Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells.
We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.
Ali
N
N
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA.
Yousufzai
S Y
SY
Abdel-Latif
A A
AA
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D053607:Adrenomedullin; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D002415:Cats; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007498:Iris / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009130:Muscle, Smooth / Q000166:cytology* / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology; D037342:Pertussis Toxin; D013997:Time Factors; D014665:Vasodilator Agents / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D010455:Peptides; D010456:Peptides, Cyclic; D014665:Vasodilator Agents; D010566:Virulence Factors, Bordetella; C068090:anantin; D053607:Adrenomedullin; D009320:Atrial Natriuretic Factor; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D006152:Cyclic GMP