Boller M, Pott L
Institut für Zellphysiologie, Ruhr-Universität Bochum, Federal Republic of Germany.
Pflugers Arch. 1989 Dec;415(3):276-88. doi: 10.1007/BF00370877.
Transient inward current (Iti) indicating Ca2(+)-release from the sarcoplasmic reticulum and L-type Ca2(+)-current (ICa) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of ICa caused by beta-adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskolin (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of Iti were used to manifest changes of sarcoplasmic Ca2(+)-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 microM EGTA spontaneous transient inward currents were recorded at a constant holding potential of -50 mV in the majority of myocytes. Superfusion with a solution containing ISO (greater than or equal to 5 x 10(-8) M) increased the amplitude of spontaneous Iti and reduced its time-to-peak. The effects of ISO on Iti developed in parallel to stimulation of ICa. In myocytes which did not show spontaneous cyclic Ca2(+)-release in the above condition, this could be evoked de novo by ISO. Spontaneous Iti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 microM. Brief (50 ms) activation of ICa by voltage steps from -50 to +10 mV usually failed to trigger Ca2(+)-release from the SR. The increase of ICa-amplitude upon administration of ISO went ahead with the induction of Ca2(+)-release by brief activation of ICa. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 3'5'-cyclic adenosine monophosphate. The effects on ICa and SR Ca2(+)-release were dependent o the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca2(+)-release events per number of Ca2(+)-currents elicited and a shortening of time-to-peak of Iti's. The stimulating effects of ISO or FORSK on Ca2(+)-release were only partially due to an increase of the triggering ICa. Ca2(+)-currents too small to trigger Ca2(+)-release before beta-adrenergic stimulation could evoke Ca2(+)-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on ICa were reversible, the stimulatory effects on Ca2(+)-release persisted after washing out the substances. The results give support to the hypothesis that beta-adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca2(+)-release.
采用全细胞膜片钳技术，研究成年豚鼠心脏心房和心室肌细胞中的瞬时内向电流（Iti），该电流指示肌浆网释放Ca2+，以及L型Ca2+电流（ICa）。使用异丙肾上腺素（ISO）进行β-肾上腺素能刺激或相关实验操作（如用福斯可林（FORSK）灌注）引起的ICa增加，被用作细胞内cAMP增加的定性监测指标。Iti的变化用于表明肌浆网Ca2+释放的变化。在用含有100μM乙二醇双四乙酸（EGTA）的柠檬酸盐基（60mM）移液管填充溶液透析的肌细胞中，在大多数肌细胞中，于-50mV的恒定钳制电位下记录到自发瞬时内向电流。用含有ISO（≥5×10-8M）的溶液灌注可增加自发Iti的幅度并缩短其峰值时间。ISO对Iti的作用与对ICa的刺激同时出现。在上述条件下未显示出自发周期性Ca2+释放的肌细胞中，ISO可重新诱发这种释放。通过将透析溶液中EGTA的浓度增加到200μM，大多数细胞中的自发Iti受到抑制。从-50mV到+10mV的电压阶跃对ICa进行短暂（50ms）激活通常无法触发肌浆网释放Ca2+。给予ISO后ICa幅度的增加先于通过短暂激活ICa诱导Ca2+释放。ISO的作用可被FORSK或用3',5'-环磷酸腺苷进行细胞内透析模拟。对ICa和肌浆网Ca2+释放的作用取决于刺激物质的浓度。在给定细胞中，将灌注从低浓度ISO或FORSK改为高浓度，导致每引发的Ca2+电流中Ca2+释放事件的数量增加，以及Iti峰值时间缩短。ISO或FORSK对Ca2+释放的刺激作用仅部分归因于触发ICa的增加。在β-肾上腺素能刺激之前太小而无法触发Ca2+释放的Ca2+电流，在细胞内cAMP增加后可引发Ca2+释放。虽然ISO和FORSK对ICa的作用是可逆的，但在洗去这些物质后，对Ca2+释放的刺激作用仍然存在。这些结果支持了以下假设：β-肾上腺素能受体介导的正性肌力和致心律失常作用至少部分归因于一种依赖环磷酸腺苷的调节机制，该机制调节肌浆网Ca2+释放。
