Calmodulin stimulation and calcium regulation of smooth muscle adenylate cyclase activity

The Ca²⁺-dependent regulation of the adenylate cyclase activity associated with microsomes isolated from bovine aortic smooth muscle has been studied. Calmodulin content of microsomal membranes employed in these studies was 80 ± 14 ng/mg as determined by specific radioimmunoassay. In the absence of exogenous calmodulin, Ca²⁺ concentrations greater than 0.8 μM inhibited adenylate cyclase activity with one-half-maximal inhibition occurring at 2.5 μM Ca²⁺. In the presence of 5 or 9 μM bovine testis calmodulin, Ca²⁺ stimulated smooth muscle adenylate cyclase activity with one-half-maximal stimulation occurring at 0.2 μM for both 5 and 9 μM calmodulin. Calmodulin stimulation was observed between 0.1 and 0.8 μM Ca²⁺. Despite the presence of calmodulin, Ca²⁺ concentrations greater than 0.8 μM were inhibitory to smooth muscle adenylate cyclase activity. However, calmodulin reduced the sensitivity of the enzyme to inhibition by Ca²⁺. Trifluoperazine (100 μM) reversed both the calmodulin-dependent-stimulation of cyclase activity and the calmodulin-induced decrease in sensitivity to the inhibitory actions of Ca²⁺. Trifluoperazine alone shifted the curve describing Ca²⁺ inhibition of cyclase activity to the left. The value of Ca²⁺ for one-half-maximal inhibition decreased from 2.9 to 1.2 μM. The trifluoperazine-induced shift was reversed by exogenous calmodulin. These data suggest: 1) Ca²⁺, at physiological concentrations, can stimulate as well as inhibit smooth muscle adenylate cyclase activity; 2) the stimulation of adenylate cyclase activity is medidated by calmodulin; 3) the Ca²⁺-calmodulin complex reduces the sensitivity of smooth muscle adenylate cyclase to the inhibitory actions of Ca²⁺; and 4) the level of calmodulin associated with smooth muscle adenylate cyclase may modulate the response (both stimulatory and inhibitory) of the enzyme to Ca²⁺.