Effects of Ranitidine on Gastric Vesicles Containing H+, K+-Adenosine Triphosphatase in Rats

Background: To ascertain the mechanism for rebound acid hypersecretion after treatment with an H₂-receptor blocker, we investigated the effects of ranitidine on gastric H⁺, K⁺-adenosine triphosphatase (ATPase) in rats, Methods: Male Wistar rats received ranitidine (1-50mg/kg body weight intraperito-neally twice a day for 5 days). The rats were starved for 15 h after the last treatment and then killed, and gastric vesicles containing H⁺, K⁺-ATPase were prepared. Results: Treatment with ranitidine dose-dependently increased protein content in the gastric vesicular fraction purified from the gastric mucosa without changing total protein content. Ranitidine also increased the content of a 94,000-dalton protein, the catalytic subunit of H⁺, K⁺-ATPase. On the other hand, ranitidine did not affect the specific activity of the enzyme (μmol/min/mg of the gastric vesicular protein). Since gastric vesicles in the fasting state mainly consist of the tubulovesicular membrane, these results suggest that ranitidine administration increases total tubulovesicular H⁺, K⁺-ATPase content (μmol/min/rat) by increasing the number of tubulovesicles per parietal cell. The ranitidine-induced increase in total tubulovesicular H⁺, K⁺-ATPase activity was still evident 1 week after treatment and returned to control level 1 month later. Conclusions: All these findings suggest that the increased content and total activity of tubulovesicular H⁺, K⁺-ATPase after ranitidine treatment may contribute to the mechanism for acid rebound after H₂-blocker therapy.     

Nitric Oxide-Mediated Regulation of Gastric H+, K+-ATPase and Alcohol Dehydrogenase Following Ethanol-Induced Injury in Rats

The mucosal protective effect of nitric oxide(NO) was examined by usingN^G-nitro-L-arginine methyl ester (L-NAME) asnitric oxide synthase (NOS) inhibitor and nitroprusside(NP) as NO donating agent, in ethanol-induced rat gastric lesion model.The results are summarized as follows: (1) As gastrictissue samples were examined by light microscopy,intragastric exposure of ethanol was demonstrated to induce gastric injury, which was more prominentin female rats. The depletion of NO by L-NAME treatmentexacerbated the ethanol-induced gastric lesion but NPtogether with ethanol promoted repair of the mucosal injury, especially in female rats. (2)Gastric H⁺, K⁺-ATPase enzymeactivity, which was responsible for acid secretion,seemed not to be effected by ethanol treatment. Togetherwith ethanol, L-NAME treatment activated, whereas NP treatmentinhibited, the enzyme activity in female rats. (3)Ethanol treatment inhibited gastric alcoholdehydrogenase (ADH) activity, which was responsible forthe first-pass metabolism of ethanol. Together with ethanol,L-NAME did not effect the enzyme activity whereas NPtreatment disappeared the inhibitory effect of ethanolin both gender. Hydroxyl radical (OH) scavenger activity was found to increase in ethanol andethanol + NP groups in both sexes, but superoxideradical (O₂ ^-) scavengeractivity did not change. The results indicate that NOmay ameliorate the damaging effect of ethanol possibly by regulating acidsecretion, ethanol metabolism, and antioxidant contentin rat gastric mucosa.     

Inhibition of Na+, K+-ATPase Activity by the Metabolites Accumulating in Homocystinuria

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na⁺, K⁺-ATPase, and Mg²⁺-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na⁺, K⁺-ATPase but not Mg²⁺-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na⁺, K⁺-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na⁺, K⁺-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.     

Platelet Na+,K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria

Thein vitro effects of phenylalanine or alanine alone or combined on Na⁺,K⁺-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na⁺,K⁺-ATPase activity by 20–50% [F(4,25)=11.47; p<0.001]. Alanine had no effect on Na⁺,K⁺-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na⁺,K⁺-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na⁺,K⁺-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.     

Alanine Prevents the Decrease of Na+,K+-ATPase Activity in Experimental Phenylketonuria

Our objective was to investigate the effect of alanine administration on Na⁺,K⁺-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 mol alanine or 5.2 mol phenylalanine plus 2.6 mol -methylphenylalanine per g body weight or phenylalanine plus -methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na⁺,K⁺-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na⁺,K⁺-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.     

Proline Administration Decreases Na+,K+-ATPase Activity in the Synaptic Plasma Membrane from Cerebral Cortex of Rats

Buffered proline was injected subcutaneously into rats twice a day at 8 h intervals from the 6^th to the 28^th day of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation 12 h after the last injection. Cerebral cortex was used for the determination of Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities. Body, whole brain and cortical weights were similar in the two groups. Na⁺,K⁺-ATPase activity was significantly reduced (by 20%) in membranes from the proline-treated group compared to the controls, whereas Mg²⁺-ATPase activity was not affected by proline. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 29-day-old rats and incubated with proline at final concentrations ranging from 0.1 to 2.0 mM. Na⁺,K⁺-ATPase activity, but not Mg²⁺-ATPase activity, was inhibited by 20-30%. Since proline concentrations in plasma of chronically treated rats and of type ll hyperprolinemic children are of the same order of magnitude as those tested in vitro, the results suggest that reduction of Na⁺,K⁺-ATPase activity may contribute to the neurological dysfunction found in some patients affected by type ll hyperprolinemia.     

Intrastriatal Administration of Guanidinoacetate Inhibits Na+, K+-ATPase and Creatine Kinase Activities in Rat Striatum

Guanidinoacetate methyltransferase deficiency (GAMT deficiency) is an inherited neurometabolic disorder clinically characterized by epilepsy and mental retardation and biochemically by accumulation of guanidinoacetate (GAA) and depletion of creatine. Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not yet established. In the present study, we investigated the effect of intrastriatal administration of GAA on Na⁺, K⁺-ATPase activity, total (tCK), cytosolic (Cy-CK), and mitochondrial (Mi-CK) creatine kinase (CK) activities in rat striatum. We verified that Na⁺, K⁺-ATPase, tCK, and Mi-CK activities were significantly inhibited by GAA, in contrast to Cy-CK which was not affected by this guanidino compound. Since these enzyme activities can be affected by reactive species, we also investigated the effect of intrastriatal administration of GAA on thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in rats. We found that this metabolite significantly increased this oxidative stress parameter. Considering the importance of Na⁺, K⁺-ATPase and CK activities for brain metabolism homeostasis, our results suggest that the inhibition of these enzymes by increased intracerebral levels of GAA may contribute to the neuropathology observed in patients with GAMT-deficiency.     

Helicobacter pylori augments the acid inhibitory effect of omeprazole on parietal cells and gastric H(+)/K(+)-ATPase

BACKGROUND: In duodenal ulcer patients, intragastric acidity during omeprazole treatment is significantly lower before Helicobacter pylori eradication than after cure. AIMS: To determine if H pylori enhances the acid inhibitory potency of omeprazole in isolated parietal cells and on H(+)/K(+)-ATPase. METHODS: Rat parietal cells and pig gastric membrane vesicles enriched in H(+)/K(+)-ATPase activity were incubated with H pylori and the H pylori fatty acid cis 9,10-methyleneoctadecanoic acid (MOA), and the inhibitory effects of omeprazole on parietal cell acid production, H(+)/K(+)-ATPase enzyme activity, and ATPase mediated proton transport were assessed. RESULTS: In isolated parietal cells, H pylori and MOA increased the acid inhibitory potency of omeprazole 1.8 fold. H pylori did not affect the inhibitory potency of omeprazole on H(+)/K(+)-ATPase enzyme activity. In proton transport studies, H pylori (intact bacteria and sonicate) and MOA accelerated the onset of the inhibitory effect of omeprazole and enhanced the proton dissipation rate in response to omeprazole. H. pylori itself increased proton permeability at the vesicle membrane. CONCLUSION: Our results show that H pylori augments the acid inhibitory potency of omeprazole in parietal cells and enhances omeprazole induced proton efflux rate from gastric membrane vesicles. We suggest that omeprazole unmasks the permanent effect of H pylori on proton permeability at the apical parietal cell membrane, which is counteracted in the absence of a proton pump inhibitor by a reserve H(+)/K(+)-ATPase capacity.     

Famotidine,a histamine-2-receptor antagonist,inhibits the increase in rat gastric H+/K+-ATPase mRNA induced by intravenous infusion of gastrin 17 and histamine

We examined the effects of gastrin and histamine on rat gastric H⁺/K⁺-ATPase, the enzyme responsible for H⁺ secretion, gene expressionin vivo. Gastrin 17 (G 17) or histamine dihydrochloride (histamine) was continuously infused through the femoral vein of anesthetized rats. Gastric H⁺/K⁺-ATPase mRNA levels were measured using northern blot analysis. Infusion of G 17 and histamine increased the H⁺/K⁺-ATPase mRNA level significantly compared with basal control level or vehicle control level (P<0.01). However, pretreatment with famotidine, a potent histamine-2 (H₂)-receptor antagonist, inhibited the increase of rat gastric H⁺/K⁺-ATPase mRNA following G 17 and histamine infusion. These findings indicate that both histamine and G 17 increase expression of H⁺/K⁺-ATPase mRNA by activating H₂ receptor on the parietal cell.     

Bufodienolides as Endogenous Na+, K+-ATPase Inhibitors: Biosynthesis in Bovine and Rat Adrenals

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [³H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. These results suggest that Na⁺, K⁺-ATPase inhibitors which interact with cuabain antibodies, but not those which interact with bufalin antibodies, are synthesized in bovine and rat adrenals     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

The sodium pump (Na⁺, K⁺-ATPase, NKA) is vital for animal cells, as it actively maintains Na⁺ and K⁺ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF₃⁻ complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K⁺-antagonism and suggest a route to isoform specific CTSs.

Under physiological conditions, Na⁺,K⁺-ATPase (NKA) actively extrudes three cytoplasmic Na⁺ ions in exchange for two extracellular K⁺ ions per ATP hydrolyzed (see Fig. 1 for a simplified reaction diagram). The established gradients for Na⁺ and K⁺ are pivotal for generating a membrane potential, regulation of cell volume, and providing chemical energy for various secondary active transporters. They are expressed in all animal cells and are finely tuned. In humans, the catalytic α-subunit exists in four isoforms. α1 is ubiquitous and best studied. α2 is most abundant in skeletal and heart muscle, whereas α3 is found in brain cells and α4 in cells of the testis. The α-subunit complexes with a β-subunit (isoforms β1–4 in humans) and a tissue specific regulatory protein FXYD (1–7 in humans) (for a recent general review, see, e.g., refs. 1, 2).Open in a separate windowFig. 1.A reaction diagram of Na⁺,K⁺-ATPase with special emphasis on the backward phosphorylation with P_i and inhibition by CTSs. CTSs can bind to at least three E2P species with different affinities. The states demonstrated to allow high-affinity binding of CTSs appear in purple letters, and those supposed to allow high-affinity binding but not demonstrated are in red letters; the state that allows low-affinity binding is in orange letters. E2P^ATP is the physiological E2P ground state (path A); E2P^Pi can be formed in the backward reaction starting from E2 with P_i (path B). This reaction places Mg²⁺ at the phosphorylation site of NKA but likely to incorporate another Mg²⁺ at site II (E2P^Pi·Mg²⁺). If the backward phosphorylation reaction starts from E2·2K⁺, E2P·2K⁺ will be (transiently) formed in which two K⁺ bind to NKA in E2P with high affinity (E2P·2K⁺_high). CTSs can stabilize a different state, in which two K⁺ are bound with (presumably) low affinity (E2P^Pi·2K⁺_low; path C). Crystal structures available are boxed and phosphate analogs used are shown below the boxes. The crystal structures obtained in this study are highlighted (yellow boxes). PDB ID codes for published crystal structures are: E1∼P·ADP·3Na⁺, 3WGU; E2P^Pi·Mg²⁺(OBN), 4HYT; E2P^Pi·Mg²⁺(DGX), 4RET; E2P^Pi·2K⁺ (BUF), 4RES; E2·P_i·2K⁺, 2ZXE; E2·P_i·2K⁺(OBN), 3A3Y.Cardiotonic glycosides, such as digoxin (DGX) and digitoxin (DTX), are specific inhibitors of NKA and have been prescribed for patients with heart failure for centuries. Canonical cardiotonic glycosides, including ouabain (OBN), the best studied member, have a tripartite structure: a central steroid core, a five-membered or six-membered lactone ring, and a carbohydrate moiety of one to four residues. Each part appears to have a different role in binding. As summarized by Glynn (3), critical features of high affinity cardiac glycosides are: 1) The unsaturated lactone ring attached in the correct configuration at C17; 2) the cis configuration of the AB and CD ring junctions in the steroid nucleus; 3) the presence of a hydroxyl group at C14; and 4) the presence of an appropriate sugar at C3. A wide range of cardiotonic steroids (CTSs), including aglycones, as the sugar at C3 does not necessarily improve the affinity, showing vastly different inhibitory properties, have been developed in order to improve their usability in the clinical setting.Indeed, several new members, such as rostafuroxin (ROS) (4) and istaroxime (IST) (5), now under clinical trials, have distinct chemical structures. ROS is proposed as a potent antihypertensive compound in ouabain-dependent models of hypertension (4). It is reported to be capable of displacing OBN from NKA at a concentration 10 times lower than that expected from its K_I, which is 1,000 times greater than that of OBN (6). IST has only a carbonyl group instead of the unsaturated lactone, and an aminoalkyloxime group instead of the sugar, but shows an inhibitory potency similar to that of digoxin (5). It is reported to have a significant inotropic effect but with a lower risk of causing cardiac arrhythmia compared to digoxin (5). Why these compounds can replace OBN at a much lower concentration than that expected from their binding affinities is paradoxical and addressed in this study.Reflecting their very long history, the accumulated literature on CTSs is huge. Numerous studies report on their inhibitory activities, but they appear rather inconsistent, partly due to differences in experimental conditions (7). It is well established that CTSs preferentially bind to NKA in the E2P ground state from the extracellular face (8). However, the E2P states formed in different routes show distinct properties. In the physiological route, in the presence of Mg²⁺ and Na⁺, the E2P state is reached through phosphorylation by ATP (path A in Fig. 1) and denoted here as E2P^ATP. The E2P state can be reached by backward phosphorylation by P_i in the presence of Mg²⁺ (path B in Fig. 1) and denoted as E2P^Pi [denoted previously as E′2P (9)]. These states show different kinetic properties. In particular, dephosphorylation of E2P^ATP is fast if K⁺ is present, whereas that of E2P^Pi is slow and hardly accelerated by K⁺ (9, 10). As this insensitivity is due to the binding of a second Mg²⁺ to the ATPase in E2P^Pi (10), it would be more appropriate to denote this state as E2P^Pi·Mg²⁺ (Fig. 1). As the affinity of Mg²⁺ in E2P^Pi is ∼0.5 mM (10), the majority of the ATPase molecules phosphorylated by P_i will be in this state. E2P^ATP has a low affinity for Mg²⁺ (not saturated at 6 mM) (10). Therefore, the transmembrane cation binding sites and, accordingly, the CTS-binding cavity will be different in the two E2P states. Indeed, the signal from RH421, a voltage-sensitive styryl dye, is clearly different (9). Then, the inhibitory properties of CTSs will also be different in these two E2P states (type I and II complexes in refs. 11, 12). Furthermore, if phosphorylation by P_i + Mg²⁺ is performed in the presence of K⁺, another type of E2P form with loosely occluded K⁺, termed E2P^Pi·2K⁺, is generated (path C in Fig. 1). This form has a high rate of dephosphorylation (9, 10). OBN is well known to have a much-reduced affinity in the presence of K⁺ (K⁺ antagonism) (e.g., ref. 13), but other CTSs have not been well characterized in this regard. Indeed, Laursen et al., reported that bufalin (BUF) requires K⁺ for high-affinity binding (14). In a recent report (15), the difference in K⁺ antagonism is attributed to the lactone ring. Therefore, systematic measurements on the inhibitory potency in the three E2P states are clearly required, in addition to the one under turnover conditions.Confusion in the literature is apparent even in structural studies. There are several crystal structures published for NKA with bound CTSs: those in E2·P_i·2K⁺ with ouabain at low affinity (2.8-Å resolution) (16), BUF in E2P^Pi·2K⁺ (3.4-Å resolution) (14), and those in E2P^Pi·Mg²⁺ with ouabain at high affinity (3.4 Å) (17) or digoxin (3.9 Å) (14). All of the crystals of the high-affinity complexes are generated in the presence of a high concentration (>100 mM) of Mg²⁺, and indeed, Mg²⁺ is observed to occupy site II for K⁺. Therefore, the E2P state stabilized by CTSs should be denoted as E2P^Pi·Mg²⁺ (Fig. 1). These crystal structures have established that the high affinity of CTSs primarily arises from complementarity between the M5 helix and the α-face of the steroid core, consistent with mutagenesis studies (18–22). However, other than this, there seems to be serious discrepancies between biochemical and structural data. For instance, ouabagenin (OBG), which lacks rhamnose attached to C3, has a 300-fold reduced affinity in binding to NKA in E2P^Pi·Mg²⁺, but Laursen et al. (17) describes that the sugar moiety in ouabain does not interact with the ATPase. Mutagenesis studies have identified residues responsible for isoform dependence (23, 24), but the crystal structure failed to explain why (24). We really do not know if any structural changes are caused by CTS binding to NKA, because no structure is available for the E2P ground state without CTS.We answer this question in this report, as we now have crystal structures of the BeF₃⁻ complex of NKA, an E2P ground state analog, free of CTS. Systematic measurements of the inhibitory properties of various CTSs, including ROS and IST, under four different conditions provide a basis for addressing their structure-activity relationships. One striking finding is that ROS shows a much higher affinity under turnover conditions than in E2P^Pi·Mg²⁺, in marked contrast to OBN. Such differences, as well as the K⁺ antagonism, are nicely explained by the crystal structures of NKA with various CTSs. The crystal structures also explain the isoform dependence unambiguously and suggest ways to confer α2 specificity on CTSs.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.     

In Vitro Homocysteine Inhibits Platelet Na+, K+-ATPase and Serum Butyrylcholinesterase Activities of Young Rats

Homocystinuria is an inborn error of sulphur amino acid metabolism, resulting in accumulation of tissue homocysteine. This disease is characterized predominantly by vascular and nervous system dysfunction. In the present study we investigated the in vitro effects of homocysteine, the main metabolite accumulated in homocystinuria, on platelet Na+,K+-ATPase and serum butyrylcholinesterase (BuChE) activities of young rats. Platelet and serum of 29-day-old Wistar rats were incubated in the absence (control) or presence of homocysteine (0.01-0.5 mM). Results showed that Na+,K+-ATPase and BuChE activities were significantly inhibited by homocysteine. It is proposed that inhibition of Na+,K+-ATPase and BuChE activities might be one useful peripheral marker for the neurotoxic effects of homocysteine.     

Mechanism of glucose-induced (Na+, K+)-ATPase inhibition in aortic wall of rabbits

Summary Hyperglycaemia decreases (Na⁺, K⁺)-ATPase activity in specific tissues by a mechanism whose effects are prevented by aldose reductase inhibitors and by raising plasma myo-inositol. This mechanism was activated and studied in vitro in normal rabbit aortic intima-media. Raising medium glucose to 10 mmol/l for 60 min inhibited a major component of (Na⁺, K⁺)-ATPase-mediated ⁸⁶Rb ⁺ /K⁺ uptake normally operative in resting aortic intima-media in medium containing normal plasma levels of glucose (5 mmol/l) and myo-inositol (70 mol/l); 20 or 30 mmol/l glucose had no greater effect. This effect occurred under conditions in which the aortic intima-media's normal myo-inositol content is not detectably decreased. The inhibition was prevented by sorbinil (10 mol/l) and by raising medium myo-inositol from 70 to 500 mol/l, which had no effect on (Na⁺, K⁺)-ATPase activity when the medium glucose remained at 5 mmol/l. Raising medium glucose selectively inhibited a component of (Na⁺, K⁺)-ATPase activity that requires medium myo-inositol, because it is maintained by a regulatory system through rapid basal phosphatidylinositol turnover in a discrete pool, which is replenished by a fraction of basal de novo phosphatidylinositol synthesis that is selectively dependent on myo-inositol uptake. Medium myo-inositol at a normal plasma level became inadequate to maintain this fraction of basal de novo phosphatidylinositol synthesis ([1,3-¹⁴C]glycerol incorporation) when the medium glucose was raised. When sorbinil was added raising medium glucose did not alter the ability of 70 umol/l medium myoinositol to maintain the (Na⁺, K⁺)-ATPase activity that requires medium myo-inositol. The inhibition caused by raising medium glucose was reproduced by a competitive inhibitor of active myo-inositol transport, scyllo-inositol (500 mol/l). The effect of medium glucose in an elevated plasma level on (Na⁺, K⁺)-ATPase activity in aortic intima-media appears to result from increased polyol pathway activity that makes myoinositol uptake at a normal plasma level inadequate to maintain the normal operation of a regulatory system.     

Age-Dependent Alterations in Na+,K+-ATPase Activity in the Central Nervous System of Spontaneously Hypertensive Rats: Relationship to the Development of High Blood Pressure

We have previously demonstrated that cerebroventricular administrations (i.c.v) of potassium chloride solutions (KCl; 0.375–1.25 μmoles/5 μl) elicit ouabain-sensitive, concentration-dependent decreases in the blood pressure and heart rates of anesthetized, normotensive Sprague-Dawley (SD) rats. These studies have suggested an inverse relationship between Na⁺-pump activity in the central nervous system (CNS) and central sympathetic outflow. Such a view is further supported by the present studies showing that i.c.v. injections of KCl failed to produce any alterations in the blood pressures of rats pretreated with an autonomic ganglionic blocker, chlorisondamine. In the present studies, depressor responses to i.c.v. potassium chloride were considered as functional indices for evaluation of neuronal Na⁺-pump activity in 8 and 12 week old (8 wk and 12 wk) SHR, WKY and Sprague-Dawley (SD) rats. Basal arterial blood pressures of 8 wk-old SD and SHR, and the responsiveness of these two groups to i.c.v. potassium chloride solutions are similar and they both are significantly greater than that of age matched WKY However, in the 12 wk-old groups, arterial pressure of SHR was significantly greater than that of WKY as well as SD, whereas the depressor responses to KCl in SHR were significantly greater than that of only WKY. Pretreatment of the rats with i.c.v. ouabain abolished the differences in the hypotensive responses to i.c.v. potassium chloride that existed between various groups but not the differences in the basal blood pressures. Evaluation of these data suggest that a) the centrally mediated hypotensive responses to K⁺ in various groups could depend upon Na⁺,K⁺-pump activity in C.N.S. and/or on basal central sympathetic discharge; b) central sympathetic activity is greater in SHR only when compared to WKY but not to SD; c) since the central Na⁺-pump activity and sympathetic tone appears to be similar in SHR and SD, mecahnisms other than the increases in sympathetic activity must play a prominent role in the development of spontaneous hypertension; d) attenuation of neuronal Na⁺-pump activity cannot account for greater sympathetic tone in SHR and SD-rats when compared to WKY.     

Angiotensin II AT1 Receptor/Signaling Mechanisms in The Biphasic Effect of The Peptide on Proximal Tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na⁺,K⁺-ATPase activity. Angiotensin II produced a biphasic effect on Na⁺,K⁺-ATPase activity: stimulation at 10^-13-10^-10M followed by inhibition at 10^-7-10^-5M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT, receptor. Angiotensin IT produced inhibition of forskolin-stimulated CAMP accumulation at 10^-13-10^-10M followed by a stimulation in basal CAMP levels at 10^-7-10^-5M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on CAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na⁺,K⁺-ATPase activity but did not affect the inhibition of Na⁺,K⁺-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na⁺,K⁺-ATPase activity. Mepacrine (l0μM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na⁺,K⁺-ATPase activity. These results suggest that the activation of AT₁angiotensin II receptors stimulates Na⁺,K⁺-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na⁺,K⁺-ATPase activity following AT₁receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2     

Elevated extracellular glucose inhibits an adenosine-(Na+, K+)-ATPase regulatory system in rabbit aortic wall

Summary The mechanism by which hyperglycaemia causes decreased (Na⁺, K⁺)-ATPase activity preventable by aldose reductase inhibitors and by raising plasma myo-inositol in specific tissues can be activated in vitro in normal rabbit aortic wall; it selectively inhibits a component of resting (Na⁺, K⁺)-ATPase activity maintained by a novel regulatory system through rapid basal phosphatidylinositol turnover (hydrolysis) in a discrete pool, which is replenished by a fraction of phosphatidylinositol synthesis that selectively requires myoinositol transport. A role for endogenously released adenosine in this regulatory system was examined. Adding adenosine deaminase or 8-phenyltheophylline, an adenosine receptor antagonist, selectively inhibited the component of (Na⁺, K⁺)-ATPase activity maintained by the regulatory system; when inhibited with adenosine deaminase this component was restored by 2-chloroadenosine, 5-N-ethylcarboxamidoadenosine, and 1-oleoyl-2-acetylglycerol, but not by forskolin (which also did not inhibit this component). Adenosine deaminase inhibited the rapid basal turnover of the discrete phosphatidylinositol pool, and 2-chloroadenosine then stimulated its turnover. Raising medium glucose from 5 to 10–30 mmol/l inhibits the regulatory system by making myo-inositol transport at a normal plasma level inadequate to maintain the replenishment of the discrete phosphatidylinositol pool. 2-Chloroadenosine stimulation of the adenosine-sensitive component of (Na⁺, K⁺)-ATPase activity was inhibited in tissue incubated with 30 mmol/l glucose and myoinositol in a normal plasma level, but this effect was demonstrable when the medium myo-inositol was raised seven-fold. Hyperglycaemia-induced decreased (Na⁺, K⁺)-ATPase activity that is preventable by aldose reductase inhibitors and by raising plasma myo-inositol results from the inhibition of a novel adenosine-(Na⁺, K⁺)-ATPase regulatory system.