Thiol group identification at or near the agonist binding site of the vascular dopamine receptor

Abstract

Cultured mesenteric artery vascular smooth muscle cells derived from male Wistar rats, expressing both beta 2-adrenoceptors and dopamine DA1 receptors, were prelabelled for 2 h with [3H]adenine. [3H]cAMP formation stimulated by the addition of dopamine (plus propranolol 10 microM), isoprenaline or forskolin, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) was then determined. Exposure of cells to the thiol-oxidizing agent DTNB (5,5'-dithiobis-2-nitrobenzoic acid) following prelabelling, and prior to cAMP assay, resulted in a time-dependent inhibition of dopamine (0.1 mM)-induced cAMP formation, which obeyed the rules of first-order kinetics, being complete by 60 min. This inhibitory effect was observed to be dose related with 50% inhibition achieved at a concentration of 0.5 mM. Exposure to DTNB (5 mM) for 45 min abolished the cAMP response to dopamine (0.1 mM) with little effect on the response to forskolin (10 microM) or isoprenaline (10 microM). Prior addition of the dopamine DA1/D1 receptor selective partial agonist (+)-SKF 38393 (1 microM) preserved the dopamine induced cAMP formation despite DTNB exposure, while its stereo-enantiomer (-)-SKF 38393 (1 microM) protected only 25% of the response. Sequential exposure of cells to DTNB (5 mM) and then either vehicle or DTT (DL-dithiothreitol; 1 mM), each for 20 min periods, resulted in a 70% inhibition of dopamine induced cAMP formation which was almost completely reversed by the disulphide bridge cleaving compound DTT.(ABSTRACT TRUNCATED AT 250 WORDS)