Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

Influence of bile acids on the (Na+−K+)-activated- and Mg2+-activated ATPase of rat colon

Summary The influence of various bile acids on the (Na⁺−K⁺)-ATPase and Mg²⁺-ATPase activity of rat colon is described. At a concentration of 0.6 mmol/l C and TC did not inhibit the (Na⁺−K⁺)-ATPase activity in contrast to GC. The taurine derivates TC, TCDC and TDC did not influence or even enhanced the (Na⁺−K⁺)-ATPase activity. All bile acids except C, TC and CDC depressed the Mg²⁺-ATPase activity. At higher concentrations only C and TC did not influence the (Na⁺−K⁺)-ATPase activity. C, GC and TC at 2.5 mmol/l decreased the (Na⁺−K⁺)-activated phosphatase with ATP as substrate. All other substrates tested did not influence the enzymic activity significantly. The results indicate that bile acids can inhibit the Na⁺-absorbing system in rat colon. Hence this inhibition can cause diarrhea.     

Na+, K+-ATPase activity in neurons and glial cells of the olfactory cortex of the rat brain during the development of long-term potentiation

Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities were studied in neurons and glial cells of the olfactory cortex of the rat by quantitative cytophotometry in conditions of long-term potentiation (LTP), and significant changes in direction and extent were found. Na⁺, K⁺-ATPase activity decreased in neurons in the first 15 min after LTP, with subsequent elevation by 30 min. Mg²⁺-ATPase activity remained unchanged in these conditions. Glial cells showed significant increases in Na⁺, K⁺-ATPase activity in the initial period after LTP, with return to control by 30 min. Again, there were no significant changes in Mg²⁺-ATPase activity. The formation and persistence of LTP in neurons and glial cells was accompanied by significant changes in Na⁺, K⁺-ATPase activity, which were reciprocal in nature. Functional Neurochemistry Laboratory (Director N. A. Emel'yanov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 3, pp. 16–20, March, 1995.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Blood pressure in essential hypertension correlates with the concentration of a circulating inhibitor of the sodium pump

Summary The influence of serum from patients with essential hypertension on the sodium efflux rate constants of human lymphocytes and on the activity of isolated (Na⁺+K⁺)-ATPase was investigated. The ouabain-sensitive sodium efflux rate constant was significantly decreased (p<0.001) in the sera of 19 hypertensives (1.92±0.11 h^–1) compared with the sera of 30 normotensives (2.44±0.07 h^–1). The ouabain-insensitive sodium efflux was unaffected. These results corresponded with a significant difference (p<0.005) of (Na⁺+K⁺)-ATPase activity (1.03±0.04 mU/ml and 0.079±0.06 mU/ml), when an isolated (Na⁺+K⁺)-ATPase was incubated with the sera of 22 normotensives or 18 hypertensives. Both the rate constant of ouabain-sensitive sodium efflux and the (Na⁺+K⁺)-ATPase activity correlated significantly with the diastolic and systolic blood pressure (p<0.001). These data, therefore, demonstrated the close relationship between essential hypertension and the concentration of a circulating inhibitor of the sodium pump.Abbreviations ATP Adenosine triphosphate - EGTA Ethyleneglycol bis(2-aminoethyl)-N,N,N,N-tetraacetic acid This paper contains an essential part of the thesis of K.M. presented to the Fachbereich Veterinärmedizin, GiessenThis work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Scho 139/16-2) and by the Fonds der Chemischen Industrie, Frankfurt/Main     

Effect of extracellular volume expansion on renal cortical and medullary Na+−K+-ATPase

Summary The role of renal Na⁺–K⁺-ATPase in the acute changes in sodium reabsorption caused by isotonic volume expansion was evaluatedin vivo andin vitro in the rat and the dog. Duringin vivo volume expansion with isotonic saline in the rat, renal medullary Na⁺–K⁺-ATPase specific activity increased, while the simultaneously determined cortical Na⁺–K⁺-ATPase specific activity and kinetics remained unchanged. Furthermore, experimentsin vitro failed to demonstrate a circulating inhibitor of renal Na⁺–K⁺-ATPase both in plasma dialysates from volume-expanded rats and in plasma dialysates concentrated 20-fold by ultrafiltration from volume-expanded dogs. These results suggest that the decreased proximal tubular reabsorption of sodium during volume expansion is not mediated by inhibition of renal cortical Na⁺–K⁺-ATPase. The acute increment in medullary Na⁺–K⁺-ATPase observed could represent an adaptive response to increased sodium reabsorption by the loops of Henle, and raises the possibility that this enzyme may participate in relatively rapid adjustments in the transport of sodium by the renal tubule.     

Erythrocyte cations and Na+,K+-ATPase pump activity in athletes and sedentary subjects

Summary The chronic effect of training on intraerythrocyte cationic concentrations and on red cell Na⁺,K⁺-ATPase pump activity was studied by comparing well-trained athletes with sedentary subjects at rest. Also the acute effect of a 50-min cross-country run on these erythrocyte measurements was studied in the athletes. At rest the intraerythrocyte potassium concentration was increased (P<0.01) in the athletes compared to that of the control subjects. The intraerythrocyte concentrations of sodium and magnesium and red cell Na⁺, K⁺-ATPase pump activity were, however, similar in the trained and the untrained subjects.As compared with the resting condition, the intraerythrocyte potassium concentration was decreased (P<0.05) after exercise in the athletes, and this was accompanied by a minor increase in the intraerythrocyte sodium concentration. Red cell Na⁺,K⁺-ATPase pump activity was slightly increased (P<0.05) after exercise.     

The rise of [Na+]i during ischemia and reperfusion in the rat heart—underlying mechanisms

Intracellular Na⁺ concentration ([Na⁺]_i) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na⁺]_i contribute to ischemic and reperfusion damage through their effects on Ca²⁺ overload. Part of the rise of [Na⁺]_i during ischemia may be caused by increased activity of the cardiac Na⁺/H⁺ exchanger (NHE1), activated by the ischemic rise in [H⁺]_i. In support of this view, NHE1 inhibitors reduce the [Na⁺]_i rise during ischemia. Another possibility is that the rise of [Na⁺]_i during ischemia is caused by Na⁺ influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na⁺ channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 μM) and TTX (300 nM) prevented the rise in [Na⁺]_i during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na⁺]_i on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na⁺ currents. At the concentrations used, TTX reduced the transient Na⁺ current (I _Na) by 11 ± 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na⁺ current (I _Na,P) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I _Na,P to 41 ± 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na⁺]_i during ischemia is by blockade of I _Na,P. The fact that a low concentration of TTX eliminated the rise of [Na⁺]_i during ischemia suggests that I _Na,P is a major source of Na⁺ influx in this model of ischemia.     

The localization of the Na+−K+-ATPase in the cells of rat kidney cortex

Summary Plasma membrane fractions of rat kidney cortex were subdivided by centrifugation on a continuous and a discontinuous sucrose gradient and by carrier free continuous electrophoresis. In the different fractions the activity of alkaline phosphatase and aminopeptidase, enzymes which are present in the brushborder membrane, as well as Mg⁺⁺-ATPase, Na⁺–K⁺-ATPase, 5-nucleotidase, acid phosphatase and glucose-6-phosphatase were determined.The distribution of alkaline phosphatase, aminopeptidase and 5-nucleotidase is identical, indicating the localization of these enzymes in the brushborder membrane. Na⁺–K⁺-ATPase does not show an identical distribution with any of the enzymes studied.Using carrier free continuous electrophoresis fractions can be obtained which are enriched in alkaline phosphatase by a factor of 15 when compared to the cortex homogenate, whereas the specific activity of Na⁺–K⁺-ATPase is reduced to one third of the starting material. On the other hand fractions can be isolated in which the specific activity of Na⁺–K⁺-ATPase is 16 times higher than in the homogenate. No enrichment of alkaline phosphatase occurs in these fractions.It is therefore concluded that the Na⁺–K⁺-ATPase is not present in the brushborder membrane nor in the lysosomes or endoplasmatic reticulum. The most probable localization of the Na⁺–K⁺-ATPase are the basal infoldings of the plasma membranes of the cells.A preliminary report has been published by Kinneet al. [28, 29].Major part of this work was done by J. E. Schmitz for his degree of M. D.     

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na⁺,K⁺-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na⁺,K⁺-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na⁺,K⁺-ATPase were used. Dose–response curves for ouabain, a specific Na⁺,K⁺-ATPase inhibitor, were obtained to ascertain which Na⁺,K⁺-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na⁺,K⁺-ATPase was studied in purified lamb kidney Na⁺,K⁺-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na⁺,K⁺-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na⁺,K⁺-ATPase. Possible interaction mechanisms are discussed.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct

The relation between transcellular Na⁺ absorption, intracellular Na⁺ concentration and Na⁺/K⁺-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na⁺ absorption was varied by perfusing the tubule with solutions containing different Na⁺ concentrations (nominally Na⁺-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K⁺ in the bathing solution was exchanged for Rb⁺. Intracellular element concentrations, including Rb⁺, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na⁺ concentration in the perfusion solution caused significant rises in intracellular Na⁺ concentration and Rb⁺ uptake of principal cells. Principal cell Na⁺ and Rb⁺ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na⁺-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na⁺ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na⁺ in the perfusion solution. In contrast, a comparable relationship between lumen Na⁺ concentration, intracellular Na⁺ concentration and basolateral Rb⁺ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na⁺ absorption. In addition, the data demonstrate that apical Na⁺ entry and basolateral Na⁺/K⁺-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na⁺ concentration leads to increased Na⁺ entry and augmented intracellular Na⁺ concentration, which then secondarily stimulates active basolateral Na⁺/K⁺(Rb⁺) exchange.     

Role of ionic transport in regulation of hemoglobin affinity for oxygen in diabetes mellitus

The rate of Na⁺/H⁺ exchange is increased by 24%, activities of Ca-dependent K+ channels is increased by 13%, and activity of erythrocyte Ca²⁺-ATPase decreased by 17% in patients with diabetes mellitus concomitant with essential hypertension in comparison with patients with essential hypertension without disorders of carbohydrate metabolism. Changes in activity of Na⁺/H⁺ exchange, Ca-dependent K⁺ channels, and erythrocyte Ca²⁺-ATPase and increased oxygen affinity of hemoglobin are due to increased glucose concentration in the plasma and are leveled by olifen.     

[Na+] i /[K+] i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression

The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na⁺,K⁺-ATPase but is independent of the inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na⁺,K⁺-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na⁺,K⁺-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin–Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK. In these cells, 6-h exposure to 3 μM ouabain led to the internalization of ∼50% of plasmalemmal Na⁺,K⁺-ATPase. Prolonged incubation in a K⁺-free medium abolished ouabain-induced Na⁺,K⁺-ATPase internalization but did not affect the cytotoxic action of ouabain seen after 18-h incubation. Previously, it was shown that CTS-induced Na⁺,K⁺-ATPase internalization is mediated by its interaction with Src within caveolae. Neither caveolae damage by cholesterol depletion with methyl-β-cyclodextrin nor Src inhibition with 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyridine affected the death of ouabain-treated C7-MDCK cells. Actinomycin D at the 0.1-μg/ml concentration almost completely abolished ribonucleic acid synthesis but did not protect C7-MDCK cells from the cytotoxic action of ouabain. Our results show that neither Na⁺,K⁺-ATPase endocytosis nor de novo gene expression contributes to -independent cell death signaling evoked by prolonged exposure to CTS.     

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium⁺ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84±2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na⁺ and HCO ₃ ^– concentrations was observed. It was followed by a sustained increase in intracellular K⁺ and Cl^– concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K⁺ accumulation significantly, whereas the H⁺ /K⁺-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19±2 mV, owing to the decrease of the ratio of Cl^– conductance to K⁺ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na⁺/H⁺ exchange in MDCK cells; (b) transient alteration of intracellular Na⁺ and pH stimulates Na⁺/K⁺-ATPase and Cl^–/HCO ₃ ^– exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl^–/K⁺ conductance ratio of the plasma membrane.     

Heterogenous Na+, K+-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissues

Na⁺, K⁺-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissue was measured using indirect immunofluorescence and confocal laser scanning microscopy. All four major sites of aggregated lymphoid tissue, i. e. Peyer's patch, sacculus rotundus, caecal patch and appendix, were studied. Na⁺, K⁺-ATPase expression was localized to the basolateral surface of cells of the follicle-associated epithelium (FAE) and adjacent villous or surface epithelia (non-FAE), where increased expression during enterocyte migration was evident. In the FAE, expression of Na⁺, K⁺-ATPase appeared to be lower in the specialized M cells than in enterocytic-type cells, although expression in both cell types was lower than in adjacent non-FAE. Quantification of immunofluorescent staining of Na⁺, K⁺-ATPase by confocal laser scanning imaging showed a reduction of expression in the FAE to approximately 20–60% relative to that in the adjacent non-FAE. These results are consistent with a primary role of the FAE in mucosal immunity with minimal involvement in active solute absorption.     

Substructure of membrane-bound Na+−K+-ATPase protein

Purified membrane-bound Na⁺–K⁺-ATPase from rat kidney outer medulla was studied by freeze-fracturing, by freeze-etching and by negative staining. Freeze-fracturing of purified Na⁺–K⁺-ATPase membranes shows intramembraneous particles with a diameter of about 100 Å. The frequency of these intramembraneous particles — as estimated from the particle densities on the two fracture faces — lies between 4700 and 5600 particles per m². Applying rotary shadowing a four partite substructure could be detected in these intramembraneous particles observed on the fracture planes. The same four partite substructure was detected in particles observed on freeze-fractured and rotary shadowed intact baso-lateral plasma membranes of the thick ascending limb of Henle's loop. Particles could be also detected on both membrane surfaces of the purified Na⁺–K⁺-ATPase. These surface particles have about the same diameter and are present at about the same frequency as those observed within the freeze-fractured membranes. Negative staining of isolated Na⁺–K⁺-ATPase membranes showed particles on both membrane surfaces with a diameter between 30 and 50 Å, at a frequency of about 19,000 per m². On aspects of membrane edges we observed structures which suggest a transmembraneous connection of the negatively stained particles on both membrane surfaces.Our results suggest that the Na⁺–K⁺-ATPase protein is composed of four units and that each unit spans the cell membrane. The native enzyme structure of the Na⁺–K⁺-ATPase protein seems to be preserved during freeze-fracturing and freeze-etching. It is proposed that the four enzyme units of the Na⁺–K⁺-ATPase complex are dissociated during the negative staining procedure.Part of this work was presented at the Frühjahrstagung of the Deutsche Physiologische Gesellschaft [6]     

Ionic storm in hypoxic/ischemic stress: Can opioid receptors subside it?

Neurons in the mammalian central nervous system are extremely vulnerable to oxygen deprivation and blood supply insufficiency. Indeed, hypoxic/ischemic stress triggers multiple pathophysiological changes in the brain, forming the basis of hypoxic/ischemic encephalopathy. One of the initial and crucial events induced by hypoxia/ischemia is the disruption of ionic homeostasis characterized by enhanced K⁺ efflux and Na⁺-, Ca²⁺- and Cl⁻-influx, which causes neuronal injury or even death. Recent data from our laboratory and those of others have shown that activation of opioid receptors, particularly δ-opioid receptors (DOR), is neuroprotective against hypoxic/ischemic insult. This protective mechanism may be one of the key factors that determine neuronal survival under hypoxic/ischemic condition. An important aspect of the DOR-mediated neuroprotection is its action against hypoxic/ischemic disruption of ionic homeostasis. Specially, DOR signal inhibits Na⁺ influx through the membrane and reduces the increase in intracellular Ca²⁺, thus decreasing the excessive leakage of intracellular K⁺. Such protection is dependent on a PKC-dependent and PKA-independent signaling pathway. Furthermore, our novel exploration shows that DOR attenuates hypoxic/ischemic disruption of ionic homeostasis through the inhibitory regulation of Na⁺ channels. In this review, we will first update current information regarding the process and features of hypoxic/ischemic disruption of ionic homeostasis and then discuss the opioid-mediated regulation of ionic homeostasis, especially in hypoxic/ischemic condition, and the underlying mechanisms.     

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat

Sodium transport correlates with varying Na⁺-K⁺-ATPase activity rates along the nephron. Whether differences in Na⁺-K⁺-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na⁺-K⁺-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na⁺-K⁺-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43±1.99 versus 2.3±0.20, respectively, P<0.01) while Na⁺-K⁺-ATPase activity was higher in mTAL (3,402±83 vs 711±158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10^–6 M decreased phosphorylation in PCT to 125±10% (P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na⁺-K⁺-ATPase. Both calcineurin inhibitor FK-506 10^–6 M and phorbol 12-myristate 13-acetate (PMA) 10^–6 M increased the degree of Na⁺-K⁺-ATPase phosphorylation (P<0.05) and inhibited Na⁺-K⁺-ATPase activity to 657±152 and 1,448±347 pmol/mm tubule per hour, respectively, in mTAL (P<0.01). Increase in [Na⁺]_i to 30, 50 and 70 mM resulted in no changes in Na⁺-K⁺-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na⁺]_i were paralleled by decreased phosphorylation (from 120±7 to 160±15% of controls, P<0.05) and increased Na⁺-K⁺-ATPase activity (from 850±139 to 1,874±203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10^–6 M decreased both Na⁺-K⁺-ATPase dephosphorylation to 41.85±9.58% (P<0.05) and Na⁺-K⁺-ATPase activity to 2,405±176 pmol/mm tubule per hour in mTAL (P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.