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(Circulation Research. 2009;104:1399.)
© 2009 American Heart Association, Inc.

Cellular Biology

Synergistic Activation of Vascular TRPC6 Channel by Receptor and Mechanical Stimulation via Phospholipase C/Diacylglycerol and Phospholipase A₂/-Hydroxylase/20-HETE Pathways

Ryuji Inoue^*, Lars J. Jensen^*, Zhong Jian, Juan Shi, Lin Hai, Andrew I. Lurie, Freja H. Henriksen, Max Salomonsson, Hiromitsu Morita, Yasuhiro Kawarabayashi, Masayuki Mori, Yasuo Mori, Yushi Ito

From the Department of Physiology (R.I., Z.J., L.H., Y.K., M.M.), Graduate School of Medical Sciences, Fukuoka University, Japan; Department of Biomedical Sciences (L.J.J., A.I.L., F.H.H., M.S.), University of Copenhagen, The Panum Institute, Denmark; Institute of Biomedical Engineering (Z.J.), School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, China; Department of Anatomy and K. K. Leung Brain Research Centre (J.S.), the Fourth Military Medical University, Xi’an, China; Special Patient Oral Care Unit (H.M.), Kyushu University Hospital, Fukuoka, Japan; Department of Synthetic Chemistry and Biological Chemistry (Y.M.), Graduate School of Engineering, Kyoto University, Japan; and Research Division for Life Sciences (Y.I.), Kumamoto Health Science University, Japan.

Correspondence to Ryuji Inoue, MD, PhD, Department of Physiology, Graduate School of Medical Sciences, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-0180, Japan. E-mail inouery{at}fukuoka-u.ac.jp

TRPC6 is a non–voltage-gated Ca²⁺ 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 µmol/L). A similar extent of mechanical potentiation was observed after activation of I_TRPC6 by GTPS 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 A₂ or pharmacological inhibition of -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 Ca²⁺ mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A₂/-hydroxylase/20-HETE pathways.

Key Words: mechanotransduction • G_q/11 protein–coupled receptor • Ca²⁺ entry channel