Dopamine as a Novel Antimigration Factor of Vascular Smooth Muscle Cells Through D 1a and D 1b Dopamine Receptors

Abstract

P82 Vascular smooth muscle cell (VSMC) migration is believed to play a key role in atherosclerosis. To elucidate the roles of rat vascular D 1A and D 1B dopamine receptors in atherosclerosis, the effect of antisense oligonucleotides (AS) to D 1A (+1-+21 of rat D 1A receptors cDNA) and D 1B (-12-+6 of rat D 1B receptors cDNA) receptors on dopamine-mediated suppression of platelet-derived growth factor (PDGF) BB-mediated VSMC migration evaluated by Boyden′s chamber method was studied. To avoid the degradation, phosphothioate-modified oligodeoxynucleotides were synthesized and purified by high-performance liquid chromatography. These oligonucleotides were added to serum free medium for 24 h before the start of PDGF BB stimulation with transfection using lipofectin reagent. Immunohistochemical studies (double staining) demonstrated the coexistence of D 1A and D 1B dopamine receptors in single VSMC derived from rat renal artery. Western blotting revealed a band of approximately 70 kD for D 1A and D 1B dopamine receptors. Increased VSMC migration by PDGF BB 5 ng/ml (16-fold) was suppressed significantly by coincubation with dopamine 0.025-10 μmol/l (15-59%). This suppression by dopamine 10 μmol/l was reversed by D 1A AS 46% and D 1B AS 51%, but by neither sense (S) nor scramble (RS) oligonucleotides to these receptors. These suppression by antisense oligonucleotides (21-51%) are dose-dependent (1-10 μmol/l) and time-dependent (0-4 hrs). Dopamine 10 μmol/l-induced cyclic AMP formation is also suppressed by D 1A AS 50% and D 1B AS 58%, but by neither S nor RS to these receptors. PDGF BB (5 ng/ml)-mediated activation of phospholipase D (PLD) activity (107%) measured by [ 3 H]-ethanolamine and protein kinase C (PKC) activity (111%) activities measured by synthetic peptide phosphorylation were significantly suppressed by coincubation with dopamine 10 μmol/l (48%, 49%), which was reversed by D 1A AS 45% and D 1B AS 50%, but by neither S nor RS to these receptors. These results suggest that vascular D 1A and D 1B receptors inhibit migration of VSMC, possibly through cAMP formation and suppression of PLD and PKC activity.