Single Ventricular Cells Of Guinea-pig Research Articles

Effects of AN-132, a novel antiarrhythmic lidocaine analogue, and of lidocaine on membrane ionic currents of guinea-pig ventricular myocytes

We studied the effects of AN-132 (10, 30 and 100 mumol/l), an analogue of lidocaine, on membrane currents and action potentials of single guinea-pig ventricular cells using whole-cell clamp techniques. The effects of lidocaine, an authentic class I antiarrhythmic agent were used for comparative purposes. (1) AN-132 decreased the Na current (INa) in a concentration-dependent manner, with a greater efficacy than seen with lidocaine. The concentration of the half maximal inhibition on INa (Kd) was 31.7 mumol/l for AN-132 and 94.9 mumol/l for lidocaine. (2) AN-132 also decreased the Ca current (ICa), concentration-dependently, while lidocaine had only a minor effect on ICa. The half maximal inhibition on ICa (Kd) was 23.1 mumol/l and 27.4 mumol/l for AN-132 and lidocaine, respectively. (3) AN-132 decreased the IK1, in a concentration-dependent manner; lidocaine was without effect. (4) AN-132 increased the unspecified steady state outward current, at positive potentials and depressed the time- and voltage-dependent outward K current (IK). Lidocaine had no effect on either current. (5) AN-132 shortened the action potential duration (APD), in a concentration-dependent manner, without altering the resting potential. From these findings, we conclude that apart from a potent inhibitory effect on INa, AN-132 had a variety of effects on other currents, properties not shared by lidocaine. Such multiple blocking effects on the membrane currents may relate to the alleged potent antiarrhythmic effect of AN-132.

Genistein Inhibits Slow Component Delayed-Rectifier K Currents via a Tyrosine Kinase-Independent Pathway

In single guinea pig ventricular cells, genistein, a potent inhibitor of protein tyrosine kinase (PTK), was found to suppress the delayed-rectifier K (IK) current. The present study was carried out to examine the underlying mechanism. Ventricular myocytes were voltage-clamped in the conventional whole-cell mode (36°C). The amplitudes of tail and steady-state (2-s pulse) currents were measured as IK. Genistein (10–100μm) reversibly inhibited both basal and intrapipette cAMP (1 mm)-enhanced IKcurrents in a concentration-dependent manner with a half-maximum inhibitory concentration (IC50) at 30μm. In contrast, lavendustin A (10μm;n=5) and tyrphostin 51 (100μm;n=5) had no effect on the currents. The inhibitory action of genistein was also seen after IKcurrents were activated by forskolin (500 nm) plus intrapipette orthovanadate (500μm). The intrapipette cAMP-enhanced IKwas also reduced to a lesser degree by daidzein, an inactive analogue of genistein. Envelope tail and short pulse protocols revealed that genistein inhibits the slow component of IK(IKs). Thus, the inhibitory action of genistein is not mediated via an inhibition of PTK but may be due to the block of IKschannels.

Angiotensin II inhibits protein kinase A-dependent chloride conductance in heart via pertussis toxin-sensitive G proteins.

Angiotensin II receptors are reported to be abundant in the guinea pig ventricle; their coupling to adenylate cyclase in the heart, however, remains controversial. Therefore, we investigated the effect of angiotensin II on Cl- conductance activated by cAMP-dependent protein kinase. After minimizing the contribution of other ionic currents, exposure of single guinea pig ventricular cells to isoproterenol (40 to 50 nmol/L; 36 degrees C) elicited a typical protein kinase A-dependent Cl- conductance. Subsequent application of angiotensin II reduced the isoproterenol-induced conductance with an IC50 of 0.24 +/- 0.08 nmol/L. Angiotensin II also inhibited the Cl- currents, which were activated through stimulation of adenylate cyclase by forskolin and histamine receptors. CV-11974 (1 mumol/L), an antagonist selective for the angiotensin type 1 receptor, prevented the effect of angiotensin II. Angiotensin II did not inhibit the current that had been persistently activated by intracellular GTP gamma S (100 mumol/L), a nonhydrolyzable guanine nucleotide, plus isoproterenol. In addition, prior incubation of myocytes with pertussis toxin prevented the angiotensin II inhibitory action. Cl- conductance, when activated directly by intracellular dialysis with cAMP (1 mmol/L), was not affected by angiotensin II. Radioimmunologic measurement of cellular cAMP in the dissociated myocytes showed that angiotensin II inhibited the isoproterenol-induced increase of cAMP. Angiotensin II receptors negatively couple to adenylate cyclase via pertussis toxin-sensitive G proteins, thereby inhibiting cardiac protein kinase A-dependent Cl- conductance.

Na +-permeable channels induced by maitotoxin in guinea-pig single ventricular cells

The characteristics of maitotoxin-induced single channel currents were studied in guinea-pig single ventricular cells using the cell-attached or inside-out configuration of the patch clamp. When the patch electrode was filled with normal Tyrode solution containing 10 nM maitotoxin, elementary currents flowing through the single channel were observed in the cell-attached patch. The amplitude of the single channel current at the resting potential was 1.6 ± 0.1 pA. The current-voltage relation of the current was linear and the single channel conductance was 16.0 ± 0.9 pS. The distribution of open times was fitted by a single exponential function (decay time constant: 27 ms), while that of closed times was fitted by the sum of two exponential functions (decay time constants: 1.6 and 34 ms). When the electrode solution was filled with the Ca 2+-free Tyrode solution, maitotoxin also induced single channel currents with parameters similar to those in the normal Tyrode solution. Under inside-out patch clamp conditions and in 150 mM Na + solution on both sides of the patch membrane, maitotoxin also induced single channel currents. Choline + could not substitute for Na +. These results indicate that maitotoxin induces single ionic channels irrespective of the presence or absence of Ca 2+ and that the charge carrier of the single channel current is Na + rather than Ca 2+. The increase in Na + permeability through maitotoxin-induced channels may be possibly responsible for its biological actions.

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes.

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal ICa significantly. The reversal potential of both carbachol- and antibody-induced extra currents were close to -80 mV, being in proximity to the calculated Ek of -90 mV. A beta-adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward ICa and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated ICa were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.

Cardiac electrophysiologic effects of parathyroid hormone in the guinea pig.

The cardiac electrophysiologic effects of parathyroid hormone (PTH) were studied in three different experimental models. Purified synthetic human parathyroid hormone (hPTH 1-84), at a dose of 30 ng/g body weight (BW), prolonged the QT interval of surface ECGs in intact guinea pigs. To further clarify the mechanisms of the QT prolongation caused by hPTH, the action potentials of guinea pig papillary muscle were recorded by conventional microelectrode techniques. hPTH (10(-7)M) prolonged the action potential duration without changing the action potential amplitude, resting potential, maximal upstroke velocity of the action potential, or maximal repolarization rate. hPTH affected only the plateau phase, in a concentration-dependent fashion. The half-maximum concentration was 6.8 x 10(-8)M for action potential duration at 30% repolarization (APD30), 3.2 x 10(-8)M for APD50, and 2.1 x 10(-8)M for APD90. In isolated single guinea pig ventricular cells, the patch clamp method was used to record the slow inward calcium current (ICa) and delayed rectifier potassium current (IK) responsible for the action potential plateau phase. hPTH increased the peak amplitude of ICa at + 10mV by an average of 42%, and increased IK at + 10mV by 85%. These results indicate that, in ventricular muscle, hPTH prolongs the action potential duration and QT interval by the balance of the prolongation effect on APD by enhancing the slow inward calcium current and the shortening effect on APD by enhancing the delayed rectifier potassium current.

Effects of cibenzoline, a new class Ia antiarrhythmic drug, on various membrane ionic currents and action potentials of guinea-pig ventricular cells.

We examined the effects of cibenzoline, a new class Ia antiarrhythmic drug, on various membrane ionic currents and action potentials of guinea-pig single ventricular cells, using patch clamp techniques in whole-cell configuration. Action potentials and the membrane currents were evoked at a clamping rate of 0.2 Hz, and all experiments were performed at 32-33 degrees C. 1) Cibenzoline (5, 10 and 30 microM) decreased the Na+ current (INa), in a concentration-dependent manner. The concentration of the half-maximal inhibition (Kd) for INa was estimated to be 7.8 microM. 2) In addition to the inhibition of INa, this drug (5, 10, and 30 microM) decreased, in a concentration-dependent manner, all other membrane currents examined, such as L-type Ca2+ current (ICa), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1). The Kd (apparent dissociation constant) values were 14.4 microM for ICa, 23.0 microM for IK, and 33.7 microM for IK1 respectively. 3) Cibenzoline (5, 10, and 30 microns) significantly shortened the action potential duration measured at both 30% and 90% repolarization without altering the resting membrane potential. From these findings, we conclude that apart from potent inhibitory effects on INa, cibenzoline possesses multiple blocking effects on other currents, e.g., ICa, IK and IK1, with a different potency (INa > ICa > IK > IK1) and with essentially the same efficacy. These effects may explain, at least in part, the alleged, potent antiarrhythmic effects of this drug.

Depressive effects of arenobufagin on the delayed rectifier K + current of guinea-pig cardiac myocytes

The effects of a bufadienolide isolated from toad venom, arenobufagin, a potent Na +/K + pump inhibitor, were studied in single guinea-pig ventricular cells in the whole-cell patch-clamp configuration. Arenobufagin (50 μM) applied extracellularly decreased the amplitude of the delayed rectifier K + current ( I dK) by 30% without affecting the gating kinetics. The L-type Ca 2+ current was also depressed, but to a lesser extent. The inward rectifier K + current was hardly affected. Ouabain and the internal dialysis of cells with the solution containing 20 mM Na + depressed I dK in a similar way as arenobufagin. On the other hand, arenobufagin also depressed I dK when the Na +/K + pump was already inhibited in the K +-free Tyrode solution. Therefore, both a direct effect on the channel and an indirect effect through the inhibition of the Na +/K + pump may be involved in the depression of I dK by arenobufagin.

Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein.

We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein.

Modulation by histamine of the delayed outward potassium current in guinea-pig ventricular myocytes.

1. Histamine receptor-mediated modulation of the delayed outward potassium current (IK) was investigated in guinea-pig single ventricular cells by the whole-cell voltage clamp. 2. Histamine increased IK in a dose- dependent manner with a half-maximum dose of 3.8 x 10(-8) M. Histamine (10(-6) M) increased IK by a factor of 3.02 without a significant change in the current kinetics. The threshold dose of histamine for increasing IK was 10(-9) M and this value was similar to that for calcium current. 3. Cimetidine decreased IK in the presence of histamine, by shifting the dose-response curve to histamine to the right. The pA2 value of cimetidine against histamine was 6.38. 4. Forskolin did not increase IK after application of 10(-6) M histamine, and histamine scarcely increased IK in the presence of a heat-stable inhibitor of cyclic AMP-dependent protein kinase (PKI). 5. We conclude that stimulation by histamine of IK is mainly by way of the H2-receptor, and is mediated by cyclic AMP-dependent phosphorylation.

Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells.

The mechanism of nicorandil-induced large inward tail current (Itail) in single guinea-pig ventricular cells was investigated using the whole-cell patch-clamp technique. In the presence of 0.5-1.0 mM nicorandil, an activator of adenosine 5'-triphosphate (ATP)-sensitive K+ current (IKATP), a depolarization pulse causing a large outward current was followed by a large inward Itail on the repolarization step to the holding potential at -85 mV. The larger the outward current, the greater the Itail. The amplitude of Itail increased as a single exponential function (tau = 74.9 ms) as the duration of preceding depolarization was prolonged. Both the outward current and Itail were inhibited nearly completely after application of glibenclamide (1 microM), a specific blocker of IKATP. Substitution of K+ with Cs+ in both the external and internal solutions resulted in a virtual elimination of Itail. Itail was well preserved under the condition where Ca2+ entry during the preceding depolarization was largely inhibited or where external Na+ was replaced by Li+. A transient positive shift of reversal potential for the net current was observed at the peak of Itail). At 30 mM external K+ concentration, Itail was almost eliminated. From these findings, its is concluded that the Itail is a K+ current associated with an alteration of the K+ equilibrium potential (EK) following a substantial K+ efflux. This EK change is most likely explained by an accumulation of K+ in transverse tubules (T-tubules) since Itail was not induced in atrial cells in which T-tubules are poorly developed.

Exponential activation of the cardiac Na+ current in single guinea-pig ventricular cells.

1. The cardiac Na+ current of guinea-pig was recorded using an improved oil-gap voltage clamp method. When a single ventricular cell was stretched between the internal and external solution compartments across an oil gap of about 40 microns in width, the sealing resistance in the oil gap was higher than 1 G omega and the time constant of the capacitive current was between 10 and 40 microseconds. Effective series resistance (Rs) was less than 50 k omega after Rs compensation. 2. The activation time course (I'Na) was separated from inactivation by dividing the digitized record of Na+ current with the inactivation variable h(t), which was obtained by fitting exponential functions to the decaying phase of current. I'Na started as a single exponential activation at time 0, which was defined by the decay of the capacitive current to 5% of its peak. 3. The Na+ tail current was recorded on repolarization after a short (1.2 ms) depolarizing pulse to -10 mV. Its single exponential decay at potentials negative to -50 mV, or its major exponential component of decay between -50 and -30 mV, was attributed to deactivation. The time constants of deactivation were similar to those of activation which were measured from I'Na on depolarization to comparable potentials. The m1 kinetics gave a better fit for Na+ activation than the m3 kinetics. 4. The time constant of deactivation was a linear function of the membrane potential on a semilogarithmic scale with an e-fold increase per 21.6 +/- 1.3 mV (n = 8) depolarization. The steady-state activation value (m(infinity)) was obtained from the amplitude of I'Na. Fitting a Boltzmann equation indicated a half-activation potential of -21.9 +/- 1.7 mV and a slope factor of 7.9 +/- 0.4 mV (n = 9). 5. m1 kinetics are more pertinent to a description of the cardiac Na+ current. Limitations in analysing the activation kinetics of Na+ current are discussed for the improved oil-gap voltage clamp method.

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.

P-061 - Sodium permeable channels induced by maitotoxin in guinea-pig single ventricular cells

P-061 - Sodium permeable channels induced by maitotoxin in guinea-pig single ventricular cells

Caffeine-induced block of Na+ current in guinea pig single ventricular cells.

Effects of caffeine on Na+ current (INa) were investigated in single ventricular cells from guinea pigs using the whole cell clamp method. With a Ca(2+)-containing internal solution (pCa 8.2), 10 mM caffeine blocked INa by 17.5 +/- 4.6% at a -120-mV holding potential. It was accompanied by 3- to 5-mV shifts of the steady-state inactivation curve and time constant-voltage relationship toward hyperpolarization. The inactivation kinetics spontaneously shifted toward hyperpolarization at 0.30 +/- 0.17 mV/min. The spontaneous shift was accompanied by a similar negative shift of the threshold potential, whereas caffeine did not affect it. Caffeine retarded the recovery of INa from inactivation, and a 4-mV positive shift in the recovery potential produced a similar retardation in INa recovery without caffeine. The INa block by caffeine was not influenced by reinforcing the internal buffering capacities using internal solutions containing 40 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid or by pretreating the cell with 1 microM ryanodine. Neither pretreatment with isoproterenol or H 7 nor prestimulation of Gs protein by nonhydrolyzable GTP (GTP gamma S) altered the effects of caffeine on INa. It is concluded that caffeine inhibits INa and shifts the inactivation kinetics without being mediated by changes in intracellular ionic composition or intracellular signaling systems. Direct action on the channel proteins may be involved.

Beta‐adrenergic and muscarinic regulation of the chloride current in guinea‐pig ventricular cells.

1. Single guinea-pig ventricular cells were voltage clamped using the patch clamp method combined with the pipette-perfusion technique. The voltage-dependent current systems were mostly blocked, and the background membrane conductance was measured by applying ramp pulses. 2. beta-Adrenergic effectors and related substances such as adrenaline, isoprenaline, forskolin or internal application of cyclic AMP induced a current component which showed a reversal potential near the expected Cl- equilibrium potential as well as an outward rectification in the I-V relation. It is suggested that the activation of this Cl- current was due to phosphorylation of the channel protein or related structure by the cyclic AMP-dependent protein kinase. Coincidentally with the activation of the Cl- current, the membrane capacitance of the cell decreased reversibly. 3. Acetylcholine (ACh) depressed the responses induced by beta-adrenergic stimulation and forskolin, but failed to interfere with the one induced by cyclic AMP. 4. The dose dependence of the Cl- current activation by isoprenaline or forskolin was fitted by the Hill equation, with a coefficient of 1.9 and a half-maximum concentration K 1/2 = 13 nM for isoprenaline, and with a Hill coefficient of 3 and a K 1/2 = 1.2 microM for forskolin. In the presence of 5.5 microM-ACh the dose-response relation shifted to higher doses; K 1/2 was 65 nM for isoprenaline and 3.6 microM for forskolin. 5. Washing out ACh in the presence of isoprenaline frequently caused transient overshoots of the response. When a saturating concentration of isoprenaline was used, this rebound was not observed. 6. The internal application of cyclic GMP enhanced the response of the Cl- current induced by isoprenaline or adrenaline. 7. When cyclic AMP was applied internally, the response was small in most cells. When the cell was superfused with 20 microM-IBMX (3-isobutyl-1-methylxanthine), the Cl- current was consistently induced by the application of cyclic AMP. It is suggested that phosphodiesterase activity strongly buffered the influx of cyclic AMP through the patch pipette tip. 8. We suggest that the compensatory interaction between the beta-adrenergic stimulation and the muscarinic inhibition is at the membrane level, most probably via GTP-binding proteins in activating adenylate cyclase.

Chloride‐sensitive nature of the adrenaline‐induced current in guinea‐pig cardiac myocytes.

1. Ionic selectivity of an adrenaline-induced current was investigated in single guinea-pig ventricular cells by recording whole-cell currents using the patch clamp technique combined with internal perfusion. Other ionic currents and exchange currents known in ventricular cells were suppressed by appropriate inhibitors and the adrenaline-induced current was defined as a difference between currents obtained in the presence and absence of adrenaline. 2. The adrenaline-induced current was time independent and its I-V relation showed saturation of the inward current in the negative voltage range. 3. The reversal potential was approximately -20 mV with 140 mM-NaCl external solution and Cs(+)-rich internal solution containing 51 mM-Cl-. Replacing Na+ with various monovalent and divalent cations (Li+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+) produced no appreciable change in the reversal potential. 4. Varying the external Cl- concentration ([Cl-]o) in exchange for aspartate or benzenesulphonate greatly changed the reversal potential. The relationship between the reversal potential and log[Cl-]o indicated a slope of 59.5 or 53.6 mV per tenfold change in [Cl-]o in the presence of 51 or 102 mM-Cl- in the internal solution, respectively. 5. Anion substitutions did not appreciably affect the I-V relation before application of adrenaline, suggesting that the cell membrane had a low Cl- conductance in the control state. 6. 4.4'-Dinitrostilbene-2-2'-disulphonic acid (DNDS: 1-10 mM), a specific inhibitor of membrane chloride permeability, depressed the adrenaline-induced current without changing the reversal potential. 7. The results suggest strongly that the adrenaline-induced current is carried mainly by Cl-. However, the development of this current appears to depend also on external cations, since the magnitude of the adrenaline response varied depending on the external cations species, with no response in Tris-HCl or TEA-Cl solution. The external cations may facilitate the adrenaline response with a sequence of efficacy of Na+ greater than K+, Rb+ greater than Cs+, Li+, divalent cations.

Effect of OPC-8490 on the membrane potentials and membrane currents of single guinea-pig myocytes

The direct actions of OPC-8490 on mammalian myocardium were examined by determination of the drug's effects on the action potentials of isolated guinea-pig single ventricular cells and on the underlying ionic currents. OPC-8490 (10(-6) to 10(-4) M) did not alter the resting membrane potential, but rather produced a dose-dependent prolongation of the duration of the action potential. The amplitude of the action-potential plateau was also increased by OPC-8490. Whole-cell voltage clamp experiments revealed that OPC-8490 blocks myocardial delayed outward K+ current (IK), which regulates repolarization of the action potentials. However Ik1, which regulates the resting membrane potential, was not changed by OPC-8490. Ca current (ICa) was increased by OPC-8490 in a dose-dependent and reversible manner. These results suggest that OPC-8490 augments the plateau amplitude and increases the duration of the action potentials by not only increasing ICa, but also by decreasing delayed outward K+ currents. Moreover, OPC-8490 did not affect the intracellular concentration of cyclic AMP in single cells. The OPC-8490 increase in ICa was thus unlikely to be mediated by a process involving cyclic AMP.

Calcium-sensitive delayed rectifier potassium current in guinea pig ventricular cells

The calcium sensitivity of the delayed rectifier K+ current (IK) was investigated in guinea pig single ventricular cells using the whole cell configuration of the patch-clamp technique with a cell dialysis method. The concentration-response curve of IK for intracellular Ca2+ indicated that IK started to increase at intracellular Ca2+ concentration [Ca2+]i of 10(-8) M (pCa 8) and it increased threefold at pCa 7. At lower [Ca2+]i than pCa 9, IK remained unchanged. A shift of the activation curve of IK by the elevation of [Ca2+]i was not observed. Although [Ca2+]i had little effect on time constants of the activation and deactivation of IK, it predominantly increased the amplitudes of the fast components in the activation process and the slow component in the deactivation process. In the ensemble noise analysis, the elevation of [Ca2+]i increased the number and open probability of functional IK channels but not the unit amplitude of IK channel. These results suggest that the elevation of [Ca2+]i enhances IK, probably by increasing the number and open probability of functional IK channels. Ca2(+)-sensitive IK in cardiac cells is a class of current different from Ca2(+)-activated K+ current in other tissue because the activation curve of Ca2(+)-sensitive IK was not shifted by the [Ca2+]i elevation, and the single channel conductance of IK was smaller than the one of Ca2(+)-activated K+ current.

Mechanism of receptor‐mediated modulation of the delayed outward potassium current in guinea‐pig ventricular myocytes.

1. Receptor-mediated modulation of the delayed outward potassium current (IK) was investigated in guinea-pig single ventricular cells by using whole-cell voltage clamp and intracellular dialysis. 2. Isoprenaline increased IK in a dose-dependent manner with a half-maximum dose of 1.8 X 10(-8) M. Isoprenaline (10(-6) M) maximally increased IK by a factor of 2.85. This effect did not depend on the concentration of intracellular Ca2+ [( Ca2+]i). 3. External application of 10(-5) M-forskolin and internal application of 5 X 10(-5) M-cyclic AMP or 5 X 10(-6) M of the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) also increased IK about 3-fold. The effect of isoprenaline on IK was masked by previous application of cyclic AMP. 4. All the above phosphorylating agents increased the amplitude of IK without a significant change in the current kinetics. 5. In the presence of 10(-5) M-forskolin, an additional application of 10(-8) M-12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C (PKC), produced a further increase in IK, suggesting that the active sites of PKA and PKC on the IK channel are different. 6. Acetylcholine (10(-6) M) suppressed IK when the current was previously enhanced by 2 X 10(-8) M-isoprenaline, but had little effect in the absence of isoprenaline. 7. We conclude that beta-adrenergic modulation of IK is mediated by cyclic AMP-dependent phosphorylation but not by an increase in [Ca2+]i, that PKA and PKC enhance IK independently, and that acetylcholine antagonizes beta-adrenergic stimulation of IK most probably by inhibiting adenylate cyclase.