Calmodulin stimulation of the rat cerebral cortical adenylate cyclase is required for the detection of guanine nucleotide- or hormone-mediated inhibition.
Functional interactions between the inhibitory guanine nucleotide-regulatory component (Ni) and the adenylate cyclase catalytic subunit (C) from cerebral cortex have been investigated. The inhibition of adenylate cyclase activity by guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p] was used as a functional measurement of Ni-C interactions in membranes and cholate extracts. Calmodulin stimulation of C activity was required for the detection of Gpp(NH)p inhibition in these preparations. A similar calmodulin requirement was observed for adenosine receptor-mediated inhibition in membranes. The requirement for calmodulin was demonstrated directly in membranes from which calmodulin had been removed by washing and centrifugation. Adenylate cyclase activity in these preparations was not stimulated by free Ca2+ (1 microM). However, upon the readdition of calmodulin (1 microM), these preparations were stimulated 4-fold by Ca2+. Under these assay conditions, Gpp(NH)p- and adenosine receptor-mediated inhibition was absolutely dependent on Ca2+-calmodulin stimulation. However, forskolin stimulation of activity also restored Gpp(NH)p-mediated inhibition. The following experiments were used to implicate the role of calmodulin in detergent-solubilized preparations: (i) by demonstrating that free Ca2+ was required to observe Gpp(NH)p-mediated inhibition, and (ii) by demonstrating that the calmodulin antagonist, calmidazolium, abolished Gpp(NH)p-mediated inhibition while concomitantly decreasing basal activity. As observed in membranes, detection of guanine nucleotide-mediated inhibition required calmodulin stimulation of the detergent-solubilized adenylate cyclase. These results suggest that stimulation of cerebral cortical C activity by either calmodulin or forskolin is required for Ni-mediated inhibition.
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002540:Cerebral Cortex / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051381:Rats
D000067956:Adenylyl Cyclase Inhibitors; D002147:Calmodulin; D007093:Imidazoles; D046911:Macromolecular Substances; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; C031938:calmidazolium; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium