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 μM), NE (0.1-10 μM) or isoproterenol (ISO; 1 μM) 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-I: 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 μM 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 μM NE + ET-1; 619 ± 64 nM) compared with ET-1 alone, and NE's effect could be reversed by propranolol (β-adrenergic antagonist) treatment. Neither CCH nor NE was able to completely abolish ET-1's ability to mobilize calcium in HCSM cells. Isoproterenol (a β-agonist) mimicked NE's effect on ET-1-induced [Ca²⁺](i) (1 μM 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 β-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.