Ca2+-Induced Inhibition of Adenylyl Cyclase in Turkey Erythrocyte Membranes

Abstract

We investigated the effects of calcium ions (Ca²⁺) on the adenylyl cyclase activity in purified turkey erythrocyte membranes. Results showed the following: (i) Ca²⁺ inhibits cAMP accumulation stimulated by isoproterenol (1 µmol/l), NaF + AlCl₃ (10 mmol/l + 20 µmol/l) or forskolin (10 µmol/l) in EGTA-washed turkey erythrocyte membranes. IC₅₀ of free [Ca²⁺] is approximately 0.1 mmol/l in the presence of Mg²⁺ (2.5 mmol/l) and isobutylmethylxanthine (1 mmol/l). (ii) The potency of Ca²⁺ to inhibit cAMP accumulation is independent of the type of stimulus used to activate the adenylyl cyclase. We also evaluated the calcium sensitivity of the basal cAMP accumulation in the presence of GTP (10 µmol/l) and Mg²⁺ (2.5 mmol/l) which was also inhibited by Ca²⁺ with the same potency. (iii) The inhibition pattern of cAMP accumulation is not affected by the presence of added calmodulin (100 nmol/l). (iv) Ca²⁺ is ineffective on the binding of isoproterenol to the β-adrenoceptors. (v) Increasing the concentration of Ca²⁺ does not induce an observable activation of cyclic nucleotide phosphodiesterase in the present experimental conditions. Thus, we concluded that the inhibition of cAMP accumulation is due to an inhibition of the adenylyl cyclase rather than the activation of phosphodiesterase(s). The presence of a yet unidentified isoform of adenylyl cyclase that can be directly inhibited by Ca²⁺ or a G_i protein that can be activated by Ca²⁺ seems to explain these results. In either case, these results provide an additional mode of cross-talk that can take place between the Ca²⁺- and cAMP-signaling systems.