心钠素对高血压大鼠动脉平滑肌细胞离子泵活性和基因表达的影响

目的 探讨心钠素(ANP)对自发性高血压大鼠(SHR)动脉平滑肌细胞膜(ASMC)Na+,K+-ATP酶、Ca2+-ATP酶活性及Na+,K+-ATP酶α,亚单位、Ca+-ATP)酶亚型1(PMCA1)mRNA表达的影响.方法 对SHR大鼠,予不同浓度ANP和血管紧张素Ⅱ(Ang Ⅱ)干预,通过放射免疫、生化酶学和逆转录-聚合酶链反应等方法,检测ASMC的ANP、AngⅡ含量,ATP酶活性及其mRNA表达变化并设WKY大鼠为对照.结果 SHR大鼠ANP含量比WKY大鼠下降(7.3±2.4)pg·10-6比(19.3±3.3) Pg·10-6,P＜0.01],Ang Ⅱ含量增加(57±4)pg·10-6比(44±4) pg·10-6,P＜0.01],Na+,K+-ATP酶、Ca2+-A11)酶活性及Na+,K+-ATP酶α1亚单位、PMCA1 mRNA表达均显著降低Na+,K+-ATP:(4.3±0.8) μmol·h-1·mg-1比(5.3±1.0) μmol·h-1·mg-1,Ca2+-ATP酶:(3.2±0.7)μmol·h-1·mg-1比(4.5±0.7) μmol·h-1·mg-1,α1亚单位:0.524±0.025比0.704±0.116,PMCA1:0.193±0.030比0.547±0.045](P＜0.05～P＜0.01).ANP可增加SHR大鼠Na+,K+-ATP酶、Ca2+-ATP酶活性及Na+,K+-ATP酶α1,亚单位及PMCA1 mRNA表达(均P＜0.01),Ang Ⅱ则抑制Ca2+-ATP酶活性和PMCA1 mRNA表达(P＜0.05～P＜0.01),仅1×10-7 mol/L AngⅡ抑制Na+,K+-ATP酶活性及α1亚单位mRNA表达,ANP能拮抗AngⅡ对两种ATP酶活性及其mRNA表达的效应.ANP也能拮抗AngⅡ对WKY大鼠Ca2+-ATP酶活性及PMCA1mRNA表达的效应,对Na+,K+-ATP酶活性及α1亚单位mRNA表达无影响(P＞0.05).结论 高血压大鼠ASMC两种ATP酶活性和基因表达下降与局部ANP和AngⅡ分泌异常有关,ANP能拮抗AngⅡ对两种ATP酶活性和基因表达的效应.

Effects of ANP upon ion pump activity and gene expression in aortic smooth muscle cells from spontaneously hypertensive rats

Objective To explore the effects of atrial natriuretic peptide (ANP) upon the activities of Na+,K+-ATPase,Ca2+-ATPase and mRNA expression levels of Na+,K+-ATPase α1-subunit and plasma membrane Ca2+-ATPase isoform 1 (PMCAI) in cultured thoracic aortic vascular smooth muscle cells (ASMCs) isolated from spontaneously hypertensive rats (SHR).Methods ASMCs isolated from 14-weekold male SHR and Wistar-Kyoto (WKY) rots were interference-cultured in different doses of ANP and Angiotensin Ⅱ (Ang Ⅱ).The contents of ANP and Ang Ⅱ in supernatant from ASMCs were measured by radioimmunoassay.The activities of the above two ATPases were measured by biochemistry and enzymology.RT-PCR assay was employed to determine the relative levels of Na+,K+-ATPase α1-subunit and PMCA1 mRNA in ASMCs.Results The ANP level of supematant in SHR ASMCs was significantly lower than those from WKY control (7.3±2.4) pg·10-6 cells vs (19.3±3.3) pg·10-6 cells,P＜0.01]while the content of Ang Ⅱ in SHR ASMCs was significantly higher than those from WKY control (57±4) pg·10-6 cells vs (44±4) pg·10-6 cells,P＜0.01].The activity of Na+,K+-ATPase (4.3±0.8) μmol·h-1·mg-1 vs (5.3±1.0) μmol·h-1·mg-1],Ca2+-ATPase (3.2±0.7) μmol·h-1·mg-1 vs (4.5±0.7) μmol·h-1·mg-1]in ASMCs from SHR were significantly lower than those from WKY control (both P＜0.01).The mRNA expression of Na+,K+-ATPase α1-subunit (0.524±0.025 vs 0.704±0.116),PMCAI (0.193±0.030 vs 0.547±0.045) significantly decreased in ASMCs from SHR versus the WKY control (both P＜0.01).As compared with SHR control,exogenous ANP improved obviously the activities of Na+,K+-ATPase,Ca2+-ATPase and expression of α1-subunit,PMCA1 mRNA in a does-dependent manner (P＜0.05-P＜0.01).Exogenous AngⅡ(1×10-9,1×10-8,1×10-7 mol/L) significantly repressed activities of Ca2+-ATPase and attenuated the expression of PMCA1 mRNA (P＜0.05-P＜0.01).Only AngⅡ(1×10-7 mol/L) significantly inhibited the activity of Na+,K+-ATPase and attenuated the expression of Na+,K+-ATPase α1-subunit mRNA (both P＜0.05).ANP antagonized the effects of AngⅡ (1×10-7 mol/L) upon the activities of two ATPases and the expression of Na+,K+-ATPase α1-subunit PMCA1 mRNA (P＜0.05-P＜0.01).AngⅡ(1×10-7 mol/L) increased the Na+,K+-ATPase activity and the expression of Na+,K+-ATPase α1-subunit mRNA,repressed the Ca2+-ATPase activity and the expression of PMCA1 mRNA in ASMCs from WKY rat (P＜0.05-P＜0.01).ANP antagonized the effects of AngⅡ(1×10-7 mol/L) upon the activity of Ca2+-ATPase and the expression of PMCA1 mRNA (P＜0.05-P＜0.01),but did not antagonize the effects of Ang Ⅱ(1×10-7 mol/L) upon the activity of Na+,K+-ATPase and the expression of α1-subunit mRNA in ASMCs from WKY rats (P＞0.05).Conclusion The decreased activities of Na+,K+-ATPase and Ca2+-ATPase may be related to the abnormal autocrine of ANP and AngⅡ in ASMC of SHR.ANP can antagonize the effects of Ang Ⅱ upon the activities of two ATPases and the expression of Na+,K+-ATPase α1-subunit PMCA1 mRNA.