Hinton J M, Hill P, Jeremy J, Garland C
Cardiovascular Research Laboratories, School of Medical Sciences, University of Bristol, Bristol, UK.
J Vasc Res. 2000 Nov-Dec;37(6):457-68. doi: 10.1159/000054078.
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.
兔肾动脉血管平滑肌细胞（VSMCs）原代培养物的潜能被评估为一种手段，用于研究与天然动脉中5-羟色胺（5-HT）5-HT(1B)/5-HT(1D)受体相关的信号通路。在去除内皮的肾动脉段中，用酮色林和哌唑嗪（各1 microM）孵育，并先用20 mM K(+) Krebs缓冲液预刺激，5-HT和5-HT(1B)受体激动剂CP 93,129引起浓度依赖性收缩。5-HT(1B)/5-HT(1D)受体拮抗剂GR 127935在纳摩尔浓度下显著拮抗5-HT引起的收缩。对分离的肾动脉平滑肌细胞以及同一动脉VSMCs原代培养物的mRNA进行逆转录聚合酶链反应（RT-PCR），两种制剂中均表达了5-HT(1B)受体和5-HT(1D)受体的mRNA转录本。PCR片段的序列与这些受体的已知序列相符。5-HT的应用在培养细胞和完整动脉中均引起浓度依赖性、百日咳毒素（PTx）敏感的环磷酸腺苷（cAMP）减少（10 microM 5-HT时，cAMP浓度分别比基础值降低65.53 +/- 3.33%和52.65 +/- 5.34%）。在存在20 mM K(+)的情况下，10 microM 5-HT对cAMP的作用增强（分别降低82.50 +/- 2.50%和87.54 +/- 3.97%）。在完整动脉中，磷脂酰肌醇3-激酶抑制剂（渥曼青霉素）和活化的丝裂原活化蛋白激酶（MAPK）、MEK（U0126）可显著减弱通过5-HT(1B)/5-HT(1D)受体的收缩。在培养的VSMCs中，5-HT刺激后通过免疫细胞化学和免疫印迹鉴定出活化的MAPK，但仅在刺激开始时存在20 mM K(+)的情况下。这些数据提供了首个直接证据，表明5-HT(1B)/5-HT(1B)受体与MAPK的激活有关，并表明肾VSMCs原代培养物可提供一个模型系统，以进一步研究与这些受体相关的信号通路。
