Alpha 1-adrenergic stimulation of phospholipase C activity in purified cardiac sarcolemmal membranes.
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.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
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D058646:Adrenergic alpha-1 Receptor Agonists; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000032:analysis; D008297:Male; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D012508:Sarcolemma / Q000201:enzymology / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D058646:Adrenergic alpha-1 Receptor Agonists; D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D002118:Calcium