Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.     

Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.     

Determination of basolateral Na(+)/H(+) exchange activity in MDCK cells using a multiwell-multilabel reader

Na(+)/H(+) exchangers (NHE) are important membrane transport proteins involved in transepithelial transport and cellular pH homeostasis. NHE-1, important for cellular pH and volume homeostasis, is expressed in the basolateral membrane of epithelial cells. We evaluated the use of a multiwell-multilabel reader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a filter perfusion system. Using the multiwell-multilabel reader we obtained virtually the same values for steady-state pH and NHE-1 activity under control conditions compared to the imaging system. With both setups Na(+)-dependent pH recovery after an acid load occurred virtually only after basolateral addition of Na(+). Furthermore, Na(+)-dependent pH recovery was reduced by >80% in the presence of the NHE-1 inhibitor HOE642. In addition, we detected an almost identical increase of NHE-1 activity with the two methods after stimulation of protein kinase C using phorbol myristate acetate. In summary, our data indicate that multiwell-multilabel readers are suitable to investigate physiology and regulation of basolateral NHE. Thus, multiwell-multilabel readers offer a valid and convenient alternative to investigate basolateral transporters.     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Role of NH3 and NH4+ transporters in renal acid-base transport

Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.     

Ferroportin is expressed on the mucous granule membrane of a subpopulation of goblet cells in the duodenum of the rat

Ferroportin is a basolateral transporter involved in the release of iron from cells. In addition to expression on the basolateral membrane of enterocytes, ferroportin is also seen on the microvillus membrane. This led us to consider that ferroportin might be expressed by other cells of the intestine where it contributes to iron metabolism. Ferroportin gene and protein expression in rat duodenum was studied by in situ hybridisation and immunohistochemistry, respectively in rats with different efficiencies of iron absorption. Ferroportin mRNA localised to enterocytes of the villus only. Ferroportin was demonstrated in enterocytes and in 30% of goblet cells. In goblet cells it localised to the mucous granule membrane. In iron-loaded intestine some goblet cells contained iron suggesting that ferroportin may transport iron into the mucous granule where it would be lost during discharge of mucous. The finding of ferroportin in iron deficient goblet cells also suggests an additional role to iron excretion.     

Immunological detection of Na(+)/H(+) exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus

Na(+)/H(+) exchangers (NHE) are a family of ion exchangers with diverse functions that are well defined in mammals. NHE-1 is expressed in the plasma membrane of most mammalian cells where it regulates intracellular pH, and usually in the basolateral membrane of epithelial cells. It has also been detected in teleost gills where it may participate in systemic pH regulation. NHE-3 is usually expressed in the apical membrane of mammalian epithelial cells where it helps reabsorb Na(+) and HCO(3)(-); it has also been detected in teleost gills. We used Western blotting and heterologous antibodies to screen for expression of NHE-1 and NHE-3 in gills of an agnathan (Myxine glutinosa) and an elasmobranch (Raja erinacea), and NHE-3 in gills of a teleost (Fundulus heteroclitus). Positive NHE-1 bands were detected in gills from the agnathan and elasmobranch. Using the NHE-3 antibody, bands were detected in the gills of the elasmobranch and teleost. These data are some of the first direct evidence of NHEs in the gills of an agnathan and elasmobranch, and confirm the presence of NHEs in the gills of teleosts.     

Modulation of liver cell membrane NHE-1, Na+-K+ ATPase, and GLUT-2 protein content after cold preservation and rewarming

Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+ -H+ exchanger isoform NHE-1 and the Na+ -K+ ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37 degrees C did not further reduce this parameter. On the other hand, expression of Na+ -K+ ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins.     

Expression and sub cellular localization of the sodium hydrogen exchanger isoform-1 in rat tissues: A possible functional relevance

In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K⁺-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.     

Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH- sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti- ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Molecular and functional evidence for electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters in murine duodenum

Inward Na(+)-HCO(3)(-) cotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na(+)-HCO(3)(-) cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pH(i)) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl(-) increased pH(i), whereas an acidification was observed on removal of Na(+) or both Na(+) and Cl(-). The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na(+)-HCO(3)(-) cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO(3)(-) transport as well as basolateral electrogenic HCO(3)(-) transport in proximal duodenal villus cells.     

Expression of the ammonia transporter proteins Rh B glycoprotein and Rh C glycoprotein in the intestinal tract

Ammonia metabolism is important in multiple aspects of gastrointestinal physiology, but the mechanisms of ammonia transport in the gastrointestinal tract remain incompletely defined. The present study examines expression of the ammonia transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) in the mouse gastrointestinal tract. Real-time RT-PCR amplification and immunoblot analysis identified mRNA and protein for both RhBG and RhCG were expressed in stomach, duodenum, jejunum, ileum, and colon. Immunohistochemistry showed organ and cell-specific expression of both RhBG and RhCG. In the stomach, both RhBG and RhCG were expressed in the fundus and forestomach, but not in the antrum. In the forestomach, RhBG was expressed by all nucleated squamous epithelial cells, whereas RhCG was expressed only in the stratum germinativum. In the fundus, RhBG and RhCG immunoreactivity was present in zymogenic cells but not in parietal or mucous cells. Furthermore, zymogenic cell RhBG and RhCG expression was polarized, with apical RhCG and basolateral RhBG immunoreactivity. In the duodenum, jejunum, ileum, and colon, RhBG and RhCG immunoreactivity was present in villous, but not in mucous or crypt cells. Similar to the fundic zymogenic cell, RhBG and RhCG expression in villous epithelial cells was polarized when apical RhCG and basolateral RhBG immunoreactivity was present. Thus the ammonia transporting proteins RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they function cooperatively to mediate gastrointestinal tract ammonia transport.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.     

Stable Polarized Expression of hCAT-1 in an Epithelial Cell Line

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.