Ileal Brush-border Vesicles Research Articles

Pre-steady-state and steady-state function of the ileal brush border SO4(2-)-OH- exchanger.

Rapid-sampling analysis of the detailed time course of 35SO4(2-) uptake under pH-gradient (pHin = 7.5; pHout = 5.5) conditions converged to a model of an initial burstlike pre-steady-state with relaxation to linear steady-state uptake across pig ileal brush border vesicles. A model of low affinity transport (K(m) = 7.7 mM) plus an unsaturable component described the steady-state component of 35SO4(2-) uptake. Steady-state transport was maximal under pH-gradient conditions and sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Significant steady-state 35SO4(2-) transport sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was found under acidic (pHin = pHout = 5.5) and neutral (pHin = pHout = 7.5) pH-equilibrated conditions. Varying conditions from pH gradient to neutral pH equilibrated had no effect on the amplitude of the pre-steady-state burst or on the time constant for relaxation from pre-steady-state to steady-state conditions. However, maximal amplitude was obtained under acidic pH-equilibrated conditions. These results are incorporated into a model for the low affinity ileal brush border SO4(2-)-OH- exchanger whereby, under 0-trans (0 internal substrate concentration) conditions, translocation of inner-facing to outer-facing conformations defines the overall rate-limiting step of the transporter. Provision for slippage of the unloaded carrier could account for 35SO4(2-) transport under acidic pH-equilibrated conditions.

Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.

Effect of inhibitors on sodium and chloride transport in brush border vesicles from human jejunum and ileum.

Brush border vesicles were isolated from surgically resected pieces of human jejunum and ileum using a Mg2+/EGTA precipitation method. When compared to the homogenate, the final membrane preparation contained alkaline phosphatase at a 14 times higher concentration and almost no (Na++K+)-stimulated adenosine triphosphatase. An Na+/H+ antiport could be demonstrated in the jejunum by imposing a pH gradient between the interior and the outside of the vesicles (pHinside 5.2, pHoutside 7.2). In the presence of amiloride or harmaline, Na+/H+ antiport was inhibited by 60 +/- 5% (p less than 0.05) or 65 +/- 5% (p less than 0.05), respectively. In vesicles of human ileum we found an Na+/H+ antiport and in contrast to the jejunum a Cl-/OH- antiport could be demonstrated by imposing a pH gradient (pHinside 5.2, pHoutside 7.2). Besides this double-exchange mechanism for sodium and chloride, a Na+/Cl- cotransport and a Cl- conductive pathway could be detected in ileal brush border vesicles. In the presence of the anion transport inhibitors, furosemide, SITS and DIDS activities of Cl-/OH- antiport and Na+/Cl- cotransport were suppressed by 30 +/- 5% (p less than 0.05), 35 +/- 5% (p less than 0.05) and 40 +/- 5% (p less than 0.05), respectively. We conclude that absorption of sodium and chloride in the absence of organic solutes is mediated through different transport mechanisms at the luminal plasma membrane, which are in part subject to regulation by sodium and chloride transport inhibitors.

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.

Short-term studies of taurocholate uptake by ileal brush border membrane vesicles: Anion effects

Experiments allowing Na +-dependent short-term uptake measurements by ileal brush border vesicles were described. Glucose uptake was compared with taurocholate uptake in the presence of NaCl, NaSCN and Na 2SO 4. In contrast to the observation made with glucose, taurocholate transport was the same for the three electrolytes, indicating electroneutral taurocholate transport.

Bile acid transport in isolated brush border vesicles

BILE ACID TRANSPORT IN ISOLATED BRUSH BORDER VESICLES. F.A. Wilson and L.L. Treanor. Department of Medicine, Vanderbilt University, Nashville, Tennessee Bile acids(BA) are transported across the intact jejunum and ileum via passive and sodium-dependent active processes, respectively. Previous studies in our laboratory indicated no significant difference in total BA binding betweenjejunal and ileal brush borders (J.Memb. Bio!. 33:213,1977). One possible explanation is that such a demonstration requires the presence of an energy source. Active glucose transport occurs in brush border vesicles (BBV) when free energy is supplied by the asymmetric distribution of Nat and an electrical potential difference across the vesicle wall (Hopfer et al. J. Biol. Chem. 248:25, 1973). To determine whether BA transport differences exist, BBV were prepared from rat iejunum and ileum. Purity of preaarations was assessed byelectronmicroscopy and sucrase activity. Vesicle uptake was determined by membrane filtration. Validity studies revealed that d-glucose displayed the overshoot phenomenon of stimulated uptake in the presence, but not absence, of a Nat gradient. Maximal d-glucose uotake was higher in jejunal than ileal BBV. The time of maximal d-glucose uptake correlated with the absolute ratio of maximal/equilibrium uptake. Lglucose failed to show the overshoot. Transport of BA in ileal BBV was linearwith resoect to membrane protein and demonstrated the overshoot phenomenon with resoect to time in the presence of a Na+ gradient, but not after Nat equilibrium. The maximal uptake of glycodeoxycholate (GDC) occurred at 10 set and the ratio of maximal/equilibrium uptake was > 1.9. The overshoot phenomenon was not seen for GDC in the presence or absence of a Nat gradient across the wall of jejunal BBV. GDC uptake at 15 min (Nat equilibrium) was subtracted from maximal uptake at 10 set (Nat gradient) to yield a saturable transport component. Kinetic analysis revealed a Km of O.lmM, a value similar to that described for isolated whole epithelial cells (Biochim.Biophys. Acta. 406: 280, 1975). These studies support the existence of a transport system in the brush border membrane that: 1) reflects the kinetics and characteristics of BA transport in isolated epithelial cells and intact intestinal tissue and 2) catalyzes the co-transport of Nat and BA across this membrane in a manner analogous to d-glucose transport.