Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (Plenary Lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(P_i)-homeostasis. In both epithelia, apical sodium (Na⁺)/P_i-cotransport across the luminal (brush border) memi brane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/P_i-cotransporters have been identified: (i) type I cotransporter(s) - present in the proximal tubule - also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type IIa)and in the small intestine (type IIb), isoform determines Na⁺-dependent transcellular P_i-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in P_i-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered P_i-intake, levels of 1.25 (OH₂) vitamin D₃, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/P_i-cotransporters operate (as functional monomers) in a 3 Na⁺:1 P_i stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na⁺, slippage)and of the fully loaded carriers (3 Na⁺, 1 P_i). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na⁺-interaction or specific pH-dependence of the IIa and IIb cotransporters. For the COOH-terminal tail of the IIa Na/P_i -cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Effect of Two Tyrosine Mutations on the Activity and Regulation of the Renal Type II Na/P i -Cotransporter Expressed in Oocytes

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY₄₀₂FAM matching the consensus sequence GYXXZ, and Y₅₀₉RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated ³²P _i -uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/P _i -cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter. Received: 3 November 1998/Revised: 20 January 1999     

Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (plenary lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(Pi)-homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/Pi-cotransporters have been identified: (i) type I cotransporter(s)--present in the proximal tubule--also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type Ila) and in the small intestine (type IIb), isoform determines Na+-dependent transcellular Pi-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in Pi-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered Pi-intake, levels of 1.25 (OH2) vitamin D3, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/Pi-cotransporters operate (as functional monomers) in a 3 Na+:1 Pi stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na+, slippage) and of the fully loaded carriers (3 Na+, 1 Pi). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na+-interaction or specific pH-dependence of the IIa and IIIb cotransporters. For the COOH-terminal tail of the IIa Na/Pi-cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10^–8 m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10^–8 m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10^–8 m) resulted in a rapid and transient increase in [Ca²⁺] _i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca²⁺ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P _i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.     

Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytes

Xenopus laevis oocytes have been used for the expression of Na/P_i-cotransport activity by injections of poly(A)⁺ RNA (mRNA) isolated from an established renal cell line (OK cells). 3–5 days after mRNA injection, Na-dependent phosphate (P_i) uptake by oocytes was increased in a dose-dependent manner; there was no increase in Na-independent P_i uptake. Sucrose density-gradient fractionation indicated that the mRNA species encoding this activity is 2.4–2.8 kb in length. In Northern blots, using a cDNA probe related to human kidney-cortex Na/P_i-cotransport activity (NaPi-3), hybridization with a mRNA-species of 2.4–2.6 kb was obtained. Kinetic characterization ([P_i], [Na]) showed that expressed transport activity has properties similar to apical Na/P_i cotransport in OK cells.     

Expression of the Na/P<Subscript>i</Subscript>-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

In mammals the type IIb Na/P_i-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/P_i-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/P_i-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K _ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/P_i-cotransporter can be purified by Ni-NTA chromatography.     

Upregulation of the Na+-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF

B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I _P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal I_P. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased I_P in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal I_P. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.     

The Basis of Higher Na+ Transport by Inner Medullary Collecting Duct Cells from Dahl Salt-Sensitive Rats: Implicating the Apical Membrane Na+ Channel

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α₁ subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na⁺ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na⁺, and K⁺, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na⁺ transport rates. The analysis of the steady-state Na⁺ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na⁺ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na⁺. They suggest that the epithelial Na⁺ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996     

Lipopolysaccharides stimulate Na-dependent transport in alveolar cells and protect against oxidant injury

We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium-coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H₂O₂ (2.5 mM; 20 min). LPS (10 μg/ml, 24 h) induced a significant increase in the Na-dependent component of alanine and phosphate uptake while they decreased Na, K-ATPase activity measured by ouabain-sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS-treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H₂O₂-induced decrease of Na-dependent alanine and phosphate uptake. This protective effect of LPS against H₂O₂ injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H₂O₂ clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium-coupled transport by a process involving protein synthesis and partially prevent H₂O₂-induced decrease of Na-coupled transport without discernible change in antioxidant activities. © 1995 Wiley-Liss, Inc.     

Apical and basolateral effects of PTH in OK cells: Transport inhibition,messenger production,effects of pertussis toxin,and interaction with a PTH analog

Summary The cellular distribution (apicalvs. basolateral) of parathyroid hormone (PTH) signal transduction systems in opossum kidney (OK) cells was evaluated by measuring the action of PTH on apically located transport processes (Na/P_i cotransport and Na/H exchange) and on the generation of intracellular messengers (cAMP and IP₃).PTH application led to immediate inhibition of Na/H-exchange without a difference in dose/response relationships between apical and basolateral cell-surface hormone addition (halfmaximal inhibition at 5×10^–10 m). PTH required 2–3 hr for maximal inhibition of Na/P_i cotransport with a half-maximal inhibition occurring at ×10^–12 m for apical application. PTH addition to either side of the monolayer produced a dose-dependent production of both cAMP and IP₃. Half-maximal activation of IP₃ was at about 7×10^–12 m PTH and displayed no differences between apical and basolateral hormone addition, while cAMP was produced with a half maximal concentration of 7×10^–9 m for apical PTH application and 10^–9 m for basolateral administration.The PTH analog [nle^8.18, tyr³⁴]PTH(3-34), (nlePTH), produced partial inhibition of Na/P_i cotransport (agonism) with no difference between apical and basolateral application. When applied as a PTH antagonist, nlePTH displayed dose-dependent antagonism of PTH inhibition of Na/P_i cotransport on the apical surface, failing to have an effect on the basolateral surface. Independent of addition to the apical or basolateral cell surface, nlePTH had only weak stimulatory effect on production of cAMP, whereas high levels of IP₃ could be measured after addition of this PTH analog to either cell surface. Also an antagonistic action of nlePTH on PTH-dependent generation of the internal messengers, cAMP and IP₃, was observed; at the apical and basolateral cell surface nlePTH reduced PTH-dependent generation of cAMP, while PTH-dependent generation of IP₃ was only reduced by nlePTH at the apical surface.Pertussis toxin (PT) preincubation produced an attenuation of both PTH-dependent inhibition of Na/P_i cotransport and IP₃ generation while producing an enhancement of PTH-dependent cAMP generation; these effects displayed no cell surface polarity, suggesting that PTH action through either adenylate cyclase or phospholipase C was transduced through similar sets of G-proteins at each cell surface.It is concluded that apparent receptor activities with high and low affinity for PTH exist on both cell surfaces; those with apparent high affinity seem to be coupled preferentially to phospholipase C and those with apparent low affinity to adenylate cyclase. The differences in apparent affinity of receptor events coupled to adenylate cyclase and the differences in PTH/nlePTH interaction on the two cell surfaces are suggestive of the existence of differences in apparent PTH-receptor activities on the two cell surfaces.     

Relationships among sodium current,permeability, and Na activities in control and glucocorticoid-stimulated rabbit descending colon

Summary Effects of a potent synthetic glucocorticoid, methylprednisolone (MP), on transepithelial Na transport were examined in rabbit descending colon. Current-voltage (I–V) relations of the amiloride-sensitive apical Na entry pathway were measured in colonic tissues of control and MP-treated (40 mg im for 2 days) animals. Tissues were bathed mucosally by solutions of various Na activities, (Na)_m, ranging from 6.2 to 75.6mm, and serosally by a high K solution. TheseI–V relations conformed to the constant field flux equation permitting determination of the permeability of the apical membrane to Na,P _Na ^m , and the intracellular Na activity, (Na)_c. The following empirical relations were observed for both control and MP-treated tissues: (i) Na transport increases hyperbolically with increasing (Na)_m obeying simple Michaelis-Mentin kinetics; (ii)P _Na ^m decreased hyperbolically with increasing (Na)_m, but was unrelated to individual variations in (Na)_c; (iii) (Na)_c increased hyperbolically with (Na)_m; (iv) both spontaneous and steroid-stimulated variations in Na entry rate could be attributed entirely to parallel variations inP _Na ^m at each mucosal Na activity. Comparison of these empirical, kinetic relations between control and MP-treated tissues revealed: (i) maximal Na current andP _Na ^m were greater in MP tissues, but the (Na)_m's at which current andP _Na ^m were half-maximal were markedly reduced; (ii) (Na)_c was significantly increased in MP tissues at each (Na)_m while the (Na)_m at half-maximal (Na)_c was unchanged. These results provide direct evidence that glucocorticoids cause marked stimulation of Na absorption across rabbit colon primarily by increasing the Na permeability of the apical membrane. While the mechanism for the increased permeability remains to be determined, the altered relation betweenP _Na ^m and (Na)_m suggests possible differences in the conformation or environment of the Na channel in MP-treated tissues.     

Physical characterization of high-affinity gastrointestinal Cu transport in vitro in freshwater rainbow trout Oncorhynchus mykiss

This study investigated the transport of copper (Cu) in the gut of trout. Examination of the spatial distribution of Cu along the digestive tract and a physical characterization of the uptake process was carried out using an in vitro gut sac technique and ⁶⁴Cu as a tracer. Unidirectional Cu uptake was highest in the anterior intestine followed in decreasing order by the posterior intestine, mid intestine and the stomach. Cu uptake was resistant to hypoxia and appeared to be fueled equally well by Cu(II) or Cu (I) at Cu concentrations typically found in the fluid phase of the chyme in vivo in the trout intestine. Transport demonstrated saturation kinetics (e.g. K _m = 31.6 μM, J _max = 17 pmol cm⁻² h⁻¹, in mid intestine) at low Cu levels representative of those measured in the chyme in vivo, with a diffusive component at higher Cu concentrations. Q ₁₀ analysis indicated Cu uptake is via diffusion across the apical membrane and biologically mediated across the basolateral membranes of enterocytes. The presence of l-histidine but not d-histidine stimulated both Cu and Na uptake suggesting a common pathway for the transport of Cu/Na with l-histidine.     

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO₃ secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Ca-sensitive sodium absorption in the colon of Xenopus laevis

Summary Transepithelial electrogenic Na transport (I_Na) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (I_sc) of the voltage-clamped tissue was 24.2±1.8 A·cm^-2 (n=10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca²⁺ from the mucosal Ringer solution stimulated I_Na by about 120%. I_Na was not blockable by amiloride (0.1 mmol·l^-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol·l^-1 lanthanum (La^3-). No Na-self-inhibition was found, because I_Na increased linearly with the mucosal Na concentration. A stimulation of I_Na by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is typically found in the colon of vertebrates.Abbreviations G _T transepithelial conductance - I _sc short circuit current - I _Na transepithelial Na-current - m mucosal - s serosal - PDS power density spectrum - f frequency - f _c corner frequency of the Lorentzian component of the PDS - S(f) power density of the Lorentzian component of the PDS - S_o plateau value of the Lorentzian component of the PDS     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)