Lu C, Kumar R, Akita T, Joyner R W
Todd Franklin Cardiac Research Laboratory, Department of Pediatrics, Emory University, Atlanta, GA 30323.
Pflugers Arch. 1994 Jul;427(5-6):389-98. doi: 10.1007/BF00374252.
We used whole-cell voltage clamp to compare the modulation of calcium current density (ICa, picoampere per picofarad) of freshly isolated, adult and newborn rabbit heart in response to intracellular application of microcystin and okadaic acid, both of which block phosphatase activity of phosphatase type 1 and 2A. Newborn cells showed a much larger response to the intracellular application of either microcystin or okadaic acid than did adult cells. In newborn cells, the application of microcystin produced an increase in ICa which appeared to maximize ICa, as shown by the rise in ICa to levels which could be reached by application of 10 microM forskolin or by the intracellular application of 200 microM 3',5'-cyclic adenosine monophosphate (cAMP). In adult cells, the maximal response to microcystin was considerably less than that obtainable with forskolin or cAMP. After achieving a maximal response with microcystin, the addition of forskolin increased ICa further in adult cells but elicited no additional response in newborn cells. The treatment of cells with 0.1 microM isoproterenol, a concentration approximately equal to that required for a half-maximal response, strongly potentiated the effect of microcystin in newborn cells, but not in adult cells. We propose that newborn rabbit heart cells compared with adult rabbit heart cells have a greater level of protein phosphatase activity (perhaps combined with a somewhat greater kinase activity), a greater proportion of the protein phosphatase activity in the form of protein phosphatase type 1 (which is inhibited by isoproterenol) and a greater dependence on the inhibition of protein phosphatase as a mechanism of action of isoproterenol, compared with the increase in kinase activity on calcium channels.
我们使用全细胞膜片钳技术，比较了新生和成年兔新鲜分离心肌细胞的钙电流密度（ICa，皮安/皮法）对细胞内应用微囊藻毒素和冈田酸的反应，这两种物质均可阻断1型和2A型磷酸酶的磷酸酶活性。与成年细胞相比，新生细胞对细胞内应用微囊藻毒素或冈田酸的反应要大得多。在新生细胞中，应用微囊藻毒素可使ICa增加，且似乎达到了ICa的最大值，这表现为ICa升高至应用10μM福斯高林或细胞内应用200μM 3',5'-环磷酸腺苷（cAMP）所能达到的水平。在成年细胞中，微囊藻毒素的最大反应明显小于福斯高林或cAMP所能引起的反应。在用微囊藻毒素达到最大反应后，添加福斯高林可使成年细胞的ICa进一步增加，但对新生细胞无额外反应。用0.1μM异丙肾上腺素处理细胞（该浓度约等于产生半数最大反应所需的浓度），可强烈增强微囊藻毒素对新生细胞的作用，但对成年细胞无此作用。我们提出，与成年兔心肌细胞相比，新生兔心肌细胞具有更高水平的蛋白磷酸酶活性（可能与稍高的激酶活性相结合），以1型蛋白磷酸酶形式存在的蛋白磷酸酶活性比例更高（该酶可被异丙肾上腺素抑制），并且与钙通道上激酶活性的增加相比，对蛋白磷酸酶抑制的依赖性更大，这是异丙肾上腺素作用的一种机制。
