Crucial Role of Type 1, but Not Type 3, Inositol 1,4,5-Trisphosphate (IP ₃ ) Receptors in IP ₃ -Induced Ca ²⁺ Release, Capacitative Ca ²⁺ Entry, and Proliferation of A7r5 Vascular Smooth Muscle Cells

Abstract

Abstract —Stimulation of G protein– or tyrosine kinase–coupled receptors regulates cell proliferation through intracellular Ca 2+ ([Ca 2+ ] i ) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP 3 ) mediates vasopressin (VP)-evoked Ca 2+ release from intracellular stores and showed that types 1 (IP 3 R 1 ) and 3 (IP 3 R 3 ) IP 3 receptors were expressed. Using antisera selective for IP 3 R 1 or IP 3 R 3 and another that interacted equally well with both subtypes, together with membranes from Sf 9 cells expressing only single IP 3 R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP 3 R 1 and 19% IP 3 R 3 . To elucidate the contributions of IP 3 R 1 and IP 3 R 3 to Ca 2+ signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (−90 to +9) corresponding to the two IP 3 R subtypes were selected. Mild inhibition of IP 3 R 1 (71±8% of control level) slightly attenuated the IP 3 -evoked Ca 2+ release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca 2+ 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 IP 3 R 3 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 IP 3 R 1 and IP 3 R 3 . A high dose of heparin had a similar effect to complete inhibition of IP 3 R 1 expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP 3 R 1 , but not IP 3 R 3 , is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.