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

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.