K. B. Pustovit

Diadenosine tetra- and pentaphosphates affect contractility and bioelectrical activity in the rat heart via P2 purinergic receptors

Diadenosine polyphosphates (Ap(n)As) are endogenously produced molecules which have been identified in various tissues of mammalian organism, including myocardium. Ap(n)As contribute to the blood clotting and are also widely accepted as regulators of blood vascular tone. Physiological role of Ap(n)As in cardiac muscle has not been completely elucidated. The present study aimed to investigate the effects of diadenosine tetra- (Ap4A) and penta- (Ap5A) polyphosphates on contractile function and action potential (AP) waveform in rat supraventricular and ventricular myocardium. We have also demonstrated the effects of A4pA and Ap5A in myocardial sleeves of pulmonary veins (PVs), which play a crucial role in genesis of atrial fibrillation. APs were recorded with glass microelectrodes in multicellular myocardial preparations. Contractile activity was measured in isolated Langendorff-perfused rat hearts. Both Ap4A and Ap5A significantly reduced contractility of isolated Langendorff-perfused heart and produced significant reduction of AP duration in left and right auricle, interatrial septum, and especially in right ventricular wall myocardium. Ap(n)As also shortened APs in rat pulmonary veins and therefore may be considered as potential proarrhythmic factors. Cardiotropic effects of Ap4A and Ap5A were strongly antagonized by selective blockers of P2 purine receptors suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), while P1 blocker DPCPX was not effective. We conclude that Ap(n)As may be considered as new class of endogenous cardioinhibitory compounds. P2 purine receptors play the central role in mediation of Ap4A and Ap5A inhibitory effects on electrical and contractile activity in different regions of the rat heart.

Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD + ) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart

In rat sinoatrial node, NAD+ (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fi bers, NAD+ (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fi bers, NAD+ reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD+ on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fi bers) are probably related to activation of P1 and P2 purinoceptors.

Effects of Nicotinamide Adenine Dinucleotide (NAD+) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs

Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD+; diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD+ and Ap4A in concentrations of 10−5 and 10−4 M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD+ and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10−4 M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD+ and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors.

The role of diadenosine pentaphosphate and nicotinamide adenine dinucleotide (NAD + ) as potential nucleotide comediators in the adrenergic regulation of cardiac function

The functioning of the heart is under the tight control of the sympathetic division of the autonomous nervous system. The terminals of the postganglionary fibers release both noradrenaline (NA), which is the major sympathetic neurotransmitter, and comediators that can contribute to the fine “tuning” of the adrenergic control of cardiac function. Purine compounds, such as diadenosine pentaphosphate (Ар5А), as well as nicotinamide adenine dinucleotide (NAD+), can act as comediators. The distinctive features of the effects of these compounds on the heart have been incompletely characterized. It is not clear whether these compounds can act as comediators, i.e., to modulate the sympathetic (adrenergic) activity in the heart. Exogenous extracellular Ар5А was shown to reduce the contractility of the ventricular myocardium and to suppress conduction in the atrioventricular (AV) junction in a Langendorff-perfused isolated rat heart. Extracellular NAD+ had a weak effect on inotropy but induced a negative dromotropic effect in the AV junction, similarly to Ар5А. We found that Ар5А and NAD+ suppress both the positive inotropic effect of noradrenaline in the ventricular myocardium and the positive dromotropic effect of NA in the AV junction. The “inhibitory” effects of both purine compounds were more pronounced in the case of combined application with NA. In addition, the influence of Ар5А and NAD+ on the effects of noradrenaline was shown to depend on the timing of the application of these compounds. Our results suggest that the role of the sympathetic comediators NAD+ and Ар5А may consist of the limitation of noradrenaline effects and/or the effects of sympathetic stimulation in the heart.

Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors.

BackgroundRecently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms.MethodsThe standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+.ResultsExtracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10–100xa0μM NAD+ produced two opposite effects in LA and RA – quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NADu2009+u2009−induced alterations of electrical activity. P2X receptors may be responsible for NADu2009+u2009−induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects.ConclusionsExtracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.

Effects of diadenosine polyphosphates on inward rectifier potassium currents in rat cardiomyocytes

Diadenosine polyphosphates are now considered a novel class of endogenous paracrine signal compounds. The putative role of these compounds in pathogenesis of myocardial infarction was proposed, since the concentration of diadenosine polyphosphates increases in the cardiac tissue following the ischemic lesion and myocardial necrosis. Therefore, possible effects of diadenosine polyphosphates on cardiac electrical activity and their ionic mechanisms are of considerable interest.In the present study, we have investigated the effects of diadenosine pentaphosphate (Ap5A), diadenosine tetraphosphate (Ap4A), and NAD+ on transmembrane currents belonging to the family of potassium inward rectifiers: background inward rectifier (IKI), ATP-dependent potassium current (IKATP), and acetylcholinedependent current (IKACh). Experiments were performed using the whole-cell patch-clamp technique on isolated atrial and ventricular rat cardiomyocytes. We have demonstrated that none of the tested adenine compounds affects IKI and IKACh. Ap5A (10–5 M) induces considerable decrease of both inward and outward component of IKATP by 22.1 and 19% of control value, respectively. Higher concentration of Ap5A (3 × 10–5 M) induces stronger suppression of IKATP—by 47.5% and 37.8%, respectively. However, IKATP was found to be insensitive to Ap4A and NAD+.

Effects of Extracellular Diadenosine Tetraphosphate on Action Potentials in the Atrial and Ventricular Myocardium of the Rat Heart during Early Postnatal Ontogenesis

Diadenosine tetraphosphate (AP4A) belongs to a wide group of naturally derived endogenous purine compounds that have recently been considered as new neurotransmitters in the autonomic nervous system. It has been shown that AP4A induces inhibitory effects and modulates adrenergic control in the heart of adult mammals. Nevertheless, the physiological significance of AP4A in early postnatal development, when sympathetic innervation remains yet immature, has not been investigated. The aim of the present study was to elucidate the effects of AP4A on the heart bioelectrical activity in early postnatal ontogenesis. Action potentials (AP) were recorded using the standard microelectrode technique in multicellular isolated right atrial (RA), left atrial (LA), and ventricle (RV) preparations from male Wistar rats at postnatal days 1, 14, and 21 and from 60-day animals that were considered as adults. The application of AP4A caused significant reduction of AP duration in atrial (RA and LA) preparations from rats of all ages. Also, AP4A caused significant AP shortening in RV preparations from rats of various ages; however, the effect was more pronounced in 21-day-old and adult rats. AP4A failed to alter automaticity of RA preparations from the rats at postnatal days 1, 14, and 21 and weakly decreased spontaneous rhythm in RA preparations from the adult rats. The effect of AP4A was partially abolished by P2-receptor blocker PPADS in LA preparations from both 21-dayold and adult rats, while it failed to suppress AP4A-caused AP shortening in preparations from 1- and 14-dayold animals. Thus, extracellular AP4A causes shortening of AP both in the atrial and ventricular myocardium in the rats of early postnatal ontogenesis and in adults. The effect of AP4A depends on age only for ventricular myocardium where it may be attributed with growing contribution of diadenosine polyphosphates to the control of myocardium inotropy.

Negative inotropic effects of diadenosine tetraphosphate are mediated by protein kinase C and phosphodiesterases stimulation in the rat heart

ABSTRACT Extracellular diadenosine polyphosphates (ApnA) are recently considered as an endogenous signaling compounds with transmitter‐like activity which present in numerous tissues, including heart. It has been demonstrated previously that extracellular ApnA cause alteration of the heart functioning via purine receptors in different mammalian species. Nevertheless, principal intracellular pathways which underlie ApnA action in the heart remain unknown. In the present study the role of the P2Y‐associated intracellular regulatory pathway in the mediation of diadenosine tetraphosphate (Ap4A) effects in the rat heart has been investigated for the first time. Extracellular Ap4A caused significant decreasing of the ventricular inotropy. Ap4A evoked reduction of the left ventricle contractility in the isolated Langendorff‐perfused rat hearts, decreasing of the Ca2+ transients in the enzymatically isolated ventricular cardiomyocytes and induced shortening of action potentials in the ventricle multicellular preparations. The inhibitory effects of Ap4A in the rat heart were significantly attenuated by protein kinase C (PKC) inhibitor chelerythrine but these effects were not affected by NO‐synthase inhibitor L‐NAME and guanylyl cyclase (sGC) inhibitor ODQ. In addition, substantial attenuation of Ap4A‐caused negative inotropy in the left ventricle was produced by nonselective phsophodiesterase (PDE) inhibitor IBMX, while PDE type 2 inhibitor EHNA was ineffective. In conclusion, our results allow suggesting that Ap4A‐induced inhibitory effects in the rat heart are mediated by PKC, but not by NO/sGC/PKG‐related signaling pathway. In addition, PDE stimulation may contribute to Ap4A‐caused inhibition of the rat heart contractility.

Extracellular NAD+ Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD+ against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD+, ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD+ against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of ICaL, which can be the main mechanism of NAD+ action on rat heart.

Effect of Purine Co-Transmitters on Automatic Activity Caused by Norepinephrine in Myocardial Sleeves of Pulmonary Veins

We studied the effect of extracellular purine nucleotides (NAD+ and ATP) on spontaneous arrhythmogenic activity caused by norepinephrine in myocardial sleeves of pulmonary veins. In pulmonary veins, NAD+ and ATP reduced the frequency of action potentials and their duration at regular type of spontaneous activity caused by norepinephrine. NAD+ and ATP lengthened the intervals between spike bursts at periodic (burst) type of spontaneous activity. In addition, ATP shortened the duration of spike bursts and the number of action potentials in the “bursts” caused by norepinephrine in the pulmonary veins. It was hypothesized that NAD+ and ATP attenuate the effects of sympathetic stimulation and when released together with norepinephrine from sympathetic endings in vivo, probably, reduce arrhythmogenic activity in myocardial sleeves of pulmonary veins.