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(Circulation Research. 2001;88:202.)
© 2001 American Heart Association, Inc.

Cellular Biology

Crucial Role of Type 1, but Not Type 3, Inositol 1,4,5-Trisphosphate (IP₃) Receptors in IP₃-Induced Ca²⁺ Release, Capacitative Ca²⁺ Entry, and Proliferation of A7r5 Vascular Smooth Muscle Cells

Yuepeng Wang, Jie Chen, Yue Wang, Colin W. Taylor, Yasunobu Hirata, Hisashi Hagiwara, Katsuhiko Mikoshiba, Teruhiko Toyo-oka, Masao Omata, Yoshiyuki Sakaki

From the Human Genome Center (Yuepeng Wang, H.H., Y.S.), the Department of Molecular Neurobiology (K.M.), Institute of Medical Science, the Second Department of Internal Medicine (J.C., Yue Wang, Y.H., M.O.), the Health Service Center (T.T.), University of Tokyo, Tokyo, Japan; Department of Pharmacology (C.W.T.), University of Cambridge, Cambridge, UK.

Correspondence to Yuepeng Wang, Laboratory of Functional Genomics, the Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail srwang-tky{at}umin.ac.jp

Abstract—Stimulation of G protein– or tyrosine kinase–coupled receptors regulates cell proliferation through intracellular Ca²⁺ ([Ca²⁺]_i) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP₃) mediates vasopressin (VP)-evoked Ca²⁺ release from intracellular stores and showed that types 1 (IP₃R₁) and 3 (IP₃R₃) IP₃ receptors were expressed. Using antisera selective for IP₃R₁ or IP₃R₃ and another that interacted equally well with both subtypes, together with membranes from Sf9 cells expressing only single IP₃R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP₃R₁ and 19% IP₃R₃. To elucidate the contributions of IP₃R₁ and IP₃R₃ to Ca²⁺ signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP₃R subtypes were selected. Mild inhibition of IP₃R₁ (71±8% of control level) slightly attenuated the IP₃-evoked Ca²⁺ release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca²⁺ entry (CCE) and proliferation. Moderate inhibition (34±6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP₃R₃ expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP₃R₁ and IP₃R₃. A high dose of heparin had a similar effect to complete inhibition of IP₃R₁ expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP₃R₁, but not IP₃R₃, is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Key Words: inositol 1,4,5-trisphosphate receptors • IP₃-induced Ca²⁺ release • capacitative Ca²⁺ entry • proliferation • vascular smooth muscle cell

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