title: 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 microM), 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 microM 5-HT, respectively). The effect of 10 microM 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 (U0126). 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.
authors.affiliation: Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, Bristol, UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Hinton
authors.forename: P
authors.initials: P
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Jeremy
authors.forename: C
authors.initials: C
authors.lastname: Garland
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007650:Ketanserin / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D009570:Nitriles / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010879:Piperazines / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology; D011817:Rabbits; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000502:physiology; D012077:Renal Artery / Q000166:cytology / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012701:Serotonin / Q000494:pharmacology*; D012702:Serotonin Antagonists / Q000494:pharmacology*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002070:Butadienes; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D007650:Ketanserin; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009570:Nitriles; D010069:Oxadiazoles; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010879:Piperazines; D011224:Prazosin; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; D012701:Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin; C113580:U 0126; C090701:GR 127935