You Y, Pelzer D J, Pelzer S
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Biophys J. 1997 Jan;72(1):175-87. doi: 10.1016/S0006-3495(97)78656-9.
Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.
在用双（邻氨基苯氧基）乙烷 - N，N，N'，N'-四乙酸（BAPTA）透析的心肌细胞中，预计Ca2+通道孔附近的游离Ca2+浓度低于用乙二醇双（β - 氨基乙基） - N，N，N'，N'-四乙酸（EGTA）透析的细胞，因为BAPTA能更快速地螯合进入的Ca2+。在电压钳制的豚鼠心室肌细胞中，研究了BAPTA（0.2 - 60 mM）和EGTA（0.2 - 67 mM）对全细胞L型Ca2+电流（ICa,L）的细胞内Ca2+缓冲作用的影响；使用荧光Ca2+指示剂indo-1监测大量细胞质游离Ca2+（Cac2+）。当细胞透析液中的BAPTA从0.2 mM增加到50 mM时，ICa,L增加了约12倍（在31 mM时达到半最大刺激），而将细胞内EGTA从0.2 mM提高到67 mM时，ICa,L仅增加了约2倍。当细胞内BAPTA或EGTA≥20 mM时，Cac2+＜20 nM。虽然高达67 mM的EGTA对3 μM福斯可林刺激ICa,L的作用只有微不足道的抑制作用，但在50 mM BAPTA透析的心肌细胞中，ICa,L对福斯可林诱导的腺苷3'，5'-环磷酸（cAMP）升高不敏感；相反，cAMP负载细胞中的ICa,L对BAPTA透析无反应。用BAPTA而非EGTA进行细胞透析加速了ICa,L失活的慢成分（tau S），而不影响其快成分（tau F），类似于cAMP依赖性磷酸化的作用。BAPTA刺激的ICa,L受到乙酰胆碱和cAMP依赖性蛋白激酶（PKA）阻滞剂H-89的抑制。这些结果表明，BAPTA诱导的通道周围Ca2+浓度降低通过解除对Ca(2+)敏感的腺苷酸环化酶的抑制来刺激cAMP合成和通道磷酸化。
