Katsube Y, Yokoshiki H, Nguyen L, Sperelakis N
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, OH 45267-0576, USA.
Eur J Pharmacol. 1996 Dec 19;317(2-3):391-400. doi: 10.1016/s0014-2999(96)00745-5.
The developmental changes in the isoproterenol stimulation of the L-type calcium current (ICa(L)) were studied in freshly isolated neonatal (3-5-day-old) and adult (2-3-month-old) rat ventricular myocytes using whole-cell voltage clamp (at room temperature). ICa(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300 ms pulse from a holding potential of -40 mV. The pipette solution was Cs(+)-rich and Ca(2+)-free. The external solution was Na(+)-free and K(+)-free. Isoproterenol stimulated ICa(L) in a dose-dependent manner. The concentrations of isoproterenol for half-maximal effect were 6.8 nM in neonatal and 13.3 nM in adult. The maximal stimulation of ICa(L) was 147 +/- 14% in neonatal and 97 +/- 7% in adult. The steady-state inactivation curves were not affected by isoproterenol, whereas the steady-state activation curve was shifted to the left in both neonatal and adult. Forskolin (10 microM) increased ICa(L) by 105 +/- 10% in neonatal and 90 +/- 12% in adult. After stimulating ICa(L) by forskolin, the addition of isoproterenol produced a further increase of ICa(L) by 99 +/- 27% in neonatal, but only by 19 +/- 3% in adult. The presence of an inhibitor of cAMP-dependent protein kinase in the pipette did not affect this marked difference between neonatal (87 +/- 23%) and adult (11 +/- 8%). We conclude that, in rat ventricular myocytes, (1) stimulation of ICa(L) by the beta-adrenoceptor agonist, isoproterenol, is already fully developed in the neonatal stage and actually decreases during development; (2) there is evidence for a cAMP-independent stimulation of Ca2+ channels by isoproterenol, and this is greater in neonatal than in adult. We believe that the cAMP-independent pathway is the direct pathway mediated by Gs alpha protein.
利用全细胞膜片钳技术（在室温下），研究了新生（3 - 5日龄）和成年（2 - 3月龄）大鼠心室肌细胞中异丙肾上腺素对L型钙电流（ICa(L)）刺激的发育变化。通过从 - 40 mV的钳制电位施加300 ms的脉冲，在 + 10 mV（或 + 20 mV）的测试电位下测量ICa(L)作为内向电流峰值。电极内液富含Cs(+)且无Ca(2+)。细胞外液无Na(+)且无K(+)。异丙肾上腺素以剂量依赖性方式刺激ICa(L)。半最大效应时异丙肾上腺素的浓度在新生大鼠中为6.8 nM，在成年大鼠中为13.3 nM。ICa(L)的最大刺激在新生大鼠中为147±14%，在成年大鼠中为97±7%。稳态失活曲线不受异丙肾上腺素影响，而稳态激活曲线在新生和成年大鼠中均向左移动。福斯高林（10 μM）使新生大鼠的ICa(L)增加105±10%，成年大鼠增加90±12%。在福斯高林刺激ICa(L)后，加入异丙肾上腺素使新生大鼠的ICa(L)进一步增加99±27%，而成年大鼠仅增加19±3%。电极内存在环磷酸腺苷依赖性蛋白激酶抑制剂并不影响新生（87±23%）和成年（11±8%）之间的这种显著差异。我们得出结论，在大鼠心室肌细胞中，（1）β - 肾上腺素能受体激动剂异丙肾上腺素对ICa(L)的刺激在新生阶段已充分发育，且在发育过程中实际下降；（2）有证据表明异丙肾上腺素对钙通道有不依赖环磷酸腺苷的刺激作用，且这种作用在新生大鼠中比成年大鼠中更强。我们认为不依赖环磷酸腺苷的途径是由Gsα蛋白介导的直接途径。
