Dopamine as a Novel Antimigration Factor of Vascular Smooth Muscle Cells Through D1a and D1b Dopamine Receptors
Vascular smooth muscle cell (VSMC) migration is believed to play a key role in atherosclerosis. To elucidate the roles of rat vascular D1A and D1B dopamine receptors in atherosclerosis, the effect of antisense oligonucleotides (AS) to D1A (+1-+21 of rat D1A receptors cDNA) and D1B (-12-+6 of rat D1B 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 D1A and D1B dopamine receptors in single VSMC derived from rat renal artery. Western blotting revealed a band of approximately 70 kD for D1A and D1B 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 D1A AS 46% and D1B 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 D1A AS 50% and D1B 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 [3H]-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 D1A AS 45% and D1B AS 50%, but by neither S nor RS to these receptors. These results suggest that vascular D1A and D1B receptors inhibit migration of VSMC, possibly through cAMP formation and suppression of PLD and PKC activity.