Cimetidine transport in rat renal brush border and basolateral membrane vesicles

The transport of cimetidine by rat renal brush border and basolateral membrane vesicles has been studied in relation to the transport system of organic cation. Cimetidine inhibited [3H]tetraethylammonium uptake by basolateral membrane vesicles in a dose dependent manner, and the degree of the inhibition was almost the same as that by unlabeled tetraethylammonium. In contrast, cimetidine inhibited the active transport of [3H]tetraethylammonium by brush border membrane vesicles more strongly than unlabeled tetraethylammonium did. In agreement with the transport mechanism of tetraethylammonium in brush border membranes, the presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of cimetidine uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by unlabeled tetraethylammonium. These results suggest that cimetidine can share common carrier transport systems with tetraethylammonium in renal brush border and basolateral membranes, and that cimetidine transport across brush border membranes is driven by an H+ gradient via an H+-organic cation antiport system.     

Biology of the 2Na+/1H+ antiporter in invertebrates

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.     

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.     

Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Streptozotocin diabetes and sugar transport by rat ileal enterocytes: evidence for adaptation caused by an increased luminal nutrient load.

Preparations of villus enterocytes and brush border membrane vesicles have been used to study the effects of streptozotocin-induced diabetes mellitus in rats on sugar transport across the brush border and basolateral membranes of ileal epithelial cells. In isolated cells, diabetes increased Na(+)-dependent galactose transport across the brush border of mid-villus but not upper villus cells. Galactose transport across the basolateral membrane was, however, enhanced by diabetes in both cell populations. Kinetic analysis of vesicle data suggested the presence of two transporters for Na(+)-dependent glucose transport. Diabetes induced a 5-fold increase in both KT and Vmax of the high-affinity/low-capacity system together with a 2-fold increase in the Vmax of the low-affinity/high-capacity transporter. Glucose was almost undetectable in the lumen of the upper and lower ileum in control animals but was present at high levels (26.1 +/- 4.3 mM and 6.5 +/- 1.3 mM) in diabetic rats. The possible significance of these changes in luminal sugar concentration in relation to the adaptation of transport across ileal enterocytes is discussed.     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

Sodium and sulfate ion transport in yeast vacuoles

The intra-luminal acidic pH of endomembrane organelles is established by a proton pump, vacuolar H(+)-ATPase (V-ATPase), in combination with other ion transporter(s). The proton gradient (DeltapH) established in yeast vacuolar vesicles decreased and reached the lower value after the addition of alkaline cations including Na(+). As expected, the uptake of (22)Na(+) was coupled with DeltapH generated by V-ATPase. Disruption of NHX1 or NHA1, encoding known Na(+)/H(+) antiporters, did not result in the loss of (22)Na(+) uptake or the alkaline cation-dependent DeltapH decrease. Upon the addition of sulfate ions, the V-ATPase-dependent DeltapH in the vacuolar vesicles increased, but the membrane potential (DeltaPsi) decreased. Consistent with this observation, radioactive sulfate was transported into the vesicles with a K(m) value of 0.07 mM. The transport activity was unaffected upon disruption of the putative genes coding for homologues of plasma membrane sulfate transporters. These results indicate that the vacuoles exhibit unique Na(+)/H(+) antiport and sulfate transport, which regulate the luminal pH and ion homeostasis in yeast.     

Transport of sulfate and phosphate in small intestine and renal proximal tubule: methods and basic properties

1. Isolated brush border membrane vesicles, basolateral membrane vesicles, and cultured renal epithelial cells provide good material for studying transport systems. 2. The vesicle systems have been used to study the transport of labeled phosphate, sodium/phosphate cotransport, sodium/sulfate cotransport, basolateral transport of sulfate and basolateral transport of phosphate via anion exchange. 3. Cultured renal cells show sodium/phosphate cotransport and parathyroid dependent inhibition of phosphate transport.     

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.     

Transport of C(4)-dicarboxylates in Wolinella succinogenes

C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

Increase of (Na+ + K+)-activated ATPase activity of basolateral plasma membranes from intestinal mucosa of diabetic rats

A significant increase of the (Na+ + K+)-activated ATPase was found in mucosal homogenates of rat small intestine under conditions of alloxan and streptozotocin diabetes. From studies with isolated plasma membranes it has been shown that the activity changes were caused by that part of the (Na+ + K+)-activated ATPase only which is localized in the basolateral plasma membranes, whereas the enzyme activity in the brush border region remains unchanged. In connection with the enhanced capacity of ion, nonelectrolyte and water absorption in experimental diabetes, our findings support a concept of intestinal transport mechanism which suggest that the basolateral part of the (Na+ + K+)-activated ATPase is responsible for metabolic energy supply. The luminal part of the enzyme may be involved in regulation of passive Na+ influx.     

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.     

Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.     

Proton fluxes associated with erythrocyte membrane anion exchange.

Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942. Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloride-sulfate exchange. The pH rise can be reversed by the addition of HCO(-3) (4 muM) or 2,4-dinitrophenol (90 muM). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloride exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972, In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Rorth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.     

Lysine uptake and exchange in Corynebacterium glutamicum.

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.