Gomez J P, Fares N, Potreau D
Laboratoire de Physiologie Générale URA CNRS 1869, Faculté des Sciences, Poitiers, France.
J Mol Cell Cardiol. 1996 Oct;28(10):2217-29. doi: 10.1006/jmcc.1996.0213.
We examined the effects of the dihydropyridine agonist Bay K 8644 on L-type calcium channels in newborn rat ventricular cardiomyocytes during their development in primary culture. Experiments were performed at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. In the presence of racemic Bay K 8644 (10(-6) M), L-type calcium current (ICa-L) density recorded by perforated patch-clamp technique was increased by 127 +/- 4% (n = 8) in 2-day-old cells. The increase was only 103 +/- 5% (n = 10) in 7-day-old cells, resulting in a current density similar to that observed in freshly-dissociated adult cells (90 +/- 7%; n = 10). At every stage of the culture Bay K 8644 increased ICa-L with a 10-mV shift of the peak current towards hyperpolarized levels but without change in activation threshold and reversal potential. This shift can be explained by the corresponding change in steady-state activation and inactivation relationships towards negative potentials. The potentiating effect of Bay K 8644 was further studied as a function of phosphorylation levels of calcium channels. When calcium channels were phosphorylated by dibutyryl cyclic AMP (2 x 10(-4) M), increase of ICa-L density by Bay K 8644 was comparable at every stage of cell culture. However, direct activation of beta-receptors by isoproterenol did not increase ICa-L density in 2-day-old cultured cells as it did in 7-day-old cells, while direct activation of adenylate cyclase by forskolin similarly increased ICa-L, at both stages of culture. From these results, it can be suggested that the higher increase of ICa-L density by Bay K 8644 in 2-day- than in 7-day-old cultured cells could be interpreted as the result of a difference in the phosphorylation level of calcium channels for each stage of development. The possible increase in the basal phosphorylation level of calcium channels during culture of neonatal cardiac cells is discussed with respect to changes in functional properties of calcium channels during postnatal maturation of these cardiac cells.
我们研究了二氢吡啶激动剂Bay K 8644对原代培养新生大鼠心室心肌细胞发育过程中L型钙通道的影响。实验分别在培养的第2天和第7天进行，这两个时间点分别代表了我们实验条件下分离细胞的出生后早期和发育成熟阶段。在消旋Bay K 8644（10(-6) M）存在的情况下，用穿孔膜片钳技术记录的2日龄细胞的L型钙电流（ICa-L）密度增加了127±4%（n = 8）。7日龄细胞中的增加仅为103±5%（n = 10），导致电流密度与新鲜分离的成年细胞中观察到的相似（90±7%；n = 10）。在培养的每个阶段，Bay K 8644都增加了ICa-L，使峰值电流向超极化水平偏移10 mV，但激活阈值和反转电位没有变化。这种偏移可以用稳态激活和失活关系向负电位的相应变化来解释。进一步研究了Bay K 8644的增强作用与钙通道磷酸化水平的关系。当钙通道被二丁酰环磷腺苷（2×10(-4) M）磷酸化时，Bay K 8644对ICa-L密度的增加在细胞培养的每个阶段都是相当的。然而，异丙肾上腺素直接激活β受体在2日龄培养细胞中并没有像在7日龄细胞中那样增加ICa-L密度，而福斯可林直接激活腺苷酸环化酶在两个培养阶段都同样增加了ICa-L。从这些结果可以推测，Bay K 8644在2日龄培养细胞中比在7日龄培养细胞中对ICa-L密度的更高增加可以解释为每个发育阶段钙通道磷酸化水平差异的结果。关于这些心脏细胞出生后成熟过程中钙通道功能特性的变化，讨论了新生心脏细胞培养过程中钙通道基础磷酸化水平可能的增加。
