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Molecular Pharmacology

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Abstract

Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells.

C W Lin, T R Miller, D G Witte, B R Bianchi, M Stashko, A M Manelli and D E Frail
Molecular Pharmacology January 1995, 47 (1) 131-139;
C W Lin
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T R Miller
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D G Witte
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B R Bianchi
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M Stashko
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A M Manelli
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D E Frail
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Abstract

Dopamine (DA) D1 receptors are generally known to couple only to Gs 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 (1992)]. To further evaluate second messenger systems that could be activated by the D1 receptor, we examined the effects of DA, (R)-(+)-SKF-38393, and DA antagonists on cAMP production and calcium release in human embryonic kidney 293 cells stably expressing three different levels (Bmax = 0.12, 1.4, and 23 pmol/mg of protein) of the human D1 receptor. DA and (R)-(+)-SKF-38393 activated cAMP production and calcium release in all three D1-293 clones, and their potency was proportional to receptor density. The efficacy of SKF-38393 was also increased with receptor 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'-tetraacetic acid-sensitive plateau level above the base-line (>5 min). The effect of DA on cAMP and calcium release was selectively inhibited by SCH23390, a selective D1 antagonist, and not by spiperone, a selective D2 antagonist. DA did not induce PI hydrolysis in any of the three receptor-expressing clones. A 24-hr pretreatment with cholera toxin (2 micrograms/ml) greatly attenuated the effect of DA on cAMP formation and calcium release. To address how DA could activate calcium release without enhancing PI hydrolysis, the effects of forskolin, thapsigargin, and isoproterenol (Iso) were studied. Similarly to the effects of DA, forskolin 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 calcium release from the inositol-1, 4, 5-trisphosphate-sensitive calcium store and attenuated the response to subsequent addition of DA. Carbachol stimulated PI hydrolysis and calcium release but had little effect on cAMP production. Prestimulation with carbachol abolished the calcium response to DA, Iso, or forskolin. These studies indicate that D1 receptor-mediated calcium mobilization in 293 cells is dependent on cAMP production and the cAMP-dependent calcium store is part of the inositol-1,4,5-trisphosphate-sensitive calcium pool.

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Molecular Pharmacology
Vol. 47, Issue 1
1 Jan 1995
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Abstract

Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells.

C W Lin, T R Miller, D G Witte, B R Bianchi, M Stashko, A M Manelli and D E Frail
Molecular Pharmacology January 1, 1995, 47 (1) 131-139;

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Abstract

Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells.

C W Lin, T R Miller, D G Witte, B R Bianchi, M Stashko, A M Manelli and D E Frail
Molecular Pharmacology January 1, 1995, 47 (1) 131-139;
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