Denis V. Abramochkin

Modulation of rabbit sinoatrial node activation sequence by acetylcholine and isoproterenol investigated with optical mapping technique

Aims:  Changes in the rabbit sinoatrial node (SAN) activation sequence with the cholinergic and adrenergic factors were studied. The correlation between the sinus rhythm rate and the leading pacemaker site shift was determined. The hypothesis concerning the cholinergic suppression of nodal cell excitability as one of the mechanisms associated with pacemaker shift was tested.

Non‐quantal release of acetylcholine from parasympathetic nerve terminals in the right atrium of rats

Acetylcholinesterase (AChE) inhibitors provoke typical cholinergic effects in the isolated right atrium of the rat due to the accumulation of acetylcholine (ACh). Our study was designed to show that in the absence of vagal impulse activity, ACh is released from the parasympathetic nerve fibres by means of non‐quantal secretion. The conventional microelectrode technique was used to study changes in action potential (AP) configuration in the right atrium preparation of rats during application of AChE inhibitors. Staining with the lipophilic fluorescent dye FM1‐43 was used to demonstrate the presence of endocytosis in cholinergic endings. The AChE inhibitors armin (10−7–10−5 m) and neostigmine (10−7 to 5 × 10−6 m) caused a reduction of AP duration and prolonged the cycle length. These effects were abolished by atropine and were therefore mediated by ACh accumulated in the myocardium during AChE inhibition. Putative block of impulse activity of the postganglionic neurons by tetrodotoxin (5 × 10−7 m) and blockade of ganglionic transmission by hexomethonium (2 × 10−4 m), as well as blockade of all forms of quantal release with Clostridium botulinum type A toxin (50 U ml−1), did not alter the effects of armin. Experiments with FM1‐43 dye confirmed the effective block of exocytosis by botulinum toxin. Selective inhibition of the choline uptake system using hemicholinium III (10−5 m), which blocks non‐quantal release at the neuromuscular junction, suppressed the effects of AChE inhibitors. Thus, accumulation of ACh is likely to be caused by non‐quantal release from cholinergic terminals. We propose that non‐quantal release of ACh, shown previously at the neuromuscular junction, is present in cholinergic postganglionic fibres of the rat heart in addition to quantal release.

Normobaric, intermittent hypoxia conditioning is cardio- and vasoprotective in rats.

Favorable versus detrimental cardiovascular responses to intermittent hypoxia conditioning (IHC) are heavily dependent on experimental or pathological conditions, including the duration, frequency and intensity of the hypoxia exposures. Recently, we demonstrated that a program of moderate, normobaric IHC (FIO2 9.5–10% for 5–10 min/cycle, with intervening 4 min normoxia, 5–8 cycles/day for 20 days) in dogs afforded robust cardioprotection against infarction and arrhythmias induced by coronary artery occlusion–reperfusion, but this protection has not been verified in other species. Accordingly, in this investigation cardio- as well as vasoprotection were examined in male Wistar rats completing the normobaric IHC program or a sham program in which the rats continuously breathed atmospheric air. Myocardial ischemia and reperfusion (IR) was imposed by occlusion and reperfusion of the left anterior descending coronary artery in in situ experiments and by subjecting isolated, perfused hearts to global ischemia–reperfusion. Cardiac arrhythmias and myocardial infarct size were quantified in in situ experiments. Endothelial function was evaluated from the relaxation to acetylcholine of norepinephrine-precontracted aortic rings taken from in situ IR experiments, and from the increase in coronary flow produced by acetylcholine in isolated hearts. IHC sharply reduced cardiac arrhythmias during ischemia and decreased infarct size by 43% following IR. Endothelial dysfunction in aorta was marked after IR in sham rats, but not significant in IHC rats. Similar findings were found for the coronary circulations of isolated hearts. These findings support the hypothesis that moderate, normobaric IHC is cardio- and vasoprotective in a rat model of IR.

The Effect of Hydrogen Sulfide on Electrical Activity of Rat Atrial Myocardium

Changes in the configuration of action potentials and in the frequency of pacemaker discharges in a preparation of isolated rat right atrium under the effect of sodium hydrosulfide degrading in water solution with hydrogen sulfide release were studied by intracellular recording of action potentials in the myocardium. Sodium hydrosulfide in concentrations of 100–500 μM markedly reduced the duration of action potentials at the level of 50 and 90% repolarization and decelerated the sinus rhythm. Moreover, sodium hydrosulfide shortened action potentials in the preparations working in the forced rhythm. Glybenclamide (potassium ATP-dependent channel blocker; 10 μM) reduced the effect sodium hydrosulfide (200 μM) by more than 60%, which suggested the involvement of potassium ATP-dependent current in the realization of the effect of hydrogen sulfide on configuration of action potentials. Hence, hydrogen sulfide, recently described as a signal compound, modulates many electrophysiological parameters of the myocardium.

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model - protoplasts obtained from lily pollen grains at the early germination stage - to reveal the effect of H2 O2 on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS-sensitive currents on the pollen grain plasma membrane: the hyperpolarisation-activated calcium current, which is strongly enhanced by H2 O2 , and the outward potassium current, which is modestly enhanced by H2 O2 . We used low concentrations of H2 O2 that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.

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.

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium-calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K(+) currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (IK1) and the carbacholine-induced inward rectifier (IKACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal IK1 of ventricular myocytes was blocked with apparent IC50-values of 4.6×10(-6) M and 3.5×10(-6) M for rat and fish, respectively. IKACh was almost an order of magnitude more sensitive to KB-R7943 than IK1 with IC50-values of 6.2×10(-7) M for rat and 2.5×10(-7) M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9-3×10(-6) M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order IK1~INCX<IKACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular IKACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.

Carbon monoxide affects electrical and contractile activity of rat myocardium

BackgroundCarbon monoxide (CO) is a toxic gas, which also acts in the organism as a neurotransmitter. It is generated as a by-product of heme breakdown catalyzed by heme oxygenase. We have investigated changes in electrical and contractile activity of isolated rat atrial and ventricular myocardium preparations under the influence of CO.MethodsStandard microelectrode technique was used for intracellular registration of electrical activity in isolated preparations of atrial and ventricular myocardium. Contractions of atrial myocardial stripes were registered via force transducer.ResultsCO (10-4 - 10-3 M) caused prominent decrease of action potential duration (APD) in working atrial myocardium as well as significant acceleration of sinus rhythm. In addition CO reduced force of contractions and other parameters of contractile activity. Inhibitor of heme oxygenase zinc protoporphyrin IX exerts opposite effects: prolongation of action potential, reduction of sinus rhythm rate and enhancement of contractile function. Therefore, endogenous CO, which may be generated in the heart due to the presence of active heme oxygenase, is likely to exert the same effects as exogenous CO applied to the perfusing medium. In ventricular myocardium preparations exogenous CO also induced shortening of action potential, while zinc protoporphyrin IX produced the opposite effect.ConclusionsThus, endogenous or exogenous carbon monoxide may act as an important regulator of electrical and contractile cardiac activity.

Cholinergic modulation of activation sequence in the atrial myocardium of non-mammalian vertebrates

Cholinergic changes of electric activity were studied in isolated atrium preparations from fishes (cod and carp), amphibians (frog) and reptilians (lizard) using the microelectrode technique and high-resolution optical mapping. Perfusion of isolated atrium with acetylcholine (10(-6)-5.10(-5) M) caused gradual suppression of action potential generation and, eventually, completely blocked the excitation in a part of the preparation. Other regions of atrium, situated close to the sinoatrial and atrioventricular junctions, remained excitable. Such cholinergic suppression of electric activity was observed in the atrial myocardium of frog and in both fish species, but not in reptilians. Ba(2+) (10(-4) M), which blocks the acetylcholine-dependent potassium current (I(KACh)), prevented cholinergic reduction of action potential amplitude. In several preparations of frog atrium, cholinergic suppression of excitation coincided with episodes of atrial fibrillation. We conclude that the phenomenon of cholinergic suppression of electric activity is typical for atria of fishes and amphibians. It is likely to be caused by I(KACh) activation and may be important for initiation of atrial arrhythmias.

Mechanisms of Cardiac Muscle Insensitivity to a Novel Acetylcholinesterase Inhibitor C-547

We compared the effects of the novel acetylcholinesterase (AChE) inhibitor C-547 on action potential configuration and sinus rhythm in the isolated right atrium preparation of rat with those of armin and neostigmine. Both armin (10−7, 10−6, and 10−5 M) and neostigmine (10−7, 10−6, and 5 × 10−6 M) produced a marked decrease in action potential duration and slowing of sinus rate. These effects were abolished by atropine and are attributable to the accumulation of acetylcholine in the myocardium. The novel selective AChE inhibitor C-547 (10−9 to 10−7 M), an alkylammonium derivative of 6-methyluracil, had no such effects. The inhibition constant of C-547 on cardiac AChE is 40-fold higher than that on extensor digitorum longus muscle AChE. These results suggest that C-547 might be employed to treat diseases such as myasthenia gravis or Alzheimer disease, without having unwanted effects on the heart.