Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Membrane (Na+ + K+)-ATPase of canine brain,heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures

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-dependent 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.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.     

NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

The nuclear magnetic resonance (NMR) spectrum of Na⁺ is suitable for qualitative and quantitative analysis of Na⁺ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na⁺ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na⁺ gives no detectable NMR spectrum. This is probably due to complexation of Na⁺ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na⁺ interacts with ion exchange resin. NMR also indicates that about 60% of Na⁺ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na⁺. NMR studies show that Na⁺ is complexed by actomyosin, which may be the molecular site of complexation of some Na⁺ in muscle. The same studies indicate that the solubility of Na⁺ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na⁺ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na⁺ become appropriate.     

A rapid method for preparation of sarcolemma from frog skeletal muscle

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na⁺, K⁺ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and K⁺ -activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney

The rates of association of [³H]ouabain to Na⁺,K⁺-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na⁺,K⁺-ATPase being the fastest. Although the major determinant of the affinity of the Na⁺,K⁺-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na⁺,K⁺-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I₅₀ (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na⁺,K⁺-ATPase activity, approached the value calculated from [³H]ouabain binding. The ratio of the I₅₀ value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na⁺,K⁺-ATPase is not the reason for the faster dissociation rate of this enzyme.     

Effects of vanadate on Na+,K+-ATPase and on the force of contraction in guinea-pig hearts.

Vanadate has been shown to be a potent inhibitor of isolated Na⁺,K⁺-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na⁺,K⁺-ATPase. Vanadate inhibited isolated Na⁺,K⁺-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K⁺-stimulated phosphatase activity was more sensitive than Na⁺,K⁺-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [³H] oubain binding in the presence of Mg²⁺, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive ⁸⁶Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na⁺,K⁺-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na⁺,K⁺-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.     

Evidence for a role for cardiac myosin in regulating the contractile response.

Dinitrophenylated bovine cardiac myosin incorporates 1.3 mol of 1-fluoro-2,4-dinitro-benzene per 5 × 10⁵ g of protein. Concomitantly there was an activation of the Ca²⁺-ATPase activity and an inhibition of the K⁺(EDTA)-ATPase activity. The dinitrophenyl group is located in the smallest active proteolytic fragment, subfragment 1. Virtually all of the labeling occurs in the region containing the heavy chains of cardiac myosin as judged by dissociation experiments in sodium dodecyl sulfate. Dinitrophenylated myosin failed to form calcium-sensitive actomyosin when tested in an ATPase assay system containing actin, tropomyosin, troponin and ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid. Thiolysis of the dinitrophenyl group from myosin with 2-mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. The Ca²⁺ and K⁺(EDTA)-ATPase activities were also restored to control values. These results indicate that cardiac myosin participates in the regulation of the interaction between the contractile proteins.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

(Na (+) + K (+)-stimulated ATPase of human kidney, normal and adenocarcinoma. Phosphorylation and inhibition by antitumor proteins

(Na⁺+K⁺)-ATPase was purified from human kidney of normal and tumor tissue with specific activities of 100.0 and 16.6 μmol Pi/mg/h, respectively. The antitumor proteins, macromomycin, largomycin, and NSC 327459 (50 μg/ml each) caused 70 to 90% inhibition of (Na⁺+K⁺)-ATPase from tumor tissue, whereas auromomycin had no effect. (Na⁺+K⁺)-ATPase from both sources could be phosphorylated by rabbit muscle protein kinase; there was 3 to 6-fold stimulation of phosphorylation by cyclic AMP. Phosphorylation resulted in 70 to 80% decrease in (Na⁺+K⁺)-ATPase activity, and caused the normal enzyme to become sensitive to inhibition by macromomycin.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A

(Na⁺ + K⁺)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na⁺ + K⁺)-ATPase activity, while their ouabain-insensitive Mg²⁺-ATPase is markedly lowered. A solubilized (Na⁺ + K⁺)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21μmol P_i/h per mg protein). Concanavalin A has no effect on these partially purified (Na⁺ + K⁺)-ATPase, while it inhibits (40%) this activity in less purified fractions which still contain Mg²⁺-ATPase activity.     

Sulfatide content and (Na++K+)-ATPase activity of skin and gill during larval development of the chilean frog,Calyptocephalella caudiverbera

Summary The sulfatide content, phospholipid concentration, and (Na⁺+K⁺)-ATPase activity from skin and gills of different stages of larval development ofCalyptocephalella caudiverbera (a Chilean frog) were analyzed. Additionally, the short-circuit current in skin was studied. When skin and gills, depending on the stage of larval development, present (Na⁺+K⁺)-ATPase activity, they have a high ratio of sulfatide to amount of membrane and the phosphatidylserine concentration remains unchanged. Sulfatide content and (Na⁺+K⁺)-ATPase activity in skin are in direct relationship with the level of sodium flux present during development. The specific enzymatic hydrolysis of sulfatide with partially purified arylsulfatase of pig kidney inhibits 100% of the ouabain-sensitive (Na⁺+K⁺)-ATPase. The ouabain-insensitive ATPase remains virtually unchanged with the treatment, even with a high concentration of arylsulfatase or with ouabain present in the medium. These experiments strongly suggest a role of sulfatides in the (Na⁺+K⁺)-ATPase activity and, as a consequence, in sodium ion transport.     

Effect of ethylisopropyl amiloride,a Na+-H+ exchange inhibitor,on cardioprotective effect of ischaemic and angiotensin preconditioning

The present study is designed to investigate the role of Na⁺-H⁺ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na⁺-H⁺ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na⁺-H⁺ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na⁺-H⁺ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning.     

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

The activity of a partially purified bovine heart Na⁺,K⁺-ATPase is inhibited by DL- and L- palmitylcarnitine (I₅₀=44–48μM). Palmitylcarnitine with a I₅₀ of 25μM also markedly inhibits K⁺-phosphatase activity. Palmityl-CoA decreases Na⁺,K⁺-ATPase activity, but to a lesser extent (I₅₀=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na⁺,K⁺-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na⁺ + K⁺)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na⁺ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na⁺ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na⁺ transport sensitively. This technique relies on the presence of Na⁺-Ca²⁺ exchange and ATP-driven Na⁺ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na⁺ uptake is monitored by a subsequent Na_i⁺-dependent Ca²⁺ uptake reaction (Na⁺-Ca²⁺ exchange) using ⁴⁵Ca²⁺. We present evidence that the Na⁺-Ca²⁺ exchange will be linearly related to the prior active Na⁺ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na⁺ isotopes. Applying this method, we measure cardiac ATP-dependent Na⁺ transport and (Na⁺ + K⁺)-ATPase activities in identical ionic media. We find that the (Na⁺ + K⁺)-ATPase and the Na⁺ pump have identical dependencies on both Na⁺ and ATP. The dependence on [Na⁺] is sigmoidal, with a Hill coefficient of 2.8. Na⁺ pumping is half-maximal at [Na⁺] = 9 mM. The K_m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na⁺ pumping. This approach should allow other new investigations on on ATP-dependent Na⁺ transport across cardiac sarcolemma.