Calmodulin activated adenylyl cyclase in ciliary processes: Additivity of calcium and cyclic adenosine monophosphate signals on intraocular pressure response of the rabbit eye

Purpose. The regulation of adenylyl cyclase by multiple signal systems was investigated by biochemical studies of the enzyme in ciliary processes and by in vivo intraocular pressure responses in the rabbit eye. Methods. Adenylyl cyclase enzyme activity was determined by radiometric assay using α-32 P- ATP as substrate. Drugs were administered to the rabbit eye by intravitreal injection or topical application, and intraocular pressure was measured by pneumatonography. Results. Adenylyl cyclase activity in the membrane/particulate fraction of the rabbit ciliary process was activated by calmodulin in the presence of Ca²⁺, Co²⁺, or Mn²⁺, and inhibited by the calmodulin antagonist calmidazolium. The activity was additive to stimulations of adenylyl cyclase by the activating G-protein (G(s)) via isoproterenol or vasoactive intestinal peptide receptors, and by forskolin. The biochemical findings were supported by in vivo correlation experiments with intravitreal injection of MnCl₂, and by topical treatment with the Ca²⁺-mobilizing α₁-adrenergic agonist phenylephrine and with agents affecting cyclic adenosine monophosphate levels (forskolin, isobutylmethylxanthine). The intraocular pressure response was augmented by combining threshold doses of phenylephrine with threshold doses of either forskolin or isobutylmethylxanthine. The maximal and most prolonged response (a decrease in intraocular pressure of 4 mm Hg for up to 8 hr) was obtained by combination treatment with all three agents at doses that produced no significant effect by themselves. Conclusions. These findings provide a biochemical mechanism for ocular hypotensive drugs having both a Ca²⁺- mobilizing activity as well as increasing cyclic adenosine monophosphate levels by receptor-coupled stimulation of adenylyl cyclase.