Meij J T, Dhalla V, Panagia V
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Receptor. 1994 Summer;4(2):109-19.
Cardiac sarcolemmal (SL) phospholipase C (PLC) is a key enzyme in the signal transduction of several cardiac receptors. Thus, the earlier described Ca(2+)-stimulated SL PLC activity may represent variously coupled enzymes. The present study was undertaken to delineate the alpha 1-adrenoceptor/G protein-stimulated PLC activity in purified cardiac SL vesicles. Although certain detergents and membrane pore formers enhanced SL PLC activity, measured as formation of 3H-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] from 3H-phosphatidylinositol 4,5-bisphosphate, alpha 1-adrenoceptor stimulated activity was not observed. When SL vesicles were preincubated (0-4 degrees C) with substrate in detergent-free buffer, subsequent incubation (37 degrees C; in mM: 100 NaCl, 2 EGTA, 1.8 CaCl2, 10 LiCl) resulted in a time-dependent production of 3H-Ins(1,4,5)P3, that was increased in the presence of 100 microM GTP gamma S. GTP gamma S stimulation of SL PLC activity required the presence of Mg2+ and Ca2+, but was lost at (sub)millimolar concentrations of these bivalent cations. Mg2+ (0.01-10 mM) promoted a 2,3-diphosphoglycerate-insensitive phosphatase activity. GTP gamma S enhanced the sensitivity of SL PLC to Ca2+, but did not increase the maximum Ca2+ (0.1-1 mM) stimulated SL PLC activity. At 5 microM Ca2+, GTP gamma S induced a concentration-dependent rise in inositol phosphates production, which was further elevated by the alpha 1-agonist, phenylephrine (PhE). The PhE-effect was inhibited by the alpha 1-antagonist prazosin, but not by the beta-antagonist atenolol. These results show that the components necessary for the alpha 1-adrenoceptor transmembrane signal are associated with the SL membrane and can be functionally coupled.
心肌肌膜（SL）磷脂酶C（PLC）是几种心脏受体信号转导中的关键酶。因此，先前描述的Ca(2+)刺激的SL PLC活性可能代表多种偶联酶。本研究旨在阐明纯化的心肌SL囊泡中α1-肾上腺素能受体/G蛋白刺激的PLC活性。尽管某些去污剂和膜孔形成剂可增强SL PLC活性（以从3H-磷脂酰肌醇4,5-二磷酸形成3H-肌醇1,4,5-三磷酸[Ins(1,4,5)P3]来衡量），但未观察到α1-肾上腺素能受体刺激的活性。当SL囊泡在无去污剂缓冲液中与底物预孵育（0-4℃），随后孵育（37℃；以mM计：100 NaCl、2 EGTA、1.8 CaCl2、10 LiCl）会导致3H-Ins(1,4,5)P3随时间产生，在100μM GTPγS存在时增加。GTPγS对SL PLC活性的刺激需要Mg2+和Ca2+的存在，但在这些二价阳离子的（亚）毫摩尔浓度下会丧失。Mg2+（0.01-10 mM）促进了一种对2,3-二磷酸甘油酸不敏感的磷酸酶活性。GTPγS增强了SL PLC对Ca2+的敏感性，但未增加最大Ca2+（0.1-1 mM）刺激的SL PLC活性。在5μM Ca2+时，GTPγS诱导肌醇磷酸产生浓度依赖性升高，α1-激动剂去氧肾上腺素（PhE）可进一步提高该升高幅度。PhE的作用被α1-拮抗剂哌唑嗪抑制，但不被β-拮抗剂阿替洛尔抑制。这些结果表明，α1-肾上腺素能受体跨膜信号所需的成分与SL膜相关且可功能偶联。
