Signalling Pathways Activated by 5-HT1B/5-HT1D Receptors in Native Smooth Muscle and Primary Cultures of Rabbit Renal Artery Smooth Muscle Cells

Abstract

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_1B/5-HT_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⁺ Krebs buffer, 5-HT and CP 93,129, a 5-HT_1B receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT_1B/5-HT_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_1B receptor and the 5-HT_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⁺ (reduced by 82.50 ± 2.50 and 87.54 ± 3.97%, respectively). In intact arteries, contraction through 5-HT_1B/5-HT_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⁺ was present at the onset of stimulation. These data provide the first direct evidence that 5-HT_1B/5-HT_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.