Eble D M, Cadre B M, Qi M, Bers D M, Samarel A M
Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153, USA.
J Mol Cell Cardiol. 1998 Jan;30(1):55-60. doi: 10.1006/jmcc.1997.0571.
[Ca2+]i transients, and the activation of Ca(2+)-sensitive kinases have been considered potential signaling mechanisms regulating ANF gene expression in cultured neonatal rat ventricular myocytes (NRVM). However, it is unclear whether [Ca2+]i is directly involved, or is indirectly involved by generating additional mechanical signals via contractile activity. Primary cultures of spontaneously contracting NRVM (CON), and NRVM treated for 48 h with verapamil (V, 10 microM), KCl (50 mM), or 2,3-butanedione monoxime (BDM, 7.5 mM) were used to delineate the affects of contractile activity v [Ca2+]i. Verapamil, a calcium, channel blocker, inhibits contraction and decreases [Ca2+]i. High [K+]o causes membrane depolarization, loss of contraction, and elevates [Ca2+]i; whereas BDM strongly inhibits contractile activity but only modestly reduces [Ca2+]i transients. ANF production, as assessed by radioimmunoassay, was significantly reduced upon contractile arrest independently of [Ca2+]i levels. Northern blotting analysis demonstrated that contractile arrest also reduced ANF mRNA levels. Transient transfection of a 3003 bp ANF promoter-luciferase expression plasmid in CON, V, KCl, and BDM-treated NRVM demonstrated marked down-regulation of ANF promoter activity in all of the contractile arrested myocytes. These results indicate that the activation of Ca(2+)-sensitive processes alone are insufficient to maintain high levels of ANF gene expression and peptide production in NRVM.
细胞内钙离子浓度（[Ca2+]i）瞬变以及钙敏激酶的激活被认为是调节培养的新生大鼠心室肌细胞（NRVM）中利钠肽（ANF）基因表达的潜在信号传导机制。然而，尚不清楚[Ca2+]i是直接参与，还是通过收缩活动产生额外的机械信号间接参与。使用自发收缩的NRVM（CON）的原代培养物，以及用维拉帕米（V，10 microM）、氯化钾（KCl，50 mM）或2,3-丁二酮单肟（BDM，7.5 mM）处理48小时的NRVM来描述收缩活动对[Ca2+]i的影响。维拉帕米是一种钙通道阻滞剂，可抑制收缩并降低[Ca2+]i。高细胞外钾离子浓度（[K+]o）会导致膜去极化、收缩丧失并升高[Ca2+]i；而BDM强烈抑制收缩活动，但仅适度降低[Ca2+]i瞬变。通过放射免疫测定评估，无论[Ca2+]i水平如何，收缩停止后ANF的产生均显著减少。Northern印迹分析表明，收缩停止也降低了ANF mRNA水平。在CON、V、KCl和BDM处理的NRVM中瞬时转染3003 bp的ANF启动子-荧光素酶表达质粒，结果表明所有收缩停止的心肌细胞中ANF启动子活性均显著下调。这些结果表明，仅钙敏过程的激活不足以维持NRVM中高水平的ANF基因表达和肽产生。
