Zhang Gui-hai, Shang Qian-hui, Jiang Qian-feng, Wu Ze-bing, Liu Zu-lin, Wan Wei-hong
Institute of Clinical Medicine Research, Zunyi Medical College, Department of Cardiology, Affiliated Hospital of Zunyi Medical College, Guizhou, China.
Zhonghua Yi Xue Za Zhi. 2009 Nov 3;89(40):2862-6.
To explore the effects of atrial natriuretic peptide (ANP) upon the activities of Na(+), K(+)-ATPase, Ca(2+)-ATPase and mRNA expression levels of Na(+), K(+)-ATPase alpha(1)-subunit and plasma membrane Ca(2+)-ATPase isoform 1 (PMCA1) in cultured thoracic aortic vascular smooth muscle cells (ASMCs) isolated from spontaneously hypertensive rats (SHR).
ASMCs isolated from 14-week-old male SHR and Wistar-Kyoto (WKY) rats were interference-cultured in different doses of ANP and Angiotensin II (AngII). The contents of ANP and AngII 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 alpha(1)-subunit and PMCA1 mRNA in ASMCs.
The ANP level of supernatant in SHR ASMCs was significantly lower than those from WKY control [(7.3 +/- 2.4) pg x 10(-6) cells vs (19.3 +/- 3.3) pg x 10(-6) cells, P < 0.01] while the content of AngII in SHR ASMCs was significantly higher than those from WKY control [(57 +/- 4) pg x 10(-6) cells vs (44 +/- 4) pg x 10(-6) cells, P < 0.01]. The activity of Na(+), K(+)-ATPase [(4.3 +/- 0.8) micromol x h(-1) x mg(-1) vs (5.3 +/- 1.0) micromol x h(-1) x mg(-1)], Ca(2+)-ATPase [(3.2 +/- 0.7) micromol x h(-1) x mg(-1) vs (4.5 +/- 0.7) micromol x h(-1) x 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 alpha(1)-subunit (0.524 +/- 0.025 vs 0.704 +/- 0.116), PMCA1 (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, Ca(2+)-ATPase and expression of alpha(1)-subunit, PMCA1 mRNA in a does-dependent manner (P < 0.05-P < 0.01). Exogenous AngII (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L) significantly repressed activities of Ca(2+)-ATPase and attenuated the expression of PMCA1 mRNA (P < 0.05-P < 0.01). Only AngII (1 x 10(-7) mol/L) significantly inhibited the activity of Na(+), K(+)-ATPase and attenuated the expression of Na(+), K(+)-ATPase alpha(1)-subunit mRNA (both P < 0.05). ANP antagonized the effects of AngII (1 x 10(-7) mol/L) upon the activities of two ATPases and the expression of Na(+), K(+)-ATPase alpha(1)-subunit PMCA1 mRNA (P < 0.05-P < 0.01). AngII (1 x 10(-7) mol/L) increased the Na(+), K(+)-ATPase activity and the expression of Na(+), K(+)-ATPase alpha(1)-subunit mRNA, repressed the Ca(2+)-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 x 10(-7) mol/L) upon the activity of Ca(2+)-ATPase and the expression of PMCA1 mRNA (P < 0.05-P < 0.01), but did not antagonize the effects of AngII (1 x 10(-7) mol/L) upon the activity of Na(+), K(+)-ATPase and the expression of alpha(1)-subunit mRNA in ASMCs from WKY rats (P > 0.05).
The decreased activities of Na(+), K(+)-ATPase and Ca(2+)-ATPase may be related to the abnormal autocrine of ANP and AngII in ASMC of SHR. ANP can antagonize the effects of AngII upon the activities of two ATPases and the expression of Na(+), K(+)-ATPase alpha(1)-subunit PMCA1 mRNA.
探讨心房利钠肽（ANP）对自发性高血压大鼠（SHR）胸主动脉血管平滑肌细胞（ASMCs）中Na(+)、K(+)-ATP酶、Ca(2+)-ATP酶活性以及Na(+)、K(+)-ATP酶α(1)亚基和质膜Ca(2+)-ATP酶同工型1（PMCA1）mRNA表达水平的影响。
从14周龄雄性SHR和Wistar-Kyoto（WKY）大鼠分离出ASMCs，用不同剂量的ANP和血管紧张素II（AngII）进行干预培养。采用放射免疫分析法测定ASMCs上清液中ANP和AngII的含量。通过生物化学和酶学方法测定上述两种ATP酶的活性。运用RT-PCR检测法测定ASMCs中Na(+)、K(+)-ATP酶α(1)亚基和PMCA1 mRNA的相对水平。
SHR的ASMCs上清液中ANP水平显著低于WKY对照组[（7.3±2.4）pg×10(-6)细胞对（19.3±3.3）pg×10(-6)细胞，P<0.01]，而SHR的ASMCs中AngII含量显著高于WKY对照组[（57±4）pg×pgpg×10(-6)细胞对（44±4）pg×10(-6)细胞，P<0.01]。SHR的ASMCs中Na(+)、K(+)-ATP酶活性[（4.3±0.8）μmol×h(-1)×mg(-1)对（5.3±1.0）μmol×h(-1)×mg(-1)]、Ca(2+)-ATP酶活性[（3.2±0.7）μmol×h(-1)×mg(-1)对（4.5±0.7）μmol×h(-1)×mg(-1)]显著低于WKY对照组（均P<0.01）。与WKY对照组相比，SHR的ASMCs中Na(+)、K(+)-ATP酶α(1)亚基mRNA表达（0.524±0.025对0.704±0.116）、PMCA1 mRNA表达（0.193±0.030对0.547±0.045）显著降低（均P<0.）。与SHR对照组相比，外源性ANP明显改善了Na(+)、K(+)-ATP酶、Ca(2+)-ATP酶活性以及α(1)亚基、PMCA1 mRNA的表达，且呈剂量依赖性（P<0.05-P<0.01）。外源性AngII（1×10(-9)、1×10(-8)、1×10(-7)mol/L）显著抑制Ca(2+)-ATP酶活性并减弱PMCA1 mRNA的表达（P<0.05-P<0.01）。仅AngII（1×10(-7)mol/L）显著抑制Na(+)、K(+)-ATP酶活性并减弱Na(+)、K(+)-ATP酶α(1)亚基mRNA的表达（均P<0.05）。ANP拮抗了AngII（1×10(-7)mol/L）对两种ATP酶活性以及Na(+)、K(+)-ATP酶α(1)亚基、PMCA1 mRNA表达的影响（P<0.05-P<0.01）。AngII（1×10(-7)mol/L）增加了WKY大鼠ASMCs中Na(+)、K(+)-ATP酶活性和Na(+)、K(+)-ATP酶α(1)亚基mRNA的表达，抑制了Ca(2+)-ATP酶活性和PMCA1 mRNA的表达（P<0.05-P<0.01）。ANP拮抗了AngII（1×10(-7)mol/L）对Ca(2+)-ATP酶活性和PMCA1 mRNA表达的影响（P<0.05-P<0.01），但未拮抗AngII（1×10(-7)mol/L）对WKY大鼠ASMCs中Na(+)、K(+)-ATP酶活性和α(1)亚基mRNA表达的影响（P>0.05）。
Na(+)、K(+)-ATP酶和Ca(2+)-ATP酶活性降低可能与SHR的ASMCs中ANP和AngII的自分泌异常有关。ANP可拮抗AngII对两种ATP酶活性以及Na(+)、K(+)-ATP酶α(1)亚基、PMCA1 mRNA表达的影响。
