Basolateral Membrane Fractions Research Articles

SWATH-Based Comprehensive Determination of the Localization of Apical and Basolateral Membrane Proteins Using Mouse Liver as a Model Tissue.

The purpose of this study was to develop a method to comprehensively determine the localization of apical and basolateral membrane proteins, using a combination of apical/basolateral membrane separation and accurate SWATH (Sequential Window Acquisition of all THeoretical fragment ion spectra) proteomics. The SWATH analysis of basolateral and apical plasma membrane fractions in mouse liver quantified the protein expression of 1373 proteins. The basolateral/apical ratios of the protein expression levels were compared with the reported immunohistochemical localization for 41 model proteins (23 basolateral, 11 apical and 7 both membrane-localized proteins). Three groups were perfectly distinguished. Border lines to distinguish the apical-, both- and basolateral localizations were determined to be 0.766 and 1.42 based on probability density. The method that was established was then applied to the comprehensive determination of the proteins in mouse liver. The findings indicated that 154 and 125 proteins were localized in the apical and basolateral membranes, respectively. The levels of receptors, CD antigens and integrins, enzymes and Ras-related molecules were much higher in apical membranes than in basolateral membranes. In contrast, the levels of adhesion molecules, scaffold proteins and transporters in basolateral membranes were much higher than in apical membranes.

Quantitative Targeted Absolute Proteomics for 28 Transporters in Brush-Border and Basolateral Membrane Fractions of Rat Kidney

The purpose of the present study was to determine the absolute protein expression levels of various transporters in renal brush-border membrane (BBM) and basolateral membrane (BLM) fractions, in order to understand the quantitative differences in average transport activities among different transporters at each cellular membrane. BBM and BLM fractions of rat kidney were prepared and digested with trypsin, and simultaneous absolute quantification of 28 transporters and a BLM marker, Na+/K+-ATPase, was performed using our established quantitative-targeted absolute proteomics (QTAP) technique. In BBM fraction, the protein expression levels of bcrp, urat1, mate1, octl1, mrp4, mdr1a, and abca3 were 40.3, 22.2, 8.90, 4.85, 4.69, 3.22, and 0.976 fmol/μg protein, respectively. In BLM fraction, the protein expression levels of oat1, oat3, oct1, mrp6, and mrp1 were 10.6, 10.2, 4.59, 0.724, and 0.271 fmol/μg protein, respectively. The expression levels of abca2, abca4, abca5, abca12, abcb4, mrp5, abcc9, abcg1, abcg5, lat1, ntcp, pgt, oatp2b1, oatp1b2, oatp3a1, and oct3 were under the limit of quantification in both fractions. The quantitative transporter protein expression profiles at these membranes, as determined by QTAP analysis, should be helpful to understand the contributions of individual transporters to renal excretion of xenobiotics and endogenous compounds.

Lipid Polarity Is Maintained in Absence of Tight Junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.

Nervonic Acid (24:1n-9) is a Dominant Unsaturated Fatty Acid in the Intestinal Brush Border of Atlantic Cod

Atlantic cod is a coldwater teleost of commercial importance. The intestinal epithelium is a large organ in vertebrates serving an important role in nutrient selection and uptake as well as an immunological barrier. Here, we perform lipid and fatty acid analysis of the plasma membrane from the cod intestinal enterocytes after separation of the brush border membrane and the basolateral membrane fractions. Our results show that both membrane fractions contain an unusually high amount of cholesterol and glycolipids but low levels of glycerophospholipids compared with other reported studies on fish. Sphingomyelin was the dominant lipid in the brush border fraction and was also prominent in the basolateral fraction where phosphatidylcholine was the dominant glycerophospholipid. Furthermore, our results show a distinct difference in fatty acids content, where monounsaturated fatty acids (MUFA) were more abundant than polyunsaturated fatty acid (PUFA). Nervonic acid (24:1n-9) was a prominent fatty acid in the BBM at ~50% of the total MUFA. We hypothesize that the high cholesterol content and the presence of this rare fatty acid may serve to maintain membrane fluidity in the cold environment.

Role of basolateral efflux transporter MRP4 in the intestinal absorption of the antiviral drug adefovir dipivoxil

Adefovir dipivoxil is a diester prodrug of the antiviral drug adefovir, with much greater oral bioavailability than adefovir. Evidence shows that the prodrug is metabolized to adefovir in the enterocytes during intestinal absorption. However, it is unknown how the highly charged and hydrophilic adefovir crosses the basolateral membrane in the intestine. This study determines the role of specific basolateral transporter(s) in the egress of adefovir across the basolateral membrane when formed from adefovir dipivoxil in Caco-2 cells, a model for intestinal epithelium. Multidrug resistance-associated protein 4 (MRP4) plays an important role in renal secretion of adefovir. Immunofluorescence images showed that MRP4 is localized in the basolateral membrane of Caco-2 cells. This localization was further confirmed by Western blotting of the apical and basolateral membrane fractions that were isolated by a novel method involving biotinylation of respective membrane proteins and affinity enrichment. MRP4-knockdown Caco-2 cells were produced by stable transfection with MRP4-specific siRNA expression plasmid. These cells showed reduced MRP4 protein expression and corresponding reduction in the basolateral egress of adefovir when adefovir dipivoxil was dosed on the apical side. A comparison of these data with the reduction in the basolateral egress of adefovir by the general MRP inhibitor indomethacin established that MRP4, among MRPs, plays a predominant role in the basolateral egress of adefovir in Caco-2 cells. The results highlight the importance of MRP4 in oral absorption of adefovir dipivoxil, and suggest that significant drug–drug interactions can occur if an MRP4 inhibitor is co-administered with adefovir dipivoxil.

Overexpression of the Na-K-ATPase α2-subunit improves lung liquid clearance during ventilation-induced lung injury

Mechanical ventilation with high tidal volumes (HV(T)) impairs lung liquid clearance (LLC) and downregulates alveolar epithelial Na-K-ATPase. We have previously reported that the Na-K-ATPase alpha(2)-subunit contributes to LLC in normal rat lungs. Here we tested whether overexpression of Na-K-ATPase alpha(2)-subunit in the alveolar epithelium would increase clearance in a HV(T) model of lung injury. We infected rat lungs with a replication-incompetent adenovirus that expresses Na-K-ATPase alpha(2)-subunit gene (Adalpha(2)) 7 days before HV(T) mechanical ventilation. HV(T) ventilation decreased LLC by approximately 50% in untreated, sham, and Adnull-infected rats. Overexpression of Na-K-ATPase alpha(2)-subunit prevented the decrease in clearance caused by HV(T) and was associated with significant increases in Na-K-ATPase alpha(2) protein abundance and activity in peripheral lung basolateral membrane fractions. Ouabain at 10(-5) M, a concentration that inhibits the alpha(2) but not the Na-K-ATPase alpha(1), decreased LLC in Adalpha(2)-infected rats to the same level as sham and Adnull-infected lungs, suggesting that the increased clearance in Adalpha(2) lungs was due to Na-K-ATPase alpha(2) expression and activity. In summary, we provide evidence that augmentation of the Na-K-ATPase alpha(2)-subunit, via gene transfer, may accelerate LLC in the injured lung.

Altered expression of rat renal cortical OAT1 and OAT3 in response to bilateral ureteral obstruction

Bilateral ureteral obstruction (BUO) is characterized by the development of hemodynamic and tubular lesions. However, little is known about the expression of organic anion renal transporters. The objective of this work was to study the renal excretion of p-aminohippurate (PAH) and the cortical renal expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) in BUO rats. Male Wistar rats underwent bilateral obstruction of the proximal ureters for 24 hours (BUO) or sham operation. After 24 hours of ureteral releasing, the following studies were performed: PAH renal excretion employing conventional clearance techniques and OAT1 and OAT3 abundance (homogenates, intracellular and basolateral plasma membrane fractions from renal cortex) using immunoblotting and immunocytochemical techniques (light microscopic and confocal immunofluorescence microscopic analysis). BUO rats showed a lower renal excretion of PAH. In obstructed kidneys, immunoblotting revealed a significant decrease in the abundance of both OAT1 and OAT3 in basolateral plasma membranes from renal cortex. An increase of OAT1 expression was observed in homogenates and in intracellular membrane fractions in kidneys from BUO rats compared with sham-operated ones, indicating an internalization of this carrier. Immunocytochemical techniques confirmed these results. On the contrary, OAT3 expression was reduced both in homogenates and in intracellular membrane fractions in obstructed kidneys. BUO was associated with down-regulation of OAT1 and OAT3 in basolateral plasma membranes from proximal tubule cells, thus these carriers may play important roles in the impaired organic anion excretion displayed in the obstructed kidney.

Selective permeability barrier to urea in shark rectal gland

Elasmobranchs such as the dogfish shark Squalus acanthius achieve osmotic homeostasis by maintaining urea concentrations in the 300- to 400-mM range, thus offsetting to some degree ambient marine osmolalities of 900-1,000 mosmol/kgH(2)O. These creatures also maintain salt balance without losing urea by secreting a NaCl-rich (500 mM) and urea-poor (18 mM) fluid from the rectal gland that is isotonic with the plasma. The composition of the rectal gland fluid suggests that its epithelial cells are permeable to water and not to urea. Because previous work showed that lipid bilayers that permit water flux do not block flux of urea, we reasoned that the plasma membranes of rectal gland epithelial cells must either have aquaporin water channels or must have some selective barrier to urea flux. We therefore isolated apical and basolateral membranes from shark rectal glands and determined their permeabilities to water and urea. Apical membrane fractions were markedly enriched for Na-K-2Cl cotransporter, whereas basolateral membrane fractions were enriched for Na-K-ATPase. Basolateral membrane osmotic water permeability (P(f)) averaged 4.3 +/- 1.3 x 10(-3) cm/s, whereas urea permeability averaged 4.2 +/- 0.8 x 10(-7) cm/s. The activation energy for water flow averaged 16.4 kcal/mol. Apical membrane P(f) averaged 7.5 +/- 1.6 x 10(-4) cm/s, and urea permeability averaged 2.2 +/- 0.4 x 10(-7) cm/s, with an average activation energy for water flow of 18.6 kcal/mol. The relatively low water permeabilities and high activation energies argue strongly against water flux via aquaporins. Comparison of membrane water and urea permeabilities with those of artificial liposomes and other isolated biological membranes indicates that the basolateral membrane urea permeability is fivefold lower than would be anticipated for its water permeability. These results indicate that the rectal gland maintains a selective barrier to urea in its basolateral membranes.

Cloning and Characterization of a Novel Na+-dependent Glucose Transporter (NaGLT1) in Rat Kidney

To identify novel transporters in the kidney, we have constructed an mRNA data base composed of 1000 overall clones by random sequencing of a male rat kidney cDNA library. After a BLAST search, approximately 40% of the clones were unknown and/or unannotated and were screened by measuring the uptake of various compounds using Xenopus oocytes. One clone stimulated the uptake of alpha-methyl-d-glucopyranoside and therefore was termed rat Na(+)-dependent glucose transporter 1 (rNaGLT1). The rNaGLT1 cDNA (2173 bp) has an open reading frame encoding a 484-amino acid protein, showing <22% homology to known SGLT and GLUT glucose transporters. alpha-Methyl-d-glucopyranoside uptake by rNaGLT1 cRNA-injected oocytes showed saturability, with an apparent K(m) of 3.7 mm and a coupling ratio of 1:1 with Na(+). rNaGLT1 mRNA was expressed predominantly in the kidney upon Northern blot analysis and reverse transcription-PCR. Reverse transcription-PCR in microdissected nephron segments revealed that rNaGLT1 mRNA was primarily localized in the proximal tubules. A clear signal corresponding to rNaGLT1 protein was recognized in the brush-border (but not basolateral) membrane fraction by immunoblot analysis. The rNaGLT1 mRNA level in the kidney was significantly higher than rat SGLT1 and SGLT2 mRNA levels. These findings suggest that rNaGLT1 is a novel Na(+)-dependent glucose transporter with low substrate affinity that mediates tubular reabsorption of glucose.

Possible involvement of myosin‐X in intercellular adhesion: Importance of serial pleckstrin homology regions for intracellular localization

Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)-immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell-cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell-cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.

Regulation of L-leucine transport in rat kidney by dexamethasone and triiodothyronine.

We have investigated the transport mechanisms involved in the stimulation of renal tubular reabsorption of large amino acids by glucocorticoids in vivo through the examination of activity and expression of specific transport systems L and y(+)L for L-leucine in membrane preparations of rat kidneys. Kidneys were removed from adult female Wistar rats treated with dexamethasone or triiodothyronine, and the fractions of brush-border and basolateral membranes were isolated by density gradient centrifugation. Functional analysis of L-leucine uptake using rapid filtration technique revealed induction of a sodium-dependent, arginine-inhibitable system y(+)L transport component in the basolateral membrane in the dexamethasone-treated group. A minor sodium-independent, BCH-inhibitable, system L transport component was unaffected by glucocorticoids. L-leucine uptake remained unaffected in the triiodothyronine-treated group. Expression of both subunits of the system y(+)L transporter was increased in dexamethasone-treated rat kidneys: Western blot analysis showed a significant (46%) increase of 4F2hc protein abundance in the basolateral membrane fraction and competitive RT-PCR revealed an almost 4-times induced expression of y(+)LAT1 mRNA. Our results indicate that system y(+)L in rat kidney is regulated by glucocorticoids. We suggest that enhancement of both 4F2 heavy chain and y(+)LAT1 light chain is necessary for induction of this transport system in the kidney.

Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.

Sodium absorption, volume control and potassium channels: in tribute to a great biologist.

It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.

Beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats.

beta-Adrenergic agonists accelerate the clearance of alveolar fluid by increasing the expression and activity of epithelial solute transport proteins such as amiloride-sensitive epithelial Na(+) channels (ENaC) and Na,K-ATPases. Here we report that adenoviral-mediated overexpression of a human beta(2)-adrenergic receptor (beta(2)AR) cDNA increases beta(2)AR mRNA, membrane-bound receptor protein expression, and receptor function (procaterol-induced cAMP production) in human lung epithelial cells (A549). Receptor overexpression was associated with increased catecholamine (procaterol)-responsive active Na(+) transport and increased abundance of Na,K-ATPases in the basolateral cell membrane. beta(2)AR gene transfer to the alveolar epithelium of normal rats improved membrane-bound beta(2)AR expression and function and increased levels of ENaC (alpha subunit) abundance and Na,K-ATPases activity in apical and basolateral cell membrane fractions isolated from the peripheral lung, respectively. Alveolar fluid clearance (AFC), an index of active Na(+) transport, in beta(2)AR overexpressing rats was up to 100% greater than sham-infected controls and rats infected with an adenovirus that expresses no cDNA. The addition of the beta(2)AR-specific agonist procaterol to beta(2)AR overexpressing lungs did not increase AFC further. AFC in beta(2)AR overexpressing lungs from adrenalectomized or propranolol-treated rats revealed clearance rates that were the same or less than normal, untreated, sham-infected controls. These experiments indicate that alveolar beta(2)AR overexpression improves beta(2)AR function and maximally upregulates beta-agonist-responsive active Na(+) transport by improving responsiveness to endogenous catecholamines. These studies suggest that upregulation of beta(2)AR function may someday prove useful for the treatment of pulmonary edema.

Sulindac is excreted into bile by a canalicular bile salt pump and undergoes a cholehepatic circulation in rats

Background & Aims: Dihydroxy bile acids induce a bicarbonate-rich hypercholeresis when secreted into canalicular bile in unconjugated form; the mechanism is cholehepatic shunting. The aim of this study was to identify a xenobiotic that induces hypercholeresis by a similar mechanism. Methods: Five organic acids (sulindac, ibuprofen, ketoprofen, diclofenac, and norfloxacin) were infused into rats with biliary fistulas. Biliary recovery, bile flow, and biliary bicarbonate were analyzed. Sulindac transport was further characterized using Tr − rats (deficient in mrp2, a canalicular transporter for organic anions), the isolated perfused rat liver, and hepatocyte membrane fractions. Results: In biliary fistula rats, sulindac was recovered in bile in unconjugated form and induced hypercholeresis of canalicular origin. Other compounds underwent glucuronidation and were not hypercholeretic. In the isolated liver, sulindac had delayed biliary recovery and induced prolonged choleresis, consistent with a cholehepatic circulation. Sulindac was secreted normally in Tr − rats, indicating that its canalicular transport did not require mrp2. In the perfused liver, sulindac inhibited cholyltaurine uptake, and when coinfused with cholyltaurine, induced acute cholestasis. With both basolateral and canalicular membrane fractions, sulindac inhibited cholyltaurine transport competitively. Conclusions: Sulindac is secreted into bile in unconjugated form by a canalicular bile acid transporter and is absorbed by cholangiocytes, inducing hypercholeresis. At high flux rates, sulindac competitively inhibits canalicular bile salt transport; such inhibition may contribute to the propensity of sulindac to induce cholestasis in patients. GASTROENTEROLOGY 1999;117:962-971

Dietary lipid modulation of Na +/glucose co-transporter (SGLT1), [formula omitted] ATPase, and ornithine decarboxylase gene expression in the rat small intestine in diabetes mellitus

Nutrient transport is increased in streptozotocin-induced diabetes mellitus (DM) in rats. Variations in dietary lipid composition have been shown to modulate nutrient transport during intestinal adaptation in DM. Here, we examined Na +/glucose cotransporter (SGLT1), Na +,K +-ATPase and ornithine decarboxylase (ODC) gene expression in the small intestine in DM. One week after the animals had been given streptozotocin or injected with vehicle, they were randomly allocated to receive either standard chow or semisynthetic isocaloric diets with triglycerides enriched with polyunsaturated fatty acid (PUFA) or saturated fatty acid (SFA). Two weeks later they were killed and brush border membrane (BBM) and basolateral membrane (BLM) fractions and RNA were isolated from the mucosa of the proximal and distal small intestine. Western and Northern blotting were performed with antibodies and cDNA probes specific to SGLT1, the Na +,K +-ATPase α 1 and β 1 subunit isoforms, and ODC. Increased levels of immunodetectable SGLT1, Na +,K +-ATPase α 1 and β 1 subunit, and ODC expression and their corresponding mRNAs were observed in DM versus control rats fed chow. All of these parameters were increased in diabetic and in nondiabetic rats fed SFA as compared with PUFA. These findings suggest that the increased glucose uptake in diabetes, and in response to feeding a saturated as compared with a polyunsaturated diet, is achieved by transcriptional events that modulate transport function and cell proliferation.

Intrahepatic biliary cholesterol and phospholipid transport in humans: effect of obesity and cholesterol cholelithiasis.

The mode of transport of biliary lipids within the hepatocyte and the role of the bile canalicular membrane (BCM) in biliary lipid secretion are not well understood. We hypothesized that biliary cholesterol and phospholipid are co-transported across the hepatocyte in vesicular form from the endoplasmic reticulum to the bile across the BCM. We obtained wedge liver biopsies and fasting gallbladder bile from 15 cholesterol gallstone patients and 10 control subjects. BCM, basolateral membrane (BLM), and many microsomal vesicular fractions were isolated by centrifugation. One of the vesicular fractions (V3) was enriched in both the microsomal and the BCM marker enzymes and had a high phosphatidylcholine proportion in its phospholipid with a fatty acid pattern similar to biliary phosphatidylcholine. Moreover, its cholesterol content was increased in the obese cholesterol gallstone subjects, who had an increase in cholesterol synthesis, as indicated by the increased activity of the HMG-CoA reductase. The cholesterol content correlated with HMG-CoA reductase activity. A direct correlation was found between cholesterol/phospholipid ratio in V3, BCM, and in bile but not in the BLM. These data are in agreement with the assumption that this vesicular fraction is involved in the transport of cholesterol and phospholipid from the endoplasmic reticulum to the site of secretion in the BCM, and thence to bile, and that this transport is enhanced in obese gallstone patients.

The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum.

The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expressed in the basolateral membrane (BLM) of the human intestine. BBM and BLM were isolated by fractionation of human jejunum. BBM were enriched with alkaline phosphatase activity by over 9-fold relative to a crude jejunal homogenate and contained immunoreactive sucrase-isomaltase and GLUT5 proteins. By contrast the BBM fraction was substantially depleted of immunoreactive a1 subunits of the Na,K-ATPase and GLUT2 glucose transporters which were abundantly present in the BLM fraction. This BLM fraction was enriched by over 11-fold in potassium-stimulated phosphatase activity relative to the crude homogenate; BLM also reacted to immunological probes for GLUT5 but showed no observable reactivity with antibodies directed against sucrase-isomaltase. Quantitative immunoblotting revealed that the BBM and BLM contained near equal amounts of GLUT5 per mg of membrane protein. Immunogold localization of GLUT5 on ultrathin sections of human jejunum showed that GLUT5 was present in both apical BBM and BLM. This gold labelling was absent when antiserum was pre-incubated with the antigenic peptide corresponding to a specific C-terminal sequence of human GLUT5. Quantitative analyses of the number of gold particles per unit length of BBM and BLM indicated that the mean density of gold labelling was marginally greater in the BBM (0.399 gold particles/micrometer) than in the BLM (0.293 gold particle/micrometer). The localization of GLUT5 in the BLM of the human jejunum may suggest that it specifically participates in the transfer of fructose across the basal membrane of the enterocyte.

Binding proteins for cyclosomatostatins and bile acids in basolateral plasma membranes of rat liver

The bile acids cholate and taurocholate on the one hand and the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, on the other hand, display mutually competitive transport inhibition into isolated rat hepatocytes. This indicates a common transport system for bile acids and cyclosomatostatins in sinusoidal rat liver plasma membranes. In order to identify and isolate common binding and/or transport proteins for bile acids and the cyclopeptides by affinity chromatography, the bile acid derivative 4′-amino-7-benzamidotaurocholate (ABATC) and the cyclosomatostatin-analog 008 were attached to a gel matrix. Two methods were used to prepare integral membrane proteins: (1) alkaline EDTA extraction and (2) Triton X-114 phase separation. Octyl glycoside solubilized, alkaline EDTA-extracted integral basolateral membrane proteins with apparent molecular masses of 52 and 48 kDa bound specifically to the ABATC affinity matrix. Two-phase Triton X-114 separated integral membrane proteins of the same molecular masses bound specifically to the cyclosomatostatin ligand. The 48 kDa ABATC and 008 binding protein was shown to be present in the basolateral plasma membrane fraction and in the microsomal fraction. The isolated 52 kDa ABATC binding protein was localized only in basolateral plasma membranes and could not be found in isolated microsomes.

Cryoprotection of Aplysia gut basolateral membranes by trehalose

Cl −-ATPase activity resides in the basolateral membrane of gut cells. The sugar alcohols sorbitol, mannitol and glycerol could not maintain any cryoprotection of the basolateral membrane fraction for at least 12hr. The sugar, mannose, could not maintain any cryoprotection of basolateral membrane for at least 12 hr, while sucrose could only maintain 20% of basolateral membrane Cl −-ATPase activity after 12 hr. Trehalose cryoprotected basolateral membrane Cl −-ATPase activity by 98% of control up to 48 hr.