Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.     

Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.     

A Na+-independent,phloretin-sensitive monosaccharide transport system in isolated intestinal epithelial cells

A monosaccharide transport system in addition to the active Na+-dependent system characteristic of the brush border surface of vertebrate intestinal tissue has been identified in isolated chick intestinal epithelial cells. The newly described system differs in several characteristics from the Na+-dependent process, including function in the absence of Na+; a high sensitivity to phloretin, relative insensitivity to phlorizin; different substrate specificity; and a very high KT and Vmax. The system apparently functions only in a facilitated diffusion manner so that it serves to move monosaccharide across the cell membrane down its chemical gradient. An appreciable fraction of total sugar efflux occurs via the Na+-independent carrier from cells which have accumulated sugar to a steady state. Phloretin selectively blocks this efflux so that a normal steady-state sugar gradient of seven- to eightfold is transformed to a new steady-state gradient which is greater than 14-fold. Locus of the new system is tentatively ascribed to the serosal cell surface where it would serve for monosaccharide transfer between enterocyte and lamina propria of the villus.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

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.     

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.     

Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

Thyroid hormones stimulate calcium transport systems in rat intestine

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca(2+) influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca(2+) into BBMV as well as Ca(2+) efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca(2+) uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K(m)), indicating a conformational change in Ca(2+) transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca(2+) efflux across enterocytes was attributed to sodium-dependent Ca(2+) exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca(2+)-ATPase activity of BLMs of all groups. Kinetic studies of Na(+)/Ca(2+) exchanger revealed that alteration in Na(+)-dependent Ca(2+) efflux was directly associated with maximal velocity (V(max)) of exchanger among all the groups. cAMP, a potent activator of Na(+)/Ca(2+) exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca(2+) influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na(+)/Ca(2+) exchange in enterocytes possibly via cAMP-mediated pathway.     

Comparison of glucose transport mechanisms at opposing surfaces of the renal proximal tubular cell

This review contrasts the glucose transport mechanisms at opposing surfaces of the renal proximal convoluted tubule: the Na+-dependent D-glucose transporter localized at the brush border membrane and the Na+-independent transporter localized at the basolateral surface. The two sugar transport mechanisms are discussed from the point of view of their specificity, kinetic, and regulatory behaviors. Recent results focussing on molecular characterization of these different carrier proteins are also described, including some newer information on purification of the Na+-dependent glucose carrier from the brush border membrane.     

Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics

The interaction of two renin inhibitors, S 86,2033 and S 86,3390, with the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86,3390 and 2.5 mM of S 86,2033 led to a half-maximal inhibition of the H(+)-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86,2033 and S 86,3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of S 86,3390 was stimulated by an inwardly directed H(+)-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86,2033 and 86,3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent Mr 127,000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and beta-lactam antibiotics.     

Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

Intestinal brush border calcium uptake in spontaneously hypertensive rats and their genetically matched WKY rats

The current studies were designed to characterize calcium transport by intestinal brush border membrane in the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. The biochemical and functional purity of the intestinal brush border membranes in SHR and WKY rats was validated by marker enzymes and the ability to transiently transport D-glucose in the presence of Na+ gradient. Calcium transport into duodenal and jejunal vesicles represented a minor binding component and transmembrane movement as evident by initial rate studies, A23187 studies, and lanthanum displacement experiments. Initial rate and time course of calcium uptake was lower in SHR compared with WKY rats. Kinetic analysis of calcium uptake by the jejunum (total uptake minus binding component) showed a Vmax of 6.98 +/- 0.2 and 1.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.76 +/- 0.04 and 0.87 +/- 0.1 mM for WKY rats and SHR, respectively. Similar kinetic analysis of calcium uptake by the duodenal segments showed a Vmax of 10.3 +/- 0.8 and 2.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.01). Km values were 0.7 +/- 0.2 and 0.3 +/- 0.06 mM (P greater than 0.05). Vmax of calcium uptake in the 2-week-old rats (prehypertensive period) was 6.0 +/- 0.3 and 3.53 +/- 0.3 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.60 +/- 0.07 and 0.5 +/- 0.01 mM, respectively. These results suggest that calcium binding and uptake by duodenal and jejunal intestinal brush border membranes of SHR is significantly decreased compared with WKY rats. The decrease in transmembrane calcium uptake is secondary to decrease in Vmax and is present before the appearance of hypertension, implying a genetically determined defect in calcium uptake in intestinal brush border membranes of the SHR.     

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.     

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.     

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.     

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.     

Isolation and reconstitution of the intestinal Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.