Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

Effects of rubratoxin B on the kinetics of cationic and substrate activation of (Na+-K+)-ATPase and p-nitrophenyl phosphatase.

Rubratoxin B, a lactone-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dose-dependent manner with an estimated IC50 of 6 x 10(-6) M. Hydrolysis of ATP was linear with time and enzyme concentration, with or without rubratoxin in reaction mixtures. Altered pH and activity curves for (Na+-K+)-ATPase demonstrated comparable inhibition by rubratoxin in buffered acidic, neutral, and alkaline pH ranges. Kinetic studies of cationic-substrate activation of (Na+-K+)-ATPase indicated classical competitive inhibition for Na+ and K+. Results also showed competitive inhibition for K+ activated p-nitrophenyl phosphatase as demonstrated by altered binding site parameters without change in the catalytic velocity of dephosphorylation of the enzyme . phosphoryl complex. Noncompetitive inhibition with regards to activation by ATP and p-nitrophenyl phosphate was indicated by altered Vmax values with no change in Km values. Inhibition was partially restored by repeated washings. Preincubation with sulfhydryl agents protected the enzyme from inhibition. Cumulative inhibition studies with rubratoxin and ouabain indicated possible interaction between the two inhibitors of (Na+-K+)-ATPase. Rubratoxin appeared to exert its effects on (Na+-K+)-ATPase by interacting at Na+ and K+ sites.     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Schistosoma mansoni: preparation, characterization of (Na+ + K+)ATPase from tegument and carcass

The preparation and some biochemical properties of a (Na+ + K+)ATPase from male adult Schistosoma mansoni are described. After incubation in a membrane disruption medium, the tegument and carcass of the worms were separated and treated to obtain fractions enriched in (Na+ + K+)ATPase. The activity of the tegumental ouabain sensitive (Na+ + K+)ATPase at 37 C was 20.3 mumole Pi X mg-1 protein X hr-1 and represented 32% of the total ATPase activity. The (Na+ + K+)ATPase prepared from the carcass had a lower specific activity (3.7 mumole Pi X mg-1 protein X hr-1) but a higher relative activity (55%). Similar concentrations of Na+ and K+ activated the enzymes from both sources, and both enzymes were inhibited by similar concentrations of calcium. However, the enzyme from carcass was ten times more sensitive to ouabain than the enzyme from tegument. Comparison with results obtained on the (Na+ + K+)ATPase of human heart showed that the enzymes from the worms were more resistant to ouabain. The half maximal inhibitory concentration of dihydroouabain compared to that of ouabain was also different in the enzymes from human and worm. We conclude that (1) there exists at least one structural difference between the (Na+ + K+)ATPase of S. mansoni and that of the human host, and (2) it is useful to separately study the enzymes from tegument and carcass because they differ in sensitivity to cardiac glycosides.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Inhibitory action of opioid peptides on ouabain-sensitive Na+-K+ and Ca2+-dependent ATPase activities in bovine cardiac sarcolemma

The present study demonstrates that morphine (10(-6) and 10(-5) M), methionine-enkephalin or leucine-enkephalin (10(-10), 10(-8), and 10(-6) M) were able to inhibit significantly, in a dose-dependent manner, both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. The inhibitory action of these opioids on the two ATPases was not antagonized by preincubation with naloxone (10(-6) M). Naloxone alone (10(-8), 10(-6) and 10(-5) M) did not affect both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. Heat-denatured methionine-enkephalin (10(-6) M) or leucine-enkephalin (10(-6) M) also unaffected both the ATPases. The possibility is also discussed that opioid peptides may regulate myocardial contractility by modulating the movement of ions across the heart sarcolemma.     

Effect of vanadate on (Na+-K+)-stimulated ATPase activity in the brain of rats of different ages

Experiments were carried out on infant rats aged five days and on adult rats (of both sexes) to investigate vanadate inhibition of (Na+-K+)ATPase activity in various parts of the brain. Vanadate was administered in 10(-5), 10(-7), 10(-8), 10(-9) and 10(-10) mol/l concentration. The enzyme activity and the effect of vanadate were studied in the tissue of the cerebral cortex, subcortical formations and the medulla oblongata. It was demonstrated that an inhibitory effect of vanadate on ouabain-sensitive ATPase could be determined in the brain of very young rats, i.e. in the immature nervous tissue. It was further demonstrated that the inhibitory effect of vanadate (in low concentrations) was significantly more potent in the nervous tissue of adult rats than in the CNS tissue of 5-day-old animals. Lastly, attention is drawn to certain differences in the sensitivity of ouabain-sensitive ATPase to the action of vanadate indifferent parts of the CNS in both the given age groups.     

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.     

Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

Effect of lysophosphatidylcholine on K+ transport in rat heavy gastric membranes enriched with (H+-K+)-ATPase

K+- and ATP-dependent H+-accumulation in rat heavy gastric membrane vesicles enriched with (H+-K+)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3-14 at concentrations of 10(-5) M. Their stimulatory effect was dependent on K+-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H+-K+)-ATPase. Lysophosphatidylcholine at the optimal dose (3 X 10(-5) M) showed dual effects on K+-dependent membrane functions; it stimulated the rate of K+-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K+-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H+-pumping through (H+-K+)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K+-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limiting step for operation of the ATPase is the availability of K+ ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K+-transport in rat heavy gastric membranes through specific interactions with the putative K+-transporter.     

Ischemia-reperfusion alters gene expression of Na+-K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.     

Rat brain (Na+-K+)ATPase: modulation of its ouabain-sensitive K+-PNPPase activity by thimerosal

1. The (Na+ + K+) ATPase activity of a rat brain synaptic membrane preparation was inhibited by 10(-5) M thimerosal. 2. The ouabain inhibitable K+-PNPPase activity of thimerosal treated membranes was compared with that of untreated membranes with respect to sensitivity to temperature, ouabain, K+ and ATP. 3. All those kinetic characteristics were substantially altered by treatment with thimerosal.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Human neuromuscular fatigue is associated with altered Na+-K+-ATPase activity following isometric exercise.

The purpose of this study was to investigate the hypothesis that reductions in Na+-K+- ATPase activity are associated with neuromuscular fatigue following isometric exercise. In control (Con) and exercised (Ex) legs, force and electromyogram were measured in 14 volunteers [age, 23.4 +/- 0.7 (SE) yr] before and immediately after (PST0), 1 h after (PST1), and 4 h after (PST4) isometric, single-leg extension exercise at ~60% of maximal voluntary contraction for 30 min using a 0.5 duty cycle (5-s contraction, 5-s rest). Tissue was obtained from vastus lateralis muscle before exercise in Con and after exercise in both the Con (PST0) and Ex legs (PST0, PST1, PST4), for the measurements of Na+-K+-ATPase activity, as determined by the 3-O-methylfluorescein phosphatase (3-O-MFPase) assay. Voluntary (maximal voluntary contraction) and elicited (10, 20, 50, 100 Hz) force was reduced 30-55% (P < 0.05) at PST0 and did not recover by PST4. Muscle action potential (M-wave) amplitude and area (measured in the vastus medialis) and 3-O-MFPase activity at PST0-Ex were less than that at PST0-Con (P < 0.05) by 37, 25, and 38%, respectively. M-wave area at PST1-Ex was also less than that at PST1-Con (P < 0.05). Changes in 3-O-MFPase activity correlated to changes in M-wave area across all time points (r = 0.38, P < 0.05, n = 45). These results demonstrate that Na+-K+- ATPase activity is reduced by sustained isometric exercise in humans from that in a matched Con leg and that this reduction in Na+-K+-ATPase activity is associated with loss of excitability as indicated by M-wave alterations.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

HPLC method for measuring (Na(+)-K(+)) ATPase and (Ca(++)-Mg(++)) ATPase in erythrocytes from different species of mammals]

(Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase are enzymes located in erythrocyte plasma membranes, driving back ions against the electrochemical gradient; (Na(+)-K+)ATPase transports 3 Na+ ions out of the cell, and 2 K+ ions into it for each hydrolyzed ATP molecule, whereas the Ca(2+)-pump transports Ca2+ ions out of the cells, by utilizing still the ATP hydrolysis. The method used to test the activity of the above mentioned enzymes is based on the measuring of the ADP quantity released during the reaction by HPLC, that is High Performance Liquid Chromatography; the chromatographic type is a Ion-Pair Reversed-Phase. This method presents the following important advantages for the assay of the enzymes we analysed: 1) It is reproducible through time; 2) It is perfectly linear; 3) It is extremely sensitive. This method allowed us to carry out a comparative study of (Na(+)-K+)ATPase and (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of several species of mammalia: man, horse, rabbit, lamb, rat. We recovered different values in ATPase activity; (Ca(++)-Mg++)ATPase shows a higher activity than Na(+)-K+)ATPase; moreover, some differences exist in the various Mammalia considered, with relation to each pump: the lamb shows the lowest activity for both pumps, whereas the rabbit shows the highest one. At present, the different values obtained are being interpreted and analysed. This method is also very versatile, since it allowed us to assess the Km value for Ca++ of the (Ca(++)-Mg++)ATPase in erythrocyte plasma membranes of rabbit. The value resulted to be 100 microMs, thus 10 times higher than the human Km value for the Ca++.     

Alterations in jejunal transport and (Na+-K+)-ATPase in an experimental model of hypoxia in rats

Hypoxia was induced by exposing rats to an atmosphere of 93% N2, 7% O2 for 4-48 hr. The animals became hypoxic as indicated by a decreased blood PaO2 (mean +/- SEM: 48 +/- 10 mm Hg). Hypoxia was accompanied by metabolic acidosis (pH 7.22 +/- 0.02) and decreased serum bicarbonate levels (9.0 +/- 4.0 meq/liter). Hypoxic rats also showed evidence of tissue hypoxia; liver tryptophan oxygenase levels were increased to 21 +/- 2 nmole/min/mg protein. In the hypoxic animals there was decreased jejunal mucosal (Na+-K+)-ATPase activity and an inhibition of active intestinal transport of sodium, glucose, 3-O-methylglucose, galactose, tyrosine, phenylalanine, and glycine as determined by in vivo perfusion studies. Jejunal fructose transport, which has a large passive component, was unaffected by hypoxia. The electrolyte, carbohydrate, and amino acid transport alterations produced by hypoxia were seen in the absence of an effect on jejunal cell number, DNA synthesis, or cell turnover. There was also no evidence of histological or ultrastructural damage. Furthermore, studies with a luminal macromolecular tracer, horseradish peroxidase, indicated that the jejunal lumen-to-blood barrier to macromolecules was also unaltered in these hypoxic animals. In vitro local oxygenation of the jejunum, by bubbling of 95% O2:5% CO2, markedly improved sodium and glucose (but not 3-O-methylglucose) absorption in hypoxic rats and control rats. The (Na+-K+)-ATPase activity of the jejunal mucosa of hypoxic rats was significantly enhanced by the local bubbling of 95% O2:5% CO2. Overall, our data indicate that during relatively mild conditions of hypoxia there is an inhibition of jejunal (Na+-K+)-ATPase activity and related transport processes that is prevented by in situ oxygenation.     

Buffer systems variably affect the interaction of norepinephrine with brain Na+-K+ ATPase

The reported effects of norepinephrine (NE) on brain Na+-K+ ATPase are quite variable. Different investigators have reported activation, inhibition, or no effect. An investigation of the importance of reaction conditions on brain Na+-K+ ATPase activity was undertaken to resolve some of these discrepancies. Using porcine cerebral cortical Na+-K+ ATPase and rat brain synaptosomal membrane preparations, it was observed that NE strongly inhibited brain Na+-K+ ATPase in Tris-HCl buffer. This inhibition of the enzyme was reversed by the addition of EDTA. In contrast, NE did not significantly inhibit Na+-K+ ATPase in imidazole-glycylglycine and Krebs-Ringer-phosphate buffers. This buffer dependence of NE inhibition of the enzyme was consistently demonstrated with three different established methods for phosphate measurement. Kinetic analysis indicated that NE, in Tris-HCl buffer, inhibited the enzyme noncompetitively at high affinity, and competitively at low affinity, ATP substrate sites.     

19(S)-hydroxyeicosatetraenoic acid is a potent stimulator of renal Na+-K+-ATPase

Omega- and omega-1 hydroxylations are the major pathways by which arachidonic acid is metabolized in cortical and outer medullary microsomes of rat and rabbit kidneys. It is a cytochrome P450-dependent oxidation leading to the formation of 20-hydroxy- and 19-hydroxyeicosatetraenoic acids. In this study, we compared the effects of the synthetically prepared omega- and omega-1 metabolites of arachidonic acid on the activity of the renal Na+-K+-ATPase partially purified from rat renal cortical microsomes. 19(S)-hydroxyeicosatetraenoic acid caused a dose related stimulation of Na+-K+-ATPase activity with an EC50 of 3 x 10(-7) M. In contrast, neither 19(R)-hydroxyeicosatetraenoic acid, 20-hydroxyeicosatetraenoic acid nor arachidonic acid at 10(-6) M had any effect on Na+-K+-ATPase activity. In the same preparation, ouabain at 10(-3) M and 12(R)-hydroxyeicosatetraenoic acid at 10(-6) M inhibited the enzyme activity by 75% and 60%, respectively. We conclude that 19(S)-hydroxyeicosatetraenoic acid is a specific stimulator of renal Na+-K+-ATPase. Therefore, the formation of 19(S)-hydroxyeicosatetraenoic acid by renal cortical cytochrome P450 omega-1-hydroxylase may contribute to the regulation of renal function by regulating Na+-K+-ATPase which is essential for transtubular transport processes.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.