RESUMO
1. The properties of the low-threshold calcium current, ICa,T, were investigated in bullfrog isolated atrial cardiomyocytes using the whole-cell, patch-clamp technique under control conditions and during beta-adrenergic stimulation. 2. The intracellular application of GTP gamma S or adenosine-5'-O-3-thiotriphosphate (ATP gamma S), poorly hydrolysable analogues of GTP and ATP, respectively, barely affected ICa,T amplitude in control conditions. beta-Adrenergic stimulation effects were more marked in the presence of ATP gamma S. 3. The intracellular application of GDP beta S and ADP reduced ICa,T amplitude. In cells pretreated with pertussis toxin, ICa,T amplitude was significantly increased. In both conditions, the addition of isoprenaline was without effect. 4. Under both control and beta-adrenergic-stimulated conditions, a conditioning prepulse to +70 mV did not fully inactivate ICa,T; rather ICa,T facilitation often occurred after beta-adrenergic stimulation. 5. In GTP gamma S- and ATP gamma S-dialysed cells, ICa,T facilitation was generally observed after a prepulse; it was larger in the ATP gamma S dialysis. Facilitation was sustained but ended immediately upon cessation of conditioning prepulses. After beta-adrenergic stimulation, facilitation was more marked in GTP gamma S- than in ATP gamma S-dialysed cells. 6. ICa,T facilitation was prevented by the intracellular application of GDP beta S and by pertussis toxin pretreatment. 7. ICa,T facilitation developed markedly in the presence of intracellular cyclic AMP. This effect was prevented by pertussis toxin pretreatment of the cells. 8. It is thus proposed that ICa,T is under a double antagonistic control by both a Gs and a Gi protein. Furthermore, the double-pulse-induced facilitation of ICa,T results from a voltage-dependent relief of the Gi protein inhibitory tone. Such an effect is increased by protein kinase A-dependent phosphorylation, presumably of the Gi protein.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Átrios do Coração/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , AMP Cíclico/farmacologia , Átrios do Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Rana catesbeianaRESUMO
Tetrandrine is a natural alkaloid classified as a calcium antagonist. However, its precise actions on Ca(2+)-currents in cardiac cells have not been fully characterized. In the present study, we have investigated the mechanism of action of tetrandrine on the Ca(2+)-currents of single bullfrog cardiac cells, using the patch-clamp technique. Tetrandrine slightly increased ICaL from negative holding potentials (-100 mV) at low concentrations (10 nM-1 microM) and inhibited it at higher concentrations. At depolarized holding potentials (-50 mV) only an enhanced inhibition was seen. Tetrandrine blockade of the L-type Ca(2+)-current (ICaL) was mostly tonic. This is similar to ICaL blockade by nifedipine but not by verapamil, the latter being mostly use-dependent. Use-dependent effects of tetrandrine and nifedipine were evident at high rates. Availability curves were shifted leftwards (10-12 mV) by tetrandrine (10 microM) and nifedipine (1 microM). The T-type Ca(2+)-current (ICaT), although less sensitive, was decreased by both agents in a voltage-independent way. Tetrandrine (10-30 microM) but not nifedipine (1-10 microM), depressed the Na(+)-current (INa) in tonic, use- and voltage-dependent manners. We conclude that tetrandrine and nifedipine share some common actions on cardiac Ca(2+)-channels, while showing differences in their actions on Na(+)-channels. The depression of INa by tetrandrine suggests it could be effective on supraventricular tachycardias.