Model of anion and monovalent cation transport as neutral ion pairs through lipophilic water channels of the Na,K ATPase complex

A model of anion and monovalent cation transport through a lipophilic water channel of the Na,K ATPase complex is presented. Literature data for the Na,K ATPase cation binding sites are combined with data for the anion binding sites of Band 3 to obtain adjacent cation and anion combining sites at the inner and outer channel mouths. Cations and anions form neutral ion pairs or undissociated acids at these sites and then partition much more favorably into lipophilic channel water, passing through the channel in diffusive fashion. Cation movements in an "uphill" direction occur without an enzyme translocating moiety and its specific energetic requirement. The pertinent factors are the exclusion of unpaired cations by the tight channel and the site selectivity or pickup ratios for Na/K at each side which dominate over bulk and transmembrane concentration ratios. ATP hydrolysis provides phosphate for ion pairing.     

Distribution of (Na+ + K+)ATPase and sodium channels in skeletal muscle and electroplax

Summary The distributions of (Na⁺ + K⁺)ATPase and sodium channels in skeletal muscle fibres and electrocytes were determined by immunofluorescent and immunoelectron microscopic techniques using antibodies against rat and eel (Na⁺ + K⁺)ATPase and the eel electric organ sodium channel. The extrajunctional sarcolemma of skeletal muscle was uniformly stained by polyclonal antibodies against (Na⁺ + K⁺)ATPase and the sodium channel. The T-tubule system of skeletal muscle was also labelled heavily for both (Na⁺ + K⁺)ATPase and the sodium channel. The terminal cisternae of the sarcoplasmic reticulum was stained for (Na⁺ + K⁺)ATPase but not sodium channels. At the motor endplate, (Na⁺ + K⁺)ATPase-like immunoreactivity was present along the plasmalemma of motor nerve terminals but not along the postsynaptic junctional sarcolemma. Paradoxically, a monoclonal antibody that binds to the form of the catalytic subunit of (Na⁺ + K⁺)ATPase from rat hepatocytes and renal tubule cells did not label the enzyme in rat skeletal muscle. In electrocytes, (Na⁺ + K⁺)ATPase-like irnmunoreactivity was concentrated primarily along the plasmalemma and calveolae of the non-innervated face. In contrast, sodium channel-like immunoreactivity was concentrated along the plasmalemma of the innervated face except in the clefts of the postsynaptic membrane. Thus, we conclude that at endplates both the (Na⁺ + K⁺)ATPase of rat skeletal muscle and sodium channels of eel electrocytes are not concentrated in the juxtaneuronal postsynaptic membrane. We also interpret the failure of the monoclonal anti- (Na⁺ + K⁺)ATPase antibodies to bind to the enzyme in muscle to indicate that the catalytic subunit of skeletal muscle (Na⁺ + K⁺)ATPase displays different epitopes than does the a subunit of kidney and liver.     

A model of fluid, erythrocyte, and solute transport in the lung

A mathematical model of fluid, solute, and red cell transport in the lung has been developed that includes the effects of simultaneous changes in lung vascular and interstitial volumes. The model provides separate arterial, microvascular, and venous pulmonary regions and a systemic vascular region in addition to a pulmonary interstitial compartment. Pressure, volume, hematocrit, flow, and concentration of up to 12 solutes and tracers can be computed in each compartment. Computer code is written in the C programming language, with Microsoft Excel serving as a user interface. Implementation is currently on PC-486 microcomputer systems, but the core program can easily be moved to other computer systems. The user can select different models for the blood-interstitial barrier (e.g. multiple pore, nonlinear Patlak equation), osmotic pressure-concentration relationships (e.g., Nitta, Navar-Navar), solute reflection coefficients, interstitial macromolecule exclusion, or lymph barrier characteristics. Each model parameter or a combination of parameters can be altered with time in a predetermined fashion. The model is particularly useful in interpreting lung experimental data where simultaneous changes occur in vascular and extravascular compartments. Several applications are presented and discussed, including interpretation of optical filtration experiments, venous occlusion experiments, external detection of macromolecular exchange, and blood-lymph studies that use exogenous tracers. A number of limitations of the model are identified and improvements are proposed. A major strength of the model is that it is specifically designed to incorporate newly discovered relationships as the field of lung physiology expands.     

Effect of salt intake on jejunal dopamine,Na+,K+‐ATPase activity and electrolyte transport

The present study addresses the question of the relevance of salt intake on jejunal dopamine, Na⁺,K⁺‐ATPase activity and electrolyte transport. Low salt, but not high salt, intake for 2 weeks increased dopamine levels in the jejunal mucosa accompanied by a marked decrease in L ‐3,4‐dihydroxyphenylalanine tissue levels. By contrast, in rats fasted for 72 h the effect of refeeding for 24 h with a low salt diet failed to change dopamine tissue levels, although it significantly increased those of L ‐3,4‐dihydroxyphenylalanine. By contrast, high salt intake markedly increased the tissue levels of both dopamine and L ‐3,4‐dihydroxyphenylalanine, without changes in dopamine/ L ‐3,4‐dihydroxyphenylalanine tissue ratios. Tissue levels of both L ‐3,4‐dihydroxyphenylalanine and dopamine in control conditions (normal salt intake for 2 weeks) were markedly higher (P < 0.05) than in rats submitted to 72 h fasting plus 24 h refeeding. The effect of fasting for 72 h followed by 24 h refeeding was a marked decrease in jejunal Na⁺,K⁺‐ATPase activity, particularly evident for rats fed a normal salt and high salt diets during the refeeding period. Basal short circuit current was similar in rats fed a normal salt diet for 2 weeks and 24 h, and the type of diet failed to alter basal short circuit current after refeeding with normal, low and high salt diets. On the other hand, the effect of prolonged low salt intake was a marked decrease in jejunal Na⁺,K⁺‐ATPase activity and basal short circuit current, whereas high salt intake failed to alter enzyme activity and basal short circuit current. In rats fed for 2 weeks a high salt diet ouabain was found to be more potent in reducing jejunal short circuit current than in rats fed normal and low salt diets. The effect of furosemide was more marked in rats fed for 2 weeks high and low salt diets than in animals receiving a normal salt intake. Dopamine (up to 1 μmol L^–1) was found not to alter Na⁺,K⁺‐ATPase and basal short circuit current in jejunal epithelial sheets, in rats fed with normal, low and high salt diets for 2 weeks and 24 h.     

Vascular tissue (Na,K)ATPase activity and aging in the F344 rat

Vascular tissue (heart, thoracic aorta and tail artery) was removed from Fischer 344 rats, 12, 18 and 27 months of age. The intact tissue was then used to determine total, ouabain-sensitive and ouabain-insensitive 86Rubidium (86Rb) uptakes, to provide a reflection of (Na,K)ATPase activity. These studies indicate no change in total, ouabain-sensitive nor ouabain-insensitive 86Rb uptakes into cardiac tissue isolated from these rats. However, tail artery total and ouabain-sensitive 86Rb uptake decreased with age (a 61% decrease in ouabain-sensitive 86Rb uptake in 12 vs. 27-month-old rats) without significant changes in the ouabain-insensitive 86Rb uptake. This pattern was repeated in aortic tissue with a 56% decrease in ouabain-sensitive 86Rb uptake in 12 vs. 27-month-old rats. The results of these studies support an age-related decline in (Na,K)ATPase activity in aortic and tail artery tissue without a significant change in cardiac (Na,K)ATPase activity between 12, 18 and 27-month-old Fischer 344 rats.     

Normal expression and inflammation-induced changes of Na and Na/K ATPase activity in spinal dorsal horn of the rat

We tested whether that peripheral inflammation induces changes in the spinal dorsal horn ATPase activity. Adult Sprague-Dawley rats were anesthetized (thiobarbital), the left hind paw (inflammation group; n = 15) was immersed in water at 60 degrees C for 60s, which induced a local inflammation. A control group (n = 12) was tested with water at room temperature. After 60 min of peripheral inflammation left (LDH) or right lumbar dorsal horn (RDH) were processed for total, Na/K, Na and remanent ATPase activities (nM P(i) (mgprotein)(-1) min(-1)). In control animals isoenzymatic activities were: Na (31.2%); Na/K (20.6%) and remanent (48.2%) from total ATPase activity. No LDH-RDH asymmetry was found. The inflammation group presented an ipsilateral increase of total ATPase activity in LDH (X+/-S.E.M.; 4798.9+/-601) over the RDH (3982.2+/-451; Delta+817; P<0.05). This is due to an increase in Na ATPase activity (1609.3+/-297) over RDH (1164.2+/-166; Delta+445; P<0.05). ATPase activities were increased in LDH from inflamed over the control group as follows: total (4798.9+/-601; Delta+840; P<0.05), Na/K (1298.1+/-301; Delta+483; P<0.05) and Na (1609.3+/-297; Delta+373; P<0.05). These increased ATPase activities, induced in a short time, can be considered a functional marker of nociceptive neuronal activity.     

The influence of catecholamines on Na,K transport in slow- and fast-twitch muscles of the rat

The effects of catecholamines on active sodium and potassium transport was compared in slow- (SOL) and fast-twitch (EDL) skeletal muscles of the rat. Stimulation of active Na+-extrusion and K+-uptake induced by adrenaline (6-30 mumol . l-1) and isoprenaline (1-40 mumol . l-1) was markedly greater in slow- than in fast-twitch muscle. In sodium-preloaded muscles the maximal stimulation of 24Na-efflux induced by adrenaline was about 2-fold higher in SOL than in EDL. Isoprenaline caused a 2.4-fold increase in ouabain-sensitive 86Rb influx in SOL muscle, but failed to alter the ouabain-sensitive influx in EDL. The stimulating action of isoprenaline on 86Rb influx in EDL was due to an increase in the ouabain-insensitive fraction of Rb uptake. The effects of catecholamines of fast- and slow-twitch muscles were probably due to the accumulation of cyclic AMP, however the fact that there were no significant differences between the nucleotides levels in fast- and slow-twitch muscle suggests the participation of other mechanism as well. The results presented suggest that cyclic AMP-induced stimulation of ouabain-insensitive transport of cation in the isolated EDL muscle of the rat is similar to that of barnacle muscle.     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+‐ATPase

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on α‐adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na⁺,K⁺‐adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na⁺,K⁺‐ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the α‐adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM . The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na⁺,K⁺‐ATPase on the Ser23 residue. The level of PKC induced Na⁺,K⁺‐ATPase phosphorylation was determined using an antibody that only recognizes Na⁺,K⁺‐ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na⁺,K⁺‐ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na⁺,K⁺‐ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na⁺,K⁺‐ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium‐dependent protein phosphatase, calcineurin, increased the state of Na⁺,K⁺‐ATPase phosphorylation. The results imply that phosphorylation of renal Na⁺,K⁺‐ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.     

Na−K ATPase activity and intracellular ion concentrations in the lactating guinea pig mammary gland

Summary The intracellular sodium, potassium and chloride concentrations in slices of lactating guinea pig mammary gland have been determined by chemical analysis and the use of appropriate values for extracellular space. These ion concentrations after 1 hr incubation at 37° C in a Krebs-Ringer bicarbonate solution are 45 mM Na⁺, 138 mM K⁺ and 44 mM Cl^–, which values are in agreement with those found in fresh mammary gland slices. Inhibition of the Na–K activated ATPase cation pump system of the tissue by 10^–4 M ouabain, anoxia or cooling to 0°C causes a gain of Na⁺ and an equimolar loss of K⁺ without a significant change in chloride concentration. The effect of cooling (0°C) is reversible by reincubation at 37°C. Water content of the tissue (76.5% of wet weight) and extracellular space (40.5%) do not change under these conditions. The results permit the conclusion that the Na–K activated ATPase system is responsible for the maintenance of the intracellular Na⁺ and K⁺ concentrations, but do not support the presence of a chloride pump.     

Stimulation of Xenopus oocyte Na(+),K(+)ATPase by the serum and glucocorticoid-dependent kinase sgk1

The serum and glucocorticoid-dependent kinase-1 (sgk1) is expressed in a wide variety of tissues including renal epithelial cells. As it is up-regulated by aldosterone, it is considered to participate in the regulation of renal Na(+) reabsorption. Indeed, co-expression of sgk1 with the renal epithelial Na(+) channel (ENaC) augments Na(+) channel activity. The aim of the present study was to examine possible effects of sgk1 on Na(+)/K(+)-ATPase activity. To this end dual-electrode voltage-clamp experiments were performed in Xenopus oocytes expressing the active kinase (S422D)sgk1 or the inactive mutant (K127N)sgk1. Na(+)/K(+)-ATPase activity was estimated from the hyperpolarization (delta V(m)) and the outwardly-directed current ( I(P)) created by addition of extracellular K(+) in the presence of K(+) channel blocker Ba(2+). Both delta V(m) and I(P) were significantly larger in oocytes expressing (S422D)sgk1 than in those expressing (K127N)sgk1 or having been injected with water. I(P) was fully inhibited by ouabain. Ion-selective microelectrodes showed that the stimulation of pump current was not the result of altered cytosolic Na(+) activity or pH. The present results thus point to an additional action of sgk1 that may participate in the regulation of renal tubular Na(+) transport. Moreover, sgk1 may be involved in the regulation of Na(+)/K(+)-ATPase in extrarenal tissues.