Salt intake and intestinal dopaminergic activity in adult and old Fischer 344 rats

We have earlier shown that the renal dopaminergic system failed to respond to high salt (HS) intake in old (24-month-old) Fisher 344 rats (Hypertension 1999;34:666-672). In the present study, intestinal Na+,K+-ATPase activity and intestinal dopaminergic tonus were evaluated in adult and old Fischer 344 rats during normal salt (NS) and HS intake. Basal intestinal Na+,K+-ATPase activity (nmol Pi/mg protein/min) in adult rats (142+/-6) was higher than in old Fischer 344 rats (105+/-7). HS intake reduced intestinal Na+,K+-ATPase activity by 20% (P<0.05) in adult, but not in old rats. Dopamine (1 microM) failed to inhibit intestinal Na+,K+-ATPase activity in both adult and old Fischer 344 rats (NS and HS diets). In adult animals, co-incubation of pertussis toxin with dopamine (1 microM) produced a significant inhibitory effect in the intestinal Na+,K+-ATPase activity. L-DOPA and dopamine tissue levels in the intestinal mucosa of adult rats were higher (45+/-9 and 38+/-4 pmol/g) than those in old rats (27+/-9 and 14+/-1 pmol/g). HS diet did not change L-DOPA and DA levels in both adult and old rats. DA/L-DOPA tissue ratios, an indirect measure of dopamine synthesis, were higher in old (1.1+/-0.2) than in adult rats (0.6+/-0.1). Aromatic L-amino acid decarboxylase (AADC) activity in the intestinal mucosa of old rats was higher than in adult rats. HS diet increased the AADC activity in adult rats, but not in old rats. It is concluded that intestinal dopaminergic tonus in old Fisher 344 rats is higher than in adult rats and is accompanied by lower basal intestinal Na+,K+-ATPase activity. In old rats, HS diet failed to alter the intestinal dopaminergic tonus or Na+,K+-ATPase activity, whereas in adult rats increases in AADC activity were accompanied by decreases in Na+,K+-ATPase activity. The association between salt intake, increased dopamine formation and inhibition of Na+,K+-ATPase at the intestinal level was not as straightforward as that described in renal tissues.     

Effect of CDP-choline on hippocampal acetylcholinesterase and Na+,K(+)-ATPase in adult and aged rats

The aim of this study was to investigate the effect of different cytidine-5'-diphosphocholine (CDP-choline) concentrations (0.1-1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg(2+)-ATPase activities in homogenates of adult and aged rat hippocampi. Tissues were homogenised, centrifuged at 1000 x g for 10 min and in the supernatant, AChE activity and Na+,K(+)-ATPase and Mg(2+)-ATPase activities were determined according to Ellman's method and Bowler's and Tirri's method, respectively. After an 1-3 h preincubation of the homogenised tissue with CDP-choline, a maximal AChE stimulation of about 25% for both adult and aged rats (p < 0.001) and a Na+,K(+)-ATPase activation of about 50% for adult rats (p < 0.001) and about 60% for aged rats (p < 0.001) were observed, while hippocampal Mg(2+)-ATPase activity was not influenced in either adult or aged animals. It is suggested that: CDP-choline can restore hippocampal AChE and Na+,K(+)-ATPase activities in the aged rat and thus it may play a role in improving memory performance which is impaired by aging and some neuronal disturbances.     

Effect of hypoosmotic stimulation of the gastrointestinal mucosa on the activity of NA+, K+-atpase of epithelial cells in the small intestine and kidney in rats]

The experiments on Wistar rats have shown that intragestral injection with mannitol hypotonic solution (20 mmol/l) causes the significant activation of Na+, K(+)-ATPase of duodenum and distal intestine epithelial cells, kidney cortex cells, but does not affect the brain cortex of Na+, K(+)-ATPase activity. Simultaneously the activator of enzyme (AE) enters blood serum of rats, its activity is revealed by blood serum addition to homogenates of tissues of control rats. It is assumed that AE is produced in duodenal and intestinal mucosa released to blood after stimulation of mucosal surface by hypotonic solutions and included into the osmoregulation processes on the Na+, K(+)-ATPase level.     

State of the Na+, K+-ATPase system of the female rat brain cerebral cortex in the postnatal period during ethanol consumption in pregnancy and lactation

The ontogenetic development of the rat brain cortex Na+, K(+)-ATPase and Mg(2+)-ATPase activities under female ethanol (20% v/v) consumption in the third trimester of gestation or in postpartum period was studied. The weight characteristics (body, whole brain and cortex weight) of viable rats on the first day after birth were not affected critically by prenatal alcohol exposure. It is revealed that the delay of postnatal rat growth 10 days after birth under translactational ethanol consumption is accompanied by reliable decrease of plasma membrane Na+, K(+)-ATPase activity in comparison with control animals. The comparable decrease in activities was observed for the ouabain-sensitive and ouabain-resistant Na+, K(+)-ATPase components (isoform species). From the 20th day the differences in enzyme activity were not revealed. Mg(2+)-ATPase increases in postnatal period independent of Na+, K(+)-ATPase activity and it remains insensitive to postnatal maternal alcohol intake. It is suggested, the first ten day period of lactation is critical for ethanol effect on the developmental control of the brain Na+, K(+)-ATPase functional expression and the course of adaptive processes in the rat organism.     

Comparative study of in vitro and in vivo effect of ouabain and calixarene C107 on Na+,K(+)-ATPase activity in plasma membranes of rat hepatocytes

The comparative study of influence of ouabain and calixarene C107, and the structure component of this calixarene--fragment M3, in the conditions of in vitro and chronic action in vivo on Na+, K(+)-ATPase activity was carried out on the fractions of plasmatic membranes (PM) of the rat hepatocytes. A general property in the conditions in vitro is the ability of calixarene C107 and ouabain (both substances were in the concentration of 1 mM) to inhibit PM Na+, K(+)-ATPase of rat hepatocytes. However, in the case of activities of calixarene C107 and ouabain in the conditions in vivo heterogeneous action on Mg2(+)-ATPase and Mg2+, Na+, K(+)-ATPase activities takes place: total activity in the conditions of injection of increased concentrations of ouabain remains without changes, but Mg2(+)-ATPase activity significantly grows; in analogous conditions under the action of calixarene C107 both these activities decrease twice in comparison with control. Both under the in vitro and in vivo conditions, M3 fragment (the structural component of C107) does not change the values of investigated enzymatic activities. The biochemical mechanisms of calixarene C107 action on Na+, K(+)-ATPase activity in PM of rat hepatocytes are discussed.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

The effect of antimembrane testicular antibodies on Na+, K(+)-ATPase activity in the testicles of rats of different ages]

The effect of large and small doses of rabbit antibodies specific to plasma membranes of the rat testicle cells has been studied in the experiments on Wistar rats of three age groups (preadolescent--aged 20 days, puberal--aged 5-7 months and old--aged 24-26 months). It is stated that incubation of plasma membranes by IgG fraction isolated from antimembrane testicular serum (IgG-ATCSm) in a large dose (43 g of protein G per 125 g of protein of membrane fraction) caused statistically reliable inhibition of Na+, K(+)-ATPase activity in the membranes of testicle cells of puberal and old rats. Preadolescent rats exhibit only a tendency to decrease the activity of this enzyme. Incubation of plasma membranes of testicle cells in rats of different age by small doses of IgG-ATCSm (0.43 g of protein G per 125 g of membrane protein) induced a statistically reliable increase of Na+, K(+)-ATPase activity in puberal and old animals and its slight increase in preadolescent rats. The IgG fraction isolated from normal rabbit serum (IgG-NRS) exerted a less pronounced effect on Na+, K(+)-ATPase activity parallel with retention of a tendency to a decrease of activity under the influence of large doses of the drug and to an increase with introduction of small doses.     

Adenosine triphosphatase activity in sciatic nerve tissue of streptozocin-induced diabetic rats with and without high dietary sucrose: effect of aldose reductase inhibitors.

The ability of aldose reductase inhibitors to prevent the decline in neural Na+,K(+)-ATPase activity in diabetic rats has not been confirmed by all laboratories. In this study, the efficacy of two structurally different aldose reductase inhibitors was evaluated under different experimental conditions. Na+,K(+)-ATPase activity was measured in sciatic nerves from streptozocin-induced diabetic rats fed normal rodent chow or a chow supplemented with 68% sucrose. Nerve homogenates from chow-fed rats were prepared with a Dounce tissue grinder, whereas homogenates from the sucrose-fed rats were prepared with an Ultra-Turrax disperser. In the chow-fed rats, 4 weeks of untreated diabetes resulted in an increase in neural sorbitol and fructose, a decrease in myoinositol, and a 54% decline in Na+,K(+)-ATPase activity. Sorbinil administration (20 mg/kg/day) completely prevented the rise in sorbitol and fructose and the depletion of myoinositol, but did not prevent the decline in Na+,K(+)-ATPase activity. In diabetic rats fed the sucrose diet for 4, 6, and 8 weeks, the neural sorbitol and fructose levels were elevated, the myoinositol concentration declined, and the Na+,K(+)-ATPase activity was 26 to 28% below the control. Prevention or intervention treatment with sorbinil (20 mg/kg/day) or tolrestat (50 mg/kg/day) for 4 to 6 weeks prevented the alterations in sorbitol, fructose, and myoinositol, and also prevented the decline in Na+,K(+)-ATPase activity. In conclusion, prevention and intervention therapy with aldose reductase inhibitors prevented the decline in Na+,K(+)-ATPase in sciatic nerves of sucrose-fed streptozocin-diabetic rats that were homogenized with an Ultra-Turrax disperser, but not in sciatic nerves from streptozocin-diabetic rats fed normal rodent chow that were homogenized with a Dounce tissue grinder. These findings indicate that the assessment of aldose reductase inhibitor efficacy is dramatically affected by the type of nerve preparation assayed and/or the diet.     

Cortical Na+,K+-ATPase of immature rats following bicuculline-induced seizures

The Na+,K+-ATPase activity was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats in which seizures were induced by systemic (i.p.) administration of bicuculline. Na+,K+-ATPase activities in control animals increased during postnatal development, but they were not significantly influenced by seizure activity when determined under optimal conditions in vitro. Although the ratio of neuronal vs. non-neuronal cells in cortical samples of 7-, 12- and 18-day-old rats was different, there was a remarkable similarity in the activation curves for K+, obtained for Na+,K+-ATPase of all age groups under normal conditions; 50% of enzyme activities were attained at 1 mmol.l-1 K+ and the maximal activities were found around 10 mmol.l-1 K+. The activation curves for K+ in rats with bicuculline-induced seizures were not significantly different from those of the controls.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Age-Related Differences in Synaptosomal Peroxidative Damage and Membrane Properties

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.     

Sodium-potassium pump inhibitor in the mechanism of one-kidney, one wrap hypertension in dogs.

Using ouabain sensitive 86Rb uptake by the vessel wall, we previously showed that sodium-potassium pump activity is decreased in the arteries and veins, and that the sodium-potassium pump inhibitor (SPI) is increased in the plasma of dogs with one-kidney, one wrap (1-K, 1W) hypertension, a low renin model of hypertension. We also showed in rats with a similar type of hypertension that the membrane potential of vascular smooth muscle cells in arteries is decreased, and that this decrease can be reproduced in arterial cells in arteries from normal rats by applying plasma from the hypertensive animals. One endogenous SPI in human plasma has been reported to be ouabain or its isomer. In this study, we used a newly available Dupont ouabain enzyme immunoassay kit to examine plasma and kidneys for SPI in dogs with 1-K, 1W hypertension. We also examined 1) the inhibiting activity of plasma of Na+, K(+)-ATPase obtained from normal kidneys, and 2) the Na+, K(+)-ATPase activity of the kidneys from these hypertensive animals. 1-K, 1W hypertension was produced in dogs by wrapping the left kidney in a silk bag and removing the right kidney. The removed kidney was kept at -70 degrees C till assayed. After 4 weeks of hypertension, the remaining kidney was removed and stored at -70 degrees C till assayed. Blood samples were drawn before and at weeks 3 and 4 of hypertension. Plasma levels of "ouabain" and Na+, K(+)-ATPase inhibitory activity were increased at weeks 3 and 4 of hypertension, compared to pre-hypertension levels. Renal tissue "ouabain" levels were also increased at week 4 of hypertension. However, renal Na+, K(+)-ATPase activity was unchanged. These findings, using two different assays, confirm our 1980 conclusion that SPI is elevated in the plasma of dogs with 1-K, 1W hypertension. The absence of renal Na+, K(+)-ATPase inhibition, despite increased plasma and renal SPI in these animals, may have important implications for the development of this type of hypertension.     

Age-related characteristics of the Na+,K+-ATPase activity of synaptic membranes from the rat brain

The study of albino rats aged 6-7 months and 25-27 months revealed the age-related increase of maximal activity (V) of Na+, K+-ATPase of synaptosomal plasma membranes, separated from the cerebral cortex, while the level of Km remained stable. It is shown that in old rats as compared to the adult ones the affinity of Na+, K+-ATPase to sodium ions increases and the character of the ATP hydrolysis schedule changes in the presence of different ration of ions-activators. There are no significant changes in the inhibiting effect of strophantidin K on Na+, K+-ATPase activity of synaptosomal plasma membranes.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.     

Na+,K(+)-ATPase activity in human colorectal carcinoma

Na+,K(+)-ATPase activities in macroscopically unchanged mucosa (conditionally normal tissue) and human colorectal carcinoma (mainly low-grade and moderately differentiated adenocarcinomas) have been investigated. Microsomal fractions are similar by dimensions of the membrane fragments detected by photon correlation spectroscopy analysis. The activation optima under digitonin pretreatment of the membrane fractions differ significantly for Na+,K(+)-ATPase and concomitant Mg(2+)-ATPase activity, but are the same in conditionally normal and cancerous tissues. This allows to detect correctly total levels of the Na+,K(+)-ATPase activity in the detergent-pretreated preparations. The moderate decrease of the Na+,K(+)-ATPase activity is revealed in carcinomas. It is concluded that a decrease of activity of the ouabain-sensitive human Na+,K(+)-ATPase is characteristic of colorectal carcinoma.     

Temporal changes in intestinal Na+, K(+)-ATPase activity and in vitro responsiveness to cortisol in juvenile chinook salmon

Seasonal changes in endogenous Na+, K(+)-ATPase activity were measured in pyloric ceca and posterior intestine of juvenile chinook salmon (Oncorhynchus tshawytscha) maintained in fresh water over 18 months. In tissues from these same fish, the in vitro responsiveness of Na+, K(+)-ATPase activity to 10 microg cortisol/ml was assessed. There were pronounced increases in endogenous Na+, K(+)-ATPase activity in summer for both intestinal regions, in underyearlings and yearlings. In pyloric ceca, a significant positive response of Na+, K(+)-ATPase activity to cortisol, in vitro, was restricted to the months preceding increases in endogenous Na+, K(+)-ATPase and the month afterward. Na+, K(+)-ATPase activity of the posterior intestine was only responsive to cortisol in underyearlings in the period before the peak in endogenous enzyme activity. At a time when explants were responsive to cortisol, in vitro exposure to 0.1-10 microg cortisol/ml resulted in dose-dependent elevations of Na+, K(+)-ATPase activity over controls (0 microg cortisol/ml). The results show that the intestine exhibits increased enzymatic potential for water absorption that is indicative of parr-smolt transformation. Alterations in tissue responsiveness to cortisol may contribute to these changes in Na+, K(+)-ATPase activity of pyloric ceca.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.