Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.     

Bufodienolides as Endogenous Na+, K+-ATPase Inhibitors: Biosynthesis in Bovine and Rat Adrenals

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [³H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. These results suggest that Na⁺, K⁺-ATPase inhibitors which interact with cuabain antibodies, but not those which interact with bufalin antibodies, are synthesized in bovine and rat adrenals     

Effects of age on lipids and sensitivity of Na+,K+-ATPase to ouabain in rat spinal cord

Reports have suggested that variation in phosopholipid and cholesterol content and/or ratio affects cellular membrane fluidity. Quantitative changes in these lipids may provide evidence of age-associated changes in membrane structure which, in turn, may influence functional parameters of the cell. Male, Fischer 344 rats (ages 3, 6, 12, 24, 28 months) were sacrificed and spinal cords removed. Lipids were extracted and phospholipid and cholesterol content was measured. Significant increases in the content of both lipids occurred between 3 and 6 months and between 24 and 28 months of age. The ratios of phospholipids to cholesterol decreased significantly between 3 and 6 months of age. From 6 to 28 months of age the increase in phospholipids generally paralleled those of cholesterol having a smaller effect on the ratio. The altered lipid content was accompanied by increased sensitivity of spinal cord microsomal Na⁺, K⁺-ATPase to the cardiac glycoside ouabain. The changes in absolute content as well as ratios of phospholipids and cholesterol, in addition to increased sensitivity of Na⁺,K⁺-ATPase to cardiac glycosides, suggest that membrane structure in rat spinal cord may change as a result of age.     

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.     

Evidence for Imbalanced Furosemide-Sensitive Na+, K+ Cotransport in Hereditary Stomatocytosis

The red cells from 5 related patients with hereditary stomatocytosis were investigated. Maximal rate constant of Na⁺ passive permeability was increased while that of K⁺ passive permeability was nearly normal. Ouabain-sensitive Na⁺ efflux was elevated. The Na⁺ component of furosemide-sensitive Na⁺, K⁺ cotransport was also increased. However, its K⁺ component, determined in 2 patients, remained within normal limits, thus departing from the strict 1:1 stoichiometry of the Na⁺, K⁺ cotransport system. Yet, intracellular Na⁺ and K⁺ concentrations displayed limited and inconstant changes. A variety of abnormally-shaped cells, including stomatocytes, were observed in scanning electron micrographs. Upon differential centrifugation, reticulocytes usually concentrated in the most dense region of the gradient. Red cell deformability, as studied by ektacytometry, was reduced. Membrane phosphatidylcholines and sphingomyelins were increased and decreased, respectively, whereas fatty acid distribution was unchanged. Membrane microviscosity was normal.     

Intrastriatal Administration of Guanidinoacetate Inhibits Na+, K+-ATPase and Creatine Kinase Activities in Rat Striatum

Guanidinoacetate methyltransferase deficiency (GAMT deficiency) is an inherited neurometabolic disorder clinically characterized by epilepsy and mental retardation and biochemically by accumulation of guanidinoacetate (GAA) and depletion of creatine. Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not yet established. In the present study, we investigated the effect of intrastriatal administration of GAA on Na⁺, K⁺-ATPase activity, total (tCK), cytosolic (Cy-CK), and mitochondrial (Mi-CK) creatine kinase (CK) activities in rat striatum. We verified that Na⁺, K⁺-ATPase, tCK, and Mi-CK activities were significantly inhibited by GAA, in contrast to Cy-CK which was not affected by this guanidino compound. Since these enzyme activities can be affected by reactive species, we also investigated the effect of intrastriatal administration of GAA on thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in rats. We found that this metabolite significantly increased this oxidative stress parameter. Considering the importance of Na⁺, K⁺-ATPase and CK activities for brain metabolism homeostasis, our results suggest that the inhibition of these enzymes by increased intracerebral levels of GAA may contribute to the neuropathology observed in patients with GAMT-deficiency.     

cAMP regulates nitric oxide production and ouabain sensitive Na+, K+-ATPase activity in SH-SY5Y human neuroblastoma cells

Summary We investigated the relation between cyclic AMP (cAMP) and nitric oxide (NO) production, as well as the effect of NO on Na⁺ , K⁺-ATPase activity in the human neuroblastoma cell line SH-SY5Y. Two cAMP agonists, dibutyryl cAMP (DBC) and beraprost sodium (BPS), increased cAMP accumulation and NO production in a time and dose dependent manner at 50 mmol/l glucose. On the other hand, cellular sorbitol and myo-inositol contents and protein kinase C activity were not altered by DBC or BPS. A specific protein kinase A inhibitor, H-89, suppressed increases in nitrite/nitrate and cyclic GMP (cGMP) and protein kinase A activity stimulated by DBC or BPS. This finding suggests that cAMP stimulates NO production by activating protein kinase A via a pathway different from the sorbitol-myo-inositol-protein kinase C pathway. We observed that an NO donor, sodium nitroprusside, and an NO agonist, L-arginine, enhanced ouabain sensitive Na⁺, K⁺-ATPase activity at 50 mmol/l glucose. We also found that a nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), inhibited Na⁺, K⁺-ATPase activity at 5 mmol/l glucose, and partially suppressed the enzyme activity stimulated by DBC or BPS. The results of this study suggest that cAMP regulates protein kinase A activity, NO production and ouabain sensitive Na⁺, K⁺-ATPase activity in a cascade fashion. The results also suggest that protein kinase A at least partially regulates Na⁺, K⁺-ATPase activity without mediation by NO in SH-SY5Y cells. We speculate that cAMP and NO are two important regulatory factors in the pathogenesis of diabetic neuropathy. [Diabetologia (1998) 41: 1451–1458] Received: 17 November 1997 and in final revised form: 27 July 1998     

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.     

Coordinated regulation of intracellular K+ in the proximal tubule: Ba2+ blockade down-regulates the Na+,K+-ATPase and up-regulates two K+ permeability pathways.

To avoid large changes in cell K+ content and volume during variations in Na+,K+-ATPase activity, Na+-transporting epithelia must adjust the rate of K+ exit through passive permeability pathways. Recent studies have shown that a variety of passive K+ transport mechanisms may coexist within a cell and may be functionally linked to the activity of the Na+,K+-ATPase. In this study, we have identified three distinct pathways for passive K+ transport that act in concert with the Na+,K+-ATPase to maintain intracellular K+ homeostasis in the proximal tubule. Under control conditions, the total K+ leak of the tubules consisted of discrete Ba2+-sensitive (approximately 65%), quinine-sensitive (approximately 20%), and furosemide-sensitive (approximately 10%) pathways. Following inhibition of the principal K+ leak pathway with Ba2+, the tubules adaptively restored cell K+ content to normal levels. This recovery of cell K+ content was inhibited, in an additive manner, by quinine and furosemide. Following adaptation to Ba2+, the tubules exhibited a 30% reduction in Na+-K+ pump rate coupled with an increase in K+ leak by means of the quinine-sensitive (approximately 70%) and furosemide-sensitive (approximately 280%) pathways. Thus, the proximal tubule maintains intracellular K+ homeostasis by the coordinated modulation of multiple K+ transport pathways. Furthermore, these results suggest that, like Ba2+, other inhibitors of K+ conductance will cause compensatory changes in both the Na+-K+ pump and alternative pathways for passive K+ transport.     

The Effect of High Salt Intake on Na+, K+-Atpase Activity of Tissues in the Rat

1. The effect of chronic feeding of high salt diet on Na⁺, K⁺-ATPase activity of heart, liver, skeletal muscle, kidney and aorta was studied in the rat.

2. Groups of rats were either given tap water or 18 g/L saline to drink. After 7 days, 3 months or 12 months, the control group and salt loaded     

Erythrocyte Na+, K+-ATPase activity in patients with congestive heart failure.

In order to determine if the Na+, K+-ATPase activity in erythrocyte membranes is altered in congestive heart failure, and to examine its clinical significance with respect to other clinical variables, erythrocyte Na+, K+-ATPase activity was measured in 51 patients with left ventricular ejection fractions <40% (coronary artery disease, n=26; dilated cardiomyopathy, n=25) and 24 control patients. Na+, K+-ATPase activity was lower in both coronary artery disease and dilated cardiomyopathy groups than control group even in the absence of digitalis use. There was a significant inverse correlation between Na+, K+-ATPase activity and plasma norepinephrine. The presence of non-sustained ventricular tachycardia was associated with a lower Na+, K+-ATPase activity in both groups with congestive heart failure without digitalis use than those without ventricular tachycardia. Plasma norepinephrine was higher in patients with non-sustained ventricular tachycardia than those without in the coronary artery disease group, but not in the dilated cardiomyopathy group. Na+, K+-ATPase activity may be helpful in predicting electrophysiologic instability in patients with heart failure.     

Continuous determination of extracellular space and changes of K+, Na+, Ca2+, and H+ during global ischaemia in isolated rat hearts

Isolated rat hearts were perfused according to the Langendorff technique and prepared to allow the measurement of left ventricular pressure (LVP), the first derivative of LVP (dP/dt), coronary perfusion pressure (CPP) and heart rate (HR). The hearts were perfused with a modified Krebs-Henseleit solution (KHS; control group) or KHS containing tetramethylammonium-chloride (TMA; 100 microM), which did not influence heart haemodynamics or extracellular potassium accumulation during global ischaemia (GI). TMA was used as a marker to determine changes in the size of the extracellular space (ECS) during 60 min of GI. Extracellular concentrations of K+, Na+, Ca2+, H+ and TMA were measured using double-barreled polyvinyl chloride (PVC) mini-electrodes. Relative changes in the ECS size and net cation movements were calculated from the extracellular TMA and cation concentrations. After 60 min of GI ECS decreased by 74% due to a water shift into the intracellular space (ICS). Within 10 s after the onset of GI extracellular potassium concentration increased in a typical triphasic pattern. A biphasic net efflux of K+ was maximal 2 and 15 min after the onset of ischaemia interrupted by a maximal uptake after 7 min, probably due to the stimulation of the Na+/K+ ATPase. The changes in extracellular sodium and calcium concentrations were biphasic; showing an initial increase occurring approximately during the first 20 min after the onset of GI followed by a decrease. Despite the initial increase of extracellular Na+ and Ca2+ maximal net influx of Na+ and Ca2+ were calculated after 2 and 19 min of GI. Accumulation of H+ in the extracellular space occurred mainly during the first 20 min of GI. It is concluded that, (1) TMA is a useful marker for continuous measurement of changes in the size of the ECS during GI; (2) Changes in extracellular ion concentration are influenced markedly by a water shift from the ECS into the ICS; (3) Stimulation of the Na+/K+ pump causes a transient net uptake of K+ and reduces the net influx of Na+; (4) Ca2+ uptake is decreased transiently probably by increased Ca2(+)-ATPase activity; (5) Energy dependent protective mechanisms for the maintainance of intracellular ionic homeostasis are exhausted after approximately 15 to 20 min of GI in isolated working rat hearts.     

Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.     

C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y

Summary This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na⁺, K⁺-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na⁺, K⁺-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na⁺ affinity of the enzyme and all subsequent studies were performed at non-saturating Na⁺ concentrations. C-peptide stimulation of Na⁺, K⁺-ATPase activity occurred in a concentration-dependent manner in the dose range 10^–8–10^–6 mol/l. The presence of neuropeptide Y, 5×10^–9 mol/l, enhanced this effect and stimulation of Na⁺, K⁺-ATPase activity then occurred in the C-peptide dose range 10^–11–10^–8 mol/l. C-peptide stimulation of Na⁺, K⁺-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca²⁺-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na⁺, K⁺-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca²⁺-dependent intracellular signalling pathways.Abbreviations PTX Pertussis toxin - NPY neuropeptide Y - PCT proximal convoluted tubule - BSA bovine serum albumin - dB cAMP dibutyryl cyclic adenosine monophosphate - PP2B Ca²⁺/calmodulin-dependent protein phosphatase 2B - PKC protein kinase C - [Ca²⁺] intracellular calcium concentration     

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.     

Inhibition of Na+, K+-adenosinetriphosphatase by endotoxin: a possible mechanism for endotoxin-induced cholestasis.

Cholestatic jaundice is one complication of nonhepatic gram-negative bacterial infection. The endotoxin of Escherichia coli has been reported to cause cholestasis by inhibiting the bile salt-independent fraction (BSIF) of bile in the perfused rat liver. Accordingly, the effects of lipopolysaccharides (LPS) of E. coli and Salmonella enteritidis on the Na+, K+-adenosinetriphosphatase (ATPase) in canalicular-enriched plasma membranes of rate liver were examined. At 20 microgram/ml, both endotoxins inhibited this enzyme by approximately 40%. Maximal inhibition (70%-80%) occurred at concentrations of greater than or equal to 120 microgram/ml. The LPS of neither organism exerted any effect on the activity of Mg++-ATPase or 5'-nucleotidase in the same preparations. Inhibition by the E. coli LPS appeared to be noncompetitive in nature, and the calculated Ki was 45 microgram/ml. Since the Na+, K+-ATPase may be responsible for the elaboration of BSIF, inhibition of this enzyme could be the underlying mechanism for the endotoxin-induced cholestasis.     

Apparent Upregulation of Na+, K+ Pump Sites in SHR Skeletal Muscle with Reduced Transport Capacity

Slow-twitch, oxidative skeletal muscles in SHR exhibit several physiological defects, including a reduced ability to maintain force during high frequency repetitive stimulation (1). Muscle fatigue may be produced by one of a variety of factors acting at different levels of the neuromuscular system. Several lines of evidence, however, suggest that SHR soleus fatigues more rapidly than WKY soleus because SHR muscles allow more K⁺ to accumulate in the extracellular space during repetitive muscle activity. An increase in extracellular K⁺ can lead to a failure in the generation or conduction of muscle action potentials. Comparison of the compound action potentials recorded from SHR and WKY muscles during repetitive stimulation provided evidence for a decrease in excitability of SHR soleus. Since the K⁺ released from muscle fibers during exercise is returned to the fiber principally via the activity of the Na⁺, K⁺ pump, the increase in extracellular K⁺ in SHR muscle may reflect a decrease in pump capacity. Measurements including intracellular K⁺ and Na⁺ content at rest, the level of hyperpolarization produced by the addition of epinephrine and insulin to SHR soleus and the post-exercise recovery of resting membrane potentials all appear to indicate that Na⁺, K⁺ pump capacity is reduced in SHR soleus muscles. Nonetheless, ouabain binding studies show a significantly greater number of pump sites in SHR muscles. The data suggest that Na⁺ pump activity is decreased in SHR soleus muscles without an apparent reduction in either the number of pump sites or in pump binding affinity.