To the Mechanism of the Antiarrhythmic Action of Compound ALM-802: the Role of Ryanodine Receptors.

The effect of the compound N1-(2,3,4-trimethoxy)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca2+ response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 µM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca2+ response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10-5 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10-5 M). The data obtained indicate that the decrease in the concentration of Ca2+ ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound.

Analysis of Electrophysiological Mechanisms of N-Deacetyllapaconitine Monochlorhydrate, the Main Metabolite of Lappaconitine Hydrobromide.

In in vitro experiments on isolated rat hippocampal neurons, we studied the electrophysiological mechanisms of the antiarrhythmic effects of N-deacetyllappaconitine monochlorhydrate (DALCh), active metabolite of lappaconitine hydrobromide (allapinin). Electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It was shown that DALCh increased the duration of both slow and fast depolarization phases and decreased the amplitude of the action potential. DALCh effectively inhibited transmembrane currents of Na+ ions and partially K+ ions through the corresponding transmembrane voltage-gated ion channels. Thus, DALCh, in contrast to lappaconitine hydrobromide, belongs not to 1C, but to the 1A class of antiarrhythmics according to the Vaughan-Williams classification.

Mechanisms Underlying the Antiarrhythmic Action of Compound ALM-802.

The mechanisms underlying the antiarrhythmic action of compound trihydrochloride N1-(2,3,4-trimethoxy)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) were studied in vitro. The experiments were performed on a culture of rat hippocampal neurons. The electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It is shown that the compound ALM-802 effectively blocks potential-dependent Na+ and K+ channels and does not affect the activity of potential-dependent Ca2+ channels. The inhibition of currents through these channels is dose-dependent; the IC50 of Na+ and K+ channels were 94±4 and 67±3 µM, respectively. These findings indicate that compound ALM-802 combines the properties of class I and class III antiarrhythmic agents according to the Vaughan-Williams classification.

Involvement of NMDA and GABA(A) receptors in modulation of spontaneous activity in hippocampal culture: Interrelations between burst firing and intracellular calcium signal.

Spontaneous burst firing is a hallmark attributed to the neuronal network activity. It is known to be accompanied by intracellular calcium [Са2+]i oscillations within the bursting neurons. Studying mechanisms underlying regulation of burst firing is highly relevant, since impairment in neuronal bursting accompanies different neurological disorders. In the present study, the contribution of NMDA and GABA(A) receptors to the shape formation of spontaneous burst -was studied in cultured hippocampal neurons. A combination of inhibitory analysis with simultaneous registration of neuronal bursting by whole-cell patch clamp and calcium imaging was used to assess spontaneous burst firing and [Са2+]i level. Using bicuculline and D-AP5 we showed that GABA(A) and NMDA receptors effectively modulate burst plateau phase and [Са2+]i transient spike which can further affect action potential (AP) amplitudes and firing frequency within a burst. Bicuculline significantly elevated the amplitude and reduced the duration of both burst plateau phase and [Са2+]i spike resulting in an increase of AP firing frequency and shortening of AP amplitudes within a burst. D-AP5 significantly decreases the amplitude of both plateau phase and [Са2+]i spike along with a burst duration that correlated with an increase in AP amplitudes and reduced firing frequency within a burst. The effect of bicuculline was occluded by co-addition of D-AP5 revealing modulatory role of GABA(A) receptors to the NMDA receptor-mediated formation of the burst. Our results provide new evidence on importance of NMDA and GABA(A) receptors in shaping burst firing and Ca2+transient spikes in cultured hippocampal neurons.