CHARACTERIZATION OF CLONED HUMAN DOPAMINE-D1 RECEPTOR-MEDIATED CALCIUM-RELEASE IN 293-CELLS

Dopamine (DA) DI receptors are generally known to couple only to G(s) and cAMP production. Recently, D1 receptors expressed in mouse Ltk(-) cells have been shown to induce cAMP production, phosphoinositide (PI) hydrolysis, and calcium mobilization [Mol. Endocrinol. 6:1815-1824 (1 992)]. To further evaluate second messenger systems that could be acti vated by the D1 receptor, we examined the effects of DA, (R)-(+)-SKF-3 8393, and DA antagonists on cAMP production and calcium release in hum an embryonic kidney 293 cells stably expressing three different levels (B-max = 0.12, 1.4, and 23 pmol/mg of protein) of the human Df recept or. DA and (R)-(+)-SKF-38393 activated cAMP production and calcium rel ease in all three D1-293 clones, and their potency was proportional to receptor density. The efficacy of SKF-38393 was also increased with r eceptor density in both cAMP and calcium studies. The effect of DA on calcium release consisted of a transient peak response (<20 sec) that declined to an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-te traacetic acid-sensitive plateau level above the base-line (>5 min). T he effect of DA on cAMP and calcium release was selectively inhibited by SCH23390, a selective D1 antagonist, and not by spiperone, a select ive D2 antagonist. DA did not induce PI hydrolysis in any of the three receptor-expressing clones. A 24-hr pretreatment with cholera toxin ( 2 mu g/ml) greatly attenuated the effect of DA on cAMP formation and c alcium release. To address how DA could activate calcium release witho ut enhancing PI hydrolysis, the effects of forskolin, thapsigargin, an d isoproterenol (Iso) were studied. Similarly to the effects of DA, fo rskolin and Iso stimulated cAMP production and calcium release from D1 -293 cells. Cells that were stimulated with Iso or forskolin showed a reduced response to subsequent addition of DA. Pretreatment of D1-293 cells with thapsigargin, a selective Ca2+-ATPase inhibitor, elicited c alcium release from the inositol-1,4,5-trisphosphate-sensitive calcium store and attenuated the response to subsequent addition of DA. Carba chol stimulated PI hydrolysis and calcium release but had little effec t on cAMP production. Prestimulation with carbachol abolished the calc ium response to DA, Iso, or forskolin. These studies indicate that D1 receptor-mediated calcium mobilization in 293 cells is dependent on cA MP production and the cAMP-dependent calcium store is part of the inos itol-1,4,5-trisphosphate-sensitive calcium pool.