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

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.

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+signals, and vasoconstriction in cerebral arteries

“…In primary cultured portal vein smooth muscle cells, IP 3 R1 and IP 3 R2 were detected by immunofluorescence, whereas IP 3 R3 was absent (32). In contrast, in primary cultured rat ureter smooth muscle cells and cultured A7r5 cells, IP 3 R1 and IP 3 R3 were expressed and IP 3 R2 was absent (32,51).…”

“…In primary cultured portal vein smooth muscle cells, IP 3 R1 and IP 3 R2 were detected by immunofluorescence, whereas IP 3 R3 was absent (32). In contrast, in primary cultured rat ureter smooth muscle cells and cultured A7r5 cells, IP 3 R1 and IP 3 R3 were expressed and IP 3 R2 was absent (32,51).IP 3 R isoforms may also regulate vascular smooth muscle Ca 2ϩ signaling in a cell-specific manner. IP 3 R2 activation contributes to ACh-induced Ca 2ϩ oscillations in cultured portal vein smooth muscle cells (32).…”

“…IP 3 R2 activation contributes to ACh-induced Ca 2ϩ oscillations in cultured portal vein smooth muscle cells (32). In contrast, IP 3 R1 was required for ACh-induced Ca 2ϩ elevations in cultured portal vein smooth muscle cells (32), ATP-induced Ca 2ϩ transients in cultured aortic smooth muscle cells (56), and vasopressinevoked Ca 2ϩ release and capacitative Ca 2ϩ entry in cultured A7r5 smooth muscle cells (51). These studies suggest that IP 3 R isoform expression may differ between smooth muscle cell types, leading to tissue-specific regulation of intracellular Ca 2ϩ signaling.…”

“…In vascular smooth muscle cells, IP 3 Rs modulate vasoconstriction and proliferation by altering spatial and temporal properties of local and global Ca 2ϩ signals (51,53). Vasoconstrictors, including norepinephrine, phenylephrine, UTP, and vasopressin, stimulate Ca 2ϩ waves and Ca 2ϩ oscillations in smooth muscle cells of portal vein, rat tail, mesenteric and cerebral arteries, and cultured A7r5 smooth muscle cells (5,7,21,23,29).…”

Calcium Mobilization via Intracellular Ion Channels, Store Organization and Mitochondria in Smooth Muscle

“…During smooth muscle proliferation IP 3 R expression and activity are increased [103][104][105] and there is a marked switch in mitochondrial phenotype from stationary to highly motile [106]. Inhibiting either IP 3 R activity [104,107] or mitochondrial motility and division [106,108] inhibits smooth muscle proliferation. The interplay between mitochondria and IP 3 R in smooth muscle thus presents an interesting potential therapeutic avenue by which pathological smooth muscle proliferation in vascular disease may be targeted.…”