Regulation of intestinal phosphate transport. II. Metabolic acidosis stimulates Na(+)-dependent phosphate absorption and expression of the Na(+)-P(i) cotransporter NaPi-IIb in small intestine

During metabolic acidosis, P(i) serves as an important buffer to remove protons from the body. P(i) is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal P(i) absorption in providing P(i) to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH(4)Cl in drinking water) for 2 or 7 days in mice increased urinary fractional P(i) excretion twofold, whereas serum P(i) levels were not altered. Na(+)-dependent P(i) transport in the small intestine, however, was stimulated from 1.89 +/- 3.22 to 40.72 +/- 11.98 pmol/mg protein (2 days of NH(4)Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na(+)-dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal P(i) absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone.     

Acid-base effects on intestinal Na(+) absorption and vesicular trafficking

We examined for vesicular traffickingof the Na⁺/H⁺ exchanger (NHE) in pH-stimulatedileal and CO₂-stimulated colonic Na⁺absorption. Subapical vesicles in rat distal ileum were quantified bytransmission electron microscopy at ×27,500 magnification. Internalization of ileal apical membranes labeled withFITC-phytohemagglutinin was assessed using confocal microscopy, andpH-stimulated ileal Na⁺ absorption was measured afterexposure to wortmannin. Apical membrane protein biotinylation of ilealand colonic segments and Western blots of recovered proteins wereperformed. In ileal epithelial cells incubated inHCO/Ringer or HEPES/Ringer solution, the number ofsubapical vesicles, the relative quantity of apical membrane NHEisoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membranefluorescence under the confocal microscope were not affected by pHvalues between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulatedileal Na⁺ absorption. In colonic epithelial apicalmembranes, NHE3 protein content was greater at aPCO₂ value of 70 than 21 mmHg, was internalized when PCO₂ was reduced, and was exocytosed whenPCO₂ was increased. We conclude that vesicletrafficking plays no part in pH-stimulated ileal Na⁺absorption but is important in CO₂-stimulated colonicNa⁺ absorption.

     

Characteristics of Na(+)-dependent intestinal nucleoside transport in the pig

Since the capacity of nucleic acid digestion and absorption appears to be comparatively high in the pig, we investigated the properties of transport of (3)H-labelled nucleosides across the porcine intestinal brush border membrane (BBM) using BBM vesicles isolated from the small intestine of slaughter pigs. In the presence of a transmembrane Na(+) gradient, uridine, thymidine and guanosine transiently accumulated in the vesicular lumen beyond the equilibrium (60 min) value suggesting the presence of Na(+)/nucleoside cotransporters in the BBM. The findings of inhibitory studies are consistent with the presence of two Na(+)-dependent nucleoside transporters with overlapping substrate specificity, one for pyrimidine nucleosides (N2) and one for purine nucleosides (N1). Guanosine appeared to be a specific substrate for N1, while this applies to thymidine for N2. Transport of thymidine and guanosine were also inhibited by 2 mmol/l D-glucose and alpha-methyl-D-glucoside. The maximal transport capacity (V(max)) for Na(+)-dependent thymidine and guanosine transport were much higher than reported for other monogastric species. Unlike in other species tested, there was no proximal-to-distal gradient, neither in nucleoside transport activity nor in the inhibition of nucleoside transport by monosaccharides in the porcine small intestine. The high intestinal nucleoside transport activity may contribute to the high digestive capacity for nucleic acids in the pig.     

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter

We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na(+)-dependent inorganic phosphate (Na(+)/P(i)) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na(+)-dependent P(i) transport, indicating that DNPI is a novel Na(+)/P(i) cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na(+)-dependent P(i) transport in the neuron-rich regions of the brain, may form a new class within the Na(+)/P(i) cotransporter family.     

Soybean impairs Na(+)-dependent glucose absorption and Cl- secretion in porcine small intestine

Recent evidence indicates that soybean, which is widely used in animal nutrition, could directly alter intestinal ion and nutrient transport. However, the mechanisms involved are still unknown. The aim of the study was to investigate the effect of three differently treated soybean products on the glucose and Cl- transport capacity in porcine small intestine by the Ussing chamber technique. Jejunal and ileal piglet epithelial tissues were pre-incubated with extracts of raw soybean flour (RSF), heated soybean flour (HSF), or ethanol heat-treated soybean protein concentrate (SPC). The Na(+)-dependent glucose co-absorption capacity was then measured as an increase in the short-circuit current (ISC) after luminal addition of D-glucose. The effect of the soybean products on cAMP-dependent Cl- secretion was measured as the increase in ISC after the addition of the phosphodiesterase inhibitor, theophylline, while nervous regulation of Cl- secretion was investigated by the addition of the enteric neurotransmitters; 5-hydroxytryptamine (5-HT), substance P and vasoactive intestinal polypeptide (VIP). Incubation with RSF and HSF induced a 30% decrease of the Na(+)-dependent glucose absorption capacity in the jejunum. The effect was similar for RSF in the ileum. Theophylline-induced secretion was decreased by 30% after incubation with RSF, HSF and SPC but only in the jejunum. 5-HT-, substance P- and VIP-induced secretion were not altered by incubation with soybean extracts except in the HSF-incubated where the substance P-induced secretion was significantly reduced. In conclusion, soybean contains ethanol-sensitive heat-insensitive compounds impairing Na(+)-dependent glucose absorption in the jejunum and ileum, and ethanol- and heat-insensitive compounds causing an acute impairment of cAMP-dependent jejunal secretion.     

Na(+)-dependent AIB transport by neuroblastoma cells.

Na(+)-dependent amino isobutyric acid transport by two neuroblastoma cell lines with and without amplification of the oncogene N-myc is studied. Surprisingly, the contribution of system A is greater in the cell line showing no N-myc amplification. Preliminary data support a role for essential tyrosine and cysteine residues in the active center of the carriers, mainly in system A.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Effect of dietary phosphate on intestinal calcium and phosphate absorption

Intestinal Ca and P absorption was investigated on rachitic chicks raised on diets with a 1% Ca and 0.3% or 1% P contents. 45Ca and 32P absorption was determined by the technique of the isolated gut sac in vivo. In addition, 32P transport was also measured by the everted gut sac procedure in vitro. Treatment with vit. D3 during 7 days increased the 45Ca absorption in animals fed diets containing 0.3% or 1% P. 32P absorption showed an increase after 2 days of treatment and a decrease afterwards. The reduction of 32P absorption was larger in animals fed diet with 1% P. Study of 32P transport with the everted gut sac technique showed an increase after vit. D3 and a loss of intracellular P, regardless the duration of treatment.     

Na(+)-dependent high-affinity uptake of L-glutamate in cultured fibroblasts.

Uptake of 1 microM [3H]L-glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0-9.6 mM). D- and L-Asparate, D- and L-threo-3-hydroxyaspartate DL-threo-3-methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of L-glutamate (including D-glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, L-trans-pyrrolidine-2,4-dicarboxylate, was also among the inhibitors. Apparent Km of the uptake was found to be less than 10 microM. The present observations indicate that Na(+)-dependent 'high-affinity' uptake of L-glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Inhibition of human intestinal brush border membrane vesicle Na+-dependent phosphate uptake by phosphophloretin derivatives

Hyperphosphatemia and II(o) hyperparathyroidism are common and severe complications of chronic renal failure. Reduced dietary phosphorus has been shown to be an effective treatment in reducing serum phosphate and serum PTH. 2(')-Phosphophloretin inhibited small intestine apical membrane Na(+)/phosphate cotransport and reduced serum phosphate in adult rats. 2(')-PP and phosphoesters of phloretin were tested for inhibition of human small intestine brush border membrane alkaline phosphatase activity and for inhibition of Na(+)-dependent phosphate uptake. The IC(50)'s for inhibition of alkaline phosphatase suggested an order of inhibitory potency of 4-PP > phloretin > 4(')-PP > 2(')-PP. Inhibition of Na(+)-dependent phosphate uptake followed the sequence 2(')-PPz.Gt;4(')-PP > 4-PP > phloretin. These results are consistent with 2(')-PP being a specific inhibitor of human intestinal brush border membrane Na(+)/phosphate cotransport.     

Electrophysiological study of L-lysine transport across Triturus proximal tubule: evidence for Na(+)-independent entry and Na(+)-dependent exit.

Cell membrane depolarization induced by intraluminal injection of lysine was entirely independent of the presence of Na+ in Triturus proximal tubule, confirming our previous observation. The amplitude of the depolarization conformed to Michaelis-Menten kinetics regardless of the presence or absence of Na+ in the perfusion solutions. pH of the intraluminal solution had no effect on the electrical response in its range from 5.5 to 8.5. In a Na(+)-free medium, particularly in a Tris-substituted medium, the depolarization induced by a constant concentration of lysine gradually decreased in its size when injection followed by washout of lysine was repetitively tested. The addition of Na+ to the peritubular side after extinction of the responsiveness resulted in a significant restoration of the voltage response to intraluminal lysine. In addition, influx of Na+ from the peritubular fluid into the cells was significantly greater in lysine-loaded tubules than in nonloaded tubules as indicated by a greater rate of increase in intracellular Na+ activity in the presence of ouabain. The data strongly suggest that lysine enters the cells via an electrogenic uniport mechanism and leaves the cells via Na+:amino acid exchange transport mechanism.     

Na(+)- and H(+)-dependent motility in the coral pathogen Vibrio shilonii

In this work, we analyzed motility and the flagellar systems of the marine bacterium Vibrio shilonii. We show that this bacterium produces lateral flagella when seeded on soft agar plates at concentrations of 0.5% or 0.6%. However, at agar concentrations of 0.7%, cells become round and lose their flagella. The sodium channel blocker amiloride inhibits swimming of V. shilonii with the sheathed polar flagellum, but not swarming with lateral flagella. We also isolated and characterized the filament–hook–basal body of the polar flagellum. The proteins in this structure were analyzed by MS. Eight internal sequences matched with known flagellar proteins. The comparison of these sequences with the protein database from the complete genome of V. shilonii allows us to conclude that some components of the polar flagellum are encoded in two different clusters of flagellar genes, suggesting that this bacterium has a complex flagellar system, more complex possibly than other Vibrio species reported so far.     

Hydrolysis by acylphosphatase of erythrocyte membrane Na+, K(+)-ATPase phosphorylated intermediate.

Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the phosphoenzyme intermediate of human red blood cell membrane Na+, K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Acylphosphatase addition to erythrocyte membranes resulted in a significant increase in the rate of Na+, K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 10 units/mg membrane protein, was of about 80% over basal value. The same acylphosphatase amount enhanced of about 40% the rate of ATP driven Na+ transport into inside out red cell membrane vesicles. Taken together these findings suggest a potential role of acylphosphatase in the control of the activity of erythrocyte membrane Na,K pump.