Ca²⁺-dependent regulation of guinea pig brain adenylate cyclase

The effect of Ca²⁺ on guinea pig brain adenylate cyclase has been studied using metal buffers to regulate the free Ca²⁺ concentration. Particulate fractions prepared in the presence of Ca²⁺ (to bind all the available calmodulin to all of the available binding sites) exhibited a biphasic response to Ca²⁺. The first was a 2-fold stimulation of basal cyclase activity (half-maximal =0.08 μm). At higher [Ca²⁺], the cyclase was inhibited to -80 to 90% of basal (half-maximal inhibition=0.3 μM). Particulate fractions prepared by exhaustive washing with EDTA and ethylene glycol bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) (in an attempt to deplete the membranes of calmodulin) exhibited no stimulation of adenylate cyclase by Ca²⁺ but did show the Ca²⁺-dependent inhibitory phase (halfmaximal inhibition=0.5 μM). The calmodulin content of this preparation (EGTA-membranes) was 0.24 μg/mg of protein. The activation phase could be restored to EGTA-membranes by addition of calmodulin. The Ca²⁺ dependence of calmodulin-dependent phosphodiesterase exhibited a half-maximal activation at [Ca²⁺]=0.3 μM, similar to the half-maximal inhibition of cyclase in the two preparations. Likewise, Sr²⁺ activation of phosphodiesterase (half-maximal stimulation=0.1 mM) occurred over the same [Sr²⁺] range as did half-maximal inhibition of cyclase (0.3 mM) in EGTA-and Ca-membranes. The Ca²⁺ and Sr²⁺ dependence of calmodulin tyrosine fluorescence (an indirect measure of Ca²⁺ and Sr²⁺ dependence of phosphodiesterase activation and the inhibition of the cyclase. These results suggest that the Ca²⁺ dependent inhibition of adenylate cyclase may be mediated either by calmodulin or by a metal binding site which has binding properties similar to calmodulin. Thus, our results show that there is an inverse relationship between adenylate cyclase activity and phosphodiesterase activity and suggest that intracellular calcium may indicate a coordinated regulation of the rates of synthesis and degradation of cyclic nucleotides.