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.
Cardiovascular Research Group, School of Clinical and Laboratory Science, Univ. of Manchester, Core Technology Facility (3floor 46 Grafton St., Manchester M13 9NT, UK.
Christopher J
CJ
Clarke
Simon
S
Forman
James
J
Pritchett
Vasken
V
Ohanian
Jacqueline
J
Ohanian
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