Wang Y, Chen J, Wang Y, Taylor C W, Hirata Y, Hagiwara H, Mikoshiba K, Toyo-oka T, Omata M, Sakaki Y
Human Genome Center, Second Department of Internal Medicine, University of Tokyo, Tokyo, Japan.
Circ Res. 2001 Feb 2;88(2):202-9. doi: 10.1161/01.res.88.2.202.
Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) (Ca(2+)) 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.
G蛋白或酪氨酸激酶偶联受体的刺激通过细胞内Ca(2+)（Ca(2+)）信号传导调节细胞增殖。在A7r5细胞中，我们证实肌醇1,4,5-三磷酸（IP(3)）介导血管加压素（VP）诱发的细胞内钙库Ca(2+)释放，并表明1型（IP(3)R(1)）和3型（IP(3)R(3)）IP(3)受体表达。使用对IP(3)R(1)或IP(3)R(3)具有选择性的抗血清以及与两种亚型相互作用同样良好的另一种抗血清，连同仅表达单一IP(3)R亚型的SF:9细胞膜用于校准免疫印迹，我们确定A7r5细胞表达81%的IP(3)R(1)和19%的IP(3)R(3)。为了阐明IP(3)R(1)和IP(3)R(3)对Ca(2+)信号传导和增殖的贡献，选择了表达与两种IP(3)R亚型相对应的启动子诱导型反义cDNA片段（-90至+9）的稳定克隆。对IP(3)R(1)的轻度抑制（为对照水平的71±8%）轻微减弱了VP诱导的IP(3)诱发的Ca(2+)释放（IICR），但显著降低了随后的容量性Ca(2+)内流（CCE）和增殖。中度抑制（34±6%）强烈降低了IICR和CCE，并进一步阻断了增殖。完全抑制几乎消除了IICR和CCE，并完全阻止了增殖。对IP(3)R(3)表达的完全抑制轻微减弱了IICR，而不影响CCE或增殖。在微注射低剂量肝素的细胞中，VP诱导的CCE比IICR对IP(3)R(1)和IP(3)R(3)的轻度抑制更敏感。高剂量肝素具有与完全抑制IP(3)R(1)表达类似的效果：它完全阻断了VP诱发的IICR，并使CCE减少90%。我们得出结论，IP(3)R(1)而非IP(3)R(3)对血管平滑肌细胞的IICR、CCE和增殖至关重要。
