Brush border membrane vesicles isolated from rabbit small intestine were used to measure the interactions between sodium and glucose transport with a rapid uptake technique. A plot of glucose uptake rate vs. increasing sodium concentration yielded a sigmoid curve. Hill analysis revealed a coefficient of 1.9 ± 0.02 (±SEM), consistent with at least two sodium ions involved in glucose transport. Transport coupling was then measured directly with double-label experiments in which the uptakes of D-glucose and sodium were determined in the presence and absence of cotransported solute. At the earliest time point, the ratio of cosubstrate-dependent sodium transport to glucose transport was 3.2 ± 0.7 (±SEM). We conclude that two or more sodium ions are coupled to glucose transport across the intestinal brush border membranes.It is generally accepted that the mechanism of concentrative glucose transport across the brush border membrane of the intestine involves coupling with an inwardly directed sodium flux down its electrochemical potential gradient (1).Isolated brush border membrane vesicles have become powerful tools for demonstrating the sodium dependence, effect of membrane potential, and independence from cellular metabolism of sodium-coupled D-glucose transport (2-5). Much work has also been done with vesicles to investigate the kinetics of coupled transport, including calculation ofthe kinetic constants (6, 7), elucidation of the reaction mechanism (ordered vs. random), and order of reactant binding and unbinding (8)(9)(10).Because an accurate determination ofcoupling stoichiometry is necessary for the precise modeling of a transport system and because previous estimates of the coupling ratio between sodium and glucose in isolated membrane vesicles have been indirect (5, 10), we have sought to demonstrate coupling stoichiometry directly. A problem with direct determination of stoichiometry has been the high permeability of the vesicles to sodium, which has made it difficult to observe glucose-dependent sodium transport. In intestinal (3) and renal (11) membranes, external D-glucose was capable of stimulating 22Na transport but only at low sodium concentrations ('5 mM). Hilden and Sacktor (11) concluded that demonstrating coupling stoichiometry directly would require extensive manipulation of the experimental conditions to optimize both glucose and sodium transport.In view of the recent success with measuring sodium-citrate cotransport in renal brush border membranes (12, 13), we have reinvestigated sodium-glucose coupling in intestinal brush border membrane vesicles. trate filter (Sartorious) of 0.45-pim pore size, and rinsing with an additional 4 ml of ice-cold stop solution. The length of incubation was timed by an electronic metronome. The filters were dissolved in 10 ml of scintillation fluid (PCS, Amersham) and the radioactivity was counted in a liquid scintillation spectrometer. Counts were corrected for filter binding, quench, and spillover, and uptakes were expressed as mol per mg of membrane prot...

Na+, Li+, K+, Rb+, Br-, Cl- and SO4(2-) transport were studied in brush border membrane vesicles isolated from rabbit jejunum. Li+ uptakes were measured by flameless atomic absorption spectroscopy, and all others were measured using isotopic flux and liquid scintillation counting. All uptakes were performed with a rapid filtration procedure. A method is presented for separating various components of ion uptake: 1) passive diffusion, 2) mediated transport and 3) binding. It was concluded that a Na+/H+ exchange mechanism exists in the jejunal brush border. The exchanger was inhibited with 300 microM amiloride or harmaline. The kinetic parameters for sodium transport by this mechanism depend on the pH of the intravesicular solution. The application of a pH gradient (pHin = 5.5, pHout = 7.5) causes an increase in Jmax (50 to 125 pmol/mg protein . sec) with no change in Kt (congruent to 4.5 nM). Competition experiments show that other monovalent cations, e.g. Li+ and NH4+, share the Na+/H+ exchanger. This was confirmed with direct measurements of Li+ uptakes. Saturable uptake mechanisms were also observed for K+, Rb+ and SO4(2-), but not for Br-. The Jmax for K+ and Rb+ are similar to the Jmax for Na+, suggesting that they may share a transporter. The SO4(2-) system appears to be a Na+/SO4(2-) cotransport system. There does not appear to be either a Cl-/OH- transport mechanism of the type observed in ileum or a specific Na+/Cl- symporter.

The transport of organic solutes (sugars, amino acids, and metabolic intermediates) and inorganic solutes (Na+/H+ exchange and Na+-SO = 4 cotransport) in renal brush border and in intestinal brush border and basal lateral membrane vesicles is preserved when the vesicles are stored in liquid nitrogen. The preservation allows comparisons among transport systems of renal and intestinal cells obtained from the same animal.

The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS-C3-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na+, K+, Rb+, and Br- were approximately 5 nanoliters/(mg protein X sec) or 10(-5) cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na+, as determined by bi-ionic potential measurements was: F-, isethionate, gluconate, choline (less than 0.1) less than Na+(1.0) less than Cl-(1.5) = NO-3(1.5) less than Br-(2.3) less than K+(2.4) less than Rb+(2.5) less than Cs+(2.6) less than Li+(3.9) less than NH+4(12) less than I-(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.

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