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.
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.
Wang
Y
Y
Human Genome Center, Second Department of Internal Medicine, University of Tokyo, Tokyo, Japan. srwang-tky@umin.ac.jp
Chen
J
J
Wang
Y
Y
Taylor
C W
CW
Hirata
Y
Y
Hagiwara
H
H
Mikoshiba
K
K
Toyo-oka
T
T
Omata
M
M
Sakaki
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000008:administration & dosage; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism* / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008845:Microinjections; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014667:Vasopressins / Q000378:metabolism / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D014667:Vasopressins; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium