Interactions Between Forskolin, Gs, and Divalent Cations on Ciliary Process Adenylyl Cyclase and Intraocular Pressure in the Rabbit Eye

The major adenylyl cyclase activity (AC) of rabbit ocular ciliary processes was investigated by dose-response analysis with respect to interactions of its regulators [the stimulatory G-protein (αs) and forskolin] under both saturating and suboptimal divalent cation (M²⁺) conditions. α_s was generated directly with fluoroaluminate or via receptors for vasoactive intestinal peptide or isoproterenol. Forskolin and α_s when liganded simultaneously to AC at suboptimal M²⁺, potentiated maximal enzyme activity (approximately twice the sum of the separate activities), while the responses were additive (no potentiation) under saturating M²⁺ conditions. The results demonstrate that the three ligands (forskolin, M²⁺ and α^s) mutually interact in a cooperative manner as follows: (1) The apparent k_act of forskolin for AC was decreased by 1·7-1·9 log units when α_s was liganded to AC. (2) Conversely, with forskolin liganded to AC the apparent kact of α_s for AC also decreased by a factor of at least 3-4. (3) The nature of the divalent cation M²⁺ had no effect on the k_act of α_s for AC alone and for the AC:FSK complex, and also did not affect the apparent k_act of forskolin for AC alone, or for the AC:α_s complex. The nature of M²⁺ (Mg²⁺ or Mn²⁺) affected only the maximal catalytic rate (E_max). (4) Potentiation of adenylyl cyclase by α_s + FSK was demonstrable in vivo by the increased ocular pressure response of the rabbit eye when a low topical dose of isoproterenol (125 ng per eye) and a subthreshold dose of forskolin (100 μg per eye) were given in combination.