Jejunal Basolateral Membrane Research Articles

926 Reduced Ferroportin Protein Expression Explains Why Intestinal Iron Absorption in the Neonatal Rat is Hyporesponsive to Stimuli That Reduce Absorption in Adult Animals

are not fully characterized, and thus, were examined in this investigation. Methods: Adult male Albino Wistar rats were used and were fed (for 4 weeks) a Lieber-DeCarli ethanol liquid-diet (36% calorie); control rats were fed the same diet but without ethanol. Carriermediated entry and exit of 3H-thiamin into and out of enterocytes was examined using purified jejunal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV), respectively. Carrier-mediated colonic uptake of 3H-thimain was determined using colonic sheets. Changes in expression of THTR1 and 2 at the protein and mRNA levels were determined byWestern blotting and by real-time PCR, respectively. Results: Carrier-mediated thiamin uptake by jejunal BBMV and BLMV from rats chronically fed the alcohol liquid diet was significantly (p<0.01) lower than uptake by BBMV and BLMV from control rats. This decrease in thiamin uptake was associated with a significant decrease in the level of expression of THTR-1 (known to be expressed at both the intestinal BBM and BLM) at the protein and mRNA levels. Expression of THTR-2 (known to be expressed exclusively at the intestinal BBM), however, was not affected. Carrier-mediated thiamin uptake by colonic sheets was also found to be significantly (p<0.01) lower in rats fed the alcohol liquid diet compared to control rats. Conclusion: These studies show that chronic alcohol consumption inhibits thiamin uptake in the small intestine (site of absorption of dietary thiamin) and the colon (site of absorption of the bacterially synthesized thiamin). Further, the inhibition appears to be differential and affect expression of THTR-1 but not THTR-2 (Supported by NIH grants DK56061-6 and AA018071).

Inhibition of gut glutathione transport by a breast cancer carcinogen

Abstract Introduction: Oral intake of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) causes breast cancer in the rat and is associated with a marked decrease in gut glutathione (GSH) efflux to the liver. We hypothesized GSH efflux is decreased by a block in either gut GSH production and/or GSH release leading to decreased liver detoxification of the carcinogen, facilitating adduct formation and carcinogenesis. Methods: GSH transport in jejunal basolateral membrane (JBLM) vesicles was investigated in female pubertal rats gavaged with 100 mg/kg DMBA (n = 15) or control (n = 15). Rats were pair-fed pre-defined chow and sacrificed one week post DMBA. Vesicles were prepared using Percoll differential centrifugation. Na+-dependent amino acid transport systems, A, ASC/B(0), and B(0,+) were examined using the inhibitors Me-AIB and Arginine. Transport velocity was determined by measuring 3H-GSH uptake. Blood and gut mucosal GSH were measured using spectrophotometric assay. Results: A significant decrease in Na+ dependent ASC/B(0) gut GSH transport velocity (42%) was seen in the DMBA group (21.89 +/− 2.63 pmol/mgprotein/min vs 35.52 +/− 4.05 pmol/mgprotein/min in controls, p Conclusions: One mechanism by which the carcinogen DMBA facilitates breast cancer induction may be through a significant decrease in gut GSH release secondary to an inhibition of the GSH ASC/B(0) transport system which would lead to increased DNA adduct formation and/or decreased carcinogen detoxification in the liver.

Evidence for a carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles.

Recent studies from our laboratory have demonstrated the presence of a pH-dependent, amiloride-sensitive, electroneutral carrier-mediated exchange for thiamine absorption in the human small intestinal brush-border membrane vesicles. However, the mechanism of thiamine transport across the human small intestinal basolateral membrane is not understood. The present study was aimed to characterize the mechanism of thiamine transport across the basolateral membranes of the human jejunum. Basolateral membrane vesicles (BLMV) were purified from mucosal scrapings of organ donors, utilizing a Percoll continuous density gradient centrifugation technique. The results showed [3H] thiamine uptake into BLMV to be: (1) markedly stimulated in the presence of an outwardly directed H+ gradient (pH 5.5in/7.5out); (2) significantly inhibited by amiloride in a dose-dependent manner; (3) sensitive to temperature and medium osmolarity and insensitive to changes in membrane potential; (4) not influenced by the addition of 1 mM Mg(2+)-ATP, inside and outside the vesicles in the presence of Na+ and K+; (5) inhibited by structural analogs-amprolium, oxythiamin, and unlabeled thiamine (100 microM); (6) not affected by organic cations, eg, TEA, N-methyl-nicotinamide (NMN), and choline; and (7) saturable as a function of concentration (apparent Km of 0.76 +/- 0.21 microM and a V(max) of 1.38 +/- 0.35 pmol/mg protein/10 sec). These results indicate the presence of a proton gradient-dependent specialized carrier-mediated exchange mechanism for thiamine transport across the human jejunum basolateral membranes.

Mechanism of thiamin transport across the human jejunal basolateral membrane vesicies (BLMVs)

Mechanism of thiamin transport across the human jejunal basolateral membrane vesicies (BLMVs)

Mechanism of thiamin transport across the human jejunal basolateral membrane vesicies (BLMVs)

Mechanism of thiamin transport across the human jejunal basolateral membrane vesicies (BLMVs)

Facilitated transport of lactate by rat jejunal enterocyte.

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.

The effect of GIP and glucagon-like peptides on intestinal basolateral membrane hexose transport.

The effect of gastric inhibitory polypeptide (GIP) and the related glucagon-like peptides-1 and -2 (GLP-1 and GLP-2) on jejunal basolateral membrane glucose transport was investigated to determine if the upregulation produced by luminal hexoses could be explained by the release of one or more of these peptides. Luminal perfusion of the rat jejunum for 4 h, under pentobarbital sodium anesthesia, with 100 mM D-glucose produced a significant increase in plasma GIP levels. Vascular infusion of saline containing 100-800 pM GIP also increased the maximal transport rate for carrier-mediated glucose uptake in jejunal basolateral membrane vesicles. The effect of vascular 400 pM GIP was maximal after 1 h and maintained out to 4 h. The effect of luminal glucose could be blocked by preinjection with anti-GIP antibodies, whereas an antineurotensin antibody had no effect. Vascular infusion with 800 pM GLP-1-(7-36) amide had no effect, but GLP-2 (400 and 800 pM) increased the D-glucose maximal transport rate. An anti-GLP antibody was able to block the response to luminal glucose.

Effect of luminal epidermal growth factor on enterocyte glucose and proline transport.

The effect of luminal epidermal growth factor (EGF; 60 ng/ml) and tyrphostin-51 (TYR; 10 microM), a tyrosine kinase inhibitor, on rabbit jejunal brush-border and basolateral membrane transport was investigated. In separate experiments, the effect of EGF, EGF and TYR, or TYR alone was examined in in vivo loops. In addition, Na+ permeability in brush-border membrane vesicles (BBMV) and the effect of Ca2+ channel blockade on EGF-stimulated glucose uptake were examined. Luminal EGF significantly (P < 0.0001) increased the maximal rate of transport (Vmax) for glucose and proline uptake in BBMV. TYR and Ca2+ channel blockade completely abolished the EGF-induced increase in glucose transport and in the case of TYR resulted in a significant reduction in Vmax compared with controls (P < 0.0001). The Michaelis-Menten constant did not differ in any experimental group. EGF had no effect on brush-border Na+ permeability or basolateral membrane glucose transport. The findings indicate a role for EGF in the acute regulation of jejunal brush-border membrane nutrient uptake. Furthermore, tyrosine kinase activity appears to be involved both in mediating EGF-induced alterations in transport function and in the maintenance of basal brush-border membrane function.

Influence of vascular and luminal hexoses on rat intestinal basolateral glucose transport.

The influence of luminal and vascular hexoses in rats on glucose transport across the jejunal basolateral membrane (BLM) was measured using isolated membrane vesicles prepared from infused animals. In vivo vascular infusions of glucose produced an increase in glucose transport across BLM vesicles. Sucrose, mannose, galactose, and fructose had no significant effect. Plasma glucose concentrations were unaffected by galactose and sucrose vascular infusions, while mannose and fructose produced a modest rise, and glucose increased plasma glucose to 20 mM. Insulin release was significantly increased by vascular infusion of glucose and fructose, while mannose produced only a small sustained rise. Sucrose and galactose had no effect. Perfusion through the lumen of the rat jejunum in vivo, for up to 4 h, with glucose, fructose, sucrose, or lactate (100 or 25 mM) produced a significant increase in the maximal rate of glucose transport (up to 4- to 5-fold) across BLMs. Galactose and mannose had no effect. Luminal glucose perfusion produced a small nonsignificant increase in glucose inhibitable cytochalasin B binding to BLM vesicles, and no change was seen in the microsomal pool of binding sites. The abundance of GLUT2 in the jejunal BLM, as determined by Western blotting, was unaffected by luminal perfusion of 100 mM glucose for 4 h. Fructose almost completely inhibited the carrier-mediated uptake of glucose in control and upregulated jejunal BLM vesicles. These results are discussed in relation to the physiological role of the upregulation of GLUT2 activity by luminal and vascular hexoses.

Evidence for a lactate-anion exchanger in the rat jejunal basolateral membrane

Background/Aims: The mechanism by which lactate, absorbed from the intestinal lumen or generated within the epithelium, crosses the basolateral membrane of the enterocyte and enters the bloodstream has not previously been characterized in detail. Methods:l-lactate uptake into and efflux from isolated jejunal basolateral membrane vesicles was investigated at room temperature using rapid filtration techniques. Results: Furosemide sensitive uptake of l-lactate was unaffected by cis sodium or proton gradients but could be stimulated by a trans gradient of bicarbonate and chloride. Kinetic analysis showed uptake to consist of a saturable component with a Michaelis constant (Km) of 3.2 mmol/L and a maximum velocity (Vmax) of 67 pmol·mg protein−1 · s−1 and a nonsaturable α-4-hydroxy-cinnamic acid insensitive component. Pyruvate, butyrate, acetate, valerate, and proprionate competitively inhibited lactate uptake into the vesicles. Efflux of lactate from preloaded vesicles was furosemide sensitive and accelerated by a trans bicarbonate gradient as well as by 10 mmol/L acetate, butyrate, and pyruvate. Conclusions: It is concluded that there is a short chain-fatty acid carrier system in the intestinal basolateral membrane, which operates as an anion exchanger.

Rheogenic Cl- conductance and Cl(-)-Cl(-)-exchange activities in guinea pig jejunal basolateral membrane vesicles

We describe a method for the simultaneous purification of apical (brush-border membrane) and basolateral membrane (BLM) vesicles from the same sample of guinea pig jejunum. We applied functional tests to demonstrate the absence of reciprocal cross contamination between the two vesicle preparations. By using the BLM vesicles and a rapid filtration technique, we quantified 36Cl uptake under conditions of equilibrated pH (pHout = pHin = 7.5). The presence of 200 mM cis of either Na+ or K+, or an equimolar mixture of both, significantly increased the initial Cl- entry rate. In the presence of K+, valinomycin further increased Cl- uptake, but no Cl- uphill transport was ever observed under any of the conditions. All the increases were abolished by voltage clamping, indicating that the alkali-metal ions act by creating an inside-positive membrane potential capable of stimulating a Cl(-)-conductance pathway. In the absence of K+, BLM vesicle preloading to obtain a [Cl-]out/[Cl-]in = 16/200 mM gradient (delta Cl-) resulted in a 500% increase in the initial 36Cl- entry rate, accompanied by a transient Cl- accumulation, with an overshoot at approximately 5 min. In the presence of both a positive-inside electrical gradient (delta psi) and a delta Cl-, the initial Cl- uptake rate was increased by 800%, indicating that the effects of delta psi and of delta Cl- are additive. The delta Cl- effect was blocked, but only partially, by short-circuiting the membrane potential with equilibrated K+ and valinomycin, thus indicating that it has both rheogenic and electroneutral components. We conclude that Cl- influx across the guinea pig intestinal BLM involves a Cl(-)-conductance pathway plus a distinct Cl(-)-Cl(-)-exchange system, exhibiting both electroneutral and rheogenic components. Alternatively, the possibility can also be entertained that the conductance and the exchange pathways share a common molecular basis, e.g., a nonobligatory Cl(-)-Cl- exchanger or rheogenic uniport.

Rat jejunal basolateral membrane Cl/HCO3 exchanger is modulated by a Na-sensitive modifier site

A Cl/HCO3 exchanger mediates HCO3 extrusion across rat jejunal basolateral membrane. Previous studies demonstrated that anion antiport activity is positively affected by Na, but evidence was given that this cation is not translocated by the carrier protein. Basolateral membranes isolated from rat jejunum were used to give more insight on Na effect. Uptake studies, performed together with vesicle sidedness determinations, indicated that the greatest stimulation of Cl-dependent HCO3 uptake occurs when Na is present at both vesicle surfaces. The kinetic dependence of Cl/HCO3 exchange on equal intra- and extravesicular Na concentration showed a hyperbolic relationship, and the calculated kinetic parameters were Vmax = 0.153 +/- 0.006 nmol mg protein-1 sec-1, Km = 23.0 mM. Ion replacement studies indicated that Na can be partially substituted only by Li and not by other monovalent cations. Results of this study suggest that Na could act as a nonessential activator of the Cl/HCO3 exchanger. A possible role of the Na-sensitive modifier site in the physiology of jejunal enterocyte is suggested.

GLUT2 is the transporter for fructose across the rat intestinal basolateral membrane

Background: The exact roles of disaccharidases and GLUT5 in the brush border membrane and GLUT2 in the basolateral membrane in the absorption of fructose across the intestine have not been fully determined. This paper describes characterization of fructose transport across the jejunal basolateral membrane using isolated membrane vesicles. Methods: Transport of fructose was measured using rapid filtration of vesicles. Luminal perfusion in vivo with glucose and fructose before vesicle preparation was used to assess modulation of GLUT2 activity. Western blotting measured the abundance of GLUT2 in the membrane. Results: The maximal rate of transport for fructose was 1100 pmol/mg protein/s and the Michaelis constant was 16 mmol/L. Fructose and glucose could completely inhibit the transport of each other. Perfusion of the intestinal lumen with fructose or glucose saline for 4 hours produced a fourfold increase in maximal fructose transport. Conclusions: These data indicate that the one transport protein, GLUT2, is responsible for moving both fructose and glucose out of the enterocyte across the basolateral membrane under basal conditions. The activity of this, or a closely related carrier, is rapidly upregulated by the presence of hexoses in the intestinal lumen, explaining the potentiation of fructose absorption by luminal glucose and obviating any need to involve apical disaccharidases.

Basolateral Cl −/HCO −3 exchange in rat jejunum: Evidence from H 14CO −3 uptake in membrane vesicles

Bicarbonate transport across basolateral membrane vesicles from rat jejunal enterocyte was studied at 28°C and pH 8.2. These experimental conditions make possible the determination of [ 14C]bicarbonate uptake. Inward gradients of Na +, K +, and Li + did not stimulate HCO − 3 uptake, suggesting that a cotransport mechanism with these cations does not occur. On the contrary a countertransport of bicarbonate driven by a Cl − gradient was evidenced. The ability of other inorganic anions to exchange with HCO − 3 was examined and results indicate that Cl − can be substituted by NO − 3, Br − and SCN −. The Cl −-dependent HCO − 3 uptake was strongly inhibited by SITS and DIDS, whereas acetazolamide was ineffective: thus transfer of labelled CO 2 is eliminated as a possible mode of HCO − 3 permeation. HCO − 3 uptake was also affected by the presence of superimposed membrane potentials, suggesting that a HCO − 3 conductive pathway is present in the jejunal basolateral membrane. These results show that there are no fundamental differences between data obtained performing H 14CO − 3 and 36Cl − (previously reported) uptake experiments.

Characterization of basolateral membrane Na/H antiport in rat jejunum

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a K m of 13.6 ± 1.4 mM and a V max of 35.4 ± 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.

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.

Intestinal Calcium Transport in Spontaneously Hypertensive Rats (SHR) and Their Genetically Matched WKY Rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.

Effect of hyperglycemia on d-glucose transport across the brush-border and basolateral membrane of rat small intestine

Experimental hyperglycemia leads to an increase in the capacity of the rat small intestine to absorb glucose. This effect occurs within hours from the onset of hyperglycemia and is thought to involve an induction of glucose transport in the brush-border and/or basolateral membrane of the intestinal epithelium. We devised a protocol for the simultaneous preparation of brush-border vesicles and basolateral vesicles from rat small intestine to determine the locus for the inductioof glucose transporter in hyperglycemic rats. A 6 h period of intravenous infusion with a 30% glucose solution had no effect on the initial rate of glucose uptake across jejunal or ileal brush-border vesicles when measured in the absence of a Na + gradient, suggesting that enhanced glucose uptake is not dependent on an increase in the number of Na +-dependent secondary active glucose transporters in the brush-border. Hyperglycemia did not effect the rate of glucose uptake across ileal basolateral vesicles but did cause a 78% increase in the initial rate of carrier-mediated d-glucose uptake across jejunal basolateral vesicles. The induction of glucose transport in the jejunal basolateral membrane was characterized by a rapid rate of glucose equilibration across the vesicles ( t 1 2 = 46 s sorbitol infused controls, 18 s hyperglycemia) and a 75% increase in the V max for carrier-mediated glucose uptake with no significant change in K t. When the rats were pretreated with cycloheximide prior to intravenous infusion, the initial rate of d-glucose uptake dropped to 13% of that seen in jejunal basolateral vesicles prepared from untreated rats. These results suggest a rapid turnover rate for the Na +-independent glucose transporter in the basolateral membrane of the enterocyte. An increase in the number of functioning glucose transporters in the basolateral membrane may play an important role in the short-term induction of glucose absorption by the jejunum of the hyperglycemic animal.