Synergistic activation of vascular TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol and phospholipase A2/omega-hydroxylase/20-HETE pathways.
TRPC6 is a non-voltage-gated Ca(2+) entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interactions of receptor and mechanical stimulations in activating this channel using the patch clamp technique. In HEK293 cells expressing TRPC6, application of mechanical stimuli (hypotonicity, shear, 2,4,6-trinitrophenol) caused, albeit not effective by themselves, a prominent potentiation of cationic currents (I(TRPC6)) induced by a muscarinic receptor agonist carbachol. This effect was insensitive to a tarantula toxin GsMTx-4 (5 mumol/L). A similar extent of mechanical potentiation was observed after activation of I(TRPC6) by GTPgammaS or a diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG). Single TRPC6 channel activity evoked by carbachol was also enhanced by a negative pressure added in the patch pipette. Mechanical potentiation of carbachol- or OAG-induced I(TRPC6) was abolished by small interfering RNA knockdown of cytosolic phospholipase A(2) or pharmacological inhibition of omega-hydroxylation of arachidonic acid into 20-HETE (20-hydroxyeicosatetraenoic acid). Conversely, direct application of 20-HETE enhanced both OAG-induced macroscopic and single channel TRPC6 currents. Essentially the same results were obtained for TRPC6-like cation channel in A7r5 myocytes, where its activation by noradrenaline or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation of receptor and mechanical stimulations may synergistically amplify transmembrane Ca(2+) mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A(2)/omega-hydroxylase/20-HETE pathways.
Inoue
Ryuji
R
Department of Physiology, Graduate School of Medical Sciences, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-0180, Japan. inouery@fukuoka-u.ac.jp
Jensen
Lars J
LJ
Jian
Zhong
Z
Shi
Juan
J
Hai
Lin
L
Lurie
Andrew I
AI
Henriksen
Freja H
FH
Salomonsson
Max
M
Morita
Hiromitsu
H
Kawarabayashi
Yasuhiro
Y
Mori
Masayuki
M
Mori
Yasuo
Y
Ito
Yushi
Y
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018679:Cholinergic Agonists / Q000494:pharmacology; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000819:agonists / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D040542:Mechanotransduction, Cellular / Q000187:drug effects*; D032342:Muscle Cells / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D054467:Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000378:metabolism; D013111:Spider Venoms / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D018679:Cholinergic Agonists; D006893:Hydroxyeicosatetraenoic Acids; D036341:Intercellular Signaling Peptides and Proteins; C411249:MTx4 protein, Grammostola spatulata; D010455:Peptides; D011976:Receptors, Muscarinic; D013111:Spider Venoms; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C492145:Trpc6 protein, rat; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D002217:Carbachol; D042926:Cytochrome P-450 CYP4A; D054467:Phospholipases A2; D010738:Type C Phospholipases