Clarke Christopher J, Forman Simon, Pritchett James, Ohanian Vasken, Ohanian Jacqueline
Cardiovascular Research Group, School of Clinical and Laboratory Science, Univ. of Manchester, Core Technology Facility (3floor 46 Grafton St., Manchester M13 9NT, UK.
Am J Physiol Heart Circ Physiol. 2008 Aug;295(2):H826-34. doi: 10.1152/ajpheart.01396.2007. Epub 2008 Jun 20.
Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.
血管收缩剂激活磷脂酶C（PLC），后者水解磷脂酰肌醇4,5-二磷酸（PIP₂），导致钙动员、蛋白激酶C激活及收缩。我们的目的是研究PLC-δ₁（一种与α₁-肾上腺素能受体信号传导及高血压发病机制有关的PLC同工型）是否参与去甲肾上腺素（NA）或内皮素（ET-1）诱导的PIP₂水解及收缩。对大鼠肠系膜小动脉进行了研究。通过压力肌动描记法测量收缩性，通过用³³Pi或肌-³H-肌醇进行放射性标记测量磷脂或肌醇磷酸，通过不连续蔗糖密度离心制备小窝/脂筏。通过免疫印迹分析对PLC-δ₁进行定位，通过递送PLC-δ₁抗体将其中和。PLC抑制剂U73122而非阴性对照U-73342显著抑制NA和ET-1诱导的收缩，但对钾或佛波酯诱导的收缩无影响，提示PLC活性参与受体介导的平滑肌收缩。PLC-δ₁存在于小窝/脂筏中，NA而非ET-1刺激这些结构域中PLC-δ₁水平迅速增加两倍。PLC-δ₁依赖于钙，去除细胞外钙可防止其因NA而与小窝/脂筏结合，同时减少NA诱导的³³P-PIP₂水解和³H-肌醇磷酸形成，但对ET-1诱导的³³P-PIP₂水解无影响。与对照抗体相比，PLC-δ₁抗体中和PLC-δ₁可阻止其与小窝/脂筏结合，并减弱对NA的持续收缩反应。相反，ET-1诱导的收缩不受PLC-δ₁抗体影响。这些结果表明小窝/脂筏定位的PLC-δ₁在完整血管组织中NA诱导的PIP₂水解及持续收缩中具有新的选择性作用。
