Phospholipase C-delta1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1.
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.
Clarke
Christopher J
CJ
Cardiovascular Research Group, School of Clinical and Laboratory Science, Univ. of Manchester, Core Technology Facility (3floor 46 Grafton St., Manchester M13 9NT, UK.
Forman
Simon
S
Pritchett
James
J
Ohanian
Vasken
V
Ohanian
Jacqueline
J
D000818:Animals; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000201:enzymology; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D006868:Hydrolysis; D015151:Immunoblotting; D066298:In Vitro Techniques; D008638:Mesenteric Arteries / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009213:Myography; D009638:Norepinephrine / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D054803:Phospholipase C delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects
D019332:Endothelin-1; D004963:Estrenes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C074854:U 73343; C518024:PLCD1 protein, human; D054803:Phospholipase C delta; C518025:Plcd1 protein, mouse; C518026:Plcd1 protein, rat; D002118:Calcium; D009638:Norepinephrine