31P NMR studies of the kinetics and regulation of oxidative phosphorylation in the intact myocardium.
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Adenosine Triphosphatases↗
Biomedical subjects
A H From
Publications and source records attributed to A H From.
31P-NMR studies of respiratory regulation in the intact myocardium.
The mechanism by which mitochondrial respiration is coupled to ATP consumption in intact tissues is unclear. We determined the relationship between high-energy phosphate levels and oxygen consumption rate in rat hearts operating over a range of workloads and perfused with different substrates. With pyruvate +glucose perfusion, ADP levels were in general very low, and varied with MVO2 yielding an apparent Km of 25 +/- 5 microM, suggesting regulation of oxidative phosphorylation through availability of ADP. In contrast, with glucose perfusion in the presence or absence of insulin, ADP levels, ADP/ATP ratio or the phosphate potential were relatively constant over the workload range examined and generally not correlated with alterations in MVO2; it is suggested that under these conditions, carbon substrate delivery to the mitochondria may control mitochondrial respiration. The common feature of both of the suggested regulatory mechanisms is substrate limitation which, however, is exercised at different metabolic points depending on the carbon substrate available to the myocardium.
Adenosine Diphosphate↗
31P NMR measurement of ATP synthesis rate in perfused intact rat hearts.
Using 31P NMR and the saturation-transfer method, the unidirectional rate of ATP synthesis was measured in isolated, Langendorff-perfused, isovolumic rat hearts operating at a rate pressure product of 25.6 +/- 2.5 (SE) X 10(3) mmHg X min-1 and consuming O2 at a rate of 35 +/- 2 mumol O2 X min-1 X (g dry wt)-1, at 37 degrees C. This rate was 7.2 +/- 0.9 mumol X s-1 X (g dry wt)-1 and was related to the rate of oxygen atom consumption by a ratio of 6.3 +/- 0.9. These data show that in the intact heart the unidirectional rate of ATP synthesis exceeds the net rate of ATP synthesis and consumption by approximately a factor of 2.
Adenosine Triphosphate↗
Measurement of an individual rate constant in the presence of multiple exchanges: application to myocardial creatine kinase reaction.
Forward [creatine phosphate (CP)----adenosine 5'-triphosphate (ATP)] and reverse (ATP----CP) fluxes of myocardial creatine kinase (CK) measured by using 31P nuclear magnetic resonance (NMR) and conventional saturation transfer (CST) methods are unequal; this is a paradoxical result because during steady state fluxes into and out of the CP pool must be the same. These measurements, however, treat the CK reaction as a two-site exchange problem and ignore the presence of the ATP gamma in equilibrium Pi exchange involving the ATPases. We have applied a method [Uğurbil, K. (1985) J. Magn. Reson. 64, 207] based on the saturation of multiple resonances, by which a single unidirectional rate constant can be measured unequivocally in the presence of multiple exchanges, to the measurement of CK fluxes in isovolumic rat hearts perfused under three different conditions; two of the three perfusion conditions showed a large discrepancy in the CK fluxes determined by CST, and one did not. In contrast, when the effect of the ATP gamma in equilibrium Pi exchange on the CK rate measurements was eliminated, multiple saturation transfer (MST) measurements on the same hearts yielded equal forward and reverse fluxes in all cases. The rate constant for the ATP gamma----CP conversion measured by MST was larger than the value obtained by the conventional methodology whereas both methods gave the same rate constant in the CP----ATP direction. These results demonstrate that the cause of the paradoxical data obtained by CST measurements of CK kinetics is the ATP gamma in equilibrium Pi exchange and that CK rates when determined rigorously are consistent with the CK reaction being in equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Cardiac glycosides. 6. Gitoxigenin C16 acetates, formates, methoxycarbonates, and digitoxosides. Synthesis and Na+,K+-ATPase inhibitory activities.
A series of 17 gitoxigenin 16 beta-formates, acetates, and methoxycarbonates was synthesized, including their 3 beta-acetates, formates, and digitoxosides. A 16 beta-formate group was generally found to increase activity 30 times, a 16 beta-acetate group 9-12 times, while a 16 beta-methoxycarbonate decreased activity by two-thirds. 3 beta-Formates and acetates had little effect on activity by themselves, but sometimes reduced the activity-increasing properties of 16 beta-formates and acetates. A 3 beta-digitoxoside increases the activity of gitoxigenin by 15 times, but the effect is less if the 16 beta-group is esterified. And finally, a 16-one decreases activity dramatically. These data suggest an important role for C16 esters and possibly the presence of a separate binding site on Na+,K+-ATPase corresponding to the cardenolide C16 position.
Animals↗
Cardiac glycosides. 7. Sugar stereochemistry and cardiac glycoside activity.
Digitoxigenin alpha-L-, beta-L-, alpha-D-, and beta-D-glucosides; alpha-L-, beta-L-, alpha-D-, and beta-D-mannosides; and alpha-L- and beta-L-rhamnosides were stereoselectively synthesized from the corresponding sugar tetrabenzyl trichloroacetimidates. The Na+,K+-ATPase receptor inhibitory activities of these glycosides (as a measure of receptor binding) were compared with those of digitoxigenin, digitoxigenin 6'-hydroxy-beta-D-digitoxoside, digitoxigenin beta-D-galactoside, and digitoxigenin beta-D-digitoxoside. The observed activities reveal that a given sugar substituent may have a role in binding of some glycoside stereoisomers, but not others. With alpha-L- and possibly beta-L-rhamnosides, the 5'-CH3 and 4'-OH appear to have a predominant role in binding to the Na+,K+-ATPase receptor. Addition of a 6'-OH to form the corresponding mannosides dramatically disrupts the effect of both the 5'-CH3 and 4'-OH in prompting receptor binding of the alpha-L isomer. However, with the beta-L isomer, some influence of 4'-OH, 3'-OH, and 2'-OH binding remains. With beta-D-glycosides, binding via the "5'-CH3 site" appears to be of little importance and addition of a 6'-OH diminishes activity only slightly. With these beta-D-glycosides, an equatorial 4'-OH, axial 3'-OH, and equatorial 2'-OH groups appear to contribute to binding.
Cardiac Glycosides↗
Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator.
The mechanism of hypoxia-induced pulmonary vasoconstriction remains unknown. To explore the possible dependence of the hypoxic response on voltage-activated calcium (Ca2+) channels, the effects of BAY K 8644 (BAY), a voltage-dependent Ca2+ channel potentiator, were observed on the pulmonary vascular response to hypoxia of both the intact anesthetized dog and the perfused isolated rat lung. In six rat lungs given BAY (1 X 10(-6)M), hypoxia increased mean pulmonary arterial pressure (Ppa) to 30.5 +/- 1.7 (SEM) Torr compared with 14.8 +/- 1.2 Torr for six untreated rat lungs (P less than 0.01). After nifedipine, the maximum Ppa during hypoxia fell 14.1 +/- 2.4 Torr from the previous hypoxic challenge in the BAY-stimulated rats (P less than 0.01). BAY (1.2 X 10(-7) mol/kg) given during normoxia in seven dogs increased pulmonary vascular resistance 2.5 +/- 0.3 to 5.0 +/- 1.2 Torr X 1(-1) X min (P less than 0.05), and systemic vascular resistance 55 +/- 4.9 to 126 +/- 20.7 Torr X 1(-1) X min (P less than 0.05). Systemic mean arterial pressure rose 68 Torr, whereas Ppa remained unchanged. Administration of BAY during hypoxia produced an increase in Ppa: 28 +/- 1.5 to 33 +/- 1.9 Torr (P less than 0.05). Thus BAY, a Ca2+ channel potentiator, enhances the hypoxic pulmonary response in vitro and in vivo. This, together with the effect of nifedipine on BAY potentiation, suggests that increased Ca2+ channel activity may be important in the mechanism of hypoxic pulmonary vasoconstriction.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
The effect of 16 beta-substitution on the structure and activity of digitoxigenin: is there an additional binding interaction with Na+,K+-ATPase?
We have studied the basis of the effect of 16 beta-substitution on the structure and activity of digitoxigenin derivatives by examining the crystal structures of these compounds and their inhibitory activity toward the receptor for these drugs, Na+,K+-ATPase. To understand the increase in inhibitory activity of the 16 beta-ester compounds and the decrease in activity of gitoxigenin (16 beta-hydroxydigitoxigenin), both with respect to digitoxigenin, we have compared the observed conformations of gitoxigenin, gitoxigenin 16 beta-formate, and other 16 beta-esters to that of digitoxigenin. Our data do not support the possibility of hydrogen bonding between the 16 beta-hydroxyl of gitoxigenin and the lactone ring, previously suggested to account for the decreased activity of gitoxigenin vis à vis digitoxigenin, but, rather, suggest that the decreased activity may be due to an intramolecular hydrogen bond between the hydroxyls on C-14 and C-16 and an unusual D-ring conformation which combine to alter the carbonyl oxygen of the lactone ring away from the putative active position. In contrast, the 16 beta-ester moiety has a preferred conformation which may serve to fix the lactone ring in the active conformation. Thus, the increased activity of the 16 beta-esters cannot be explained by altered carbonyl oxygen position and may be related to an additional receptor binding site for the ester moiety.
Animals↗
High resolution proton NMR studies of perfused rat hearts.
High resolution 1H NMR spectra of perfused rat hearts have been obtained under normoxic, ischemic and hypoxic conditions. Several myocardial metabolites including taurine, carnitine, lactate and tissue glycerides are detected in the 1H NMR spectra. Changes in oxygen availability induce perturbations in the levels of some metabolites, in particular, lactate. Experiments with fasted rats and with substrate-free perfusion suggest that the glycerides detected in 1H spectra are metabolically mobilizable but have a slow rate of turnover. These results demonstrate that utility of 1H NMR in monitoring myocardial metabolism.
Animals↗
The inotropic activity of digitalis genins is dependent upon C(17) side-group carbonyl oxygen position.
The inotropic potencies of four digitalis genins were studied utilizing cat left atrial strips. The genin concentration required to induce a 50% increase of isometric tension (T50) was found to closely correlate with the degree of displacement (D) of the C(17) side-group carbonyl oxygen from the position of that atom in digitoxigenin. The line of regression was: log T50 = 0.54D - 6.85, r2 = 0.98, P less than 0.008. These observations were related to recently reported cat ventricular Na+, K+ -ATPase inhibitory potencies of the same genins [expressed as 50% inhibitory (I50) concentrations]. I50 correlated strongly with T50: log I50 = 0.78 log T50 - 1.68, r2 = 0.99, P less than 0.003. Thus, the activity of digitalis genins towards their receptor in intact cardiac tissue is closely related to genin carbonyl oxygen position as well as to Na+, K+ -ATPase inhibitory activity. These results further support our earlier conclusions, based upon isolated Na+, K+ -ATPase studies, that the digitalis genin C(17) side-group carbonyl oxygen position versus activity relationship is biologically relevant and may prove to be a useful unifying structural model in the further elucidation of the mechanism of digitalis-receptor interactions.
Animals↗
Cardiac glycosides. 1. A systematic study of digitoxigenin D-glycosides.
A series of digitoxigenin glycosides was studied: five with beta-D-sugars varying stepwise in sugar structure from beta-D-digitoxose to beta-D-galactose, including one beta-D/alpha-D pair. I50 values for these glycosides and digitoxigenin were determined with hog kidney Na+, K+-ATPase. These data suggest a major and unexpected role for 4'-OH conformation in the sugar. All the glycosides with an equatorial 4'-OH were more active than the two with the 4'-OH axial [digitoxigenin beta-D-galactoside (6) I50 = 6.45 X 10(-8) M; digitoxigenin 2'-deoxy-alpha-D-ribo-hexopyranoside (alpha-3a) I50 = 9.33 X 10(-8) M; digitoxigenin I50 = 1.17 X 10(-7) M]. Stereochemistry of the 3'-OH had much less of an activity role than that of the 4'-OH, in contrast to existing models of "sugar-site" binding.
Animals↗
Variables affecting measurement of human red cell Na+,K+ATPase activity: technical factors, feeding, aging.
Because it has been suggested that decreased activity at the erythrocyte sodium pump might be the cause of age-related decreases in basal oxygen consumption, we have examined age-associated changes in Na+,K+-ATPase activity in red cell membranes. The initial portion of this study was directed toward elucidating possible methodological pitfalls in membrane preparation which might account for some of the variable results reported in prior erythrocyte Na+,K+-ATPase studies. We found that two of four red cell membrane fractions have substantial Mg2+-ATPase activity and contribute a significant portion of total membrane protein. As these two fractions contain little Na+,K+-ATPase activity their contamination of the other two fractions could cause significant variation in measured Na+,K+-ATPase activity. Additionally, we found that meal feeding raised Na+,K+-ATPase activity necessitating that measurements be made in the fasting state. With these methodological variables controlled, we found only a 10.8% coefficient of variation between fasting samples obtained on separate days in eight subjects. Using this methodology, we observed no correlation of Na+,K+-ATPase specific activity with age in males, and only a weak correlation in females who showed decreasing Na+,K+-ATPase specific activity occurring with advancing age. These observations suggest that changes in erythrocyte Na+,K+-ATPase activity do not cause the age-related fall in basal oxygen consumption.
Adenosine Triphosphatases↗
Photoaffinity labeling of the sodium- and potassium-activated adenosinetriphosphatase with a cardiac glycoside containing the photoactive group on the C-17 side chain.
The synthesis and properties of a radiolabeled glycoside photoaffinity probe, [3H]-(3 beta,5 beta,14 beta, 20E)-24-azido-3-[(2,6-dideoxy-beta-D-ribo-hexopyranosyl) oxy]-14-hydroxy-21-norchol-20(22)-en-23-one, containing the photoactive group at the C-17 side chain of the steroid moiety are reported. The molecule binds to the sodium- and potassium-activated adenosinetriphosphatase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl2, 3 mM phosphate, 2 mM ethylenediaminetetraacetic acid, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37 degrees C] in the dark with an equilibrium dissociation constant of (1.4 +/- 0.3) X 10(-7) M. Ultraviolet irradiation of a solution of enzyme plus 3H-labeled probe, followed by analysis of covalently incorporated radiolabel, shows ouabain-displaceable labeling exclusively of the alpha subunit of the sodium- and potassium-activated adenosinetriphosphatase. These data indicate that the binding site of the C-17 side group of cardiac glycosides is located on or near the alpha subunit of this enzyme.
Affinity Labels↗
Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.
Previous models of digitalis genin interaction with the (Na+,K+)-ATPase system (the putative receptor for such drugs) were deficient in explaining the (Na+,K+)-ATPase inhibitory activity of a number of digitalis genin analogues. With rat brain (Na+,K+)-ATPase we observed that the C-17 side chain carbonyl (C = O) oxygen distance of a given genin in relation to its position in the reference compound digitoxigenin was the primary determinant of its biological activity. With a number of genin analogues, we observed a strict correlation of this structural parameter with its binding site compatibility as well as inhibitory potency with respect to the (Na+,K+)-ATPase. In every case the correlation to inhibition data was obtained using a minimum energy conformation for the genin structure. The general applicability of that model is now proposed based on the following observations. The carbonyl oxygen position versus the biological activity relationship fully holds with (Na+,K+)-ATPase preparations from other tissues and species and also when different binding conditions are used for the enzyme genin interaction. The relationship is equally valid for the K+-p-nitrophenyl phosphatase activity. Correlations of the data obtained under these various conditions provide further support for this relationship and for the concept that altered affinities of the enzyme for a given genin under different binding conditions reflect conformational variations of a single binding site.
Animals↗
Erythrocyte Na+, K+-ATPase and serum digoxin concentrations.
Digoxin therapy has been made more rational by the measurement of serum digoxin concentrations. However, difficulties remain because of the overlap between "therapeutic" and "toxic" serum concentrations and the lack of an obvious therapeutic endpoint in many patients. An assay which measures the degree of interaction between digoxin and its putative receptor, the membrane Na+, K+-ATPase, might be capable of resolving some of these difficulties. Therefore, as a first approach in this direction we evaluated the relationship between serum digoxin concentration and the degree of inhibition of RBC ghost Na+, K+-ATPase activity in patients receiving digoxin therapy. Utilizing an improved micro-assay technique, Na+, K+-ATPase activity was determined in aliquots of RBC ghosts before and after removal of bound digoxin. In 27 patients a significant relationship was present between serum digoxin concentration and the degree of RBC ghost Na+, K+-ATPase inhibition. However, at any serum digoxin concentration, there was a variation in the magnitude of enzyme inhibition from patient to patient. This study confirms the feasibility of determining the degree of in vivo RBC Na+, K+-ATPase inhibition in man and demonstrates, for the first time, a highly significant but somewhat variable relationship between serum digoxin concentrations and the magnitude of RBC digoxin receptor inactivation. This quantitative, functional, individualized assay of digoxin effects may prove to be of clinical value in the future.
Digoxin↗
Is the erythrocyte sodium pump altered in human obesity?
Ouabain binding and electrolyte concentrations of erythrocytes, and Na+, K+-ATPase activity of red cell ghosts were measured in normal and obese subjects, ranging from 88-257% of their ideal body weight. All three independent measurements were virtually the same in obese and nonobese groups, and no correlations were found between these three variables and the percentage of ideal body weight. These results differ from previous reports of either increased or decreased sodium pump function and suggest that Na+, K+-ATPase does not directly influence human obesity.
Adult↗
A report of Uhl's disease in identical adult twins: evaluation of right ventricular dysfunction with echocardiography and nuclear angiography.
The first example of Uhl's disease affecting identical adult twins is reported, offering support for the contention that a congenital developmental defect or hereditable tendency is the responsible cause. In one case, echocardiography and nuclear angiography proved to be valuable in making the diagnosis. Uhl's disease in the adult should be suspected among cases of isolated right ventricular enlargement and failure complicated by ventricular dysrhythmias. The diagnosis may be made using a combination of noninvasive tests, obviating the need for cardiac catheterization.
Adipose Tissue↗
Serum creatine kinase MB isoenzyme activity in long-term hemodialysis patients.
Serum creatine kinase MB isoenzyme (CK-MB) activity was determined in 46 male long-term hemodialysis patients without evidence of myocardial infarction. Thirteen (28.3%) showed mild elevations. The abnormality persisted in seven of eight patients on repeated measurement at three- to eight-month intervals. There was a significant correlation between serum CK-MB and CK-MN activity, and the activity of both enzymes rose after intramuscular injection. The reason for the abnormality is not known. It is possible that skeletal muscle is the source of elevated enzyme activity. Caution should be exercised in the interpretation of serum CK-MB activity in the diagnosis of acute myocardial infarction in this patient population.
Adult↗