Medical Center, Los Angeles, California 90024, U.S.A. (Received 27 July 1970)

Transcription

1 J. Physiol. (1971), 212, pp With 1 text-figure Printed in Great Britain GALACTOSE TRANSPORT ACROSS THE HAMSTER SMALL INTESTINE; THE EFFECT OF SODIUM ELECTROCHEMICAL POTENTIAL GRADIENTS By ANTHONY P. SMULDERS AND ERNEST M. WRIGHT From the Department of Physiology, University of California Medical Center, Los Angeles, California 90024, U.S.A. (Received 27 July 1970) SUMMARY 1. Measurements of galactose unidirectional fluxes across the isolated mucosa of the hamster small intestine were made under a variety of experimental conditions designed to investigate the interrelationships between galactose transport, the sodium ion and electrical potentials. 2. Galactose transport was inhibited by lowering the sodium concentration in the mucosal and/or serosal solutions and by increasing the p.d. across the tissue. This inhibition of galactose transport was judged to be irreversible. 3. Transport was unaffected by decreasing the p.d. across the tissue. 4. These results are discussed in relation to the model proposed to explain galactose transport across the intestine. INTRODUCTION Sugars are actively absorbed from the alimentary canal into the bloodstream by the epithelial cells of the small intestine. This transport phenomena occurs in at least two stages: (i) the active accumulation of the sugar within the epithelial cell and (ii) the subsequent, presumably passive, downhill movement of sugar from the cell into the blood. An ingenious hypothesis has been put forward to explain the active accumulation of sugars within the cell in terms of the cotransport of sugar and sodium across the brush border (for reviews see Crane, 1968). Briefly, this hypothesis requires that sugar and sodium enter the cell from the lumen of the intestine via a 'sugar-sodium-carrier' complex and that it is the difference in the sodium electrochemical potential energy (A/,Na) between the lumen and cell which provides the energy for the 'active' uphill transport of sugar. It is also suggested that the electrochemical potential gradient is maintained by a sodium pump located at the serosal and/or lateral membranes of the cell.

2 278 A. P. SMULDERS AND E. M. WRIGHT There is a substantial body of experimental evidence which lends support to this hypothesis, e.g. (1) sugar transport requires the presence of sodium in the external solutions (Ricklis & Quastel, 1958; Csaiky & Thale, 1960; Bihler & Crane, 1962); (2) sugars stimulate the active transport of sodium across the intestine (Schultz & Zalusky, 1964a; Barry, Smyth & Wright, 1965); (3) there is a 1/1 relationship between the sugar influx and the sugar dependent sodium influx across the brush border (Goldner, Schultz & Curran, 1969); (4) the actively transported sugars generate electrical potentials across the intestine (Barry, Dikstein, Matthews, Smyth & Wright, 1964). The purpose of the present paper is to further investigate the interrelationships between galactose transport, the sodium ion and electrical potentials. METHODS The principle of the experiments. Our approach to this problem is to measure sugar transport while varying the difference in sodium electrochemical potential (Aft~a) across the intestinal epithelium. This AjUtw was varied in two ways: (1) by a voltage clamp technique and (2) by changing the sodium concentration in the mucosal or serosal solutions. These manipulations might be expected to disturb the Aiia across the brush border directly and, in addition, indirectly by varying the rate at which sodium is pumped out of the cell into the serosal solution. A positive A is expected to inhibit and a negative AjiNa is expected to enhance sugar transport. Calculation shows that the free energy available from the A\Uwa across the brush border of the hamster epithelium is about 1500 cal/mole. So in these experiments we have varieda/fta across the epithelium by about ± 1000 cal/mole. The change in A7l1a produced by varying the sodium concentration in the mucosal solution is to be equated with the change in AuiNa across the brush border since it has been shown that the trans-epithelial diffusion potentials generated under these conditions in the tortoise intestine are due solely to the change in p.d. across the brush border (Gilles- Baillien & Schoffeniels, 1967). Likewise the change in Ai7Na produced by varying the serosal sodium concentraton is equated with the change in Aj4na across the serosal (or lateral) membranes. With regard to the voltage clamp experiments we do not know at present the relative distribution of the voltage at the brush border and serosal membranes. Unidirectional trans-epithelial fluxes are used in these experiments as a measure of sugar transport. Experimental procedure. In this study we have chosen to study galactose transport across the hamster small intestine. It is well documented that galactose is actively transported across this epithelium (Wilson, 1962). Male golden hamsters, weighing between 80 and 100 g, were maintained in the laboratory for at least three weeks prior to use. Water and food were made available ad libitum. The hamsters were anaesthetized with sodium phenobarbitone and the combined jejunum and ileum was removed as described by Barry et al. (1964) for the rat. The serosal muscle layers were removed from short segments of the mid-intestine (mid-jejunum) and the isolated mucosa was clamped between two lucite half chambers similar to those described by Schultz & Zalusky (1964b). The area of the window between the two half chambers was 1-13 cm2. Each surface of the tissue was perfused with 10 ml. physiological saline recirculated by a 95 % oxygen 5 % carbon dioxide gas lift. The temperature of the solutions was maintained at 370 C. The

3 INTESTINAL GALACTOSE TRANSPORT 279 composition of the saline was NaCl 119 mm, KCl 4 mm, CaCl2 2-5 mm, MgSO mm, NaHCO3 25 mm, K2HPO4 1-2 mmi and KH2PO4 0-2 mm. The galactose concentration in both the mucosal and serosal fluids was 20 mm except for a few experiments mentioned specifically in the text. When the sodium chloride in the saline was varied mannitol was used to maintain the osinolarity. As sugar transport is insensitive to the anion composition of the incubation media (Bihler & Crane, 1962) the effect of varying the sodium chloride is mainly due to the sodium ion alone. Unidirectional transmural fluxes of galactose were determined using the [14C]sugar. The radioactive samples were then assayed by conventional liquid scintillation counting techniques where each sample was counted to at least 1% counting efficiency. Galactose fluxes were calculated in t,-mol/cm2 hr. The p.d. across the tissue was recorded by connecting an electrometer (Keithley, Model 6lOB) to the mucosal and serosal solutions via calomel half cells and salt bridges. The output of the electrometer was displayed on a chart recorder (Varian, Model G11A) and the p.d.s were corrected for the asymmetry of the electrical circuit (< 0 5 mv). The salt bridges contained saturated KCI in 3 00 agar and the tips were placed 1-3 mm from each side of the epithelium. The use of saturated KCl bridges can introduce significant errors in evaluating potential differences between asymmetrical solutions (see Barry & Diamond. 1970). However, direct measurements have shown that under our experimental conditions the junction potential corrections were less than 1 mv. In some experiments the voltage across the intestinal epithelium was controlled by passing a direct current across the tissue. The current, tapped off potentiometrically from a battery, was measured on a Keithley 600A electrometer and was passed to opposite sides of the tissue by means of Ag/AgCl electrodes and salt bridges. In these experiments the p.d.s were corrected for the resistance drop across the saline between the salt bridges used to monitor the p.d. and the tissue. RESULTS To show that this preparation of the hamster small intestine is capable of active sugar transport we have measured the galactose unidirectional fluxes across the tissue. These results are shown in Table 1 where, in addition, we have included for comparison data obtained from experiments where we did not remove the serosal muscle layers. In both preparations there was a net flux of the sugar from the mucosal to the serosal solutions. The net flux in the absence of the muscle was twice that in the intact preparation, the difference being largely due to a higher unidirectional flux from mucosa to serosa. The greater transport capacity of the stripped intestine is also associated with a higher transmural p.d. and lower resistance. The resistance measurements show that about 25 % of the resistance of the intact intestinal wall is due to the presence of the muscle layers. Thus at least part of the increase in the mucosa to serosa flux must be due to the removal of this diffusion barrier, i.e. there is a reduction in the backflux of actively transported sugar. This interpretation is supported by the finding that the half-times for the build up and decay of the diffusion potentials resulting from changes in the composition

4 280 A. P. SMULDERS AND E. M. WRIGHT of the serosal fluid are reduced by some 30 % on removal of the serosal tissue. These experiments show that galactose is actively transported across this preparation of the hamster intestine. TABLE 1. A comparison between the stripped and intact intestinal preparations. The unidirectional fluxes were average values determined at 10 min intervals from 15 to 100 min after setting up the experiments. The resistances were measured during the initial 15 min and the p.d.s are the maximum values obtained during each experiment Mucosa to Flux Serosa to serosa mucosa Resistance p.d. (/z-mole/cm2. hr) (fl/cm2) (mv) Stripped 3-7 ± 041 (112) 0 3 ± 0 1 (34) (24) (26) Intact 1F7±0-1 (47) (16) 42+2 (6) (6) Effect of sodium concentrations In these experiments we have investigated the effect of the external sodium concentration on theunidirectional galactose fluxes. Galactose fluxes were first measured under normal conditions and when the sodium concentration in the mucosal and/or serosal solutions was varied between 25 and 144 m-equiv/l. In Table 2 we show the sodium concentrations in the mucosal and serosal solutions, the unidirectional galactose fluxes and the transmural p.d.s. The first point to observe is that there is a significant reduction in the galactose mucosa to serosa flux when the sodium concentration is reduced from 144 to 25 m-equiv/l. in (a) the mucosal solution, (b) the serosal solution and (c) in both the mucosal and serosal solutions. The second point to note is that changing the sodium concentration had no significant effect on the galactose serosa to mucosa flux. We also observed that when the sodium concentration was returned to 144 m- equiv/l. on both sides of the epithelium (after 30 min in the low sodium solutions) there was little if any reversal of the inhibition of sugar transport. Bosackova & Crane (1965) also found that sugar accumulation was impaired after incubations in sodium-free mannitol solutions. These effects are probably not due to mannitol alone as we find there is no inhibition to galactose transport when high concentrations of mannitol are added to solutions containing normal sodium concentrations. The response of the transmural p.d.s to the variations in the external sodium concentration is that expected for diffusion potentials across the epithelium (Wright, 1966a, b).

5 INTESTINAL GALACTOSE TRANSPORT 281 C)o._ O o C _ 00V0 x 6 A O >- ; - l+ D,_ 0 0S f :. 00 _'' Io +1 - ci) I x PL ci 0-- ci 0 c:z em x -t c..~ c ce C -4- I- - T < 00 +l L 2? co,!l0 C C I+ I:Z. ~- + I+ z F--f cre Cn 0 0I C) 1-4 C) C) I C I Ct C) I-)

6 282 A. P. SMULDERS AND E. M. WRIGHT Effect of voltage clamping In the control experiments (Table 2) the average p.d. across the intestinal mucosa was 6-5 mv, serosal solution positive with respect to the mucosal solution. The relationship between the transepithelial p.d. and sugar transport was investigated by clamping the p.d. at either + 40 or -40 mv (serosa with respect to mucosa). The epithelium was clamped at these voltages so that the sodium electrochemical potential difference across the tissue approximates the maximum obtained when the sodium concentration was varied in either the mucosal or serosal solutions (Table 2). Some of the experimental results obtained are shown in Table 3. TABLE 3. The effect of the p.d. on the galactose flux. The p.d. across the intestine was clamped at -40 or + 40 mv and the galactose mucosa to serosa flux was compared with the flux obtained when the p.d. was left at open circuit. The All7 was calculated from the difference in the p.d. between the open circuit and the voltage clamp conditions according to the equation AANa = RTln{[Na],/ [Na]l} + FAVf ATIXa Flux p.d. (mv) (cal/mole) (,t mole/cm2. hr) P (12) < (52) (8) <0.7 When the p.d. across the preparation was clamped at + 40 mv (serosa positive) we observed a very significant reduction in the unidirectional flux of galactose from the mucosal to the serosal solutions. This is also illustrated in an individual experiment shown in Fig. 1 a. In contrast, there was no significant change in the sugar flux when the potential was clamped at -40 mv. However, when the -40 mv clamp was removed we observed a significant reduction in the sugar flux. This is illustrated for one experiment in Fig. 1b where it can be seen that removing the voltage clamp reduced the rate of appearance of [14C]galactose in the serosal fluid. These experiments show that the flux of galactose is reduced by increasing the p.d. across the epithelium, i.e. increasing the p. d. from either -40 to + 6*5 mv or from + 6'5 to + 40 mv. As noted in the experiments where galactose transport was inhibited by low external sodium concentrations there was little evidence ofreversibility. We also failed to observe an increase in the galactose mucosa to serosa flux at a p.d. of -40 mv even when the sodium and galactose concentrations were reduced to 50 m-equmv/l. and 5 mm respectively. There was no significant effect of voltage clamping on the galactose serosa to mucosa backflux. In the rat intestine it can be seen that increasing the p.d. also

7 INTESTINAL GALACTOSE TRANSPORT 283 produced a slight decrease in the net transport of galactose (Barry, Smyth & Ude, 1969). 60 a,, b _60 a * d10p / x 40 X I~.-150/ 5/.S30- E E ~~~~~~~~~~~~~E l'-100/ E l 0. = Open -40 u u-+0m-.u 50- mv circuit ~mv 10 0 I I Time (min) Time (min) Fig. 1. Experiments showing the effect of voltage clamping on galactose transport across the intestinal mucosa. Isotope was added to the mucosal reservoir and the rate of appearance of radioactivity in the serosal solution was monitored. The ordinate shows the serosal counts/ml. and the abscissa shows the time in minutes. In the experiment shown in Fig. 1 a the rate of appearance of the galactose isotope in the serosal solution was initially determined in the open circuit condition, secondly when the tissue was clamped at + 40 mv and finally under open circuit conditions. In the experiment shown in Fig. 1 b the tissue was clamped at -40 mv before allowing transport to proceed under open circuit conditions and finally the tissue was clamped at -40 mv. The galactose and sodium concentrations in the external solutions were 20 mm and 144 m-equiv/l. respectively in both experiments. The dashed lines in the two Figures show control experiments where the rate of appearance of isotope was determined in the absence of voltage clamping. DISCUSSION These experiments show that the net transport of galactose across the tissue was inhibited by adverse electrochemical potential gradients as predicted and the degree of inhibition was much the same when the gradient was changed by lowering the mucosal solution sodium concentration or by clamping the tissue at + 40 mv. In contrast we were unable to enhance sugar transport with favourable -7Na gradients, i.e. by decreasing the serosal sodium concentration to 25 m-equiv/l.; or by clamping the tissue at -40 mv. Lowering the serosal sodium concentrations also apparently fails to stimulate sugar transport across the toad small intestine (Csaiky & Thale, 1960). Contrary to expectations lowering the serosal sodium concentration actually reduced the net transport of galactose.

8 284 A. P. SMULDERS AND E. M. WRIGHT How can these anomalous observations in the hamster intestine be reconciled with the model proposed to explain sugar transport? This can be accomplished by making two assumptions about the properties of the epithelium. The first is that exposure of the cell to low external sodium concentrations inhibits metabolism in addition to the direct effect on sugar transport. The second is that when current is passed across the epithelium the major voltage drop is at the serosal (or lateral) membrane i.e. the effective resistance of the serosal membrane is much greater that that of the brush border. This latter assumption is supported by the finding that in the rabbit ileum the effective resistance of the brush border membrane is less than 10 % of the total epithelium (S. G. Schultz, personal communication). The first assumption would explain the irreversible effect of low sodium concentrations on sugar transport and the inhibition of sugar transport when the sodium concentration in the serosal fluid alone is reduced to 25 m-equiv/l. Further evidence to support this suggestion comes from the in vivo studies of sugar absorption in the rat where Csa6ky & Zollicoffer (1960) found that inhibition of glucose transport produced by sodium free solutions was overcome only after prolonged perfusions of the intestine with normal sodium solutions. Pre-incubations of in vitro preparations of the rat small intestine in sodium free media also reduces oxygen uptake and glucose utilization (Jordana & Ponz, 1969) and abolished the active transport of L-phenylalanine (Robinson, 1967). The second assumption requires that the effect of voltage clamping is not due to a direct change in the AllNa across the brush border, but that the effect is due to a change in the intracellular sodium concentration. Voltage clamping the tissue would be expected to change the efficiency of the sodium pump at the serosal face of the cell and so change the intracellular sodium concentration. For example, when the tissue is clamped at + 40 mv a lower efficiency of the pump would increase the intracellular sodium concentration and so reduce AiiNa across the brush border indirectly. The apparent irreversible nature of the effect could be due to the inability of the cell to compensate for large changes in the intracellular sodium concentration. This second assumption could also explain the failure to obtain an increase in the net transport of galactose when the p.d. is clamped at -40 mv. A potential of this polarity would increase the efficiency of the sodium pump but it is likely, however, that the increase in the efficiency is balanced by the leakage of sodium into the cell. A more precise interpretation of the voltage clamp experiments is complicated owing to the possibility that the passage of current across the epithelium may change ion concentrations both within the cell and adjacent to the cell membranes. Such changes in salt concentration have

9 INTESTINAL GALACTOSE TRANSPORT 285 been observed by Wedner & Diamond (1969) in the gall bladder during electroosmosis experiments where they are thought to originate from the transport number effect (Barry & Hope, 1969). The magnitude of the concentration changes depends on the current pathway and the ionic species which carry the current in the extracellular fluid, the intracellular fluid and across the cell membranes. The effect is small if the tight junctions represent a low resistance pathway to current flow as has been suggested in other epithelial membranes (see Barry & Diamond, 1970). We conclude that the results obtained in this investigation do not provide definitive proof for the model proposed to explain sugar transport across the intestine. It is possible to rationalize our results in terms of the model if we assume that metabolism is inhibited by low external sodium concentrations and that the effective resistance of the serosal membrane is much greater than the resistance of the brush border membrane. However, this investigation does not rule out an additional or an alternative source of energy for sugar transport, e.g. the energy may be obtained directly from a sodium dependent ATPase in the brush border membrane. We wish to record our thanks to Drs P. Barry, J. Diamond, and S. Schultz for their comments on the manuscript. This work was supported by a program project from the U.S. Public Health Service (HE 11351) and a grant from the Los Angeles County Heart Association. One of us (A. P. S.) was a predoctoral fellow under the U.S. Public Health Service training grant GM REFERENCES BARRY, P. H. & DiAmoD, J. M. (1970). Junction potentials, electrode standard potentials, and other problems in interpreting electrical properties of membranes. J. Membrane Biol. (in the Press). BARRY, P. H. & HoPE, A. B. (1969). Electroosmosis in membranes: effects of unstirred layers and transport numbers. I. Theory. Biophy8. J. 9, BARRY, R. J. C., DiKsTEiN, S., MATTHEWS, J., SmyTri, D. H. & WRIGHT, E. M. (1964). Electrical potentials associated with intestinal sugar transfer. J. Physiol. 171, BARRY, R. J. C., SMYTH, D. H. & WRIGHT, E. M. (1965). Short-circuit current and solute transfer by rat jejunum. J. Physiol. 181, BARRY, R. J. C., SMYTH, D. H. & UDE, J. F. (1969). Hexose and sugar transfer in the rat jejunum. Life Sci. Oxford 8, BIHLER, I. & CRANE, R. K. (1962). Studies on the mechanism of intestinal absorption of sugars. V. The influence of several cations and anions on the active transport of sugars, in vitro, by various preparations of hamster small intestine. Biochim. biophy8. Acta 59, BOSAC'OVA, J. & CRANE, R. K. (1965). Studies on the mechanism of intestinal absorption of sugars. VIII. Cation inhibition of active sugar transport and 22Na influx into hamster small intestine, in vitro. Biochim. biophy8. Acta 102, CRANE, R. K. (1968). Absorption of sugars. In: Handbook of Phy8iology, ed. CODE, C. F. vol. 3. Washington: American Physiological Society.

10 286 A. P. SMULDERS AND E. M. WRIGHT CscKY, T. Z. & THALE, M. (1960). Effect of ionic environment on intestinal sugar transport. J. Physiot. 151, CSkKY, T. Z. & ZOLLICOFFER, L. (1960). Ionic effect on intestinal transfer of glucose in the rat. Am. J. Physiol. 198, GITTES-BAILLIEN, M. & SCHOFFENIELS, E. (1967). Bioelectric potentials in the intestinal epithelium of the Greek tortoise. Comp. Biochem. Physiol. 23, GOLDNER, A. M., SCHULTZ, S. G. & CURRAN, P. F. (1969). Sodium and sugar fluxes across the mucosal border of the rabbit ileum. J. yen. Physiol. 53, JORDANA, R. & PONZ, F. (1969). Effects of the pre-incubation in a Na-free medium on the 02 uptake and glucose utilization by the intestine. Revta esp. Fisiol. 25, RIKLIs, E. & QUASTEL, J. H. (1958). Effects of cations on sugar absorption by isolated surviving guinea pig intestine. (Can. J. Biochem. Physiol. 36, ROBINSON, J. W. L. (1967). The loss of intestinal transport capacity following preincubation in sodium free media in vitro. Pfliiger Arch. yes. Physiol. 294, SCHULTZ, S. G. & ZALUSKY, R. (1964a). Ion transport in isolated rabbit ileum. II. The interaction between active sodium and active sugar transport. J. gen. Physiol. 47, SCHULTZ, S. G. & ZAIusKY, R. (1964b). Ion transport in isolated rabbit ileum. I. Short circuit current and Na fluxes. J. yen. Physiol. 47, WEDNER, H. J. & DIAMOND, J. M. (1969). Contribution of unstirred layer effects to apparent electrokinetic phenomena in the gall bladder. J. Membrane Biol. 1, WILSON, T. (1962). Intestinal Absorption. Philadelphia: Saunders. WRIGHT, E. M. (1966 a). The origin of the glucose dependent increase in the potential difference across the tortoise small intestine. J. Physiol. 185, WRIGHT, E. M. (1966b). Diffusion potentials across the small intestine. Nature, Lond. 212,