Regulation of lens cyclic nucleotide metabolism by Ca²⁺ plus calmodulin

Adenylate cyclase activity was identified in membranes isolated from bovine lens fiber cells. Basal activity, in the presence of μM Ca²⁺ was stimulated by either sodium fluoride, guanosine 5'-[α,β-imido]triphosphate (Gpp(NH)p), or forskolin; ethylene glycolbis(2-aminoethylether) tetraacetic acid (EGTA) markedly inhibited both the basal activity and the extent of stimulation by these agents. Exogenous calmodulin enhanced the Ca²⁺-dependent stimulation of adenylate cyclase activity. In the presence of optimal concentrations of Ca²⁺ plus calmodulin, adenylate cyclase activity was approximately 15 times greater than that in the presence of EGTA. Adenylate cyclase activity was not stimulated by a number of potential agonists that included carbachol, serotonin, prostaglandin E₁ (PGE₁) prostaglandin E₂ (PGE₂), adenosine, isoproterenol, epinephrine, dopamine, and phenylephrine. The presence of the N(s) and N(i) guanine nucleotide regulatory complexes was indicated by two observations: Cholera toxin catalyzed the adenosine diphosphate (ADP) ribosylation of a number of lens membrane proteins, including a 46,500-dalton component (likely the α-subunit of N(s)), and Pertussis toxin catalyzed the ADP ribosylation of a single 41,000-dalton lens membrane component (likely the α-subunit of N(i)). However, that Gpp(NH)p did not inhibit either the forskolin-activated or the calmodulin-activated adenylate cyclase activities does not indicate a role for N(i) in regulating this enzyme. Both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterase activities were identified in a supernate fraction derived from bovine lens. The cAMP phosphodiesterase activity appeared to be predominantly the low K(m) form of the enzyme. The cGMP phosphodiesterase activity, which was Ca²⁺-dependent, was partly inhibited maximally by 7 μM R24571, indicating its probable calmodulin dependence. These studies indicate that Ca²⁺ plays a major role in the metabolism of lens fiber cell cyclic nucleotides.