Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats

Na+,K+-ATPase activity, phosphorylation, and [3H]ouabain binding in sarcolemma isolated from spontaneously hypertensive rat (SHR) hearts were compared to the same parameters in sarcolemma from normotensive rat (WKY) hearts. Sarcolemma prepared from SHR heart contained significantly less ouabain-inhibitable ATPase activity than sarcolemma from WKY heart. No significant differences in sarcolemmal protein content or recovery were noted between the two groups. The numbers of phosphorylation sites and ouabain binding sites were lower for SHR hearts than for WKY hearts. The KD values for ouabain binding were the same (0.30 muM) in cardiac sarcolemma of SHR and WKY. The I50 values for inhibition by ouabain of Na+,K+-ATPase were also the same for both groups (SHR = 49 microM; WKY = 44 microM). These data suggest that the decrease of cardiac sarcolemmal Na+,K+-ATPase activity in SHR hearts is due to a decrease in the number of active sites.     

alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable 86Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and K1/2 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type. The increase in abundance of the Na+ pump is associated with a 4-fold increase in abundance of the mRNA for the alpha 1 subunit of the Na+,K+-ATPase. We could not detect mRNA for alpha 2 or alpha 3 or for the beta subunits. The increase in abundance of the A system and Na+,K+-ATPase is associated with a negligible increase in intracellular Na+ concentration. We propose that the increase in the abundance of the A system and the Na+,K+-ATPase is the result of a mutation in regulatory gene R1 that controls the A system and the Na+,K+-ATPase and is not due to a primary effect of a possible initial increase in Na+ concentration.     

3H]ouabain binding of red blood cells in whites and blacks

In a previous study, we demonstrated that the red blood cell Na+ concentration and Na+,K+-ATPase activity are sex-dependent and race-dependent: a higher intracellular Na+ concentration in blacks and men was associated with a lower Na+,K+-ATPase activity. To examine whether the low Na+,K+-ATPase activity is due to a decreased number of enzyme units, altered structure of the enzyme, or the presence of an endogenous digoxinlike substance, ouabain binding studies were performed on the same subject group. The measurements included displacement of [3H]ouabain from its specific binding sites by unlabeled ouabain or potassium. The results demonstrate that groups with lower enzyme activity manifest lower numbers of total specific ouabain binding sites on the surface of the red blood cell (mean +/- SD: blacks, 654 +/- 24.4; whites, 806 +/- 18.3; women, 806 +/- 26.9; men, 728 +/- 21.2). Other kinetic parameters of [3H]ouabain displacement appear to be the same among the groups. The respective red blood cell Na+ and K+ concentrations were negatively and positively correlated with the number of ouabain binding sites. Our findings suggest that the lower activity of red blood cell Na+,K+-ATPase in blacks and men is a function of a lower number of Na+-K+ pump units. The results also indicate that sex and race should be considered when red blood cell ouabain binding is examined.     

Possible mechanism responsible for mechanical dysfunction of ischemic myocardium: a role of oxygen free radicals

It has been proposed that a major target organelles damaged by the ischemic process, probably by the oxygen free radicals generated, is the portion of the excitation-contraction coupling system that regulates Ca2+ delivery (the sarcoplasmic reticulum and sarcolemma) to the contractile proteins. We tested this hypothesis by studying the effect of in vitro generation of oxygen free radicals from xanthine-xanthine oxidase system or dihydroxyfumarate (DHF)/Fe3+-ADP system on Ca2+ flux behavior of canine cardiac sarcoplasmic reticulum (SR); sarcolemmal (Na+, K+)-ATPase and Na+-Ca2+ exchange activities; and myofibrillar (Ca2+, Mg2+)-ATPase activity. Generation of oxygen free radicals by xanthine oxidase acting on xanthine as a substrate increased the passive Ca2+ efflux and decreased intravesicular Ca2+ with no effect on active Ca2+ influx (Ca2+-ATPase) of SR vesicles. Similar exposure of sarcolemmal vesicles to xanthine plus xanthine oxidase stimulated Na+-Ca2+ exchange activity. When sarcolemmal vesicles were incubated with DHF plus Fe3+-ADP, (Na+, K+)-ATPase activity was decreased. It is postulated that the SR Ca2+ efflux pathways but not catalytic activity of the Ca2+ pump and sarcolemmal (Na+, K+)-ATPase involving Na+-Ca2+ exchange activity are altered by oxygen free radicals, and such changes may partly account for the occurrence of intracellular Ca2+ overload during the course of myocardial ischemia. Interestingly, oxygen free radicals from xanthine-xanthine oxidase system had no effect on myofibrillar pCa-ATPase curve. From this set of observations we would hypothesize that the SR and sarcolemma may be the principal target organelles of oxygen free radicals attack in the ischemic injury and not the contractile proteins per se.     

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.     

Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme.

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.     

Studies on the specificity of the effects of oxygen metabolites on cardiac sodium pump

Isolated myocytes of rat heart, and sealed sarcolemmal vesicles of bovine heart, were used to examine the selectivity of the effects of partially reduced oxygen species (generated by a mixture of xanthine and xanthine oxidase) on cardiac sodium pump and several other ion transporters of the plasma membrane. When myocytes were exposed to xanthine plus xanthine oxidase, there were time-dependent inhibitions of ouabain-sensitive 86Rb+ uptake and (Na+ + K+)-ATPase activity that could be prevented by allopurinol, or by catalase and superoxide dismutase; suggesting the involvements of H2O2 or oxygen free radicals in the inhibition of the pump. This inhibition preceded any significant decrease in cellular ATP or in the number of viable cells. While ouabain increased 45Ca2+ uptake by myocytes as expected, exposure to xanthine plus xanthine oxidase decreased 45Ca2+ uptake; suggesting that the Na+, Ca2(+)-exchanger of the intact myocytes is also inhibited by oxygen metabolites. Simultaneous inhibitions of the pump, the Na+, Ca2(+)-exchange, the Na+, H(+)-exchange, and the Na+, Pi-cotransport activities also occurred in sarcolemmal vesicles that were treated with xanthine plus xanthine oxidase. These findings indicate that inactivations of the sodium pump and other sarcolemmal ion carriers are early events in the oxidant-induced damage to the cardiomyocyte. In the rat heart myocytes, a fraction of (Na+ + K+)-ATPase that seems to be more sensitive to ouabain, was inactivated more rapidly upon exposure of myocytes to xanthine plus xanthine oxidase; raising the possibility of the existence of different pump populations with different sensitivities to extracellularly generated oxygen metabolites.     

Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.     

Intact vesicles of canine cardiac sarcolemma: evidence from vectorial properties of Na+, K+-ATPase.

Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmalion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K+ of Na+, K+-ATPase and its associated K+-phosphatase activities, using as substrates ATP or p-nitrophenylphosphate (pNPP) in the presence of Mg2+. Optimal concentrations of K+ alone (10 mM) stimulated p-nitrophenylphosphatase (pNPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na+ plus K+ concentrations (100 mM and 10 mM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 +/- 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg2+-ATPase activity by 70-90%. The K+-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K+ fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg2+-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by oubain, are consistent with the hypothesis that permeability-intact vesicles of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesicles highly permeable to the ions, substrates, and ouabain.     

Inhibition of Na+,K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function

BACKGROUND & AIMS: To determine how interferon (IFN)-gamma inhibits epithelial barrier and ion transport functions, intestinal T84 cells were studied. METHODS: Acute and chronic effects of IFN-gamma on T84 barrier function, Na+,K+-adenosine triphosphatase (ATPase) activity, and certain ion transport and tight junctional proteins were determined. To assess the role of Na+,K+-ATPase and intracellular Na+, similar studies with the Na+,K+-ATPase inhibitor ouabain and Na+ ionophore monensin were performed. To determine the role of nitric oxide (NO), the NO donor SPER-NO was used. RESULTS: IFN-gamma acutely (<6 hour) decreased cellular Na+,K+-ATPase activity, followed later (>24 hours) by decreases in expression of Na/K/2Cl, the alpha subunit of Na+,K+-ATPase, occludin, and ZO-1. In contrast, cystic fibrosis transmembrane conductance regulator or the Na+ pump beta subunit were unchanged. Ouabain and monensin caused nearly identical changes to IFN-gamma. Incubation in low Na+ media significantly blunted the chronic effects of IFN-gamma. Hypotonic-induced cell swelling, in contrast, had effects similar to IFN-gamma but did not alter the expression of the Na+ pump alpha subunit. The NO donor SPER-NO rapidly inhibited Na+,K+-ATPase and also down-regulated transport and barrier proteins. CONCLUSIONS: IFN-gamma inhibition of Na+,K+-ATPase activity acutely causes increases in intracellular Na(i) concentration and cell volume, which are distinct signaling events that ultimately result in a leaky and dysfunctional epithelium associated with chronic inflammation.     

The search for a hypothalamic Na+,K+-ATPase inhibitor

Accumulating experimental evidence suggests that natriuresis in response to intravascular volume expansion is promoted by an endogenous regulator of Na+,K+-adenosine triphosphatase (ATPase). Efforts to purify this substance by a number of laboratories have as yet been unsuccessful. The properties of partially purified inhibitors from plasma, urine, and tissue often fail to possess the characteristics thought to be consistent with those of a physiological regulator. These include potency (Ki of approximately 1 nM), reversibility of inhibition, specificity for Na+,K+-ATPase, and responsiveness to relevant physiological stimuli. Two rather different candidate substances, extracted from urine and hypothalamus, have been purified to a high degree. Neither is a peptide, and both are of low molecular weight and resistant to acid hydrolysis. The substance from urine is rather nonpolar and interacts with digoxin-specific antibodies, while that from hypothalamus is polar and does not appear to share epitopes with the cardiac glycosides. On the serosal surface of the toad urinary bladder, the hypothalamic substance causes a reversible inhibition of Na+ transport, inhibits rubidium uptake in red blood cells by acting on the membrane's exterior surface, inhibits binding of ouabain to purified Na+,K+-ATPase, and reversibly inhibits hydrolysis of adenosine 5'-triphosphate by the enzyme with a Ki of 1.4 nM. The hypothalamic inhibitor may be differentiated from ouabain by their respective ionic requirements for optimal inhibition of enzymatic activity, and although both ouabain and the hypothalamic inhibitor fix Na+,K+-ATPase in its E2 conformation, the hypothalamic inhibitor does not promote phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. Ionic requirements for inhibition also differentiate the hypothalamic inhibitor from vanadate ion, as does the inhibitor's activity in the presence of norepinephrine. Further enzymological and physiological studies will be facilitated by structural characterizations of the inhibitory substances and by the availability of a method to measure their concentrations in physiological fluids.     

Sarcolemmal alterations during the development of genetically determined cardiomyopathy

The purpose of this study was to identify alterations in specific enzyme and Ca2+ binding activities in cardiac sarcolemmal fractions from UM-X7.1 myopathic Syrian hamsters during the development of cardiomyopathy. Experimental and healthy control animals were examined from 25 to 200 days of age. Sarcolemmal Na+, K+-ATPase activity was depressed in the myopathic hamsters throughout the time course of this study. Sarcolemmal ATP-independent Ca2+ binding was found to be depressed in experimental animals as early as 55 days of age. Ca2+ -stimulated, Mg2+ -dependent ATPase activity was depressed in the experimental animals by 90 days of age and this decrease in enzyme activity was accompanied by a decrease in ATP-dependent Ca2+ binding capacity of the sarcolemmal membranes. Mg2+ -ATPase and Ca2+ -ATPase activities were only affected in the latter stages of the disease (155 to 200 days old). NaF, epinephrine and Gpp(NH)p stimulation of the sarcolemmal adenylate cyclase activity was also observed to be attenuated during the latter stages of the disease. These defects in adenylate cyclase system of the sarcolemmal fraction appeared specific since basal adenylate cyclase activity was not altered at any age studied. The results demonstrate that the earliest lesions in sarcolemmal activity in myopathic hamster heart occur in Na+, K+-ATPase and ATP-independent Ca2+ binding capacity. These defects correspond temporally to the initial stages of cardiac necrotic development in this strain of myopathic hamster.     

Active calcium and sodium transport by cardiac plasma membranes in the genetically hypertensive rat

Active Na+ and Ca2+ transports by sarcolemmal vesicles from young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were compared. The effects of the calmodulin and the calcium antagonist nifedipine on Ca2+ binding ATP-dependent accumulation of Ca2+ were studied at free Ca2+ concentrations of 2.10(-8)M and 4.10(-7)M. 2.10(-7)M calmodulin stimulated Ca2+ binding to SHR membranes up to a level equivalent to that in WKY, whereas it enhanced active Ca2+ transport more in WKY than in SHR, thus suppressing the difference between the two substrains. At a 2.10(-8)M free Ca2+ concentration low concentrations of nifedipine (10(-7) to 10(-6)M) induced an increases in ATP-dependent Ca2+ transport by SHR vesicles. Inhibition of NA+, K+-adenosine triphosphatase activity by ouabain was also studied. Na+, K+ATPase activity in SHR membranes was double that in membranes from WKY (22.1 +/- 2.8 v.s. 11.3 +/- 1.1. mumole Pi/h/mg protein). These differences, observed on 3 week-old rats, before a significant rise blood pressure, may reflect genetic characteristics of these hypertensive-prone rats.     

A search for endogenous Na+,K+-ATPase inhibitor in acutely volume-expanded hog plasma led to lysophosphatidylcholine gamma-stearoyl

An Na+,K+-ATPase inhibitor possessing inhibitory activity against the specific binding of ouabain to Na+,K+-ATPase has been purified from the plasma of acutely saline-infused hogs. The purification was performed by a combination of Amberlite XAD-2 adsorption chromatography and five steps of high-pressure liquid chromatography (HPLC). Fast atom bombardment mass and proton nuclear magnetic resonance (NMR) spectrometric studies identified the purified substance as lysophosphatidylcholine gamma-stearoyl (LPCS). The ouabain-displacing activity in plasma, due to this compound, increased with time during saline infusion. The maximal level reached was approximately 12 times higher than that in the pre-infusion plasma sample. Lysophosphatidylcholines (LPCs) containing myristoyl, palmitoyl and oleoyl groups were also inhibitory to Na+,K+-ATPase and ouabain-binding to the enzyme. These LPCs were effective at 100 mumol/l concentrations in attaining 50% inhibition of the enzyme activity and ouabain-binding activity of Na+,K+-ATPase. These results suggest that LPCs containing long chain fatty acids could play an important role as a Na+,K+-ATPase inhibitors under volume-expanded conditions.     

Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation

Sarcolemmal and microsomal membranes prepared from adult canine cardiac myocytes (sarcolemmal Na+, K+-ATPase = 71.8 mumol/mg per hr and microsomal rotenone-insensitive NADH cytochrome c reductase = 114 mumol/mg per hr) were each preincubated at 37 degrees C in the presence of a free radical generating system consisting of dihydroxyfumarate and Fe -ADP; loss of the Na+, K+-ATPase and reductase activities, as well as the associated increases in lipid peroxidation, measured by malondialdehyde formation, were temporally correlated in both systems. The ATPase was inhibited 70% when the malondialdehyde was 71 nmol/mg protein at 20 minutes and 90% when malondialdehyde was 138 nmol/mg protein at 90 minutes. Inhibition of reductase activity occurred more gradually, displaying a 27% loss of activity when malondialdehyde reached 34 nmol/mg protein at 20 minutes and 60% with a malondialdehyde value of 67 nmol/mg protein at 90 minutes. The greater susceptibility of the sarcolemma to free radical-induced membrane damage may be due to the higher content of unsaturated fatty acids in this membrane, compared to microsomes.     

Ouabain-binding protein(s) from human plasma

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18 inhibit Na+ pump activity in human erythrocytes (IC50 approximately 4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (< or =0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.     

Myocardial sarcolemmal ATPase in dogs with induced mitral insufficiency.

A study of the sarcolemmal Na+K+ATPase in the left failing heart due to induced mitral insufficiency was made in dogs. The sarcolemmal Na+K+ATPase markedly increased in the failing left ventricle. There was no change in the ATPase in the nonfailing right ventricle of the same dog. It was observed that during the early period of mitral incompetence, when there was an increase in the index of myocardial contractility, there was also a decrease in the sarcolemmal Na+K+-ATPase activity. There was no change in the MPG++-ATPase of the sarcolemmal fractions of either the failing or nonfailing ventricle. These results indicate that sarcolemmal Na+K+-ATPase appears to be involved in the reduction of the myocardial contractility during heart failure.     

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.     

Sensitive assay and kinetic property of urinary inhibitor of Na+, K+-ATPase in sodium-loaded patients with essential hypertension

A sensitive assay method to evaluate the inhibitor of Na+, K+-ATPase in human urine was developed by measuring the inorganic phosphate liberated from ATP in vitro using Na+, K+-ATPase from porcine cerebral cortex. Ouabain inhibited the Na+, K+-ATPase by competing with the potassium ion (an apparent Ki = 2.6 +/- 0.89 X 10(-8) M, n = 8) under the condition of 100 mM NaCl, 4.5 mM MgSO4 and 0.56 mM ATP. The apparent Km value of KCl was 0.4 mM. Factors inhibiting Na+, K+-ATPase were detected in the post-salt fraction on Sephadex G-15 chromatography following the ethanol extraction of lyophilized fresh urine of sodium loaded human subjects (300 meq Na+/day, for 4 days) with essential hypertension. Two active fractions around the 400 daltons following salt were eluted on Sephadex G-15 chromatography. The slower eluted factor competed kinetically with potassium ion, but the inhibitory activity was lost within two days during storage at 4 degrees C. The faster-eluted inhibitor lost its activity within a day. These results indicate that the unstable inhibiting factors of Na+, K+-ATPase exist in human urine and one of these factors inhibits ouabain sensitive Na+, K+-ATPase by binding to the potassium binding site (or very close to it), which exists at the outer surface of the cell membrane of this enzyme.     

The effect of ischemia and reperfusion on sarcolemmal function in perfused canine hearts

The report deals with the effect of ischemia and reperfusion on purified sarcolemma obtained from canine myocardium of perfused supported heart preparations. Perfusion was carried out with a perfluorochemical (FC-43). Ischemia was produced by intermittent total clamping of inflow and outflow followed by release until the decrease in dP/dtmax had become stabile. Purity of sarcolemmal vesicles was ascertained with marker enzymes: succinate cytochrome c reductase (for mitochondria), K+-stimulated p-nitrophenylphosphate (K+-pNPPase), (Na+/K+)ATPase and adenylate cyclase (for SL). In addition Na+/Ca2+-exchange characteristics for SL were determined. Sidedness of vesicles was ascertained by means of adenylate cyclase activity using sarcolemmal preparations treated and untreated with alamethicin. Emphasis was placed on ATP-dependent Ca2+ uptake, phosphorylation of sarcolemmal vesicles and yield of SL proteins. Ischemia and reperfusion resulted in a significant reduction in adenylate cyclase activity. This decline was significant following ischemia and reperfusion. The yield of protein recovered from SL vesicles from ischemic-reperfused heart preparations was also significantly decreased. Both initial rate of ATP-dependent Ca2+ uptake and maximal Ca2+ uptake fell significantly following ischemia and reperfusion. The initial rate of phosphorylation also dropped significantly. These disturbances in SL Ca2+ transport following ischemia and reperfusion are probably a part of the general deficit in Ca2+ translocation.