Momen Abdul, Afroze Talat, Sadi Al-Muktafi, Khoshbin Amir, Zhang Hangjun, Choi Jaehyun, Gu Steven, Zaidi Syed H, Heximer Scott P, Husain Mansoor
Division of Experimental Therapeutics, Toronto General Research Institute , University Health Network, Toronto, Ontario , Canada and.
J Recept Signal Transduct Res. 2014 Dec;34(6):476-83. doi: 10.3109/10799893.2014.920393. Epub 2014 May 20.
Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca(2+) signalling in vascular smooth muscle cells (VSMC).
RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca(2+) concentration [Ca(2+)]i. RGS2 involvement in VSMC proliferation had not been examined.
Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca(2+)]i before and after UTP and thapsigargin. [(3)H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca(2+) transporters was performed in primary aortic VSMC.
Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca(2+) homeostasis, RGS2KO VSMC showed lower resting [Ca(2+)]i but higher thapsigargin-induced [Ca(2+)]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca(2+) ATPase-4 (PMCA4) and Na(+) Ca(2+) Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca(2+) ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca(2+) efflux activity, (45)Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La(3+), but not by replacing extracellular Na(+) with choline, implicating differences in the activity of PMCA and not NCX.
RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca(2+)-handling mechanisms in response to PDGF stimulation.
G蛋白信号调节因子2（RGS2）可抑制血管平滑肌细胞（VSMC）中Gq介导的钙（Ca2+）信号调节。
RGS2基因敲除（RGS2KO）小鼠患有高血压并表现出小动脉重塑。VSMC增殖可调节细胞内钙（Ca2+）浓度[Ca2+]i。尚未研究RGS2在VSMC增殖中的作用。
采用胸腺嘧啶核苷掺入法和3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）转化法检测细胞增殖。Fura-2比率成像法定量UTP和毒胡萝卜素处理前后的[Ca2+]i。用[3H]标记的肌醇检测磷脂酰肌醇水解。对原代主动脉VSMC进行定量RT-PCR和选定钙（Ca2+）转运体的共聚焦免疫荧光检测。
血小板衍生生长因子（PDGF）使RGS2KO小鼠VSMC进入S期和增殖的程度高于野生型（WT）对照小鼠的VSMC。与PDGF诱导的钙（Ca2+）稳态差异变化一致，与WT相比，RGS2KO VSMC的静息[Ca2+]i较低，但毒胡萝卜素诱导的[Ca2+]i较高。RGS2KO VSMC中质膜钙（Ca2+）ATP酶-4（PMCA4）和钠（Na+）钙（Ca2+）交换体（NCX）的mRNA水平较低，但肌浆网钙（Ca2+）ATP酶-2（SERCA2）的水平较高。蛋白质印迹法和免疫荧光检测显示PMCA4和SERCA2蛋白存在类似差异，而RGS2KO VSMC中NCX蛋白水平未降低。与钙（Ca2+）外流活性降低一致，RGS2KO VSMC中的（45）Ca外流率较低。这些差异可被PMCA抑制剂La3+逆转，但不能通过用胆碱替代细胞外钠（Na+）来逆转，这表明是PMCA而非NCX的活性存在差异。
RGS2缺陷的VSMC表现出更高的增殖率，并在PDGF刺激下协调钙（Ca2+）处理机制的可塑性。
