The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT[sub 1B] /5-HT[sub 1D] receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 μM), and prestimulated with 20 mM K[sup +] Krebs buffer, 5-HT and CP 93,129, a 5-HT[sub 1B] receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT[sub 1B] /5-HT[sub 1D] receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT[sub 1B] receptor and the 5-HT[sub 1D] receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 ± 3.33 and 52.65 ± 5.34% from basal with 10 μM 5-HT, respectively). The effect of 10 μM 5-HT on cAMP was increased in the presence of 20 mM K[sup +] (reduced by 82.50 ± 2.50 and 87.54 ± 3.97%, respectively). In intact arteries, contraction through 5-HT[sub 1B] /5-HT[sub 1D] receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U01296). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K[sup +] was present at the onset of stimulation. These data provide the first direct evidence that 5-HT[sub 1B] /5-HT[sub 1B] receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a model system to study further the signalling pathways linked to these receptors.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]