Signalling pathways activated by 5-HT1B/5-HT1D receptors in native smooth muscle and primary cultures of rabbit renal artery smooth muscle cells

The potential of primary cultures of rabbit renal artery vascular smooth mu scle cells (VSMCs) was assessed as a means to investigate the signalling pa thways linked to 5-hydroxytryptamine (5-HT) 5-HT1B/5-HT1D, receptors in nat ive arteries. In renal artery segments denuded of endothelium, incubated wi th ketanserin and prazosin (each 1 muM), and prestimulated with 20 mM K+ Kr ebs buffer, 5-HT and CP 93,129, a 5-HT1B receptor agonist, evoked concentra tion-dependent contractions. GR 127935, a 5-HT1B/5-HT1D receptor antagonist , significantly antagonised 5-HT-evoked contractions at nanomolar concentra tions. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA fro m smooth muscle cells from the isolated renal artery and from primary cultu res of VSMCs from the same artery expressed mRNA transcripts for the 5-HT1B receptor and the 5-HT1D receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Appl ication of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sen sitive reduction in cyclic AMP in both cultured cells and intact artery (cy clic AMP concentration reduced by 65.53 +/- 3.33 and 52.65 +/- 5.34% from b asal with 10 muM 5-HT, respectively). The effect of 10 muM 5-HT on cAMP was increased in the presence of 20 mM K+ (reduced by 82.50 +/- 2.50 and 87.54 +/- 3.97%, respectively). In intact arteries, contraction through 5-HT1B/S -HT1D receptors was significantly attenuated by inhibitors of phosphatidyli nositol 3-kinase (wortmannin) and activated mitogen-activated protein kinas e (MAPK), MEK (U01296). In the cultured VSMCs, activated MARK was identifie d by immunocytochemistry and immunoblotting after stimulation with 5-HT, bu t only if 20 mM K+ was present at the onset of stimulation. These data prov ide the first direct evidence that 5-HT1B/5-HT1B receptors are linked to th e activation of MAPK and indicate that primary cultures of renal VSMCs coul d provide a model system to study further the signalling pathways linked to these receptors. Copyright (C) 2000 S. Karger AG, Basel.