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.
|Original language||English (US)|
|Number of pages||9|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - 1987|