Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase.
We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition (approximately 45%) was observed in the presence of 10-30 microM GTP gamma S, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na+ enhanced the degree of inhibition by about 60%, whereas K+ and Li+ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A2 treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca(2+)-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.
Anand-Srivastava
M B
MB
Department of Physiology, Université de Montréal, Canada.
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000502:physiology*; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D009439:Neuraminidase; D010741:Phospholipases A; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin
D000067956:Adenylyl Cyclase Inhibitors; D002413:Cations, Divalent; D002414:Cations, Monovalent; D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D009439:Neuraminidase; D014357:Trypsin; D017461:Receptors, Atrial Natriuretic Factor; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide