Signalling Pathways Activated by 5-HT_1B/5-HT_1D 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.

Preservation of Serotonin-Mediated Contractility in Adult Sheep Pulmonary Arteries Following Long-Term High-Altitude Hypoxia

“…These results indicate that the roles of 5-HT 1B/D and 5HT 2A receptors are unaffected by LTH stress. Moreover, the relative potency of these antagonists indicates that 5-HT 2A receptors, which are commonly coupled with Gq proteins and linked to contractility via Ca 2 + -dependent signaling pathways, are of greater importance than 5-HT 1B/D receptors, which are coupled through G i mechanisms (Hinton et al, 2000).…”

5-Hydroxytryptamine-induced vasodilator responses in the hindquarters of the anaesthetized rat, involve β2-adrenoceptors

“…On the other hand, 5-HT 1D /1B receptors are negatively coupled to adenylyl cyclase, a transduction mechanism associated with the inhibition of the release of neurotransmitters, and it is known that pre-junctional 5-HT 1D /1B receptor activation inhibits noradrenaline release from vascular sympathetic nerve terminals (see Hoyer et al 1994). Recently, however, different studies have suggested that other signalling pathways different from adenylyl cyclase inhibition, such as mitogen-activated protein kinase activation, would be linked to this receptor subtype (Hinton et al 2000;Albert & Tiberi 2001;Watts et al 2001). In particular, the studies of Lin et al (2002) provide evidence that although all 5-HT 1 receptors negatively regulate adenylyl cyclase to some extent, they differentially couple to G proteins and consequently exhibit differences in coupling to cellular signals.…”

5-Hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells are extracellular signal-regulated kinase pathway dependent

“…Previous studies have shown that 5-HT induced ERK1/2 activation in rat aortic smooth muscle cells and rabbit isolated renal artery smooth muscle cells [9,10] . However, whether the intracelluar signal pathway of 5-HT in PASMCs is dependent on the ERKs activation is largely unconcerned.…”