Effects of divalent cations on the E-4031-sensitive repolarization current, I(Kr), in rabbit ventricular myocytes.

The effects of divalent cations on the E-4031-sensitive repolarization current (I(Kr)) were studied in single ventricular myocytes isolated from rabbit hearts. One group of divalent cations (Cd2+, Ni2+, Co2+, and Mn2+) produced a rightward shift of the I(Kr) activation curve along the voltage axis, increased the maximum I(Kr) amplitude (i.e., relieved the apparent inward rectification of the channel), and accelerated I(Kr) tail current kinetics. Another group (Ca2+, Mg2+ and Sr2+) had relatively little effect on I(Kr). The only divalent cation that blocked I(Kr) was Zn2+ (0.1-1 mM). Under steady-state conditions, Ba2+ caused a substantial block of I(K1) as previously reported. However, block by Ba2+ was time dependent, which precluded a study of Ba2+ effects on I(Kr). We conclude that the various effects of the divalent cations can be attributed to interactions with distinct sites associated with the rectification and/or inactivation mechanism of the channel.

Electrophysiological properties of morphologically distinct cells isolated from the rabbit atrioventricular node.

1. Experiments were conducted using the whole-cell patch clamp technique to determine the electrophysiological properties and ionic currents of ovoid and rod-shaped single isolated calcium-tolerant rabbit atrioventricular (AV) nodal cells. 2. Action potential morphologies observed in these cells were similar to those obtained previously from intracellular recordings of intact atrioventricular nodal preparations: ovoid cells had N- or NH-like action potential configurations (see below), whereas rod-shaped cells had AN-like configurations. 3. Action potential restitution in AV nodal cells was characterized by a progressive increase in overshoot potential, maximal upstroke velocity (Vmax) and action potential duration, as well as a decrease in latency from stimulus to Vmax. In rod-shaped cells, premature stimuli could induce regenerative membrane responses before full action potential repolarization, whereas ovoid cells showed only post-repolarization refractoriness. In ovoid cells stimulated at the low stimulus intensities there was no shortening of the action potential duration and the most premature action potentials were often prolonged. 4. The quasi-steady-state current-voltage relationship of ovoid cells was significantly steeper, at both depolarized and hyperpolarized potentials, than that of either the rod-shaped AV nodal cells or atrial cells. The rod-shaped AV nodal cells and the atrial cells had similar current-voltage (I-V) relationships in the positive potential range, but the I-V curves crossed over at potentials of about-90 mV. 5. A hyperpolarization-activated inward current (I(f)) was apparent in the range between -60 and -90 mV in 95% of the ovoid cells (n = 75), whereas in 88% of rod-shaped cells (n = 16) I(f) was activated at more negative potentials. The magnitude of I(f) in ovoid cells, measured at -100 mV, was approximately 25 times that in rod-shaped cells. 6. A rapid inward current (INa) greater than 1 nA was found in all rod-shaped cells (n = 16) but in only 30% of ovoid cells (n = 75). A transient outward current (I(to)) was found in 93% of rod-shaped cells (n = 14) and in 42% of ovoid cells (n = 54). The combination of I(to) and INa was found in 93% of rod-shaped cells but in only 24% of ovoid cells. 7. These results suggest that there are at least two populations of isolated AV nodal cells with distinct action potentials and ionic current profiles that may contribute to the complex electrophysiological properties observed in the intact AV node.

A quantitative description of the E-4031-sensitive repolarization current in rabbit ventricular myocytes.

We have measured the E-4031-sensitive repolarization current (IKr) in single ventricular myocytes isolated from rabbit hearts. The primary goal of this analysis was a description of the IKr kinetic and ion transfer properties. Surprisingly, the maximum time constant of this component was 0.8 s at 33-34 degrees C, which is significantly greater than the value of 0.18 s previously reported under similar conditions in the original measurements of IKr from guinea pig ventricular myocytes. The primary, novel feature of our analysis concerns the relationship of the bell-shaped curve that describes the voltage dependence of the kinetics and the sigmoidal curve that describes the activation of IKr. The midpoint of the latter occurred at approximately +10 mV on the voltage axis, as compared to -30 mV for the point on the voltage axis at which the maximum time constant occurred. Moreover, the voltage dependence of the kinetics was much broader than the steepness of the activation curve would predict. Taken together, these results comprise a gating current paradox that is not resolved by the incorporation of a fast inactivated state in the analysis. The fully activated current-voltage relation for IKr exhibited strong inward-going rectification, so much so that the current was essentially nil at +30 mV, even though the channel opens rapidly in this voltage range. This result is consistent with the lack of effect of E-4031 on the early part of the plateau phase of the action potential. Surprisingly, the reversal potential Of /Kr was ~15 mV positive to the potassium ion equilibrium potential,which indicates that this channel carries inward current during the latter part of the repolarization phase of the action potential.

Differential responsiveness of atrial and ventricular myocytes to potassium channel openers.

We examined the effects of the potassium channel openers (PCOs) pinacidil and lemakalim (BRL 38227) on action potential (AP) configurations and outward currents in atrial and ventricular myocytes isolated from rabbit and guinea pig hearts, using the whole-cell configuration of the patch clamp technique at 33 degrees-35 degrees C. The PCOs known to activate ATP-sensitive K+ current (IKATP) in various tissues, induced this current and decreased AP duration (APD) in rabbit ventricular myocytes. In contrast, in rabbit atrial myocytes, PCOs either had no effect or increased duration and plateau amplitude of the AP. The predominant outward current in rabbit atrial myocytes is a 4-aminopyridine (4-AP)-sensitive transient outward current (Ito). The PCOs caused a decrease in Ito without inducing IKATP in rabbit atria. In identical experimental conditions, PCOs activated IKATP in both guinea pig atrial and ventricular myocytes. Our results suggest that (a) a species as well as cardiac tissue difference exists in responsiveness to PCOs, and (b) the decrease in Ito without concomitant induction of IKATP can lead to changes in AP configuration opposite to that expected from IKATP activation. Different effects of PCOs on distinct parts of the heart could lead to disparity in APD and refractoriness that may contribute to arrhythmogenesis.

Inhibition of metabolism abolishes transient outward current in rabbit atrial myocytes.

Outward currents were measured in single rabbit atrial myocytes using the whole cell configuration of the patch-clamp technique in the presence of tetrodotoxin (5-10 microM) and MnCl2 (2 mM) to block inward currents. Depolarizing voltage-clamp steps from a holding potential of -80 mV elicited a predominant 4-aminopyridine (4-AP)-sensitive transient outward current (Ito). Inhibitors of oxidative metabolism, 2,4-dinitrophenol (DNP; 100 microM) and cyanide (3 mM) abolished Ito and caused a large increase in the steady-state outward current. This steady-state outward current was inhibited by glibenclamide (5 microM), a blocker of the ATP-regulated potassium current (IKATP). In the presence of DNP, glibenclamide (5 microM) not only inhibited IKATP but also partially restored Ito. Absence of ATP from the pipette produced effects on outward currents similar to those induced by DNP or cyanide. We conclude that metabolic inhibition abolishes Ito in rabbit atrial myocytes and suggest that ATP may be required for the activation of the channel.

Metabolic requirement for the inotropic effects of digitalis and BAY K 8644.

The positive inotropic effects of ouabain and of BAY K 8644 are not apparent in rabbit atria suspended in substrate free medium or in a medium containing 5 mM pyruvate. Addition of glucose in graded concentrations (1-11 mM) during continued exposure of the preparations to the inotropic agents yields graded inotropic effects. The possible involvement of the glycolytic pathway to the development of the inotropic effect of ouabain and BAY K 8644 was tested by using inhibitors of glycolysis that act at two different steps. Iodoacetic acid completely blocks the inotropic effect of ouabain in atria at 0.1 mM and papillary muscles at 0.05 mM. The inotropic effect of BAY K 8644 was blocked partially in atria and papillary muscles. Iodoacetic acid did not change the inotropic effects of isoproterenol and Ca2+. Addition of 1 mM fluoride did not affect significantly the inotropic effect of either ouabain or BAY K 8644 in atria but partially blocked the effect of BAY K 8644 in papillary muscles. The response of atria to ouabain was not changed significantly when glyceraldehyde (10 mM) was substituted for glucose. We suggest that glycolytic ATP may be important for the full inotropic effect of ouabain and BAY K 8644.

Blockade of the inotropic effect of Bay K 8644 by cytochalasin-B and phloretin.

1. The positive inotropic effect in rabbit atria and papillary muscles of Bay K 8644 is blocked by cytochalasin-B (Cyto-B) and phloretin, two compounds known to block the facilitated diffusion of glucose. These compounds do not change the concentration-response curve of calcium. 2. Cyto-B is more potent in atria than in papillary muscles, 10(-7) M having a maximal effect in atria whereas 2 x 10(-5) M was required for a maximal effect in papillary muscles. Phloretin was fully effective at 10(-4) M, the only concentration tested. 3. The inotropic effect of Bay K 8644 was virtually abolished in atria bathed in a glucose-free medium or one containing 5 mM pyruvate. The contractile response to Bay K 8644 of papillary muscles was not changed significantly in glucose-free or in pyruvate-containing medium. 4. Cyto-B (2 x 10(-5) M) caused a slight but significant increase in the KD for the binding of nitrendipine to a crude sarcolemnal preparation from rabbit ventricles. The Bmax was unchanged. 5. These results may best be explained by the hypothesis that there is a metabolic requirement for the inotropic effect of Bay K 8644.

Effects of cytochalasin-B and phloretin on digitalis inotropy.

Phloretin and cytochalasin-B are known to inhibit sugar transport across the cell membrane of many tissues. Both of these agents at concentrations of 100 and 20 microM, respectively, blocked the inotropic effects of ouabain and acetylstrophanthidin (AS) in isolated rabbit atria and papillary muscle preparations. Neither of these agents had any effect of its own on contractile force. Addition of phloretin or cytochalasin-B after the inotropic response to ouabain was fully established did not reverse the effect. The potency of cytochalasin-B was greater in atria than in papillary muscles, 1 microM of cytochalasin caused significant inhibition of the inotropic effect of ouabain in atria without significant effect in papillary muscles. Phloretin but not cytochalasin-B decreased the binding of [3H]ouabain to a semipurified sarcolemmal preparation isolated from canine left ventricular muscle. Neither ouabain nor AS had a substantial positive inotropic effect in atria suspended in substrate-free medium. Substitution of pyruvate (5 mM) for glucose did not fully support their inotropic effect in atria. Papillary muscles behaved differently, in that substrate-free as well as pyruvate media almost fully supported the inotropic effects of ouabain and of low concentrations of AS. Higher concentrations (greater than 250 ng/ml) of As produced a negative inotropic response in substrate-free medium. The possibility that an "active" sugar transport system is required for digitalis inotropy is ruled out by the observation that 2-deoxyglucose also prevents the inotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP as the second messenger for prostaglandin E in modulating suppressor cell-activation by natural suppressor/cytotoxic cells.

The M1-A5 cell line, isolated from the spleen of a tumor bearing mouse, has been shown to secrete a suppressor cell-inducing factor (SIF) which activates suppressor cells from the spleen cells of unprimed mice. In an earlier publication we demonstrated that prostaglandins of the E series (PGE) regulate the release of SIF from M1-A5 cells. In the present study we provide evidence that cyclic adenosine 3',5'-monophosphate (cyclic AMP) is the second messenger for PGE in regulating the release of SIF from M1-A5 cells. Our data show that PGE1 and PGE2 stimulate cyclic AMP production by M1-A5 cells. Furthermore, we show that drugs which: (1) elevate cyclic AMP levels (dibutyryl (db) cyclic AMP), (2) inhibit the breakdown of cyclic AMP (theophylline), or (3) stimulate adenylate cyclase (isoproterenol and histamine) all restored suppressor cell activation by M1-A5 cells in which PG synthesis had been blocked by acetylsalicylic acid (ASA). Thus, these results are consistent with the hypothesis that cyclic AMP is the second messenger for PGE in modulating the release of SIF from M1-A5 cells.

Electrophysiological effects of ethmozine in perfused rabbit hearts.

His-bundle electrocardiography was used to evaluate the effects of ethmozine on cardiac conduction in isolated perfused rabbit hearts electrically driven at cycle lengths of 320 and 250 ms. There was no significant change in conduction until high concentrations of ethmozine were reached. His-Purkinje and atrioventricular (AV) nodal conduction were slowed significantly at 0.1 microgram/mL and atrial conduction at 1.0 microgram/mL. Conduction block occurred at 10.0 micrograms/mL in all the hearts treated. Effects of the drug (0.1 and 0.01 microgram/mL) on conduction of extrasystoles were also studied in hearts driven at a basic cycle length of 270 ms. No significant change was observed in atrial conduction of extrasystoles throughout the coupling intervals tested at both concentrations. Ethmozine (0.01 and 0.1 microgram/mL) caused slowing of His-Purkinje conduction of extrasystoles but the effect of the drug did not change as a function of the coupling interval. An interval-dependent increase in AV-nodal conduction time was observed, with the maximum slowing of conduction occurring at coupling intervals close to the effective refractory period of the AV node. AV-nodal functional refractory period was increased significantly by ethmozine (0.01 and 0.1 microgram/mL). The effective refractory period was significantly increased only at the higher concentration.