PROSTAGLANDINS MEDIATE THE STIMULATORY EFFECTS OF ENDOTHELIN-1 ON CYCLIC ADENOSINE-MONOPHOSPHATE ACCUMULATION IN CILIARY SMOOTH-MUSCLE ISOLATED FROM BOVINE, CAT, AND OTHER MAMMALIAN-SPECIES

Purpose. To examine the effects and mechanisms of endothelin-1 (ET-1) on cyclic adenosine monophosphate (cAMP) accumulation, inositol 1,4,5- trisphosphate (IP3) production, and contraction in ciliary muscle (CM) isolated from bovine, cat and other mammalian species. Methods. Cilia ry muscle was incubated in the absence and presence of ET-1 for 5 minu tes. Indomethacin (Indo, 2.5 mu M) and IBMX (0.1 mM) were added 10 min utes before the addition of the peptide. Cyclic AMP accumulation and p rostaglandin E(2) (PGE(2)) release were measured by radioimmunoassay, IP3 production was measured by ion-exchange chromatography, and change s in tension were recorded isometrically. Results. First, ET-1 (0.1 mu M) increased PGE(2) release by 58% to 105% and cAMP accumulation by 9 8% to 393% in CMs isolated from bovine, cat, dog and human, and these effects were blocked completely by Indo (2.5 mu M). Unlike any other s pecies, in bovine CM, ET-1 increased IP3 production (EC(50) = 17 nM) a nd contraction (EC(50) = 13 nM), and these effects were not inhibited by Indo. Second, kinetic studies revealed that in bovine and cat CMs, ET-1 stimulated cAMP accumulation and PGE(2) release in a time- and do se-dependent manner, and these effects were inhibited by Indo in a tim e- and dose-dependent manner, and PGE(2) increased cAMP accumulation i n a dose-dependent manner (EC(50) = 0.175 mu M). The stimulatory effec t of ET-1 on cAMP accumulation is mediated through the ET(A) receptor subtype, because in contrast to ET-1, which is an ET(A) receptor agoni st, ET-3 and Sarafotoxin-S6c, two ET(B) receptor agonists, had little effect on cAMP accumulation. In addition, BQ 610, an ET(A) receptor su btype antagonist, inhibited ET-1-induced cAMP accumulation in a dose-d ependent manner (IC50s for bovine and cat were 11 and 19.5 nM, respect ively). Quinacrine, a phospholipase A(2) inhibitor, inhibited ET-1-ind uced cAMP accumulation in a dose-dependent manner (IC50s for bovine an d cat were 22 and 19 mu M, respectively). PGE(2), but not ET-1, stimul ated adenylyl cyclase activity in membranes isolated from bovine and c at CMs. Conclusions. In CMs isolated from bovine, cat, dog and human, ET-1-induced cAMP accumulation is mediated through the release of PGs. ET-1 binds to the ETA(A) receptor subtype to activate phospholipase A (2) and to release arachidonic acid for PG synthesis. PGs, such as PGE (2), may interact with the EP receptor to stimulate adenylyl cyclase. Although ET-1-induced PG release could function to modulate, through c AMP, the responses to muscarinic receptor stimulation, the precise rol e of these effects in intraocular pressure lowering and accommodation remains to be delineated.