Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.     

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.     

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.     

The calixarenes C-97 and C-107 stimulate influence of ouabain on the Na+,K+-ATPase activity in plasmatic membrane of smooth muscle cells

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97).     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

The role of the Na+/H+ exchange system in cardiac cells in relation to the control of the internal Na+ concentration. A molecular basis for the antagonistic effect of ouabain and amiloride on the heart

Cardiac cells in culture (from rat and chick heart) have a membrane Na+/H+ exchange system that is inhibited by amiloride (K0.5 = 5 microM) and by its more potent N-5-disubstituted derivatives dimethylamiloride (K0.5 = 300 nM) and ethylisopropylamiloride (K0.5 = 30 nM). The properties of the cardiac Na+/H+ exchange system are similar to those found for the Na+/H+ exchanger in other cellular types. The Na+/H+ exchange system is a major pathway for Na+ uptake by cardiac cells. Ouabain which inhibits the (Na+,K+)-ATPase, a major pathway for Na+ efflux, is known to provoke Na+ accumulation and to stimulate 45Ca2+ entry via the Na+/Ca2+ exchange mechanism, thereby producing an inotropic effect. N-5-Disubstituted amiloride derivatives, by blocking Na+ entry into cardiac cells, antagonize both ouabain-induced intracellular Na+ accumulation and the ouabain-induced acceleration of 45Ca2+ uptake.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Kinetics of the Mechanism of Action of Monoamine Oxidase in the Regulation of Na+, K+-ATPase Activity in Rat Brain

Kinetic studies on the action of monoamine oxidase (MAO) in the regulation of Na+,K+-ATPase were performed using 3-methoxy-4-hydroxybenzaldehyde (MHB), which is an analogue of 3-methoxy-4-hydroxy-phenylacetylaldehyde (product of MAO-catalysed reaction with dopamine as substrate). It was observed that at 2.6 microM MHB, the activation of Na+,K+-ATPase may be the result of the removal of the inhibitory Ca2+, thereby increasing the Vmax. Double-reciprocal plots of Pi versus MHB showed that Ca2+ counteracted the effect of the aldehyde not by changing the Km, but be decreasing the Vmax of the Na+,K+-ATPase stimulation. The removal of 3',5'-cyclic AMP-dependent protein kinase from the microsomes by sodium dodecyl sulphate treatment abolished the activation and/or inhibition of the Na+,K+-ATPase by aldehyde; it can therefore be inferred that 3',5'-cyclic AMP-dependent protein kinase is involved in the regulation of Na+,K+-ATPase.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.     

Inhibition of rat brain microsomal (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase by periodic acid

The effects of mild periodate exposure on the kinetics of (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase were studied using rat cerebral microsome preparations. Fifty percent inhibition of both enzyme activities was attained near 3 microM periodate concentrations. This inhibition was biphasic with time. Mg2+-ATPase and Mg2+-p-nitrophenylphosphatase activities were much less inhibited by periodate. Periodate inhibition was partially reversed by dimercaprol and dithiothreitol but not by diffusion. The possible reaction products formic acid, formaldehyde, glyceraldehyde, and acetaldehyde had no inhibitory effects in similar concentrations. Periodate exposure produced no detectable changes in the activation of (Na+ + K+)-ATPase by Na+, K+, Mg2+, or ATP. Residues shared by both (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase are both critical to hydrolytic function and sensitive to mild oxidation by periodate.     

Caco-2 cells in culture synthesize and degrade dopamine and 5-hydroxytryptamine: a comparison with rat jejunal epithelial cells

To explore the usefulness of Caco-2 cells in the study of intestinal dopaminergic and 5-hydroxytryptaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AADC), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B) activities in these cells using specific substrates. The activity of these enzymes was also evaluated in isolated rat jejunal epithelial cells. The results showed that Vmax values (in nmol mg protein(-1) h(-1)) for AADC, using L-DOPA as the substrate, in rat jejunal epithelial cells (127.3+/-11.4) were found to be 6-fold higher than in Caco-2 cells (22.5+/-2.6). However, Km values in Caco-2 cells (1.24+/-0.37 mM) were similar to those observed in rat jejunal epithelial cells (1.30+/-0.29 mM). Similar results were obtained when AADC activity was evaluated using L-5HTP as substrate; in rat jejunal epithelial cells Vmax values (in nmol mg prot(-1) h(-1)) were found to be 5-fold that in Caco-2 cells (16.3+/-1.0 and 3.0+/-0.2, respectively), and Km values in Caco-2 cells (0.23+/-0.08 mM) were again similar to those observed in rat intestinal epithelial cells (0.09+/-0.03 mM). Caco-2 cells were not able to O-methylate dopamine, in contrast to rat jejunal epithelial cells (Vmax = 8.6+/-0.4 nmol mg protein(-1)(h-1); Km = 516+/-57 microM). Vmax values (in nmol mg protein(-1)(h-1)) for type A and B MAO in Caco-2 cells (19.0+/-0.6 and 5.4+/-0.6, respectively) were found to be significantly lower (P<0.05) than those in rat jejunal epithelial cells (46.9+/-3.1 and 9.6+/-1.2, respectively); however, no significant differences in the Km values were observed between Caco-2 and rat jejunal epithelial cells for both type A and B MAO. In conclusion, Caco-2 cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade DA and 5-HT, though, no COMT activity could be detected in these cells.     

Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile cells

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).     

Phosphorylation of the catalytic subunit of rat renal Na+, K+-ATPase by classical PKC isoforms

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+,K+-ATPase activity (assessed by 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a nonselective PKC activator, inhibited Na+,K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKCepsilon, did not affect 86Rb+ uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+,K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.     

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.