Abstract
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 microM inhibited adenylate cyclase activity with one-half-maximal inhibition occurring at 2.5 microM Ca2+. In the presence of 5 or 9 microM bovine testis calmodulin, Ca2+ stimulated smooth muscle adenylate cyclase activity with one-half-maximal stimulation occurring at 0.2 microM for both 5 and 9 microM calmodulin. Calmodulin stimulation was observed between 0.1 and 0.8 microM Ca2+. Despite the presence of calmodulin, Ca2+ concentrations greater than 0.8 microM were inhibitory to smooth muscle adenylate cyclase activity. However, calmodulin reduced the sensitivity of the enzyme to inhibition by Ca2+. Trifluoperazine (100 microM) 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 microM. 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 mediated 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+.
Highlights
These studies was 80 2 14 nglmg as determined by calmodulin complex has been shown to stimulate the
In the absence of exoge- M C ) - ATPase associatedwith microsomal (6) andplasma nous calmodulin, Ca2+ concentrationgsreater than0.8 membranes (7),membrane phosphorylation(8), and the phosp~ inhibited adenylate cyclase activity with one-half- phorylation of myosin by myosin light chain kinase(9, 10). It maximal inhibition occurring at 2.5 p~ Ca2+
In the is thought that the stimulationof myosin light chain kinase presence of 5 or 9 p~ bovine testis calmodulin, Ca2+ activity by Ca2+ and calmodulin is a critical step in smooth stimulated smooth muscle adenylate cyclase activity with one-half-maximal stimulation occurring0.2atp~ for both5and 9p~ calmodulin
Summary
0.8 p ~ ca, lmodulin at 5 or 9 p~ stimulated adenylate cyclase activity with one-half maximal stimulation occurring a t 0.2 FM for both calmodulin concentrations. A t 23 PM was calmodulin able to induce a statistically significant stimulationof adenylate cyclase activity in the absencoef Ca2+(Fig. 3A). At 80 pM,in the absence of calmodulin (C, first bar), Ca2+produced a statistically significantinhibition of adenylate cyclase activity. These inhibitory actions were overcome in a concentration-depend-. This decrease in the sensitivityof adenylate cyclase to the inhibitory actionof Ca2+was statistically significant for calmodulin as low as 2.3 PM. As can beobserved in Fig., theability of 5 PM calmodulin (H) to stimulate adenylate cyclase activity was significantly decreased by the addition of 100 p~ trifluoperazine (A).trifluoperazine was able toblock the calmodulin-induced decrease in sensitivity of adenylate cy-
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