Calmodulin activation and calcium regulation of parotid gland adenylate cyclase

The effect of Ca²⁺ on the adenylate cyclase activity associated with membranes prepared from mouse parotid gland has been examined. Ca²⁺ stimulated then inhibited adenylate cyclase activity, with values for half-maximal stimulation and inhibition of 0.6 and 10 μM, respectively. Maximal activation (1.4-fold) was observed at 2 μM free Ca²⁺. These membranes contained 1.2 μg calmodulin/mg protein. Exogenous calmodulin (0.2-1.2 μg) activated, in a concentration-dependent manner, adenylate cyclase activity, with maximal activation being 2.5-fold at 12 μg calmodulin. Preparation of membranes in 2 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) resulted not only in a significant decrease in calmodulin levels (0.5 μg calmodulin/mg protein) but also in a loss of the ability of Ca²⁺ to stimulate the enzyme. Exogenous calmodulin restored the ability of Ca²⁺ to stimulate the adenylate cyclase activity associated with EGTA-treated membranes. Trifluoperazine (50 μM) blocked the ability of Ca²⁺ to activate adenylate cyclase activity in control membranes. The effect of trifluoperazine could be reversed by exogenous calmodulin (0.5 or 5.0 μg). These data indicate that calmoduin mediates the activation of parotid gland adenylate cyclase by Ca²⁺ and that Ca²⁺, at concentrations which stimulate and inhibit amylase secretion, can activate and inhibit adenylate cyclase activity.