Cholinergic and adrenergic modulation of the Ca2+ response to endothelin-1 in human ciliary muscle cells.

PURPOSE To determine the cholinergic (carbachol, CCH) and adrenergic (norepinephrine, NE) modulation of Ca2+ response to endothelin-1 in human ciliary smooth muscle (HCSM) cells. METHODS Intracellular calcium levels were measured using the Fura-2 calcium imaging system in HCSM cells treated either singly with endothelin-1 (ET-1; 2-200 nM), CCH (1-100 microM), NE (0.1-10 microM) or isoproterenol (ISO; 1 microM) or in combinations of CCH, NE, or ISO with ET-1. Intracellular cAMP levels after NE and ISO treatments were also measured using a radioimmunoassay. RESULTS Endothelin-1 dose-dependently increased [Ca2+]i and was characteristically biphasic (peak [Ca2+]i for ET-1: 2 nM, 517 +/- 73 nM; 20 nM, 785 +/- 65 nM; and 200 nM, 2564 +/- 359 nM). Carbachol also dose-dependently increased [Ca2+]i; however, subsequent additions of ET-1 (200 nM) resulted in lower [Ca2+]i (100 microM CCH + ET-1; 300 +/- 21 nM) compared with that observed with 200 nM ET-1 alone (2564 +/- 359 nM). Norepinephrine pretreatment also decreased ET-1-induced [Ca2+]i (10 microM NE + ET-1; 619 +/- 64 nM) compared with ET-1 alone, and NE's effect could be reversed by propranolol (beta-adrenergic antagonist) treatment. Neither CCH nor NE was able to completely abolish ET-1's ability to mobilize calcium in HCSM cells. Isoproterenol (a beta-agonist) mimicked NE's effect on ET-1-induced [Ca2+]i (1 microM ISO + ET-1; 254 +/- 56 nM). Both ISO and NE elevated [cAMP] in HCSM cells. CONCLUSIONS In HCSM cells, CCH and ET-1 can activate common as well as specific [Ca2+]i pools. The reduction in ET-1-induced [Ca2+]i after NE/ISO treatment appears to be due to elevated cAMP levels via beta-receptor activation, suggesting the existence of receptor cross talk. The ability of CCH and NE to modulate ET-1's actions on HCSM may be relevant to the regulation of ciliary muscle contraction and aqueous humor outflow.