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.     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

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.     

Reduced Na++K+-ATPase activity in peripheral nerve of streptozotocin-diabetic rats: a role for protein kinase C?

Summary Six weeks after induction of diabetes, the rate of ouabain-sensitive ⁸⁶Rb⁺ accumulation, a parameter which reflects Na⁺+K⁺-ATPase pumping activity, was significantly reduced in endoneurial preparations of sciatic nerve from untreated diabetic rats compared with that in control rats (Trial 1, 0.19±0.09 versus 0.48±0.13 pmol/min per mg wet weight of tissue, p<0.001; Trial 2, 0.27±0.16 versus 0.47±0.18, p<0.01). This decrease in ouabain-sensitive ⁸⁶Rb⁺ uptake was not observed in nerves from diabetic rats maintained on sorbinil (an aldose reductase inhibitor) or myo-inositol diets. Protein kinase C activity was demonstrated in the soluble fraction of a sciatic nerve homogenate by assaying for lipid-activated, Ca⁺-dependent phosphorylation of calf thymus histone. No significant difference in the time course of kinase C activity was observed between cytosol fractions of nerve homogenates from control and diabetic rats (control, 6.22±0.97 pmol ³²P incorporated/mg cytosol protein in 50 min; diabetic, 5.32±0.71). Three low molecular weight neural proteins (each with M_r<29,000) were identified as substrates for protein kinase C.     

The Effect of High Salt Intake on Na+, K+-Atpase Activity of Tissues in the Rat

1. The effect of chronic feeding of high salt diet on Na⁺, K⁺-ATPase activity of heart, liver, skeletal muscle, kidney and aorta was studied in the rat.

2. Groups of rats were either given tap water or 18 g/L saline to drink. After 7 days, 3 months or 12 months, the control group and salt loaded     

Transgenic overexpression of translationally controlled tumor protein induces systemic hypertension via repression of Na+,K+-ATPase

Inhibition of Na⁺,K⁺-ATPase has been implicated in the pathogenesis of hypertension via its effect on smooth muscle reactivity and myocardial contractility. We recently demonstrated that translationally controlled tumor protein (TCTP) interacts with the 3rd cytoplasmic domain of Na⁺,K⁺-ATPase α₁-subunit and acts as its cytoplasmic repressor. Therefore, we hypothesized that repression of Na⁺,K⁺-ATPase by overexpressed TCTP might underlie the development of hypertension. In the present study, we confirmed that transgenic mice overexpressing TCTP developed systemic arterial hypertension at about 6 weeks after birth. Vascular smooth muscle of TCTP-overexpressing transgenic mice also displayed augmented contractile response to vasoconstrictors and attenuated relaxation response to vasodilators. These responses seem to be caused by reduced Na⁺,K⁺-ATPase activity and increased intracellular calcium, suggesting that inhibition of Na⁺,K⁺-ATPase by overexpression of TCTP is involved in the pathogenesis of hypertension. This study provides a new link between alteration of sodium pump activity and hypertension in vivo, and suggests that TCTP might be a therapeutic target for the treatment of hypertension.     

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.     

Inhibition of Rat Na+/K+-ATPase Isoforms by Endogenous Digitalis Extracts from Neonatal Human Plasma

An unique endogeneous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (α₁, α₂and α₃) of the alpha subunits of Na⁺/K⁺-ATPase in rat

EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral α₃isoform and a steep dose-response curve with the same apparent potency for rat α₂and α₁isoforms of high (10^-7M) and low affinity (3 × 10^-5M) for ouabain. These results indicate that the Na⁺/K⁺-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain     

Gastric H+, K+-ATPase as a therapeutic target in peptic ulcer disease

The presence of unbuffered acid appears to be an essential contributory factor in the pathogenesis of peptic ulcer disease. Treatment has concentrated therefore on the reduction of acidity, and the last decade has seen the widespread and effective use of H₂ antagonists. They are, at low doses, more successful in improving the natural history of duodenal ulcer disease than of gastric or esophageal ulceration. The H₂ receptor plays a central role in activation of parietal cell acid secretion, and antagonists at this receptor block most (but not all) of the acid secretion due to even gastrinergic or muscarinic (vagal) stimulation. In hypergastrinemic states such as Zollinger-Ellison syndrome, or where acid secretion has to be inhibited by more than 20% over a 24-hr period, such as for treatment of esophagitis, NSAID damage, or gastric ulcers, the dose and frequency of administration of the currently available antagonists must be increased to achieve reliable therapy. This has led to a search for an alternative target for acid inhibitory drugs, such as the gastric acid pump, the H⁺,K⁺-ATPase. This article focuses on the function of this ATPase and suggests that inhibition of this pump will provide a more efficacious means of reduction of acid secretion by the stomach, hence improving and simplifying therapy of acid related diseases.This work was supported in part by grants from NIH and the USVA.     

Erythrocyte Na+, K+ Cotransport and Blood Pressure in Identical Twins

The erythrocyte N⁺, K⁺ cotransport system was studied in ten pairs of identical twins.

Cation fluxes were remarkably similar in each pair of twins, which supports the concept of a genetic determinant for the co-transport system.

There was, however, no apparent correlation between cotransport values and the family history of hypertension.