EFFECT OF ETHANOL ON SODIUM-DEPENDENT GLUCOSE TRANSPORT IN THE SMALL INTESTINE OF THE HAMSTER

Transcription

1 GASTROENTEROLOGY 68: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.6 Printed in U.S.A. EFFECT OF ETHANOL ON SODIUM-DEPENDENT GLUCOSE TRANSPORT IN THE SMALL INTESTINE OF THE HAMSTER P. K. DINDA, PH.D.. I. T. BEcK, M.D.. PH.D.. F.R.C.P. (C), F.A.C.P., M. BECK, L.Sc. lchim), AND T. F. McELLIGO'IT, M.D., B.CH. M.R.C. PATH., D.P.H., F.R.C.P. (C) The Division of Gastroenterology of the Department of Medicine, and the Department of Pathology, Hotel Dieu Hospital, and Departments of Medicine, Physiology, and Pathology, Queen's University, Kingston, Ontario, Canada The objective of this study was to investigate the mechanism by which ethanol inhibits intestinal absorption of sugars. In vitro experiments on hamster jejunum have shown that the presence of ethanol in the mucosal solution caused an inhibition of the net transport of water and glucose. There was also a decrease in the intracellular water content and an increase in the intracellular sodium and potassium concentration of the gut tissue. In contrast, the intracellular glucose concentration decreased in the presence of ethanol. These ethanolinduced changes were directly related to the ethanol concentration of the mucosal solution. In the presence of 450 mm (2%) ethanol in the mucosal solution, there was also a significant inhibition of transmural potential difference, estimated glucose metabolism, and both unidirectional fluxes of sodium. The net flux of sodium to the serosal side however did not decrease significantly. These effects of ethanol cannot be fully explained by its osmotic action, and it is suggested that the ethanol-induced reduction in glucose transport could be mainly the result of an interference with the carrier-mediated coupled entrance of glucose and sodium across the brush border. A depression of cellular metabolism could also have played a role in this process. Alcohol in the intestinal lumen was reported to interfere with the absorption of amino acids and glucose. 1-4 The absorption of these nonelectrolyte substances is believed to depend on the active transport of sodium across the intestine 5-8 and also on the electrolyte concentrations within the transporting cells. s-s There is no information available on the' effect of ethanol on the intestinal transport of sodium and the intracellular concentrations of electrolytes. In an attempt to understand the mechanism by which the intestinal transport of glucose is inhibited by ethanol, we have Received October 10, Accepted January 8, 1975 Address requests for reprints to: I. T. Beck, M.D., Hotel Dieu Hospital, Kingston, Ontario, Canada K7L 3H6. Supported by Grant MA 4257 of the Medical Research Council of Canada investigated the effect of alcohol on the transmural potential difference (PD), water and glucose transport, the unidirectional fluxes of sodium, and the intracellular electrolyte concentrations of the gut. Method Female hamsters, 8 to 10 weeks old were used. They were killed by decapitation. The small intestine of the animals was excised and the lumen flushed with Krebs-Ringer bicarbonate solution (KRBS). 9 A 5-cm long piece of the jejunum, beginning from the ligament of Treitz, was then everted and divided into two equal parts: the "proximal segment" and the "distal segment." Using these adjacent segments, paired experiments were performed in which one segment served as the experimental preparation and the other served as the control. In alternate experiments either the proximal or the distal segment was used as the control. Method of incubation. Each segment was

2 1518 DINDA ETAL. Vol. 68, No.6 mounted on the serosal compartment of an all-glass incubation apparatus described by us previously in detail 9 and shown schematically. in figure 1. The serosal compartment was filled with 12 ml of incubation solution (serosal (S) solution). It was weighed and then immersed into the mucosal compartment which also contained 12 ml of incubation solution (mucosal (M) solution). The assembled apparatus was placed in a water bath (maintained at 37 C) for a period of 45 min. Both sides of the intestine were gassed with a mixture of 95% 0 2 and 5% co. Experiments. Three series of experiments (series 1, 2, and 3) were performed. In series 3 there were four groups (A, B, C, and D). In series 1 the control preparation was incubated in KRBS containing 10 mm glucose (KRBSG) on both sides of the gut wall. The osmolality of the KRBSG was ± 0.9 milliosmoles per kg (mean± SE). The experimental preparation was incubated in the same manner as the control preparation, except that the mucosal surface was bathed with KRBSG containing 150 mm ethanol (0.69 g per 100 ml). The experiments of series 2 and of series 3 (all four groups) were similar to the experiments of series 1, except that the mucosal solution of the experimental A B c EB FIG. 1. Incubation apparatus. A, The serosal compartment is a glass bulb with two side arms (SA) and two funnels (F). The everted jejunum is mounted on the two side arms. The rubber stoppers (RS) have holes which hold polyethylene tubings for gassing (G) and the electrolyte bridges (EB) for the measurement of potential difference. The dotted area represents the 12-ml serosal solution (SS). B, the mucosal compartment is a glass tube. The striated area represents the 12-ml mucosal solution (MS). C, the assembled apparatus. When A is immersed into B. it is held in place by the rubber stoppers. The surface of the mucosal solution rises to the level of the serosal solution. G preparation contained 300 mm ethanol (1.28 g per 100 ml) in series 2, and 450 mm ethanol (2.07 g per 100 ml) in series 3. The addition of ethanol increased the osmolality (in millio'smoles per kg) of the mucosal solution of the experimental preparations to ± 2.6 in series 1, to ± 4.8 in series 2. and to ± 4.0 in series 3. The effect of different concentrations of ethanol on transport of glucose and water across the intestine, on the intracellular concentration of sodium, potassium and glucose was investigated in paired experiments of series 1, 2, and group A of series 3. In all of these studies the mucosal and the serosal solutions of the preparations contained 1 J.LC of [ 3 H]inulin per ml. The number of paired experiments in series 1 and in series 2 were 10 in each, whereas in group A of series 3, 22 experiments were performed. In group B of series 3, 19 paired experiments were performed to investigate the effect of ethanol on the unidirectional fluxes of sodium. The incubation solutions in these experiments were similar to those described above for series 3, except that the solutions contained no [ 3 H]inulin and tracer amounts of 22 Na were added to the incubation solution of one side of the intestine and 24 Na to the solution of the other side. The method was similar to that described previously. 10 The experiments in group C of series 3 (N = 6) were performed to investigate the viability ot the tissue after 45 min of incubation with 450 mm ethanol on the mucosal side. The intestinal segments were incubated as in other experiments in series 3, but after 45 min of incubation the mucosal solutions of both the experimental and the control preparations were replaced with prewarmed (37 C) KRBS (ethanol- and glucose-free). After 10 min, sufficient glucose (0.5 ml of a 250 mm glucose in KRBS) was added to the mucosal solutions to bring their glucose concentrations back to 10 mm, i.e., the glucose concentration of the original KRBSG. PD was measured throughout the experiments. In group D of series 3 paired experiments (N = 4) were performed to study the morphology of the tissue after 45 min of incubation with 450 mm ethanol. Measurement of water and glucose transport and intracellular electrolyte concentration. At the end of the experiment, the serosal compartment was taken out of the apparatus. It was wiped to remove the adhering mucosal solution and then weighed. The difference between the pre- and postincubation weight of this compartment was taken as a measure of net mucosal to serosal fluid transport Since the intestine was attached to the serosal compartment, the

3 June 1975 ETHANOL AND INTESTINAL TRANSPORT 1519 weight gain of this compartment represented transport of water across the epithelial layer, irrespective of whether it remained in the tissue or was transferred into the serosal solution. The mucosal and serosal solutions were separately collected and the intestinal segment was removed from the apparatus. The intestine was opened along its length, and both the mucosal and serosal surfaces were blotted. The intestine was then cut into three pieces of approximately equal sizes. Each piece was weighed separately (each weighing approximately 40 mg). One of these was used for determination of dry weight to wet weight ratio after drying at 105 C to a constant weight. The second piece was homogenized and deproteinized with ZnSO. and Ba(OH) 2 The deproteinized homogenate and suitably diluted pre- and postincubation mucosal and serosal solutions were analyzed for glucose by the glucose oxidase method. 12 The third piece was extracted for 48 hr in 3.0 ml of 15 mm LiS0 4 at 4 C as described by Schultz et a!. 6 Total tissue sodium and potassium were determined in an aliquot of the extract by using an internal standard flame photometer. An aliquot of this extract and of the suitably diluted incubation medium were assayed for [ 3 H]inulin, using a liquid scintillation spectrometer, and the intracellular glucose, sodium, and potassium concentrations were calculated by the method describeri previously. The method of measurement and calculation of the unidirectional fluxes using 22 Na and 24 Na across the hamster intestine was the same as described previously for the rat jejunum. 10 Other measurements and statistical analysis. The PD across the intestinal segments was measured by the method described by us previously The ethanol concentration of the incubation solutions was determined enzymatically. 14 The osmolality of the solutions was measured by the freezing point depression method. The values for the control and the experimental preparations were compared using the method applicable to paired experiments. 15 All data (unless otherwise stated) are expressed as mean ± SE. Tissue processing for morphological study. At the end of the 45-min incubation, the specimens were fixed within 15 sec in 3% glutaraldehyde in cacodylate buffer (ph 7.4) for 90 min. After this the tissue was routinely embedded in paraffin, cut at 5 ~-' and stained by the hematoxylinphloxine-saffran (HPS) technique. Results The effect of ethanol on the intracellular water content of the intestinal tissue and on the intracellular concentration of sodium, potassium, and glucose is shown in table 1. Ethanol caused a progressive reduction in the intracellular water content of the tissue. This reduction in intracellular water content was not significant in series 1 but became statistically significant in series 2 and group A of series 3. In all series, ethanol caused an increase in the intracellular concentration of sodium and potassium, and this increase became significant in series 3 (group A). Ethanol caused a slight and insignificant increase in the intracellular glucose concentration in the experiments of series 1. In series 2 and 3 (group A) ethanol significantly decreased the intracellular glucose concentration of the tissue. The presence of 150 mm ethanol in the mucosal solution (series 1) decreased the uptake of glucose from the mucosal side (20%) and the transfer of this sugar to the serosal side ( 18%). These changes were not statistically significant. The corresponding depressions for series 2 (300 mm ethanol) were 27% for the uptake of glucose from the mucosal solution (P < 0.01) and 24% for the transfer to the serosal solution (not significant). In the experiments with 450 mm ethanol (series 3, group A) both the uptake and the transfer of glucose decreased by 41% (P < 0.001). The actual data for this series (450 mm ethanol) are shown in figure 2. In this series ethanol also caused a depression of the estimated glucose metabolism [mucosal loss -c- (tissue glucose + serosal gain)]. This decrease was 34.3% [control 4.08 ± 0.57 Jlmoles per em, experimental 2.68 ± 0.53 Jlmoles per em (P < 0.005) ]. Water transport was depressed by ethanol with all three concentrations used, but this became statistically significant only in series 3 (group A). In this series, the depression of the net transfer of water was 57% (P < 0.001; fig. 3). In series 3 (group B) sodium fluxes were studied. The depression in M... S flux was 26%, in S... M flux 29%, and in the net M... S transport 21%. The actual data and their statistical significance are shown in figure 3. The concentrations of ethanol in the mucosal and serosal solutions after 45 min

4 1520 DINDA ETAL. Vol. 68, No.6 TABLE 1. Effect of ethanol on intracellular water content and solute concentration of the gut z..all" Intracellular Intracellular concentrations Series water content Sodium Potassium Glucose p.l/cm 11-moles/ml 1 Control 43.7 ± ± ± ± 5.57 Experimental 39.8 ± ± ± ± 6.29 Difference 3.9 ± ± ± ± 3.85 %Change Control 53.4 ± ± ± ± A Experimental 47.5 ± ± ± ± 2.58 Difference 5.9 ± ± ± ± 2.27 %Change Control 53.5 ± ± ± ± 2.95 Experimental 39.4 ± ± ± ± 2.59 Difference 14.1 ± 2.0" 7.24 ± 2.43C 8.58 ± ± 2.12 %Change "At the end of the 45-min incubation period the ethanol concentration of the mucosal (M) and the serosal (S) solutions of the experimental preparations were (mm): series 1: M = ± 1.43, S = 6.99 ± 0.64; series 2: M = ± 3.40, S = ± 1.14; series 3A: M = ± 5.25, S = ± The number of paired experiments was 10, 10, and 22 in series 1, series 2, and series 3 (group A), respectively. The data are means ± SE, and any significant difference from the control is indicated by the superscript. p < c p < d p < mol./cm r+ H- D CONTROL 0 ETHANOL 450mM r+r+- MUG. LOSS SER. GAIN GLUCOSE GLUCOSE P< P< FIG. 2. The effect of the presence of 450 mm ethanol in the mucosal solution on the loss of glucose from the mucosal side (MUC. LOSS) and on the transfer of this sugar into the serosal solution (SER. GAIN) (N = 22). of incubation are shown in footnote a to table 1. Figure 4 shows the changes in PD of the control and the experimental preparations (of series 3, group C) during the 45-min incubation, 10-min reincubation in glucose-free solution, and finally the response of the gut to the addition of glucose to the mucosal solution. In the control preparation, after initial equilibration, the PD was maintained at a steady level throughout the 45 min of incubation. Reincubation in glucose-free KRBS resulted in a fall of PD from 8.4 ± 0.4 mv to 5.8 ± 0.5 mv. The addition of glucose to the mucosal solution caused the PD to rise by 2.8 ± 0.4 mv. In the preparation incubated in a mucosal solution containing 450 mm ethanol, the PD dropped progressively to 4.8 ± 0.5 mv. Replacement of the mucosal solution with glucose- and ethanol-free KRBS had little influence on the rate of the decline of the PD, which fell to 3.8 ± 0.4 mv by 55 min. In response to the addition of glucose, the PD rose by 2.4 ± 0.2 mv. There was no statistically significant difference in the glucose-stimulated rise in PD between the

5 June 1975 ETHANOL AND INTESTINAL TRANSPORT 1521 WATER TRANSPORT,!JIIcm IJmal./cm 2 SODIUM FLUXES 0CONTROL 0 ETHANOL 450mM M--+S s-m P<O OOI P<O OI P<O OOI FIG. 3. The effect of the presence of 450 mm ethanol in the mucosal solution on water transport in microliters per em (N = 22), and on the mucosal to serosal (M-+ S), serosal to mucosal (S-+ M), and net (mucosal to serosal) fluxes of sodium in micromoles per em. N = 19. NET N.S. PD(mucoso negative) mv -10! KRBS GLUC <>-----<> CONTROL --- ETHANOL -2 ol---~----~----~ ~----~----~ ~ w ~ ~ ~ oo ro TIME IN MINUTES FIG. 4. The effect of the presence of 450 mm ethanol in the mucosal solution on transmural potential difference. Mean ± se of six experiments. Abscissa, minutes after incubation; ordinate, mucosal potential (m V) with reference to serosa; KRBS, replacement of original mucosal solution with glucose-free Krebs-Ringer bicarbonate solution, GLUC, addition of 0.5 ml of 250 mm glucose solution to the 12 ml of glucose-free mucosal solution. control and the experimental preparation. In the control preparation, the essential morphological structure of the preparation (series 3, group D) remained well preserved. The epithelial cells were intact with well defined brush borders. There was no exfoliation (fig. 5a). As compared to the control preparations there was no significant change in two of the four intestines incubated with ethanol. In the other two preparations there was a bullous separation of the epithelium with an underlying ballooning at the tip of approximately 20% of the villi. Figure 5b shows an area of bullous separation. The continuity of this elevated epithelial layer (and therefore the epithelial covering of the villus) was uninterrupted. Together with the underlying lamina propria the elevated epithelial layer appeared to circumscribe discrete spaces.

6 1522 DINDA ETAL. Vol. 68, No.6 FIG. 5. Photomicrographs of intestinal segments incubated for 45 min in a mucosal solution consisting of Krebs-Ringer bicarbonate solution, 10 mm glucose, and without (a) and with (b and c) 450 mm ethanol. a, control preparation. Note that the only change is a slight swelling of the mid and lower villous epithelial cells ( x 100). b, one of the most severely affected areas of the preparation incubated with ethanol. Note the bullous separation of the epithelium ( x 100). c, higher magnification of b to show that the epithelial layer is intact and that the brush border of the cells is well preserved. A few red blood cells are seen inside the bulli ( x 250). These spaces appeared empty after fixation but undoubtedly contained fluid at the time of the experiment. The epithelial cells in the elevated layer appeared healthy and had normal brush borders (fig. 5c). Discussion The present study was undertaken in an attempt to elucidate the mechanisms by which ethanol depresses small intestinal glucose transport. The hamster was used because the sugar transport characteristic of the hamster jejunum resembles that of the human gutt 6 (I. T. Beck et al., unpublished observation). Since it is difficult to investigate transport mechanisms in vivo, the experiments were performed in vitro. The physiological validity of the method of incubation used was previously demonstrated by us, 9-11 and confirmed in the present study by the steady PD of the control preparation (fig. 4). In a previous study we investigated the morphological integrity of the intestine incubated by the method used in the present experiments 17 and found that the preparation remains intact for 45 min, but that there was swelling of the midvillous epithelial cells. The extent of this swelling was related to water transport. In the present study the control intestinal segment again remained intact (Fig. 5a). The midvillous epithelial cell swelling, however, was less pronounced than previously reported, probably due to seasonal variation of water transport in the hamster. 18 The concentrations of ethanol used (0.69 to 2.07%, w/v) were selected because, after moderate drinking in man, the ethanol concentration in the upper jejunal content is 1 to 5% Since the experiments were performed in vitro, one may wonder whether the serosal concentrations of ethanol reached at the end of the experiment remained within physiological ranges. In the experiments with 450 mm ethanol on the mucosal side (the highest concentration used), the mean ethanol concentration of the serosal solution (18.88 mm) was lower than that found in portal blood of dogs (29.4 mm) after intragastric administration of alcohol in doses mimicking moderate drinking in man. 20 There was considerable variation in the control data from series to series (table 1). Since our experiments were carried out over a period of several months, this was probably due to the seasonal variation of the intestinal transport in the hamster. 18 To distinguish the effect of ethanol from this seasonal variation, paired experiments were performed using two adjacent segments from the same animal. The adequacy of the

7 June 1975 ETHANOL AND INTESTINAL TRANSPORT I523 method used for the determination of intracellular solute concentrations was discussed by us previously in detail. 9 The question may arise whether the changes brought about by ethanol occurred mainly in the epithelial cell layer or in other cells of the gut wall. It is to be noted that in our experiments the epithelial cell layer was in contact with a high concentration of ethanol (I50 to 450 mm), whereas the ethanol concentration of the solution bathing the muscle layer (serosal) was low (6.99 to I8.88 mm). Therefore, it is likely that most of the changes in the intracellular solute concentrations of the total tissue, brought about by ethanol, were due to changes in the epithelial cells. One may question the viability of the intestinal segment incubated with ethanol in vitro. The following points would suggest that ethanol, even at the highest concentrations used ( 450 mm), did not alter significantly the viability of our in vitro preparation. (I) Because the intracellular potassium concentration did not fall (table I), generalized cellular damage could not have occurred. (2) The intracellular glucose concentration of the ethanoltreated intestinal segments was always higher than the glucose concentration of the mucosal solution (table I). This would suggest that the tissue was capable of accumulating glucose against a concentration gradient. (3) Even at the end of a 45-min incubation the PD was 4.8 ± 0.5 mv, indicating that the tissue was still physiologically active. ( 4) Furthermore, the addition of glucose (after washout with glucose-free KRBS) produced the expected glucose induced rise in PD and this rise was of the same magnitude as in the control preparation (fig. 4). (5) The morphological changes observed in our experiments, especially the ballooning at the tip of the villi, seem to reflect subepithelial accumulation of fluid, possibly related to abnormal physiological function induced by ethanol and not to epithelial damage. Baraona et al. 23 found more extensive damage of the epithelial cells and of the villus structure after intragastric administration of alcohol to the rat in much higher concentrations (5 to 49%) than the concentration (2%) used in our experiments. The necrosis of the epithelium of the villus tip observed by Baraona et al. 23 was not seen in our preparation, although the bullous separation of the epithelial layer seen in our experiments may have represented the initial stage of the denudation brought about by higher concentrations of alcohol used by these authors. To explain the mechanism of action of ethanol on intestinal transport, one has to consider whether the effect of ethanol is the result of the high osmolality of the mucosal solution. In order to distinguish clearly the nonspecific osmotic effect from the specific action of ethanol, one would have to devise control experiments where the osmolality of the mucosal solution is increased with a compound having a Staverman reflection coefficient (u)2 4 and an oil-water partition coefficient similar to those of ethanol, but which has no specific pharmacological effect. Such a substance, to our best knowledge, is not available. We, however, have shown previously that, in response to a purely osmotic action [using mannitol (high u) in the mucosal solution] there was a reduction of water transport and a loss of cellular water, and as a result of the latter both the intracellular electrolyte and the glucose concentration increased. 9 With solutes of low u, these effects are very small, but the osmotic effect increases with the increase in the concentration of the solute in the mucosal solution. 25 Ethanol is an easily diffusable substance (low u); therefore, it is not likely to exert an appreciable osmotic pressure over the brush border. 24 It is, however, conceivable that the high osmolality (up to 760 milliosmoles per kg) produced by the levels of ethanol used by us may have had some osmotic effect. In our present experiments with ethanol, as with mannitol, there also was a decrease in water transport and intracellular water content and an increase in the intracellular concentration of sodium and potassium. This suggests that some of the effect of alcohol may have been the result of the small but noticeable osmotic pressure exerted by ethanol on the brush border. Since, however, instead of an increase (as

8 1524 DINDA ETAL. Vol. 68, No.6 was the case with mannitol) the intracellular glucose concentration decreased, the action of ethanol cannot be solely attributed to the osmotic pressure exerted by alcohol on the brush border. There is no report in the literature on the effect of ethanol on sodium fluxes across the intestine. We found a significant decrease in the M --+ S flux of this ion (fig. 3). Ethanol is known to decrease the active transport of cations in many different types of cells 26 ' 28 due to an inhibition of the Na+ -, K + -, and Mg2+ -stimulated adenosine triphosphatase27 28 which is associated with the active transport of cations. 29 Therefore, it is possible that the diminished M --+ S flux of sodium may at least partly have been the result of an ethanol-induced inhibition of the adenosine triphosphatase (sodium pump) located at the basal-lateral membrane of the epithelial cell. 30 Since the inhibition of glucose transport (40%) exceeded the inhibition of M --+ S unidirectional sodium flux (25%) or the M-+ S net flux of sodium (21% statistically not significant), and since the PD was depressed, it is likely that the diminution in glucoselinked electrogenic sodium transport was accompanied by a simultaneous increase in the chloride-linked neutral M --+ S sodium movement described by Newey and Smyth 31 and Binder and Rawlins. 32 Our results indicate that ethanol also decreased the S --+ M flux of sodium (fig. 3). This could be explained by diminished S --+ M sodium diffusion across the extracellular shunt pathway due to a decreased chemical and electrical gradient. It is believed that, due to the accumulation of actively transported sodium across the lateral membrane of the epithelial cell (M --+ S flux), the lateral intercellular space (LIS) becomes hypertonic, and the rate of the S --+ M diffusion of sodium, occurring through the extracellular shunt pathway (from LIS to mucosal solution) depends, at least partly, on the concentration gradient of sodium from LIS to the mucosal solution. 35 The ethanol-induced inhibition of the M --+ S flux of sodium probably decreased the concentration of sodium in the LIS and consequently diminished the concentration gradient of this ion from the LIS to the mucosal solution, causing a diminished S --+ M flux. In addition, a diminution in electrical gradient manifested by the depressed PD may also have contributed to the decreased S --+ M flux of sodium. A significant decrease in the net flux of sodium from the mucosal to the serosal side can occur only if the reduction in the M --+ S flux is substantially greater than the reduction of the S--+ M flux or if the S--+ M flux is greatly increased in relation to the M --+ S flux. In our experiments ethanol caused an almost similar decrease in both unidirectional fluxes of sodium (M--+ S = 26%; S--+ M = 29%) and consequently the decrease in the net M --+ S flux of this ion was small and failed to become statistically significant (fig. 3). The M --+ S net transport of water depends on the active transport of solutes In our experiments the present diminution in net water transport (57%) exceeded the decrease in them--+ S transport of glucose (40%) and sodium (21%). This proportionally higher decrease in water transport was possibly the result of a combined effect of diminished active transport of solutes and an enhanced S --+ M flux of water in response to the ethanol-induced osmotic effect discussed above. As to the mechanism of the ethanolinduced inhibition of glucose transport, it is generally believed that the mobile carrier located in the brush border translocates sugar and sodium together into the epithelial cell, and that the rate of entry of sugars across the brush border depends on the rate of active extrusion of sodium across the basal-lateral membrane of the cell. 5 Since in our experiments, in addition to glucose transport, ethanol also inhibited the M --+ S flux of sodium, the ethanol-induced inhibition of glucose absorption could have been the result of a direct interference with the carrier-mediated coupled entrance of glucose and sodium through the brush border or an indirect effect mediated through the inhibition of the active extrusion of sodium across the basal-lateral membrane. Phlorizin is a competitive inhibitor of the entrance of glucose across the brush border. 38 Recent studies in our laboratories with

9 June 1975 ETHANOL AND INTESTINAL TRANSPORT 1525 phlorizin (5 X 10-s M) have shown that this glycoside depresses glucose transport ( 49%) to a greater extent than the net transport of sodium (17%). On the basis of these data we have suggested that although phlorizin primarily inhibited the sodium linked entrance of glucose across the brush border, it also stimulated the nonelectrogenic chloride-linked sodium transport. 39 Our present results with ethanol are reminiscent of these findings with phlorizin. Here again the inhibition of glucose transport exceeded the inhibition of sodium transport. From the similarity of the results obtained by phlorizin and ethanol, it is tempting to suggest that ethanol interferes with the binding of sugar to the carrier and, if so, the primary action of ethanol on sugar transport occurs at the brush border. In addition, the inhibition of glucose metabolism may also have played a role (see Results) in the reduction of sodium and glucose transport. The present experiments do not differentiate clearly among the relative importance of the possible mechanisms discussed above and further studies are necessary to elucidate the precise mode of action of ethanol on intestinal sodiumlinked glucose transport. REFERENCES 1. Israel Y, Salazar I, Rosenmann E: Inhibitory effects of alcohol on intestinal amino acid transport in vivo and in vitro. J Nurt 96: , Israel Y, Valenzuela JE, Salazar I, et al: Alcohol and amino acid transport in the human small intestine. J Nutr 98: , Ghirardi P, Marzo A, Sardini D, et al: Changes in intestinal absorption of glucose in rats treated with ethanol. Experientia 27:61-62, Chang T, Lewis J, Glazko AJ: Effect of ethanol and other alcohols on the transport of amino acids and glucose by everted sacs of rat small intestine. Biochim Biophys Acta 135: , Crane RK: Absorption of sugars. In Handbook of Physiology, sect 6: Alimentary Canal, vol 3. Edited by CF Code, W Heidel. Washington DC, American Physiological Society, 1968, p Schultz SG, Fuisz RE, Curran PF: Amino acid and sugar transport in rabbit ileum. J Gen Physiol 49: , Goldner AM, Schultz SG, Curran PF: Sodium and sugar fluxes across the mucosal border of rabbit ileum. J Gen Physiol 53: , Schultz SG, Curran PF: The role of sodium in non-electrolyte transport across animal cell membranes. Physiologist 12: , Dinda PK, Beck M, Beck IT: Effect of changes in the osmolality of the luminal fluid on intracellular concentration of solutes in the hamster jejunum. Can J Physiol Pharmacal 50:72-82, Beck IT, Dinda PK: Sodium and water transport across the jejunum of fasted rats. Can J Physiol Pharmacal 51: , Dinda PK, Beck M, Beck IT: Effect of changes in the osmolality of the luminal fluid on water and glucose transport across the hamster jejunum. Can J Physiol Pharmacal 50:83-86, Cawley LP, Spear FE, Kendall R: Ultramicro chemical analysis of blood glucose with glucose oxidase. Am J Clin Pathol 32: , Dinda PK, Beck IT: The effect of local anesthetics on the transmural potential difference of the small intestine of the rat. Can J Physiol Pharmacal 47: , Bucher TH, Redetzki H: Eine spezifische photometrische Bestimmung von ethylalkohol auf fermentativem Wege. Klin Wschr 29: , Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa, Iowa State University Press, Stirling CE, Schneider AJ, Wong M, et a!: Quantitative radioautography of sugar transport in intestinal biopsies from normal humans and a patient with glucose-galactose malabsorption. J Clin Invest 51: , McElligott TF, Beck IT, Dinda PK, et a!: Correlation of structural changes at different levels of the jejunal villus with positive and negative net water transport in vivo and in vitro. Can J Physiol (in press), Teem MV, Phillips SF: Perfusion of the hamster jejunum with conjugated and unconjugated bile acids: inhibition of water absorption and effects on morphology. Gastroenterology 62: , Halsted CH, Robles EA, Mezey E: Distribution of ethanol in the human gastrointestinal tract. Am J Clin Nutr 26: , Beck IT, Paloschi GB, Dinda PK, et al: The effect of intragastric administration of alcohol on the ethanol concentrations and osmolality of pancreatic juice, bile, portal and peripheral blood. Gastroenterology 67: , Lyon I, Crane RK: Studies on transmural potentials in vitro in relation to intestinal absorption. I. Apparent Michaelis constants for Na+ dependent sugar transport. Biochim Biophys Acta 112: , Schultz SG, Zalusky R: Ion transport in isolated rabbit ileum. II. 'The interaction between active

10 1526 DINDA ETAL. Vol. 68, No.6 sodium and active sugar transport. J Gen Physiol 47: , Baraona E, Pirola RC, Lieber CS: Small intestinal damage and changes in cell population produced by ethanol ingestion in the rat. Gastroenterology 66: , Wright EM, Diamond JM: Patterns of non-electrolyte permeability. Proc Roy Soc Lond Bioi Sci 172: , Beck IT, Dinda PK: On the mechanism of isoosmotic transport of fluid across the small intestine. The effect of the Staverman reflection coefficient of the solute used to increase the osmolality of the mucosal solution on the composition of the absorbate. Can J Physiol Pharmacol52:96-104, Israel-Jacard Y, Kalant H: Effect of ethanol on electrolyte transport and electrogenesis in animal tissues. J Cell Comp Physiol 65: , Israel Y, Kalant H, Laufer 1: Effects of ethanol on Na, K, Mg-stimulated microsomal ATPase activity. Biochem Pharmacol 14: , Israel Y, Kalant H, LeBlanc AE: Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex. Biochem J 100:27-33, Skou JC: Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol Rev 45: , Stirling CE: Radioautographic localization of sodium pump sites in rabbit intestine. J Cell Bioi 53: , Newey H, Smyth DH: Basic concepts in intestinal absorption. In The Biological Basis of Medicine, vol. 5, chap 10. Edited bye Bitter, N Bitter. New York, Academic Press, 1969, p Binder HJ, Rawlins CL: Electrolyte transport across isolated large intestinal mucosa. Am J Physiol 225: , Diamond JM, Bossert WH: Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J Gen Physiol 50: , Machen TE, Diamond JM: An estimate of the salt concentration in the lateral intercellular spaces of rabbit gall-bladder during maximal fluid transport. J Membr Bioi 1: , Frizzell RA, Schultz SG: Ionic conductances of extracellular shunt pathway in rabbit ileum: Influence of shunt on transmural Na transport and electrical potential differences. J Gen Physiol 59: , Curran PF: Ion transport in intestine and its coupling to other transport processes. Fed Proc 24: , Dinda PK, Beck M, Beck IT: On the mechanism of isoosmotic transport across the small intestine. The composition of the absorbate transported from a mucosal solution made hypertonic by the addition of mannitol. Can J Physiol Pharmacol 51: , Alvarado F, Crane RK: Studies on the mechanism of intestinal absorption of sugars: VII. Phenylglycoside transport and its possible relationship to phlorizin inhibition of the active transport of sugars by the small intestine. Biochim Biophys Acta 93: , Dinda PK, Beck M, Beck IT: Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin induced inhibition of sugars transport. Can J Physiol Pharmacol (in press), 1975