Two functional Na+/K+-ATPase isoforms in the left ventricle of guinea pig heart (original) (raw)
Related papers
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.
Na,K-ATPase in the myocardium: molecular principles, functional and clinical aspects
PubMed, 2000
The role that Na,K-ATPase plays in Na+ and K+ antiport through the sarcolemma, in cation-homeostasis in cardiomyocytes as well as in excitation-contraction coupling and cell signalling in the myocardium is now widely recognized. It was its key importance for the cell membrane function that kept this enzyme intensively studied during the last three decades and finally brought to its discoverer the deserved Nobel Prize. Almost weekly are appearing new data concerning structure, function, regulation and role of the Na,K-ATPase in different physiological and pathological conditions. The special importance of the enzyme for heart function as well as the great amount of data that is concerned specifically with the heart Na,K-ATPase and accumulated since yet, started to call for setting them in order. The present paper updates basically important data on the cardiac Na,K-ATPase in relation to its specific properties, molecular mechanisms of function, mode of action, humoral and pharmacological modulation, adaptability, physiological role and clinical aspects.
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)
Turnover rates of the canine cardiac Na,K-ATPases
FEBS Letters, 1993
Two functional isoforms a (a,) and a' (a,) of the Na.K-ATPase catalytic subumt coexist m canine cardiac myocytes [J. Biol. Chem. (1987) 262, 8941-89431 The m vitro turnover rates of ATP hydrolysis have been determined in sarcolemma preparations by comparing ["Hlouabain-binding and Na,K-ATPase activity at various doses of ouabam (0.3-300 nM). The correlation between the occupancy of the ouabain-binding sites and the degree of Na.K-ATPase mhibition was not linear. The results showed that the form of low-affinity for ouabam (& = 300-700 nM) exhibited a lower turnover rate (88 + 10 vs. 147 ? 15 molecules of ATP hydrolyzed per second per ouabain-binding site) than the high affinity form (K, = I-8 nM). Thus our results indicate this specific isoform kmetic difference could contribute to differences in the cardiac cellular function.
Biochemical Pharmacology, 1980
We have recently reported that the heart (Na+ + K+)ATPase occurred on high 7 specific binding and low affinity of r3H10uabain to human binding sites. The dissociation constant (KD) of i'H]ouabain low affinity binding sites was close to ouabain K i, an indication that these sites are involved in the inhibition of this enzyme by the glycoside. In guinea-pig heart microsomes, only one group of [3Hlouabain binding sites were identified and the KD of these sites was close to ouabain K i (1). In intact guinea-pig heart, it was previously shown that r3H]ouabain interacts withtwo groups of specific binding sites and that the proportion of the high affinity sites is increased by increasing extracellular K + (2,3).
FACTORS AND AGENTS THAT INFLUENCE CARDIAC GLYCOSIDE‐Na+, K+‐ATPase INTERACTION*
Annals of the New York Academy of Sciences, 1974
The optimal conditions for the binding of cardiac glycosides, such as ouabain, to Na+, K+-ATPase in vitro are the simultaneous presence of ATP, Na+ and Mg++ or the presence of inorganic phosphate (Pi) and Mg++ (Reference 1). It has been postulated that in vitro binding of ouabain in the presence of ATP, Na+ and Mg++ reflects the binding of cardiac glycosides t o this enzyme system in intact animals,2 and it is this interaction that ultimately results in the production of the positive inotropic and/or arrhythmic effects3 Thus, studies on the factors and agents that influence the cardiac glycoside-Na', K+-ATPase interaction in vitro appear important for an understanding of the factors and agents that modify the magnitude of response t o cardiac glycosides in patients when the plasma concentrations of cardiac glycosides are maintained at a fixed level Moreover, such studies may shed light on the mechanism of the Na' , K+-ATPase reaction itself. Monovalent Cations Na+, K+-ATPase has been shown t o be an allosteric enzyme and its configurations are determined by monovalent cation and phosphate ligand~.~-' Therefore, it is reasonable t o assume that these ligands affect the ouabain-enzyme interactions. The binding of cardiac glycosides t o Na+, K+-ATPase in vitro in the presence of ATP, Na+ and Mg++ is markedly inhibited by K+ (Reference 1). Since K+ has been shown t o reverse cardiac glycoside-induced arrhythmias,l2> it was once assumed that K+ reduces the level of cardiac glycoside bound t o the Na+, K+-ATPase, presumably by reducing the rate of binding, increasing the rate of dissociation, or both. Supporting this hypothesis, a number of investigators has reported that K+ antagonized the ouabain-inhibition of the Na+, K+-ATPase activity in vitro, although the evidence does not quite fit simple competitive inhibition for ouabain with respect t o KCl (see Reference 14). The steady-state levels of the enzyme-bond ouabain at a given drug concentration in the medium are determined by two independent variables, i.e., the rate of binding and the rate of release. These two parameters can be studied separately. FIGURE 1A shows the results of an experiment in which the rate of [ 3 H ] ouabain binding was monitored. Na+, K+-ATPase preparations obtained from rat brain microsomal fractions following deoxycholic acid and NaI treatments were incubated at 37OC with 0.01 pM [ 3 H ] ouabain (specific activity; 13.2 Ci/mmol) in the presence of 20 mM NaCl, 5 mM Tris-ATP, and 50 mM Tris-HC1 buffer (pH 7.5). Aliquots were taken at the indicated times and the bound
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...
Biochimica Et Biophysica Acta - Biomembranes, 1978
Effects of commonly used purification procedures on the yield and specific activity of (Na ÷ + K*)-ATPase (Mg2÷-dependent, Na* + K÷-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP<tependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na*+ K*)-ATPase and the ratio between (Na*+ K*)-ATPase and Mg2÷-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na'+ K*)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na*+ K*)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain • enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter * To whom reprint requests should be sent. the properties of membrane (Na*+ K÷)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na ÷ + K÷)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.