Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues.

A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a-b-)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes.

The molecular basis for Na-dependent phosphate transport in human erythrocytes and K562 cells.

The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

Separate entry pathways for phosphate and oxalate in rat brain microsomes.

ATP-dependent (45)Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO(4) and oxalate. In the absence of divalent anions, there was a transient (45)Ca accumulation, lasting only a few minutes. Addition of PO(4) did not change the initial accumulation but added a second stage that increased with PO(4) concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 microM), 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS; 250 microM), and DIDS (3-5 microM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 microM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO(4) cotransport. An oxalate-supported (45)Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO(4) and oxalate had activating and inhibitory effects that could be explained by PO(4) inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO(4) in brain endoplasmic reticulum.

Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4.

Urea transport in the kidney is important for the production of concentrated urine and is mediated by a family of transporter proteins, identified from erythropoietic tissue (UT-B) and from kidney (UT-A). Two isoforms of the renal urea transporter (UT-A) have been cloned so far: UT-A1 and UT-A2. We used rapid amplification of cDNA ends to clone two new isoforms of the rat UT-A transporter: UT-A3 and UT-A4. UT-A3 and UT-A4 are 87% homologous. The UT-A3 cDNA encodes a peptide of 460 amino acids, which corresponds to the amino-terminal half of the UT-A1 peptide and is 62% identical to UT-A2. The UT-A4 cDNA encodes a peptide of 466 amino acids, which is 84% identical to UT-A2. Transient transfection of HEK-293 cells with the UT-A3 or UT-A4 cDNA results in phloretin-inhibitable urea uptake, which is increased by forskolin. Thus, both new isoforms encode functional urea transporters that may be vasopressin-regulated. UT-A3 and UT-A4 mRNA are expressed in the renal outer and inner medulla but not in the cortex; unidentified UT-A isoforms similar to UT-A3 may also be expressed in the testis. It is concluded that there are at least four different rat UT-A urea transporters.

Inducible expression of erythrocyte band 3 protein.

A permanent cell line with inducible expression of the human anion exchanger protein 1 (hAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable hAE1 protein by Western analysis or additional 36Cl flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4'-dinitrostilbene-2, 2'-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. There was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathway that is DNDS sensitive. The additional transport of 36Cl and 35SO4 had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 microM DNDS, 2) activation of 36Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of 36Cl efflux by external anions that was selective in the order NO3 = Cl > Br > I, and 4) activation of 35SO4 influx by external protons. Under the assumption that the turnover numbers of hAE1 were the same as in erythrocytes, there was good agreement (+/-3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expression of hAE1 in a mammalian system to track the kinetic characteristics of the native protein.

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells.

The human renal Na-PO4 cotransporter gene NaPi-3 was expressed in human embryonic kidney HEK-293 cells, and the transport characteristics were measured in cells transfected with a vector containing NaPi-3 or with the vector alone (sham transfected). The initial rate of 32PO4 influx had saturation kinetics for external Na and PO4 with K1/2Na of 128 mM (PO4 = 0.1 mM) and K1/2PO4 of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfected cells expressing the transporter. Transfection had no effect on the Na-independent 32PO4 influx, but transfection increased Na-dependent 32PO4 influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has nearly the same temperature dependence in the absence and presence of extracellular Na. The Na-dependent phosphate flux decreased with pH in sham-transfected cells but was pH independent in transfected cells. The Na-dependent 32PO4 influx was inhibited by p-chloromercuriphenylsulfonate, phosphonoformate, phloretin, vanadate, and 5-(N-methyl-N-isobutyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the study of this protein. Commensurate with the increased functional expression, there was an increase in the amount of NaPi-3 protein by Western analysis.

Urea transporters in kidney and erythrocytes.

Physiological and molecular data demonstrate that urea transport in kidney and erythrocytes is regulated by specific urea transporter proteins. The urea transporter in the terminal inner medullary collecting duct permits very high rates of regulated transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla, where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. The urea transporter in erythrocytes permits these cells to lose urea rapidly as they ascend through the ascending vasa recta, thereby preventing loss of urea from the medulla. Urea lost from the medulla would decrease concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in individuals who lack the Kidd antigen. The recent cloning of cDNAs for these two urea transporters has begun to yield new insights into the mechanisms underlying acute and long-term regulation of urea transport and should permit exciting new insights in the future. This review focuses on the physiological and biophysical evidence that established the concept of urea transporters, the subsequent cloning of cDNAs for urea transporters, and the recent integrative studies into the regulation of urea transport. We also propose a new systematic nomenclature and a new structural model for urea transporters.

Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport.

Water homeostasis is regulated in large part by the proper operation of the urinary concentrating mechanism. In the renal inner medulla, urea recycling from the inner medullary collecting duct to the inner medullary interstitium is thought to be essential for the production of a concentrated urine; however, it has not been possible to test this hypothesis in humans. Recently, a unique combination of genetic abnormalities has been described: absence of Kidd blood group antigens and absence of carrier-mediated urea transport in erythrocytes. Because animal studies indicate a similarity between urea transport in red blood cells and the nephron, it was postulated that patients without the Kidd antigen might lack facilitated urea transport in their kidneys. Hence, their ability to concentrate urine maximally was measured. Current models of nephron function would predict that in the complete absence of urea transport, the maximal concentrating ability would be around 800 to 900 mosM/kg H2O. Two homozygous patients had a moderate decrease in maximal concentrating ability (UosM,max = 819 mosM/kg H2O); a heterozygote also had some limitation. These studies raise the possibility that the erythrocyte urea transporter and the kidney urea transporter are encoded by a single gene (detected by the mutational loss of the Kidd antigen) and that a lack of facilitated urea transport impairs urea recycling in the kidney and, hence, maximal urinary concentrating ability.

Glycine transport by human red blood cells and ghosts: evidence for glycine anion and proton cotransport by band 3.

Stilbene-sensitive glycine transport was investigated in human red blood cells and ghosts. We have found that this component of glycine transport was inhibited by the stilbene derivatives 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS); the apparent constant for inhibition by DNDS was 4 microM in the presence of 150 mM chloride. DNDS-sensitive glycine influx was modulated by pH such that as pH was increased from 5.9 to 9.2, transport increased from 2.5 to 140 mumol.kg Hb-1.h-1 at 37 degrees C and 100 microM glycine. The increased transport was correlated with an increase in the amount of glycine present as the anion over this pH range (0.03-40 microM glycine anion), but, in addition, pH had a direct effect on transport. Glycine influx was studied as a function of glycine anion concentration with anion varied by changing pH at a constant total glycine concentration and by changing total glycine at a constant pH. A comparison of these data demonstrated that the stilbene-sensitive glycine anion flux is stimulated by protons with half-maximal stimulation below pH 6.5 and suggests that the glycine anion and a proton are cotransported. Inorganic anions transported by band 3, including Cl, NO3, and SO4, inhibited glycine transport. Glycine flux into resealed ghosts was inhibited by Cl with an inhibition constant of 25 mM. The similarities between the kinetic constants for transport inhibition by Cl and DNDS and the kinetic constants for Cl and DNDS binding to band 3 suggest that the DNDS-sensitive glycine anion and proton cotransport is via band 3.

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line.

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.

Urea transport deficiency in Jk(a-b-) erythrocytes.

The activity of the urea transporter was determined in human erythrocytes of the Kidd blood type Jk(a-b-) by measuring unidirectional urea and thiourea fluxes in tracer flux experiments and urea net fluxes in light-scattering experiments. When compared with control cells, Jk(a-b-) cells exhibited diminished urea and thiourea fluxes and lacked the kinetic characteristics of mediated transport, suggesting that in these cells urea and thiourea moved only by simple diffusion through the lipid bilayer. Control experiments showed that anion, water, and ethylene glycol permeabilities were the same in Jk(a-b-) and control cells. These experiments thus demonstrate that Jk(a-b-) cells lack mediated urea transport and strongly support the notion that urea transport function is separate from the transport for anions, water, and ethylene glycol, probably because different proteins are responsible for these transport functions.

Anion transport in sickle red blood cells.

Anion transport in sickle cells (SS RBC) mediated by the band 3 membrane protein was evaluated by three different measures in both oxygenated and deoxygenated conditions and compared to normal red cells. First, Cl- self-exchange measured as 36Cl- efflux at 0 degrees C was normal in SS RBC in both Vmax and dependence on extracellular Cl- concentration. There was no effect of deoxygenation on either parameter. Second, stilbene-sensitive 35SO4=; SO4= exchange, measured at 37 degrees C where morphologic sickling occurred, was also unaffected by deoxygenation and was normal compared to normal red cells. Third, conductive Cl- flux was assessed by measuring the rates of Cl(-)-limited K+ efflux in valinomycin-treated cells at 37 degrees C. Both the stilbene-sensitive and insensitive components of net Cl- flux were similar in SS RBC and normal red cells, and were unaltered by morphologic sickling. Thus, despite dramatic alterations in cation transport in SS RBC and the demonstration of interaction between band 3 protein and sickle cell, anion transport functions appear to be normal in SS RBC and are unaffected by deoxygenation. These data suggest that the majority of the anion exchangers in SS RBC are functionally normal.

Inhibition of erythrocyte anion exchange by tolrestat, an inhibitor of aldose reductase.

Tolrestat, and aldose-reductase inhibitor, was shown to be a rapid and potent inhibitor of chloride exchange on the band 3 protein of human erythrocytes. Tolrestat binds to a site distinct from the chloride transport site and binds to one half of the transporters at 5 x 10(-7) mol/L in the absence of chloride and at 3.6 x 10(-5) mol/L in physiologic chloride concentrations. Although these concentrations are 20- to 1,000-fold greater than the IC50 for aldose-reductase inhibition by tolrestat, they are achieved during routine pharmacologic therapy in humans. Consequently, Cl/HCO3 exchange rates may be reduced and there may be decreased CO2 clearance from coronary and respiratory center capillary beds and inappropriate hyperpnea. There also may be transitory intracellular alkalinization in cells with a Cl/HCO3 exchanger in their plasma membrane.

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier.

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism.

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions of inhibitors on anion transporter of human erythrocyte.

Chloride tracer efflux was measured from intact human erythrocytes into media containing different chloride concentrations and different concentrations of the inhibitors 4,4'-dinitrostilbene-2-2'-disulfonate (DNDS), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), phloretin, and sulfate. The data were analyzed to test whether these inhibitors were mutually exclusive with each other or whether they could bind at the same time. Under the assumption that mutual exclusiveness is due to steric interference, the data can be used to map out the protein surface near the outward-facing anion binding-transport site. It is concluded that there are separate domains for NAP taurine and phloretin that do not overlap with each other or with the chloride binding site. These two domains do, however, overlap with the binding domain for DNDS that, in addition, excludes the binding of chloride and sulfate.

Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.