil was shifted to the left compared with that observed at -100 mV; and semotiadil shifted the voltage-dependent inactivation curve to the left. The curve for the decay of ICa was fitted with two time constants. Semotiadil (< 1 microM) reduced the slow but not the fast time constant. The curve for the recovery from ICa inactivation also consisted of two time constants, and semotiadil (1 microM) prolonged the slow recovery. Semotiadil dissolved in deionized water more potently inhibited ICa than semotiadil dissolved in DMSO. At pH 10.0, semotiadil did not modify the voltage-dependent inactivation curve. However, recovery from the inactivation was much faster at pH 10.0 than at pH 7.3. These results indicate that the voltage-dependent inhibition of ICa by semotiadil may be due to binding of the ionized drug during the inactivated state and also inhibition of the transition from the inactivated to the resting state. Long-lasting inhibition of ICa after removal of semotiadil may be due to tight binding of semotiadil on the channel through a hydrophobic site.

Some properties of Ca(2+)-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig.

1. Late transient outward Ca(2+)-dependent K+ current (ILTO) correlated with Ca(2+)-induced Ca2+ release mechanism was studied in relation to the calcium inward current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum using the whole-cell patch-clamp technique. 2. The voltage dependencies of peak ICa and ILTO were both bell shaped. However, the I-V curve of the outward current was shifted toward more positive potentials by about 60 mV in comparison to that for ICa. 3. Reduction in the external Ca2+ concentration resulted in a decrease of peak amplitude of both ICa and ILTO. However, caffeine-induced outward current was also decreased abruptly suggesting a rapid loss of stored Ca2+ upon lowering the external Ca2+ concentration. 4. Investigation of the relation of ILTO to partially inactivated ICa showed that inactivation of ICa by approximately 65, 80 or 84% of control (produced by prepulse to -20 mV for 2 s, shifting the holding potential to -20 mV for 30 s or by the ramp voltage command from -50 to +10 mV, respectively) was without detectable effect on the ILTO generation. 5. Bath application of the Ca2+ antagonist nifedipine (300 nM) inhibited ICa by 81% without affecting ILTO peak amplitude (92.0 +/- 5.6% of control in six cells). The mean concentration-response curve for ICa inhibition was sigmoidal with the apparent dissociation constant of 86.9 nM, whereas that for the ILTO had a characteristic sharp transition indicating a definite threshold of Ca2+ influx for ILTO generation. 6. Application of Ca(2+)-free external solution during 500 ms of the time when ICa peaked inhibited the current by about 76% whereas the ILTO during such an intervention remained virtually unchanged. 7. In double-pulse experiments, with conditioning and test pulses to +10 mV from -50 mV and an interpulse interval of 600 ms, most of the cells (about 80%) showed larger outward current at the test pulse suggesting continued Ca2+ release triggered by Ca2+ influx during a short (50-200 ms) depolarizing prepulse. The outward current could also be evoked at large positive potentials (presumably near the calcium equilibrium potential) where it did not occur normally by a prepulse to +10 mV for 50 ms. The charge transferred by Ca2+ current necessary to activate Ca2+ release in most of the cells was estimated to be from 6 to 20 pC. 8. The data are interpreted to suggest that the Ca(2+)-induced Ca2+ release mechanism operates in single ileal cells in a regenerative manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.

Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4-aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1-10 microM) or angiotensin II (1-10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5-hydroxytryptamine (10 microM), Met-enkephalin (1 microM), glycine (100 microM), gamma-aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic modulation of guinea-pig cardiac L-type calcium channels by fendiline and reversal of the effects of Bay K 8644.

1. The modulation of L-type calcium channel current (ICa) by fendiline, a diphenylalkylamine type of calcium channel blocker was investigated on guinea-pig ventricular myocytes by use of the whole-cell patch-clamp technique. 2. Fendiline-induced block of ICa is accompanied by modulation of the channel kinetics in a complex manner. The time course of ICa inactivation is significantly faster and the channel availability (f infinity) curve is shifted considerably to more negative potentials by fendiline. These findings can be interpreted qualitatively in terms of a modulated receptor. 3. When the 1,4-dihydropyridine agonist (4R, 4S)-Bay K 8644 was added in presence of 30 microM fendiline a further reduction of ICa instead of the expected stimulatory effect was observed. 4. A similar 'paradoxical' inhibition of ICa was produced by the pure agonist enantiomer (4S)-Bay K 8644. Thus this novel effect of Bay K 8644 cannot be attributed to changes in affinity of the 1,4-dihydropyridine receptor site for (4R)-Bay K 8644 during fendiline action. 5. The IC50 for fendiline was reduced to 3.0 +/- 0.1 microM (control value: 17.0 +/- 2.4 microM) and the Hill slope in its presence was increased to 1.90 +/- 0.1 (control value: 1.39 +/- 0.23) by 1 microM (4R, 4S)-Bay K 8644. 6. (4R,4S)-Bay K 8644 caused the expected stimulation of ICa in the presence of verapamil, diltiazem and nifedipine, overcoming the inhibitory effect of these calcium channel blockers. 7. The 'paradoxical' inhibitory effect of the agonist Bay K 8644 can be explained in terms of an allosteric interaction between fendiline and the dihydropyridine agonist.

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).

Electrophysiologic Mechanisms Responsible for Inotropic

Inotropic and electrophysiologic effects of ketamine were investigated in cardiac preparations isolated from guinea pigs and rats. Ketamine produced a concentration-dependent negative inotropic effect in electrically driven guinea pig papillary muscles, an effect that was accompanied by a decrease in action potential duration at the 0-mV level (APD0). In contrast, ketamine produced a concentration-dependent positive inotropic effect in rat left atria in the presence of 10(-6) M propranolol. The increase in force of contraction was accompanied by an increase in APD0. Experiments using patch clamp techniques revealed that ketamine reduced the transsarcolemmal Ca2+ current (ICa) as well as the inward rectifier K+ current and delayed outward K+ current in guinea pig single ventricular cells. These results indicate that the shortening of APD0 observed in guinea pig papillary muscles might result from the suppression of ICa. In rat single ventricular cells ketamine reduced the Ca(2+)-insensitive transient outward current (Ito) and did not enhance ICa, suggesting that the ketamine-induced prolongation of APD0 observed in rat left atria is due to a decrease in Ito rather than an increase in ICa. Treatment of rat left atria with the specific Ca(2+)-insensitive Ito inhibitor 4-aminopyridine (2 mM) produced a positive inotropic effect and prolongation of APD0, and these effects were equivalent to those caused by the highest concentration of ketamine. In the presence of 4-aminopyridine, ketamine failed to induce a positive inotropic effect and instead caused a negative inotropic one. In conclusion, the negative and positive inotropic effects of ketamine may result from the suppression of ICa and Ito, respectively. The inhibitory action on these membrane currents may at least in part explain the species and tissue differences in inotropic responses to ketamine.

Novel suppression of an L-type calcium channel in neurones of murine dorsal root ganglia by 2,3-butanedione monoxime.

1. Voltage-activated currents through calcium channels in primary cultures of murine dorsal root ganglion cells (DRG) were studied with the whole-cell and cell-attached patch recording techniques. 2. The chemical phosphatase 2,3-butanedione monoxime (BDM) reversibly reduced the amplitude of L-type calcium current (ICa) in a dose-dependent manner; at a concentration of 20 mM, BDM caused a 47% suppression of ICa. 3. Application of 10 mM-8-bromo-cyclic AMP or 50 microM-isoprenaline onto DRG treated with BDM completely restored ICa to the pre-BDM level. 4. In striking contrast, bath application of Bay K 8644 (0.5-5 microM) had no effect on the BDM-suppressed ICa. As expected, Bay K 8644 alone caused a two- to threefold increase of the maximal ICa and shifted its I-V relationship to the left. Interestingly, if a cell was first exposed to Bay K 8644 further treatment with 20 mM-BDM resulted in 100% suppression of ICa. This suggests that Bay K 8644 changes the conformation of the calcium channel to one which is more sensitive or more accessible to the action of the phosphatase. 5. Pre-treatment of DRG with an activator of protein kinase C, 12-O-tetradecanoyl-phorbol-13-acetate, did not antagonize BDM's effect on ICa. 6. The depressant action of BDM on ICa was distinct from that of nifedipine in that it did not exhibit use dependence. 7. When single calcium channel currents were recorded in cell-attached patches (barium as the charge carrier), bath application of BDM reduced the percentage of time that the channel spent in the open state. 8. Superfusion with 8-bromo-cyclic AMP restored the ensemble macroscopic 'ICa' to the pre-BDM amplitude. This was due to a dramatic enhancement of the frequency of channel openings. 9. We suggest that BDM acts through the cytoplasm to alter cyclic AMP-dependent protein kinase modulation of neuronal L-type calcium channels. The brief, high-frequency openings which 8-bromo-cyclic AMP activates in the presence of BDM may reflect a rapid phosphorylation-dephosphorylation sequence which controls channel gating.

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, 0.56 on day 14, 0.90 on day 18, and 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) (but not IBa) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).

Muscarinic agonist-induced actions on potassium and calcium channels in atrial myocytes: differential desensitization.

Desensitization to the effects of acetylcholine (ACh) or carbachol (CCh) on ACh-regulated potassium current (IK(ACh)) and the voltage-dependent L-type, calcium current (ICa(L)) was studied in single guinea-pig atrial cells under voltage clamp at room temperature (22-24 degrees C). At a holding potential of -40 mV, CCh (100 microM) activated IK(ACh) repeatedly and reproducibly when applied for 15-s periods with 60-s intervals between applications. Prolonged (10 min) exposure to CCh caused a time-dependent decline of IK(ACh) and a marked reduction (desensitization) of the response to subsequent application of CCh. In the absence of guanosine triphosphate (GTP) in the pipette, only partial resensitization occurred within 20 min after CCh washout. The desensitization to CCh could also be obtained when adenosine (20 microM) was used to activate this current. Therefore, this desensitization must be heterologous. In the presence of isoproterenol (ISO, 3 microM), ICa(L) increased. Acetylcholine continued to inhibit this current at a time when its activation of IK(ACh) had become desensitized. Muscarinic inhibition of ICa(L) eventually desensitized but only after IK(ACh) desensitization had occurred. Because of its heterologous nature, as well as of the ability of ACh to desensitize in the presence of intrapipette dextran, which is reported to inhibit beta-adrenergic receptor kinase (beta ARK), it seems unlikely that beta ARK-dependent phosphorylation of the muscarinic receptor (mAChR) accounted for desensitization. The differential desensitization time course is consistent with the hypothesis that different subunits of Gi, the inhibitory guanine nucleotide binding protein, not only transduce the agonist effects of ACh on IK(ACh) and ICa(L) but are also subjected to different inactivation/regulation processes.

Properties of the late transient outward current in isolated intestinal smooth muscle cells of the guinea‐pig.

1. Whole-cell membrane currents in voltage-clamped single isolated cells of longitudinal smooth muscle of guinea-pig ileum were studied at room temperature using patch pipettes filled with either high-K+ solution or high-Cs+ solution, to suppress K+ outward current, and containing 0.3 mM-EGTA. 2. In the presence of high-K+ solution in the pipette, membrane depolarization from the holding potential of -50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above -20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds. 3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high-K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0.67 and 4.43 mM, respectively) or by high-Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM-Co2+, 0.2 mM-Cd2+ or 1 microM-nifedipine) or perfusion of 5 mM-EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca(2+)-activated K+ current. 4. There is a great discrepancy between the time course of the ICa and that of the late ILTO, which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO. 5. Bath application of caffeine (10 mM) during the development of ILTO enhanced the current. However, in the presence of caffeine ILTO was inhibited. Moderate inhibition of ICa by caffeine was also observed. 6. Ryanodine (5 microM) applied to the bathing solution completely inhibited ILTO within 3.5 min; however, it had no or little effect on the ICa. 7. Ruthenium Red (10 microM) completely blocked the ILTO and slightly and more slowly inhibited the ICa. 8. Increasing Mg2+ concentration in the pipette solution from 1 to 6 mM abolished the ILTO. 9. It was concluded that the ILTO was activated mainly by Ca2+ released from the intracellular storage sites following Ca2+ entry, presumably by a Ca(2+)-induced Ca2+ release mechanism.

The inhibitory action of caffeine on calcium currents in isolated intestinal smooth muscle cells

The patch-clamp method has been used to investigate the action of caffeine on the calcium current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum. Caffeine (10 mM) substantially inhibited ICa. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K+ in the pipette solution, application of caffeine evoked a transient Ca-dependent K+ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased ICa. Ryanodine had only little effect on ICa, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of ICa. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the ICa inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of ICa inhibition was probably due to a direct blocking action of caffeine on Ca channels.

Ca2+ influx through ATP-gated channels increments [Ca2+]i and inactivates ICa in myocytes from guinea-pig urinary bladder.

1. Whole-cell patch clamp was combined with microspectrofluometry (Indo-1) to study the effects of bath applied ATP on membrane currents and cytoplasmic Ca2+ concentration ([Ca2+]i) in single smooth muscle cells of the guinea-pig urinary bladder. Experiments were carried out at 22 degrees C and in 3.6 mM [Ca2+]o. Superimposed K+ currents were reduced by Cs+ dialysis from the patch electrode. 2. At -60 mV, ATP induced an inward current (Ins,ATP) that peaked within 0.4 s and then decayed. Ins,ATP was activated half-maximally by 1.1 microM-ATP and saturated at 50 microM-ATP to -1.1 +/- 0.2 nA (mean +/- S.E.M.). At 3.6 mM [Ca2+]o, Ins,ATP had a reversal potential (Erev) of -5 +/- 2 mV. From the shifts in Erev during changes in [Na+]o or [Ca2+]o we estimated that approximately 7% of Ins,ATP is carried by Ca2+ ions. 3. ATP (50 microM) increased [Ca2+]i transiently from resting 130 +/- 40 nM to 730 +/- 100 nM. At 22 degrees C, [Ca2+]i rose at a rate proportional to the instantaneous current amplitude of Ins,ATP. This relation was lost, however, after warming to 36 degrees C which increased the peak Ins,ATP (Q10 = 1.25) but reduced the peak of the ATP induced [Ca2+]i transient (Q10 = 0.75). We suggest that warming to 36 degrees C stimulated Ca2+ sequestration and Ca2+ efflux to such a degree that peak [Ca2+]i was attenuated significantly. 4. The contribution of Ca2+ ions to Ins,ATP was evaluated from a comparison of the increments in [Ca2+]i due to Ins,ATP and due to L-type Ca2+ channel current (ICa). For the same increment, Ins,ATP had to transport 19 times more charge than ICa. This number suggests that 5.8 +/- 0.8% of Ins,ATP is carried by Ca2+ ions which can be translated into a permeability ratio of PNa:PCa approximately 1:1. 5. During bath application of ATP, peak ICa was inhibited by 80 +/- 15%. Inhibition of ICa diminished to 20 +/- 8% after cell dialysis with 40 mM-EGTA, and it was 19 +/- 7% when extracellular Ca2+ had been substituted by Ba2+. These results are in agreement with the hypothesis of 'ICa inactivation by Ca2+'. Depletion of intracellular Ca2+ stores by pre-treatment with 20 mM-caffeine did not attenuate significantly the ATP-induced rise in [Ca2+]i or the ATP-induced inhibition of ICa. 6. The ATP-induced [Ca2+]i transients and the reduction of peak ICa recovered along a similar time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological effects of calcitonin gene‐related peptide in bull‐frog and guinea‐pig atrial myocytes.

1. Electrophysiological effects of calcitonin gene-related peptide (CGRP) on action potentials and corresponding transmembrane currents in single myocytes from bull-frog and guinea-pig atria were studied using a whole-cell voltage-clamp method. 2. CGRP at relatively low concentrations increased the height of the action potential plateau in a dose-dependent manner in both bull-frog and guinea-pig myocytes. In addition, in bull-frog cells CGRP accelerated the early phase of repolarization, thus shortening the overall duration of the action potential. In contrast, in guinea-pig myocytes CGRP prolonged the action potential duration at all concentrations that were studied. 3. Voltage-clamp measurements demonstrated that CGRP increased transmembrane calcium current (ICa) in guinea-pig myocytes without a significant change in its voltage dependence. The ED50 value for this effect on ICa was 1.28 +/- 0.55 X 10(-8) M (n = 4). The time course of the inactivation of ICa was not affected by CGRP. 4. CGRP increased the delayed rectifier K+ current (IK) at relatively low concentrations in bull-frog atria, whereas relatively high concentrations were needed to increase IK in guinea-pig myocytes. This effect was observed even after complete inhibition of ICa. 5. CGRP had no significant effect on the inwardly rectifying background K+ current, IK1, even at very high concentrations. 6. Comparison of the time course of ICa augmentation in bull-frog and guinea-pig myocytes revealed an important difference in the effect of CGRP in these two types of cells. CGRP at maximal concentrations increased ICa transiently in bull-frog myocytes, whereas this response was sustained in guinea-pig myocytes. Isoprenaline (Iso) induced sustained increase in ICa in both species. When ICa was fully activated by Iso, CGRP at high concentrations strongly inhibited ICa in the bull-frog, whereas it had little effect on ICa in guinea-pig myocytes. 7. Intracellular application of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) 10(-4) M) greatly potentiated the CGRP effect on ICa; in contrast, GDP beta S (guanosine 5'-O-(2-thiodiphosphate), 2 x 10(-3) M) partially inhibited the CGRP-induced augmentation of ICa. Taken together, these results indicate that the stimulation of ICa by CGRP is mediated by a GTP-binding protein. 8. The observed dose-dependent changes in ICa and IK in bull-frog and guinea-pig myocytes can explain the different patterns of CGRP-induced changes in action potential shape in these two myocyte preparations.

Inhibition of transient outward K+ current by DHP Ca2+ antagonists and agonists in rabbit cardiac myocytes

The 1,4-dihydropyridine (DHP) Ca2+ antagonists and agonists can inhibit a time- and voltage-dependent, but intracellular Ca(2+)-independent transient outward K+ current (It), in myocytes from rabbit atrium. In the presence of 0.3 mM CdCl2, DHPs decreased the peak It slightly and markedly accelerated its apparent rate of inactivation. When the inhibition of It was measured from integrated It records, the 50% inhibitory concentrations (IC50) of nicardipine and BAY K 8644 were 630 nM and 7 microM, respectively, and the IC50 of nicardipine for inhibition of the Ca2+ current (ICa) was only approximately fourfold lower (160 nM). The inhibition of It by nicardipine was not affected by changing holding potential from -55 to -100 mV; in contrast, the inhibitory effect on ICa was significantly reduced by this hyperpolarization. We conclude that the DHP Ca2+ antagonist nicardipine blocks It at similar doses to those that block ICa and that nicardipine blocks this K+ current by mechanism different from that for ICa inhibition. This inhibitory effect on It is shared by other DHP compounds; the rank order for potency of It inhibition is nicardipine greater than benidipine greater than nisoldipine greater than BAY K 8644 greater than nitrendipine greater than nifedipine.