No abstract

There is compelling evidence that active Cl absorption by a variety of epithelia, widely distributed throughout the animal kingdom, is the result of an electrically neutral Na-coupled transport process at the luminal membrane and that the energy for transcellular Cl movement is derived from the Na gradient across that barrier. These co-transport processes are found predominantly in "leaky" or "moderately leaky" epithelia and permit these tissues to absorb Na and Cl with high degrees of efficacy. In addition, there is a growing body of evidence that cyclic AMP and Ca-induced electrogenic Cl secretion by a wide variety of epithelia may involve electrically neutral, Na-coupled Cl entry across the contraluminal membrane and that the energy for these secretory processes is derived from the Na-gradient across that barrier. A model for electrogenic Cl secretion that accounts for the available data is presented.

The effective hydrodynamic radii of small uncharged molecules in dilute aqueous solution were determined using Einstein's classical theory of viscosity. The radii thus obtained are those of a hypothetical sphere whose hydrodynamic behavior is the same as that of the solute molecule plus that water of hydration which is too firmly bound to partake in the viscous shearing process. The results obtained compare favorably with radii determined from molecular models constructed in accordance with atomic dimensions compiled by Pauling. Although the application of the Einstein theory to molecules whose size is comparable to that of water represents a considerable extrapolation, the results suggest that this deviation from the assumptions of the theory, in the case of the molecules studied, is of second order importance.Employing the viscometric radii, we have formulated an empirical correction of the Stokes-Einstein diffusion equation. This correction is similar in form to those previously proposed by Cunningham (22) and Millikan (91) and is of particular significance when the solute molecule is comparable in size to the discontinuities of the surrounding medium. The molecular radii of a number of small organic molecules obtained by means of the corrected Stokes-Einstein equation do not differ significantly from the radii obtained from molecular models of these compounds.Few satisfactory methods are available for the evaluation of the sizes of small uncharged molecules as they exist in dilute aqueous solution. In the case of molecules not much larger than water, in the range of 3 to 5/~, the existing methods are especially unsatisfactory. Radii determined from either molecular models or crystallographic studies make no allowance for hydration in aqueous solution. Electrostriction introduces an additional theoretical drawback to calculations based on the partial molal volume of solute in aqueous solution (1). The use of the Stokes-Einstein diffusion relationship for the determination of radii of small molecules has been criticized (2),

The transmural potential difference, short-circuit current, and Na fluxes have been investigated in an in vitro preparation of isolated rabbit ileum. When the tissue is perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference in the presence of glucose averages 9 mv. Unidirectional and net Na fluxes have been determined under a variety of conditions, and in each instance, most if not all of the simultaneously measured short-circuit current could be attributed to the active transport of Na from mucosa to serosa. Active Na transport is dependent upon the presence of intact aerobic metabolic pathways and is inhibited by low concentrations of ouabain in the serosal medium. A method is described for determining whether a unidirectional ionic flux is the result of passive diffusion alone, in the presence of active transport of that ion in the opposite direction. Using this method we have demonstrated that the serosa-to-mucosa flux of Na may be attributed to passive diffusion with no evidence for the presence of carrier-mediated exchange diffusion or the influence of solvent-drag.

The relation between unidirectional influxes of Na and amino acids across the mucosal border of rabbit ileum was studied under a variety of conditions. At constant Na concentration in the mucosal bathing solution, amino acid influx followed Michaelis-Menten kinetics permitting determination of maximal influx and the apparent Michaelis constant, K~. Reduction in Na concentration, using choline as substitute cation, caused an increase in Kt for alanine but had no effect on maximal alanine influx. The reciprocal of/(~ was a linear function of Na concentration. Similar results were obtained for valine and leucine and these amino acids competitively inhibited alanine influx both in the presence and in the absence of Na. These results lead to a model for the transport system which involves combination of Na and amino acid with a single carrier or site leading to penetration of both solutes. The model predicts that alanine should cause an increase in Na influx and the ratio of this extra Na flux to alanine flux should vary with Na concentration. The observed relation agreed closely with predicted values for Na concentrations from 5 to 140 mM. These results support the hypothesis that interactions between Na and amino acid transport depend in part on a common entry mechanism at the mucosal border of the intestine.I n the p r e c e d i n g p a p e r (I), a m e t h o d for m e a s u r i n g unidirectional solute influx across the m u c o s a l b o r d e r of intestinal epithelial cells was described a n d some characteristics of L-alanine a n d N a influxes were presented. T h e s e studies p r o v i d e d the first u n e q u i v o c a l evidence t h a t the flux of a n actively t r a n s p o r t e d a m i n o acid f r o m the mucosal solution into the cell is influenced b y the N a c o n c e n t r a t i o n in the mucosal solution a n d is i n d e p e n d e n t of the b u l k cellular N a concentration. F u r t h e r study of the influx processes is necessary in order to define m o r e c o m p l e t e l y the t r a n s p o r t m e c h a n i s m s for n o n -

The unidirectional influxes of Na, K, and C1 into isolated strips of rabbit ileum are comprised of movements across the mucosal membrane of the epithelial cells and ionic diffusion into an extracellular shunt pathway. A large fraction of the Na influx across the mucosal membrane alone is inhibited by Li, suggesting the participation of a carrier mechanism in the influx process. The partial ionic shunt conductances of Na, K, and C1 account for at least 82 % of the total tissue conductance. The calculated shunt permeabilities (P) are (in centimeters per hour) P = 0.040, PN = 0.035, and Pcl = 0.019, so that Px:PN:PclI = 1.14:1.00:0.55. Diffusion potentials across the tissue resulting from isotonic replacement of NaCl in the mucosal solution with mannitol or KC1 are described by the Goldman constant-field equation together with the above permeabilities of the shunt pathway. These observations are not consistent with permeation through a fixed-charge pore but can be explained by a model featuring constant ionic partition into a neutral-polar pore that traverses the tight junction. Such a pore may be lined with either fixed dipoles or fixed dipolar ions oriented such that electronegative groups influence the permselective properties of the diffusion pathway. The essential feature of both models is that electroneutrality is maintained by means of fixed membrane components and does not depend upon the presence of mobile counterions.In recent years, increasing attention has been focused on the role of transmural, extracellular pathways in the transport of solutes and water by a variety of epithelial tissues. However, the conductance properties of these pathways have not been defined directly because studies of transmural ionic fluxes, diffusion potentials, or streaming potentials do not clearly distinguish between the properties of extracellular and transcellular routes for ion flow.

The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to scrosa. These effects arc dependent upon the active transport of the sugar per se and arc independent of the metabolic fate of the transported sugar. Furthermore, they arc inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the scrosal solution. The increase in the short-circuit current, A/e~, requires the presence of Na in the pcrfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, A/co is a saturable function of the mucosal sugar concentration which is consistent with Michaclis-Mcntcn kinetics suggesting that the increase in active Na transport is stoichiomctrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented.Previous studies (1) have indicated that when isolated segments of distal rabbit ileum are perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference (PD) in the presence of glucose averages approximately 9 my. We have further demonstrated that the transmural PD and the short-circuit current (1,0) result from the active transport of Na from mucosa to serosa, that this process is dependent upon intact aerobic metabolic pathways, and that it is inhibited by low concentrations of ouabain in the serosal solution. N aThe unidirectional serosa-to-mucosa Na flux, ~8~, on the other hand, may be attributed to passive diffusion with no evidence for the presence of exchange diffusion or the influence of solvent-drag.In the course of these studies, it was observed that the addition of actively ~o43

L-Alanine and 3-O-methyl-D-glucose accumulation by nmcosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 m~ are reached within 30 rain when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 X 10 -~ M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.In vitro preparations of m a m m a l i a n small intestine are capable of transporting sugars and amino acids from low concentrations in the mucosal solution to higher concentrations in the solution bathing the serosal side (1). Further, sugars (2) and amino acids (3, 4) are a c c u m u l a t e d by intestinal tissue and the concentrations of these solutes in tissue water m a y reach more t h a n ten times those in the surrounding media. These observations have led to the widely held view that intestinal sugar and amino acid absorption is the result of carrier-mediated processes which bring a b o u t uphill 1 transport from the mucosal solution into the cell followed by downhill exit from the cell across the serosal tissues (i).In recent years, interactions between Na and the transport of sugars and amino acids have been demonstrated. Thus, intestinal transport (transmural)The terms uphill or active transport will be used to denote movement against an electrochemical potential difference, according to the definition of Rosenberg (5). The term downhill denotes movement down an electrochemical potential difference.