Rybin V O, Steinberg S F
Department of Medicine, Columbia University, New York, NY.
Circ Res. 1994 Feb;74(2):299-309. doi: 10.1161/01.res.74.2.299.
To determine whether age-dependent differences in cardiac responses to autonomic agonists could result from developmental changes in protein kinase C (PKC) isoform expression, we probed extracts from the fetal, neonatal, and adult heart as well as cultured neonatal and isolated adult ventricular myocytes with specific antisera to calcium-dependent (alpha and beta) and calcium-independent (delta, epsilon and zeta) isoforms of the enzyme. Although PKC-beta immunoreactivity could not be detected in cultured neonatal or isolated adult ventricular myocytes, adult and neonatal myocytes expressed multiple other isoforms of PKC. Our studies revealed an age-dependent decline in the immunoreactivity for three PKC isoforms. PKC-alpha was detected in extracts from the fetal and 2-day-old neonatal heart as well as cultured neonatal rat ventricular myocytes. Only faint PKC-alpha immunoreactivity was detected in extracts from the adult heart, and PKC-alpha was not detected in extracts from isolated adult ventricular myocytes, suggesting that PKC-alpha resides in nonmyocyte elements in the adult heart. PKC-delta also was detected in greater abundance in fetal and neonatal than in adult myocardial extracts. The decline in PKC-alpha and PKC-delta expression occurred during the first 2 postnatal weeks. PKC-zeta was detected in greatest abundance in extracts from the fetal heart. PKC-zeta expression declined markedly by the second postnatal day, and only faint PKC-zeta immunoreactivity was detected in extracts from adult myocardium. Failure to detect PKC-zeta in extracts from isolated adult ventricular myocytes suggests that PKC-zeta resides primarily in nonmyocyte elements in the adult heart. PKC-epsilon was detected in all preparations, but it was detected in greatest abundance in extracts from neonatal hearts. In vitro sympathetic innervation of previously noninnervated neonatal ventricular myocytes or in vivo chemical sympathectomy of the neonatal heart did not modulate PKC isoform expression, suggesting that sympathetic innervation does not significantly regulate PKC isoform expression. PKC-alpha partitioned to the soluble fraction of unstimulated myocytes and was selectively translocated to the particulate fraction by Ca2+. In contrast, a major portion of the novel PKC isoforms partitioned to the particulate fraction of unstimulated myocytes. The subcellular distribution of novel PKC isoforms was not influenced by Ca2+. 12-O-Tetradecanoylphorbol 13-acetate (TPA, 300 nmol/L) induced translocation of soluble PKC-alpha, PKC-delta, and PKC-epsilon to the particulate fraction at 30 minutes and complete (PKC-alpha and PKC-delta) or 80% (PKC-epsilon) downregulation at 24 hours. PKC-zeta was not affected by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)
为了确定心脏对自主神经激动剂的反应中年龄依赖性差异是否可能源于蛋白激酶C（PKC）亚型表达的发育变化，我们用针对该酶的钙依赖性（α和β）及钙非依赖性（δ、ε和ζ）亚型的特异性抗血清检测了胎儿、新生儿和成年心脏提取物以及培养的新生心肌细胞和分离的成年心室肌细胞。虽然在培养的新生心肌细胞或分离的成年心室肌细胞中未检测到PKC-β免疫反应性，但成年和新生心肌细胞表达PKC的多种其他亚型。我们的研究揭示了三种PKC亚型的免疫反应性随年龄下降。在胎儿和2日龄新生心脏提取物以及培养的新生大鼠心室肌细胞中检测到PKC-α。在成年心脏提取物中仅检测到微弱的PKC-α免疫反应性，在分离的成年心室肌细胞提取物中未检测到PKC-α，这表明PKC-α存在于成年心脏的非心肌细胞成分中。在胎儿和新生儿心肌提取物中检测到的PKC-δ也比成年心肌提取物中更丰富。PKC-α和PKC-δ表达的下降发生在出生后的前两周。在胎儿心脏提取物中检测到的PKC-ζ最丰富。PKC-ζ表达在出生后第二天明显下降，在成年心肌提取物中仅检测到微弱的PKC-ζ免疫反应性。在分离的成年心室肌细胞提取物中未检测到PKC-ζ表明PKC-ζ主要存在于成年心脏的非心肌细胞成分中。在所有制剂中均检测到PKC-ε，但在新生心脏提取物中检测到的含量最高。对先前未受神经支配的新生心室肌细胞进行体外交感神经支配或对新生心脏进行体内化学交感神经切除术并未调节PKC亚型表达，这表明交感神经支配不会显著调节PKC亚型表达。PKC-α分布于未刺激的心肌细胞的可溶性部分，并被Ca2+选择性转运至颗粒部分。相比之下，新型PKC亚型的大部分分布于未刺激的心肌细胞的颗粒部分。新型PKC亚型的亚细胞分布不受Ca2+影响。12-氧十四烷酰佛波醇13-乙酸酯（TPA，300 nmol/L）在30分钟时诱导可溶性PKC-α、PKC-δ和PKC-ε转运至颗粒部分，并在24小时时导致完全（PKC-α和PKC-δ）或80%（PKC-ε）下调。PKC-ζ不受TPA影响。（摘要截断于400字）
