The properties of active sugar transport by a preparation of isolated intestinal epithelial cells have been examined with special emphasis on evaluating the sodium gradient transport hypothesis. In common with other systems, the isolated cells accumulate sugars by a sodium-dependent process which is inhibited severely by ouabain or oligomycin at concentrations known to inhibit active sodium transport.The onset of inhibition by these agents is somewhat more rapid than might be expected for dissipation of the cellular Na gradient. Moreover, cells loaded to about 50 mM intracellular Na+ are able to actively accumulate sugar within a 1-2-min interval when placed in a medium containing 20 mM Na+. It can be shown that intracellular sodium is greater than 20 mM during the entire 2-min interval. Furthermore, the increment in sugar uptake is as great during the
The unidirectional influx of alpha-methylglucoside (alpha-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na+, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance alpha-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar can be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.
Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.
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