Interactions between forskolin, GS, and divalent cations on ciliary process adenylyl cyclase and intraocular pressure...

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 (alpha S) and forskolin] under both saturating and suboptimal divalent cation (M2+) conditions. alpha S was generated directly with fluoroaluminate or via receptors for vasoactive intestinal peptide or isoproterenol. Forskolin and alpha 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 alpha 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 alpha S was liganded to AC. (2) Conversely, with forskolin liganded to AC the apparent k(act) of alpha 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 alpha 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: alpha S complex. The nature of M2+ (Mg2+ or Mn2+) affected only the maximal catalytic rate (Emax). (4) Potentiation of adenylyl cyclase by alpha 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 micrograms per eye) were given in combination.