Na+/K+-ATPase α isoforms expression in stroke-prone spontaneously hypertensive rat heart ventricles: Effect of salt loading and lacidipine treatment (original) (raw)
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Myocardial Na, K-ATPase in one-kidney, one-clip hypertensive rats**
Journal of Molecular and Cellular Cardiology, 1986
Myocardial ventricular Na, K-ATPase activity of normotensive rats was compared with that of healthy rats with chronic benign one-kidney, one-clip hypertension. The yield of protein (mg/g wet wt left plus right ventricles) in microsomal and sarcolemmal membrane fractions was the same for both normotensive and hypertensive rat ventricles. However, the yield of protein (mg]ventricle) was 26% greater in the hypertensive relative to the normotensive animals, consistent with the presence of hypertrophy, as also indicated by an increase in the ratio of ventricular to body weight and a shift in the isomyosin composition. Na, K-ATPase activity, sodium-dependent phosphorylation and ouabain binding were significantly (P < 0.05) decreased (by 20%, 40%, and 45%, respectively) in the hypertensive rat ventricles when the data were expressed in units/g tissue wet weight. However, when expressed in units per ventricle, values in normotensive and hypertensive animals were similar. The molecular activity or turnover number of ventricular (and also renal) Na, K-ATPase activity was the same in both groups of animals. These results suggest that the decrease in myocardial specific Na, K-ATPase activity in the rat made hypertensive by removing one kidney and constricting the renal artery of the other kidney is related to the presence of cardiac hypertrophy.
Life Sciences, 2002
The (Na,K)-ATPase is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembraneous efflux of Na + from cardiac cells in spontaneously hypertensive rats (SHR) with increased synthesis of nitric oxide (NO). In the investigated group of SHR the systolic blood pressure was increased by 64% and the synthesis of NO was increased by 60% in the heart. When activating the cardiac (Na,K)-ATPase with substrate, its activity was higher in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed an increase of the V max (by 37%) probably due to increased affinity of the ATP-binding site as indicated by the lowered K m value (by 38%) in SHR. During activation with Na + , we observed no change in the enzyme activity below 10 mmol/l of NaCl whereas in the presence of higher concentrations of NaCl the (Na,K)-ATPase was stimulated. The value of V max increased (by 64%), however the K Na increased (by 106%), indicating an adaptation of the Na + -binding site of the enzyme to increased [Na + ] i . Thus the (Na,K)-ATPase in our SHR group is able to extrude the excessive Na + from myocardial cells more effectively also at higher [Na + ] i , while the enzyme from controls is unable to increase its activity further. This improvement of the (Na,K)-ATPase function is supported also by increased affinity of its ATP-binding site probably due to enhanced NO-synthesis. D
Regulation of Na+,K+-ATPase α-subunit isoforms in rat tissues during hypertension
European Journal of Pharmacology: Environmental Toxicology and Pharmacology, 1995
We investigated the regulation of the protein expression of the c~ isozymes of Na+,K+-ATPase in reference to the enzyme activity in the heart, brain and skeletal muscle of rats during deoxycorticosterone acetate (DOCA)-salt hypertension. Treatment of rats with DOCA and salt for 28 days produced a significant increase in systolic blood pressure compared to the control groups which remained normotensive. Rats treated with DOCA expressed greater amounts of the immunoreactive a-1 isoform than untreated controls in whole heart membranes. However, the DOCA-induced increase in the a-1 isoform did not occur during DOCA-salt hypertension. There was a parallel change in the enzyme activity of the Na+,K+-ATPase and the protein expression of the a-1 isoform as a result of these treatments. We have also demonstrated that the hearts of DOCA-salt hypertensive rats expressed less of the a-2 isoform compared to the controls. We could not detect any alteration in the c~-1 and a-2 isoforms of the skeletal muscle and a-l, a-2 and a-3 isoforms of the whole brain Na+,K+-ATPase during salt or DOCA treatments alone or DOCA-salt hypertension. Furthermore, the Na+,K+-ATPase activity was unaltered in these tissues during these treatments. In conclusion, cardiac Na+,K+-ATPase cPsubunit protein expression appears to be regulated during DOCA-salt hypertension. In the skeletal muscle and brain, tissues not subjected directly to increased pressure, this regulation of the Na+,K+-ATPase was not apparent.
Two functional Na+/K+-ATPase isoforms in the left ventricle of guinea pig heart
European Journal of Biochemistry, 1991
Guinea pig left ventricular muscle contains two distinct molecular forms of the N a + /K+-ATPase catalytic a subunit. Sarcolemmal vesicles highly enriched in Na+/K+-ATPase were isolated by a new procedure that yielded specific activities of 60 -100 pmol Pi . h-' . mg-'. SDSjPAGE of isolated sarcolemma after reduction and alkylation of the sulfhydryl groups and identification on immunoblots with specific anti-(a subunit) antibodies indicated the presence of two major polypeptides of 100 kDa and 103 kDa, respectively. The two a subunits were functional: the dose/response curves of Na+/K+-ATPase activity with ouabain, dihydroouabain and digitoxigenin were biphasic, revealing the presence of high-affinity [concentration of drug causing 50% inhibition (IC50) = 10 nM] and low-affinity (ICs0 = 2 pM) forms with proportional contributions of 55% and 45%, respectively. The involvement of the high-affinity form in the positive inotropic effect of digitalis and of the low-affinity sites in both inotropy and toxicity are consistent with the literature data on rodents.
Molecular Cell, 1999
muscle. The ␣ subunit of the Na,K-ATPase also contains 3 Department of Molecular and Cellular Physiology the receptor site for the cardiac glycoside class of com-4 Division of Cardiology pounds (such as ouabain and digoxin), which inhibit University of Cincinnati College of Medicine the Na,K-ATPase, increase cardiac contractility, and Cincinnati, Ohio 45267 are used in the treatment of congestive heart failure (Thomas et al., 1990).
The Cellular Basis of Cardiovascular Function in Health and Disease, 1997
In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidase-diaminobenzidine methods, demonstrated α 1 isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For α 2 isoenzyme, immunoreactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For α 3 isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent α 1 isoenzyme of Na,K-ATPase. A differential as well as unique localization of α subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites. (Mol Cell Biochem 176: [107][108][109][110][111][112] 1997)
Circulation Research, 1994
There are three isoforms of the catalytic (alpha) subunit of the Na+,K(+)-ATPase, each derived from a different gene, that differ in their sensitivity to inhibition by cardiac glycosides. Antibodies specific for the three isoforms were used to study Na+,K(+)-ATPase isoform expression in ventricular myocardium, where an understanding of digitalis receptor diversity is most important. In the rat heart, there is simultaneous expression of two isoforms in adult ventricle, and immunofluorescence studies demonstrated that both isoforms are expressed uniformly in cardiomyocytes. Hypertension and hypertrophy have been reported to selectively depress alpha 2 isoform mRNA levels, and we show in the present study that alpha 2 protein levels were correspondingly depressed in rats made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate and a high-salt diet. In the human heart, where mRNA for all three alpha isoforms has been reported, we detected all three isoform prot...
Immunodetection and enzymatic characterization of the α3-isoform of Na,K-ATPase in dog heart
FEBS Letters, 1994
The expression of the canine a 2 and 3 subunit isoenxymes of NA,K-ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K-ATPase preparations, one immunoreactive signal with rat specific cc3 antisera in cardiac tissues, and two immunoreactive signals with rat a, and cc3 antisera in cerebral tissues. These findings suggested the specific expression of a3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the a 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain a, fusion protein did not cross-react with dog antigens whatever the three tissues tested. Expression of the a,-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC,, = 7 + 2 nM). A single component with low affinity to digitoxigenin (IC&, = 110 f 10 nM) characterized the a, kidney form. The mixture of a, and a3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (I& = 17 + 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac a,form (& = 10 ? 1.9 mM) and for the cerebral a2 and a3 mixture (&,, 19.6 ? 4.9 mM). The sensitivities for Na' of the low affinity sites (a,) were: 6.7 f 1.4 mM, 6.3 f 1.2 mM and 11.6 + 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the a3 subunit isoenzyme in canine cardiac plasma membranes.