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Evidence is accumulating to support a potentially important role for purinergic (P2X) receptors in heart failure (HF). We tested the hypothesis that a hydrolysis-resistant nucleotide analog with agonist activity at myocardial P2X receptors (P2XRs) improves the systolic HF phenotype in mouse and dog models. We developed a hydrolysis-resistant adenosine monophosphate derivative, (1'S,2R,3S,4'R,5'S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxymethyl] bicycle[3.1.0]hexane-2,3-diol) (MRS2339), with agonist activity at native cardiac P2XRs. Chronic MRS2339 infusion in postinfarct and calsequestrin (CSQ) mice with HF resulted in higher rates of pressure change (+dP/dt), left ventricle (LV)-developed pressure, and cardiac output in an in vitro working heart model. Heart function in vivo, as determined by echocardiography-derived fractional shortening, was also improved in MRS2339-infused mice. The beneficial effect of MRS2339 was dose-dependent and was identical to that produced by cardiac myocyte-specific overexpression of the P2X(4) receptor. The HF improvement was associated with the preservation of LV wall thickness in both systole and diastole in postinfarct and CSQ mice. In dogs with pacing-induced HF, MRS2339 infusion reduced left ventricular end-diastolic pressure, improved arterial oxygenation, and increased +dP/dt. MRS2339 treatment also decreased LV chamber size in mice and dogs with HF. In murine and canine models of systolic HF, in vivo administration of a P2X nucleotide agonist improved contractile function and cardiac performance. These actions were associated with preserved LV wall thickness and decreased LV remodeling. The data are consistent with a role of cardiac P2XRs in mediating the beneficial effect of this agonist.

Microtubules provide a chemical signaling function as well as structural support for heart cells. Microtubules modulate autonomic signaling in the heart, and their disruption by colchicine unmasks muscarinic inhibition of Ca (ICa) current. In this study, we compare the actions of the estrogen metabolite, 2-methoxyestradiol (2-ME), with those of colchicine on microtubule stability and chemical signal function in guinea pig-isolated ventricular myocytes. Like colchicine, 2-ME binds to microtubules and disrupts the cytoskeleton of cardiac myocytes. Incubation with 2-ME increased the soluble fraction of tubulin and decreased the polymerized fraction at concentrations ranging from 10 to 100 microM. 2-ME was less potent than colchicine in causing microtubular disruption. Treatment with 2-ME for up to 4 h was accompanied by a progressive increase of I(Ca) amplitude. There was no change in the rates of ICa inactivation. Carbachol, which has no effect on ICa in untreated ventricular myocytes, inhibited this current in the presence of 2-ME. The extent of inhibition increased with incubation time in 2-ME such that carbachol completely removed the increment of ICa by the estrogen metabolite. The results illustrate the important role of microtubules in modulating cardiac autonomic signaling.

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na+. Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na+ entry and modulate Na+ handling. We further determined whether P2X receptor-induced stimulation of the Na+/Ca2+ exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na+-K+-ATPase current ( Ip) and NCX current ( INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca2+ entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from −14 ± 2.3 to −25 ± 4.1 mV, causing an estimated intracellular Na+ concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca2+ entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca2+ entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na+ entry pathway through ligand-gated P2X4Rs in cardiomyocytes.

I-PC Inhibits INa/K and IK1 in Heart. introduction: The changes of the resting potential (RP) and of the current-voltage (I-V) relationship induced by l-palmitoylcarnitine (I-PC) in the presence of the IK1 blocker, cesium, or in the presence of the INa/K blocker, ouabain, were tested in guinea pig ventricular myocytes to ascertain the relative contributions of IK1 and INa/k suppression to the membrane depolarization caused by this amphiphile. Methods and Results: Ramp voltages were applied to myocytes with the whole cell, patch clamp technique. 1-PC (10 μM) produced additional membrane depolarization in the presence of either 10 mM Cs+ or 30 μM ouabain. In the presence of Cs+ 1-PC, like 3 μM ouabain, shifted current inward at potentials negative to -20 mV as a result of INa/K blockade. In the presence of 30 μM ouabain, 1-PC, like Cs+, shifted current inward at potentials between -27 and -88 mV and outward at potentials negative to -88 mV. This is attributed to IK1 block because the current was inwardly rectifying, with a reversal potential near EK. When I-PC or ouabain inhibited INa/K, the presence of an Ni2+-sensitive component attributed to INa/ca distorted the membrane I-V relationship, particularly in the presence of Cs+. The relative contributions of IK1 and INa/K block by 1-PC were voltage dependent. At the RP, 1-PC produced a greater block of INa/K than of IK1. Conclusion: I-PC depolarizes the resting membrane by inhibiting both IK1 and INa/K. It is concluded that suppression of INa/K by I-PC predominates over block of IK, to depolarize the membrane at the RP.

Objective: We studied the effect of calcium on inositol 1,4,5-trisphosphate (InsP3)-induced contractions in saponin-permeabilized chick atrial muscle (80–100 μm in diameter). Calcium has been proposed to modulate InsP3-induced response and InsP3 binding in other tissue. Methods: A transient increase in tension was used to detect the release of calcium in this multicellular preparation. Pulsed (2–3 s) superfusion of either calcium or InsP3 produced a transient contraction. Results: Pulsed coapplication of both InsP3 and calcium resulted in a contraction greater than that predicted by an additive effect of both intracellular messengers. The release of calcium by InsP3 is positively and negatively modulated by myoplasmic calcium (EC50 ∼- 0.17 μM and IC50 of approximately 1.5 μM). The peak potentiation occurred at approximately pCa = 6.3 (0.51 μM free calcium). Conclusions: These observations indicate that an increase of InsP3 in the heart, as produced by cardiac neurotransmitters and hormones, may regulate the force of contraction by virtue of its synergistic action with calcium. While calcium remains the primary trigger for calcium release during excitation-contraction (E-C) coupling, we propose that changes in cytoplasmic calcium can modulate InsP3-induced calcium release on a beat-to-beat basis. Thus, InsP3 may regulate force generation during E-C coupling by activating calcium release during a heart beat.

Carbachol (10(-6)-3X10(-4)M) induces a positive inotropic response in paced, pertussis toxin-treated fibers which is atropine-sensitive and independent of endogenous catecholamines. At the same concentrations in atria from saline-treated chicks, carbachol's negative inotropic effect on the steady state contractions (SSC) is attenuated and the rested state contraction (RSC) is increased. The RSC and SSC in pertussis toxin-treated fibers are increased by carbachol (EC50 = 30 microM) indicating that repetitive electrical depolarization is not essential for the inotropic response. The inotropic response of the SSC is frequency-independent from 0.10-1.0 Hz; however it is decreased (approximately 50%) at a high frequency (3.0 Hz). In control untreated atrial muscle, carbachol (10(-4)M) selectively increases the early component of the RSC. The late component of the RSC, representing activation of transmembrane Ca2+ inward current, is not changed. Carbachol's positive inotropic effect is perhaps exerted by enhancing Ca2+ release and/or Ca2+ content of the sarcoplasmic reticulum. The ability of various muscarinic agonists to induce a positive inotropic response was: carbachol > acetylcholine > oxotremorine. This order correlates with the ability of these agents to induce a tetrodotoxin-resistant Na+ inward current that increases intracellular Na+ and to promote phosphatidylinositol hydrolysis. These data are consistent with the hypothesis that the carbachol-induced positive inotropic response may result from greater intracellular Ca2+ availability secondary to enhanced Na-Ca exchange. The greater Ca2+ availability, together with increased production of the Ca-mobilizing messenger, inositol 1,4,5-trisphosphate (InsP3), can exert a synergistic effect to regulate force generation.

We previously reported that inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and caffeine evoked contractures in saponin-permeabilized chick atria. The magnitude of contractures evoked by maximally effective concentrations of Ins(1,4,5)P3 were half those evoked by maximally effective concentrations of caffeine. In the present report, we tested the hypothesis that these two agents may act on distinct calcium-release mechanisms by comparing the effects of ryanodine, ruthenium red, and procaine on the responses to Ins(1,4,5)P3 and caffeine. We find that procaine inhibits both responses with similar mean inhibitory concentrations in the millimolar range. Nanomolar concentrations of ryanodine selectively potentiate the contractures induced by Ins(1,4,5)P3 but have no effect on those induced by caffeine. Micromolar concentrations of ryanodine inhibit responses to both Ins(1,4,5)P3 and caffeine in a use-dependent way. Ruthenium red prevents the response to Ins(1,4,5)P3 and potentiates that to caffeine, as if ruthenium red had enhanced calcium accumulation in the caffeine-sensitive pool(s). Because we found that caffeine prevented the subsequent response to Ins(1,4,5)P3, but Ins(1,4,5)P3 had no detectable effect on the caffeine-induced contracture, we conclude that Ins(1,4,5)P3 and caffeine act on pharmacologically distinct calcium-release mechanisms that may reside in the same sarcoplasmic reticulum compartment.

Binary cardiac transgenic (Tg) overexpression of P2X4 receptors (P2X4R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X4R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca2+ transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca2+ transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca2+ transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.

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.

An early inward tail current evoked by membrane depolarization (from -80 to -40 mV) sufficient to activate sodium but not calcium current was studied in single voltage-clamped ventricular myocytes isolated from guinea pig hearts. Like forward-mode Na-Ca exchange, this early inward tail current required [Na+]o and [Ca2+]i and is thought to follow earlier reverse-mode Na-Ca exchange that triggers Ca2+ release from sarcoplasmic reticulum. The dependence of the early inward tail current on [Ca2+]i was supported by the ability of small (+10 mV) and large (+80 mV) voltage jumps from -40 mV to decrease and increase, respectively, the size of early inward tail currents evoked by subsequent voltage steps from -80 to -40 mV. As expected, tetrodotoxin selectively inhibited the early inward tail current but not the late inward tail current that followed voltage jumps to +40 mV test potentials. Although tetrodotoxin also blocked the fast Na+ current, replacement of extracellular Na+ by Li+ sustained the fast Na+ current. However, Li+, which does not support Na-Ca exchange, reversibly suppressed both the early and late inward tail currents. Inhibitors (ryanodine and caffeine) and promoters (intracellularly dialyzed inositol 1,4,5-trisphosphate) of sarcoplasmic reticulum Ca2+ release decreased and increased, respectively, the magnitude of the early inward tail current. The results substantiate the hypothesis that Ca2+ release from the sarcoplasmic reticulum participates in early Na-Ca exchange current and demonstrate that inositol 1,4,5-trisphosphate, by releasing Ca2+ from the sarcoplasmic reticulum, can promote Na-Ca exchange across the plasma membrane.

“For the best and safest method of philosophizing seems to be, first diligently to investigate the properties of things and establish them by experiments, and then later seek hypotheses to explain them… For hypotheses ought to be fitted merely to explain the properties of things and not attempt to predetermine them except so far as they can be an aid to experiments.” (I. Newton, Opera, Vol. IV, p. 314; From The Metaphysical Foundation of Modern Science. E.A. Burtt, Doubleday & Company, Inc., Garden City, New York, 1932; p.215.)

Evidence is accumulating to support the presence of P2X purinergic receptors in the heart. However, the biological role of this receptor remains to be defined. The objectives here were to determine the role of cardiac P2X receptors in modulating the progression of post-myocardial infarction ischemic heart failure and to investigate the underlying mechanism. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of P2X4R (Tg) was developed as a model. Left anterior descending artery ligation resulted in similar infarct size between Tg and wild-type (WT) mice ( P > 0.1). However, Tg mice showed an enhanced cardiac contractile performance at 7 days, 1 mo, and 2 mo after infarction and an increased survival at 1 and 2 mo after infarction ( P < 0.01). The enhanced intact heart function was manifested by a greater global left ventricular developed pressure and rate of contraction of left ventricular pressure in vitro and by a significantly increased fractional shortening and systolic thickening in the noninfarcted region in vivo ( P < 0.05). The salutary effects on the ischemic heart failure phenotype were seen in both sexes and were not the result of any difference in infarct size in Tg versus WT hearts. An enhanced contractile function of the noninfarcted area in the Tg heart was likely an important rescuing mechanism. The cardiac P2X receptor is a novel target to treat post-myocardial infarction ischemic heart failure.

Inositol 1,4,5-trisphosphate (IP3) and caffeine evoked transient, reversible, and concentration-dependent increases in tension in saponin-treated chick atrial muscle. Contractures evoked by IP3 and caffeine were detected in solutions with 70 microM EGTA at pCa 7.0. In the presence of 7 mM EGTA, neither IP3 nor caffeine was able to evoke a contracture. Maximally effective concentrations of IP3 (20 microM) and caffeine (20 mM) developed tensions to approximately 44 and 83% of that elicited by pCa 5.0 (maximum tension = 100%), respectively. The IP3- or caffeine-induced contractures were consistently reproduced when the sarcoplasmic reticulum (SR) had previously been loaded with calcium. Preexposure to caffeine suppressed the following IP3-induced response. When ryanodine (1-10 microM) was present throughout the SR-loading cycle, the responses to IP3 and caffeine were prevented. However, when ryanodine was added after the SR was loaded with calcium, neither the response to IP3 nor that to caffeine was affected. These results are consistent with the hypothesis that ryanodine inhibition requires prior activation of the SR calcium-release channel. It is concluded that both IP3 and caffeine increased tension in the SR by releasing calcium from it. The effect of IP3 is consistent with its messenger role as a calcium-mobilizing agent.

A brief outline of the ontogenesis of cholinergic neuroeffector transmission in the embryonic chick heart is given. It is important to define the development of neuroeffector transmission to evaluate the possible effects on postjunctional membrane properties. The membrane response of the aneural chick heart undergoes a qualitative change to muscarinic agonist at a time well before cholinergic innervation. This suppressed response can be revealed later in life, even in adults, when provision is made to exclude Gi/Go and or K channel activity.

In isolated guinea pig ventricular myocytes, L-palmitoylcarnitine (L-PC) produced concentration- and time-dependent changes of resting potential (RP) and action potential duration at 50% repolarization (APD50). At 10(-8) to 10(-6) M, L-PC increased APD50 without changing RP. At 10(-5) M, the amphiphile initially increased (0-10 min) and eventually decreased (greater than 10 min) APD50; the membrane depolarized when APD50 decreased. Additionally, transient depolarizations (TDs) were consistently induced in 10(-5) M L-PC within 10 min, and TD amplitude progressively increased with continued exposure to L-PC. The TDs induced in L-PC were augmented by membrane depolarization, elevated extracellular Ca2+ concentration ([Ca2+]o), and increased number of stimuli. Elevated [Ca2+]o or neuraminidase treatment also allowed TDs. In neuraminidase, the changes of RP, APD50, and TD amplitude were qualitatively similar to those seen with L-PC. These results are consistent with the hypothesis that 10(-5) M L-PC causes intracellular Ca2+ overload. The blockade of L-PC and neuraminidase-induced TDs by ryanodine is consistent with the intracellular Ca2+ overload hypothesis.

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2X4R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca2+ transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular Ca2+ transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type Ca2+ channel activation. The increased Ca2+ transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca2+ transient. Simultaneous Ca2+ transient and contraction measurements did not show any agonist-mediated change in myofilament Ca2+ sensitivity. However, activation of the overexpressed P2X4 receptor caused an enhanced SR Ca2+ loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca2+ transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR Ca2+ content, occurring in the absence of cAMP accumulation or L-type Ca2+ channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.

P2X receptors, activated by extracellular ATP, may be important in regulating cardiac function. The objective of the present study was to characterize the electrophysiologic actions of P2X4 receptors in cardiac myocytes and to determine whether they are involved in mediating the effect of extracellular ATP. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and P2X4 receptor-overexpressing transgenic (TG) mice. The P2X agonist 2-meSATP induced an inward current at -100 mV that was greater in magnitude (2-fold) in TG than in WT ventricular cells. In the presence of the P2X4 receptor-selective allosteric enhancer ivermectin (3 microM), the 2-meSATP-stimulated current increased significantly in both WT and TG ventricular cells, consistent with an important role of P2X4 receptors in mediating the ATP current not only in TG but also WT myocytes. That the current in both WT and TG cells showed similar voltage-dependence and reverse potential (approximately 0 mV) further suggests a role for this receptor in the normal electrophysiological action of ATP in WT murine cardiac myocytes. The P2X antagonist suramin was only able to block partially the 2-meSATP-stimulated current in WT cells, implying that both P2X4 receptor and another yet-to-be-identified P2X receptor mediate this current.

Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca(2+) current (I(Ca(L))) in mammalian ventricular myocytes by phosphorylating the calcium channel at a site different from that acted on by cAMP or by dephosphorylating the calcium channel through phosphatase(s)? We tested these possibilities in guinea pig ventricular myocytes superfused with Tyrode's solution (35 degrees C) and dialyzed with adenosine 5'-O-(3-thiotriphosphate) ([ATPgammaS](pip)). ATPgammaS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPgammaS](pip), I(Ca(L)) increased gradually over 20 to 25 min and then rapidly in the presence of 3-isobutyl-1-methylxanthine. 8-Bromo-cGMP (8-Br-cGMP; 1 mM) did not inhibit I(Ca(L)) significantly (-3 +/- 11.8%, n = 21) in contrast to results with ATP dialysis (). Similar results were obtained with 0.1 mM carbachol (CCh). I(Ca(L)) increased after longer dialysis (/=40 min) with ATPgammaS; again, 8-Br-cGMP had no effect. Also, isoproterenol (ISO) did not stimulate and CCh, alone or in the presence of ISO, did not inhibit I(Ca(L)). Block of CCh effect by ATPgammaS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) formation from ATPgammaS via nucleoside diphosphate kinase. GTPgammaS uncouples muscarinic and beta-adrenoceptors from intracellular effectors. Failure of 8-Br-cGMP to reduce I(Ca(L)) irreversibly excludes calcium channel phosphorylation as an inhibitory mechanism. We propose that cGMP inhibits I(Ca(L)) by activating phosphatase(s) in guinea pig ventricular myocytes.

Objective: We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current ( I Ca(L)) and contractions by muscarinic agonists. Methods: Voltage clamp pulses elicited I Ca(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode’s solution (36°C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. Results: InsP3 (1–10 μM) transiently increased isotonic contractions when applied for 4–5 min; higher concentrations (50–300 μM) caused a sustained decrease in contractions. InsP3 had no effect on I Ca(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 μM InsP3. Pentosan polysulfate (50 μg/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10–300 μM) caused sustained increases of I Ca(L) and contractions. Cyclic AMP, but not InsP3, also increased I Ca(L) when intrapipette Cs+ suppressed K+ currents. Conclusions: Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP3 in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change I Ca(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.

The effect of carbachol (CCh) on the Na/Ca exchange current ( I Na/Ca) was studied in voltage-clamped ventricular myocytes isolated from guinea pig hearts and superfused with Tyrode solution at 35°C. CCh (100 μM) increased outward current during depolarizations (10–200 ms) from −45 mV and tail current amplitude on repolarization; CCh had no effect on the L-type Ca2+current. Amplitudes of the outward and tail currents declined with increasing duration of the depolarizing clamp pulse. Ouabain produced similar current changes that are suppressed by intrapipette ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid and are characteristic of I Na/Ca. Depolarization from −80 to −30 mV elicited the rapid Na+ current followed by a slowly decaying inward I Na/Ca (J. C. Gilbert, T. Shirayama, and A. J. Pappano. Circ. Res. 69: 1632–1639, 1991.) that was reversibly increased by CCh. Atropine (1–3 μM) prevented the CCh effect. All procedures that suppressed I Na/Caalso suppressed the CCh effect. Sarcoplasmic reticulum (SR) Ca2+ release participated in generating I Na/Cabecause 10 mM caffeine or 1 μM ryanodine blocked I Na/Ca and the effect of CCh. Rapid superfusion of 10 mM caffeine induced inward I Na/Ca at −75 mV; a caffeine-induced charge transfer gives an SR Ca2+ content of 67 μM. CCh increased caffeine-induced current; SR Ca2+ content rose to 98 μM. CCh also augmented the amplitude of steady-state intracellular Ca2+ transients and contractions during a train of voltage-clamp pulses (−75 to 30 mV for 200 ms) at 1 Hz. CCh elevated intracellular Na+ (M. Korth and V. Kühlkamp. Pflügers Arch.403: 266–272, 1985) by inducing a background Na+ current [K. Matsumoto and A. J. Pappano. J. Physiol. (Lond.)415: 487–502, 1989]. Together with these data, the present results are consistent with the hypothesis that CCh, via muscarinic receptors, eventually promotes I Na/Ca at the sarcolemma through a mechanism that requires the SR and that this action accounts for the increased contractions.

1. Ca2+ release from the sarcoplasmic reticulum (SR) was examined in enzymatically isolated single guinea-pig ventricular myocytes by monitoring [Ca2+]i with fura-2 during whole-cell recording of action potentials at room temperature (23-25 degrees C). Modulation of Ca2+ release by the Na+ current (INa) was studied by manipulating Na+ influx through the Na+ channel. 2. For a comparable Ca2+ loading of the SR, brief hyperpolarizing currents applied at the peak of the action potential increased Ca2+ release, while depolarizing pulses had the opposite effect. Similar currents applied before the action potential did not affect Ca2+ release. 3. Application of tetrodotoxin (TTX; 60 microM) moderately reduced Ca2+ release from the SR, but this effect was delayed in comparison with the immediate block of INa. An early effect of TTX was to increase Ca2+ release. 4. Replacement of Na+ with Li did not reduce Ca2+ release, but led to a progressive increase in Ca2+ release, resulting in spontaneous activity. 5. Ca2+ channel blockers (CdCl2, 100 microM; nisoldipine, 20 microM; or nifedipine, 20 microM) drastically reduced Ca2+ release from the SR. 6. Voltage clamp experiments confirmed that TTX blocked INa and its associated [Ca2+]i transient during voltage steps from -90 to -50 mV. INa and its associated [Ca2+]i transient were equally suppressed following replacement of Na+ with N-methyl-D-glucamine (NMDG+), but the [Ca2+]i transient was not suppressed following replacement of Na+ with Li+. 7. The INa-associated transient was sensitive to Ca2+ channel blockers. During steps from -50 to 0 mV, it appeared that the dihydropyridine antagonists often did not provide full block of the calcium current (ICa). 8. During current clamp stimulation at 1 Hz in the presence of TTX (60 microM), the Ca2+ content of the SR was decreased, due to the changes in action potential configuration and to changes in [Na+]i. 9. Our experiments indicate that the Ca2+ entry coupled to Na+ influx via the Na+ channel does not contribute substantially to the trigger for Ca2+ release from the SR during action potentials (23-25 degrees C). However, INa modulates Ca2+ release by affecting the Ca2+ load of the SR.

We previously showed that palmitoyl-L-carnitine (L-PC) inhibits the Na/K pump current (INa/K). In the present report, we test the hypothesis that L-PC, like ouabain, should increase myocyte shortening. Membrane potentials or ionic currents were recorded simultaneously with cell shortening in single guinea pig ventricular myocytes at room temperature (22 degrees C). Like ouabain, L-PC (1 microM) reversibly depolarized the resting membrane, decreased action potential duration, and increased the amplitude of myocyte contractions. Neither L-PC nor ouabain had a significant effect on Ca current (ICa). When L-PC increased cell shortening during ramp voltage clamp, membrane current shifted inward at voltages negative to -20 mV and shifted outward at more positive voltages. Similar to toxic concentrations of ouabain, L-PC induced transient inward currents and aftercontractions. At concentrations that inhibit INa/K, L-PC acted like ouabain to produce characteristic effects on membrane potentials, currents, and cell contractions that were unrelated to significant changes in ICa. L-PC reduces surface negative charge of erythrocytes and myocytes (C. Gruver and A. J. Pappano, J. Mol. Cell. Cardiol. 25: 1275–1284, 1993), and we speculate that L-PC inhibits INa/K by this mechanism.

The time course of muscarinic effects on K and Ca currents was investigated at 22-24 degrees C in guinea-pig atrial myocytes, using the whole-cell voltage clamp. At a holding potential of -40 or -50 mV, short exposures to 100 microM acetylcholine (ACh) or carbachol (CCh) reproducibly induced outward K currents (IK,ACh). During long exposures to these agonists, IK,ACh faded with time. In cells not dialysed with guanosine triphosphate (GTP), IK,ACh could dissipate completely following 15-20 min of agonist exposure. After agonist washout, lost sensitivity was not recovered. In cells dialysed with GTP (0.2-1 mM), IK,ACh still faded but normal sensitivity to agonists was restored with washout. Fade of IK,ACh was not prevented by intracellular heparin or dextran, excluding the involvement of either beta-adrenergic or muscarinic receptor kinase. IK,ACh induced by bethanechol or adenosine also faded, and subsequent CCh application after washout revealed a diminished response. Intracellular guanosine-5'-o-(3-thiotriphosphate (GTP gamma S) induced IK,ACh which faded, and subsequent exposure to CCh was without effect. Equally, after full desensitization with CCh, GTP gamma S failed to induce IK,ACh. The Ca current (ICa) was activated by voltage steps to 0 mV and increased with 1-3 microM isoproterenol. This increase could be reversed by CCh, even when IK,ACh had completely faded. Prolonged muscarinic agonist exposure sometimes also caused fade of the effect on ICa, which always occurred after loss of IK,ACh. The results show that desensitization is heterologous and may involve the guanine nucleotide-binding (G) protein. The differential desensitization to the effects on IK,ACh and ICa suggests the involvement of two different signalling pathways in the muscarinic control of K and Ca channels.

The phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX; 100 microM) and papaverine (100 microM) increased peak L-type Ca current (ICa) more than fivefold in a way similar to isoproterenol, forskolin, or intracellular adenosine 3',5'-cyclic monophosphate in guinea pig ventricular myocytes studied with the whole cell voltage-clamp technique at 22-24 degrees C. IBMX and papaverine could also induce a chloride current. Both drugs caused an apparent increase of ICa inactivation as revealed by 1) a negative shift of the ICa inactivation curve between -40 and 0 mV and 2) a suppression of the relief from inactivation at potentials positive to 0 mV. In the presence of IBMX or papaverine, the amplitudes of both the rapidly and slowly inactivating components of ICa were increased; the effect on the fast component was more pronounced. The drugs did not accelerate the inactivation time course of either component. Carbachol (CCh; 100 microM) reversed the increase in ICa produced by IBMX or papaverine. However, ICa could not be restored to its original magnitude on washout of CCh in the presence of phosphodiesterase inhibitors. In pertussis toxin-treated cells or in the presence of Ly-83583 (1-100 microM), IBMX retained its effect but CCh was unable to reduce ICa. Dialysis with guanosine 3',5'-cyclic monophosphate (cGMP; 0.1-100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (30 microM) suppressed the increase of ICa by IBMX; the inhibition by cGMP was additive with that produced by CCh. We suggest that the major part of IBMX and papaverine effect is mediated by phosphodiesterase inhibition and involves an increase in intracellular adenosine 3',5'-cyclic monophosphate levels. CCh reversal of phosphodiesterase inhibitor action probably involves an elevation of cGMP levels and activation of cGMP-dependent protein kinase.

1-Palmitoylcarnitine (1-PC) reduced the electrophoretic mobility of human erythrocytes bathed in low ionic strength solution. Unlike divalent cations which appear to reduce electrophoretic mobility by screening surface negative charge, cationic 1-PC does so by being incorporated into the plasma membrane. Incorporation of 1-PG was assessed directly by measurement of the partition coefficients which are 1.18 × 10 5 and 1.38 × 10 5 in sarcoplasmic reticulum vesicles and erythrocyte ghosts, respectively. The hydrophobic nature of the amphiphile interaction with erythrocyte membrane was also evident as an antihemolytic effect at 1-PC concentrations that also reduced electrophoretic mobility. Moreover, the potency and efficacy of acylcarnitines to reduce electrophoretic mobility increased as the acyl chain length increased. 1-PC also reduced the electrophoretic mobility of guinea-pig ventricular myocytes in low ionic strength solution. Estimation of the zeta potential yielded positive shifts of 4mV in myocytes and 5mV on crythrocytes at 1 μM 1-PC. These voltage shifts are essentially the same as those reported for activation and inactivation of sodium and calcium currents in myocytes. Therefore, the surface charge effect of 1-PC depends upon membrane incorporation and underlies the electrophysiological actions of the amphiphile including arrhythmias.

We reinvestigated the issue of whether l-palmitoylcarnitine inhibits the Na/K pump in the heart. The effects of l-palmitoylcarnitine or ouabain on the Na/K pump current were studied with the voltage-clamp technique in isolated guinea-pig ventricular myocytes. In myocytes bathed in Tyrode's solution, l-palmitoylcarnitine shifted the current-voltage relation inward at all potentials between -80 and 20 mV. the "U"-shaped difference current seen in l-palmitoylcarnitine was maximal at -30 mV and declined at potentials more positive and negative than this. Under conditions that minimized time-dependent currents, ouabain or l-palmitoylcarnitine shifted membrane current inward in the presence of 5.4 mM extracellular potassium. Reduction of extracellular potassium to 0 mM for 2 min also shifted membrane current inward. When extracellular potassium was returned to 5.4 mM, the intracellular sodium that had accumulated was extruded and a transient outward current was generated as a result of Na/K pump stimulation. Ouabain or l-palmitoylcarnitine reversibly suppressed this transient outward current and reduced the rate constant for the decline of this current. The ability of l-palmitoylcarnitine to imitate the actions of ouabain on membrane current and on the transient outward current indicates that this amphiphile inhibits the Na/K pump current in guinea-pig ventricular myocytes. This results is consistent with the suppression by l-palmitoylcarnitine of the activity of Na/K ATPase in cardiac sarcolemmal vesicles.

Pacemaker activity of the isolated chick heart changed considerably during development. The spontaneous impulse frequency increased up to the 11th–15th incubation day. This period of acceleration was followed by a marked deceleration around the time of hatching. Both acceleration and deceleration appeared to be caused by changes intrinsic to the pacemaker. Deceleration was related to a decrease in rate of slow diastolic depolarization. The results were compared with well-known ontogenetic changes in heart rate in vivo . This comparison indicates that the deceleration of pacemaker firing around hatching is counteracted by the development of sympathetic cardiac reflexes in vivo .