Impairment of Na+,K+-ATPase activity following enterotoxigenic Campylobacter jejuni infection: changes in Na+, Cl− and 3-O-methyl-D-glucose transport in vitro, in rat ileum

Abstract Unidirectional fluxes of Na⁺, Cl⁻ and 3-O-methyl-D-glucose (3-MG) were measured in vitro across Campylobacter jejuni live culture-infected and control rat ileal short-circuited tissues by the Using Chamber technique. Net secretion of Na⁺ and enhanced secretion of Cl⁻ ions was observed in the infected animals ( P < 0.001, n =6) as compared to the net absorption of Na⁺ and marginal secretion of Cl⁻ ions in the control animals. There was a significant decrease in the mucosal-to-serosal fluxes of 3-MG in C. jejuni -infected rat ileum. The specific Na⁺,K⁺-ATPase activity when measured biochemically in the membrane-rich fraction of enterocytes was found to be significantly lower (58%) in the infected group as compared to the control group ( P < 0.001). Our results therefore suggest that infection with an enterotoxigenic C. jejuni inhibits the Na⁺,K⁺-ATPase activity in rat enterocytes. The impairment of Na⁺,K⁺-ATPase activity thus appears to induce a secondary change in Na⁺,Cl⁻ and 3-MG transport in vitro in rat ileum.     

Involvement of intracellular calcium stores in Giardia lamblia induced diarrhoea in mice

Abstract The transmucosal fluxes of Na⁺ and Cl⁻ were studied in Giardia lamblia -infected mice in the presence of absence of dantrolene (1-(5( p -nitrophenyl)furfurilidene-amino) hydantoin sodiumhydrate ). There was net secretion of Na⁺ and Cl⁻ in infected animals, while in control animals there was net absorption of these ions. The addition of dantrolene resulted in significant net increase in absorption of Na⁺ and Cl⁻ in control and experimental groups. Further, mouse intestinal epithelial cells were labelled with [³²P]Pi and then treated with G. lamblia trophozoites and their excretory secretory products separately. The optimum time for inositol triphosphate formation was 15 min in control enterocytes as well as in treated enterocytes. A plateau was formed at higher concentrations. Since raised inositol triphosphate levels mobilize Ca²⁺ from intracellular stores and dantrolene traps Ca²⁺ within intracellular calcium stores, the present study thus suggests that intracellular calcium stores are involved in G. lamblia -induced diarrhoea in mice.     

Subcellular Localization of Bioactive Cholecystokinin-Like Peptides in the Rat Cerebral Cortex

Abstract: The subcellular distribution of cholecystokinin (CCK), especially its exact localization within the synaptosome, was studied in the rat cerebral cortex. Highest CCK-like bioactivity was measured in the synaptic membrane fractions, paralleling the distribution of (Na⁺+ K⁺)-dependent ATPase. In the synaptic vesicles, which were characterized by high acetylcholine content and by the absence of (Na⁺+ K⁺)-ATPase, only minimal quantities of CCK were detected.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Tryptophan Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na⁺ gradient [Na⁺] outside > [Na⁺] inside and is maximal when both Na⁺ and Cl⁻ are present. The uptake represents transport into an os-motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo-larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na⁺, K⁺-activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4-dihydroxyphenylalanine.     

Seasonal changes in ionoregulatory variables of the glass eel Anguilla anguilla following estuarine entry: comparison with resident elvers

In the present study, glass eels Anguilla anguilla in the Minho River estuary (41·5° N, 8·5° W) decreased in size (standard length, L _S and mass, M ) from the beginning (autumn) to the end of the sampling season (summer). On the other hand elvers increased in L _S and M from spring to summer and were significantly larger than glass eels in paired comparisons. Branchial Na⁺/K⁺-ATPase and vacuolar (V-type) proton ATPase ( in vitro activities), two important ion transporting pumps, did not show significant seasonal changes in either glass eels or elvers although in glass eels Na⁺/K⁺-ATPase (activity) expression was significantly higher than in elvers. In a single month comparison Na⁺/K⁺-ATPase branchial mRNA expression was also higher in glass eels as was the protein level expression of both Na⁺/K⁺-ATPase and NKCC (Na⁺:K⁺:2Cl⁻ co-transporter). Immunofluorescence microscopy indicated apical CFTR Cl⁻ channel labelling in Na⁺/K⁺-ATPase positive chloride cell in glass eels which was absent in elvers. Whole body sodium concentration and percentage water did not show significant seasonal differences in either glass eels or elvers although there were significant differences between these two groups during some months.     

Early Signals in Serum-Induced Increases in Ouabain-Sensitive Na+-K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride-Sensitive

Abstract: The acute effects of serum on sodium-potassium (Na⁺-K⁺) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na⁺-K⁺ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and ⁸⁶Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na⁺ channels. On transfer of myotubes to Na⁺-free, choline buffer, resting Na⁺-K⁺ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na⁺-free serum, caused Na⁺-K⁺ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na⁺ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na⁺-H⁺ exchange for serum-induced increases in Na⁺-K⁺ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

The [3H]Tryptamine Receptor in Human Brain: Kinetics, Distribution, and Pharmacologic Profile

Abstract: The kinetics and distribution of [³H]tryptamine binding sites in human brain were investigated. Specific [³H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [³H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [³H]tryptamine binding site. This suggests the presence of distinct species-dependent [³H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [³H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [³H]-tryptamine in human brain.     

THE EFFECTS OF OUABAIN AND THE ACTIVATION OF NEURAL MEMBRANE ATPase BY BIOGENIC AMINES

Abstract— The effects of 10⁻⁵ m -noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) on the activities of Na⁺-K⁺ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5-HT stimulated the synaptic membrane Na⁺-K⁺ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na⁺-K⁺ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two-fold by 10⁻⁵ m -NA and 5-HT. The Na⁺-K ⁺ ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5-HT. NA, DA and 5-HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na⁺-K⁺) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Salt requirements for membrane transport and solute retention in some moderate halophiles

Abstract Many species of bacteria isolated from saline environments require Na⁺ specifically for membrane transport. Transport occurs by a Na⁺ symport process energized by an electrochemical gradient of Na⁺ ions. The gradient at neutral pH appears to be produced by a primary electrogenic extrusion of protons coupled to a secondary, outwardly directed Na⁺ pump, a Na⁺/proton antiporter. At alkaline pH Vibrio alginolyticus may also produce the gradient by an energy-dependent primary extrusion of Na⁺ ions. Alteromonas haloplanktis and Vibrio costicola require salts in the medium to retain intracellular solutes. For A. haloplanktis the effects of the salts are primarily osmotic. For V. costicola , only NaCl is effective in retaining solutes and Na⁺ is required by this organism to maintain the membrane potential. In Escherichia coli a single substitution in the nucleotide sequence of the gene coding for the melibiose transport protein changed the cation specificity of the transport system. The possible ecological significance of this finding has been considered.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

Na+-independence of the stability under alkaline conditions of the membrane of an alkalophilic Bacillus

Abstract The stability under alkaline conditions of the membrane of the alkalophile was studied. By an alkaline treatment in the absence of Na⁺ or Li⁺, the abilities of the membrane vesicles, when energized with ascorbate plus tetramethylphenylenediamine, to produce a membrane potential (negative, inside) and transmembrane pH gradient (outside > inside) were rapidly lost. The activity of cytochrome oxidase was not affected by the alkaline treatment irrespective of the presence of Na⁺. It is likely that the membrane structure is sensitive to an alkaline pH and maintained specifically by the presence of Na⁺ (or Li⁺) in the alkaline medium.     

L-Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na⁺ gradient ([Na⁺] outside > [Na⁺] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na⁺ and Cl⁻ ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na⁺+ K⁺)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K _m and V _max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.     

Limiting cytosolic Na+ confers salt tolerance to rice cells in culture: a two-photon microscopy study of SBFI-loaded cells

Rice ( Oryza sativa L.), a staple food in Asia, is very sensitive to soil salinity. However, intraspecific variations exist, with the coastal cultivar Pokkali tolerating even brackish water. This study explores cellular mechanisms that contribute to salt tolerance in rice. It is widely accepted that limiting cytosolic Na⁺ should improve the survival of plants subjected to saline stress. However, an understanding of the mechanisms by which Na⁺ levels are controlled in relatively tolerant cultivars requires monitoring cytosolic Na⁺ non-invasively and in real time, which is technically challenging. We have used two-photon excitation for the ratiometric estimation of cytosolic Na⁺ in cultured cells using sodium-binding benzofuran isophthalate. Pokkali cells maintained low cytosolic Na⁺ (approximately 25 m M ), and a viability of over 85% under high salinity , while Jaya cells were unable to maintain low cytosolic Na⁺ and suffered decreased viability even at moderate saline stress. Here we show that the permeability of the Pokkali plasma membrane to Na⁺ is significantly lower than that of Jaya, to the extent that it is comparable with permeabilities reported for halophytes. Pokkali effectively sequesters Na⁺ in intracellular compartments utilizing a Ca²⁺-regulated transport system(s). Together these cellular mechanisms allow Pokkali to maintain low cytosolic Na⁺ up to a stress of 250 m M NaCl. The findings demonstrate that differences in survival between these contrasting varieties of rice are mainly because of differences in membrane transport mechanisms and thus have significance in crop improvement.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Ion Channels and Membrane Potential in Stimulus-Secretion Coupling in Adrenal Medulla Cells

Abstract: The role of Na⁺ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)-sensitive Na⁺ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 μM). In Ca²⁺-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na⁺ with Li⁺ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na⁺ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM-100 mM-K⁺, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM-K⁺. Neither dibutyryl-cyclic AMP nor dibutyryl-cyclic GMP evokes secretion. α-Bungarotoxin (1 μM) did not inhibit nicotine-induced secretion. These studies indicate that Na⁺ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca²⁺. The membrane potential appears to affect nicotine- and veratridine-evoked secretion.