CALCIUM INHIBITION OF CARDIAC ADENYLYL CYCLASE - EVIDENCE FOR 2 DISTINCT SITES OF INHIBITION

Increasing the free calcium concentration from 10(-8) M to 10(-4) M inhibited cardiac sarcolemmal adenylyl cyclase activated by the addition of 5x10(-4) M forskolin or 1x10(-4) M GTP or Gpp(NH)p. The calcium inhibition curve in the presence of all three activators was shallow and best fit by a two site model of high affinity (< 1.0-mu-M) and low affinity (> 0.1-mM). Gpp(NH)p appeared to decrease the sensitivity of adenylyl cyclase to inhibition by calcium at the high affinity site. Similar inhibition constants were obtained with each of the activators. Calmodulin content of native freeze-thaw vesicles was 76.2 +/- 14.2 ng/mg. Treatment of the vesicles with 1 mM EGTA to remove calmodulin significantly reduced calmodulin content to 19.7 +/- 1.35 ng/mg. This treatment had no significant effect on the calcium inhibition profile. Increasing free calcium to 3x10(-6) M was shown to have no effect on the EC50 estimated for either Gpp(NH)p or forskolin but did slightly increase the EC50 estimated for Mg2+ in the presence of maximal concentrations of either activator. Nevertheless, maximally stimulating concentrations of Mg2+ were unable to overcome calcium inhibition. Pretreatment of sarcolemmal membranes with pertussis toxin was shown to have no significant effect on calcium inhibition of adenylyl cyclase. The results suggest that the overall inhibitory action of calcium was most likely calmodulin independent and involved a direct interaction with the catalytic subunit at two distinct sites of high and low affinity. At the low affinity site calcium most likely competes with Mg2+ for an allosteric divalent cation binding site. Inhibition at the high affinity site was non-competitive with respect to Mg2+, forskolin and guanine nucleotide concentration. The high affinity site may mediate feedback inhibition of cardiac adenylyl cyclase in vivo.