Effect Of Valsartan On The Expression Of Na~+, K~+-ATPase α Subunits In Left Ventricles Of SHR

Hypertension is the main cardiovascular disease in the whole world. It can not only damage vital viscera such as heart, brain and kidney, but also became the first risk of atherosclerosis, whick make it the center of cardiovascular prevention and control [1]. The Pathogenesis of essential hypertension is still not well known today. Nowadays, hypertension has been attributed to abnormalities of blood pressure control caused by lots of environmental factors based on hypertensinogenic factors. Molecular biological study developed a hypothesis called"membrane conception", which considered hypertension as a consequence of abnormalities of wide-spread cell membrane physical properties and of the multiple transport systems. Furthermore, there is evidence that the blood vascular structure, the movement of sodium and calcium in hypertension patients were all changed [2].The Na⁺,K⁺-ATPase is a ubiquitous integral membrane protein. The enzyme is composed of a catalyticαsubunit and a glycosylatedβ-subunit. Four different isoforms (α1,α2,α3,α4) are expressed in a species- and tissue-specific manner.α1 isoform is expressed ubiquitously among tissues, theα2 isoform is present largely in skeletal muscle, heart, brain, adipocytes, vascular smooth muscle, and eye, as well as a number of other tissues. Theα3 isoform is found almost exclusively in neurons and ovaries, but also occurs in white blood cells and heart of some species, such as humans. Theα4 isoform is expressed in sperm and is specifically synthesized at the spermatagonia stage, where it is required for sperm motility [3]. Theα1 isoform has a significantly lower affinity for ouabain than theα2 andα3 isoforms. The Na⁺, K⁺-ATPase couples the exchange of three cytoplasmic sodium ions for two extracellular potassium ions to the hydrolysis of one molecule of ATP and thus keeps the sodium and potassium electrochemical gradient, which is the basic for numerous body functions, such as maintaining the cellular membrane potential, the osmotic balance inside and outside cells and cell capacity, providing energy for nutrient transport [4]. In addition, it regulates cytoplasmic calcium concentration and plays a vital role in vascular tone and blood pressure regulation [5]. Dysfunction of Na⁺, K⁺-ATPase leads to sodium retention in cell cytoplasm and increased concentrations of cytoplasmic free calcium through Na⁺-Ca²⁺-exchanger, which results in increased cell excitability, vasoconstriction, increased peripheral resistance and elevated blood pressure.Now, there is increasing evidence that the activity of Na⁺, K⁺-ATPase is decreased in hypertensive patients, 1K1C hypertensive rats, DOCA-salt hypertensive rats, SHRs and many other experimental animal model of hypertension. The previous study in our laboratory has also shown the close relationship between the development of hypertension and the suppression of Na+,K⁺-ATPase activity in myocardium of 1K1C hypertensive rats and SHRs. However, substantial controversy exists according to the changes of protein and mRNA expressions of Na+,K⁺-ATPaseαsubunit in different hypertensive animal hearts.Angiotensin II is the activator of the Renin-Angiotensin- Aldosterone System (RAAS), which is converted from angiotensin I through removal of two terminal residues by the angiotensin-converting enzyme (ACE). Combined with its specific receptors in different cell membranes, angiotensin II can cause vasoconstriction, increase blood pressure, and release aldosterone from the adrenal cortex. Valsartan is an angiotensin II (AT1) receptor blocker (ARB), which blocks the activation of angiotensin II AT1 receptors and causes vasodilation, reduces secretions of vasopressin and aldosterone, and lowers blood pressure. It can also improve left ventricular afterload and reverse cardiac hypertrophy [6]. The aim of this study is to investigate the changes of mRNA and protein expression of Na+, K⁺-ATPaseαsubunits in spontaneously hypertensive rats (SHR) before and after valsartan treatment. We also analyze the role of Na+, K⁺-ATPase in the development of hypertension.Objective: The purpose of this study was to compare the expression of Na+, K⁺-ATPaseαsubunits in left ventricles of spontaneously hypertensive rats (SHR) versus normotensive Wistar rats by immunoblotting and reverse transcription- polymerase chain reaction (RT-PCR). Furthermore, we also investigate the effects of angiotensin II type 1 receptor (AT1R) blocker valsartan treatment on the expression of Na⁺, K⁺-ATPaseαsubunits in left ventricles of SHRs.Methods: Eighteen SHRs were ramdomly divided into three groups: SHR control group, SHR treated with low dose valsartan (10mg/Kg·d) group and SHR treated with high dose valsartan (30mg/Kg·d) group. Each group contains 6 animals. At the same time, 6 age-matched Wistar rats were used as normotensive control group. During the experiment, systolic blood pressure (SBP) were measured using tail-cuff LE 5001 Pressure Blood Meter every week.The protein expression of Na⁺,K⁺-ATPaseα1 andα2 isoforms were detected by Western Blot analysis. Membrane proteins were extracted from the left ventricles of different SHR groups and normotensive Wistar rats and their concentration was measured by BCA protein assay. Membrane proteins were electrophoresed into 10% SDS-PAGE gels and blotted onto PVDF membrane. Blots were then stained with Ponceau. Membranes were blocked for 1h in 5% nonfat dry milk in TBST at 25°C, then incubated overnight with Na⁺,K⁺-ATPaseα1 andα2 isoforms antibody at 4°C, washed three times with TBST, then incubated with a diluted peroxidase-labeled secondary antibody in TBST and washed with TBST. The resulting bands were finally visualized using DAB. The mRNA expressions ofα1 andα2 isoforms of Na⁺, K⁺-ATPase in left ventricular were evaluated by semiquantitative RT-PCR. Total RNA was extracted from left ventricles of different SHR groups and normotensive Wistar rats. The reverse transcription reaction was primered with Oligo(dt)15 and the subsequent cDNA was used as a template for the PCR reactions. Three primers were used successfully for PCR amplification of the twoαisoforms of Na⁺, K⁺-ATPase and GAPDH using Taq polymerase. The PCR products were size-fractionated by electrophoresis through a 2% agarose-gel containing (5μl/100ml GoldView).Results: 1. Effects of valsartan on body weight and blood presure in rats: At the beginning of the experiment, the SBP in SHR were significantly higher than that in Wistar rats group (P<0.01). The SBP of SHR treated with low dose valsartan (10mg/Kg·d) group was significantly reduced after 3 week treatment (p<0.01). The SBP of SHR treated with high dose valsartan (30mg/Kg·d) group was significantly reduced after 2 week treatment (p<0.05). After 4weeks of treatment, two SHR groups treatmented with valsartan both had significantly lower SBPs compared to that of the SHR control group (p<0.01).2. Effects of valsartan on protein expressions ofα1 andα2 isoforms in the rat left ventricles.Compared to normotensive Wistar rats,the expression ofα2 subunit protein in SHR control group showed a 10.6% decrease (p<0.01). The protein expression ofα1 isoform in SHR control group had light elevation without significant difference. Compared to SHR control group, SHR group treated with low dose valsartan had a 3.4% increase without significant difference. SHR group treated with high dose valsartan had a 15.7% increase (p<0.05), indicating a close relationship between hypertension and the decreased expression of Na⁺, K⁺-ATPaseα2 isoform in left ventricles of SHRs.3. Effects of valsartan on mRNA levels ofα1 andα2 isoforms in the left ventricles.Compared to normotensive Wistar rats , the mRNA expression ofα2 isoform in SHR control group showed a 30.4% decrease (p<0.01). The mRNA expression ofα1 isoform in SHR control group showed no significantly difference. Compared to SHR control group, SHR groups treated with low and high dose valsartan both had siginificant increase in mRNA expression ofα2 isoform, but no changes in mRNA expression ofα1 isoform, which is in accordance with the changes of protein expression ofα1 andα2 isoforms.Conclusion: The mRNA and protein expression ofα2 isoform showed significant decrease in SHR, and can be increased by hypotension treatment of valsartan. The results imply the pathogenesis of hypertension is closely related to the reduction expression of Na⁺, K⁺-ATPaseα2 isoform. The issue whether hypertension is the reason which leads to the reduction expression of Na⁺, K⁺-ATPaseα2 subunit, or the result caused byα2 subunit due to some other reasons needs further study.