Effects of Ethanol on Sodium, 3-O-Methyl Glucose, and L-Alanine Transport in the Jejunum YUH-JYH KUO, MS, and L.L. SHANBOUR, PhD

Effects of ethanol on Na +, Cl-, 3-O-methyl glucose (3-O-MG), and L-alanine fluxes were studied in the isolated rabbit jejunal mucosa. Ethanol (3% v/v present on both sides o f the mucosa) decreased electrical potential difference (PD), short-circuit current (Isc) and inhibited active transport of Na +, 3-O-MG, and L-alanine. This concentration also increased the permeability of the mucosa for Cl-, 3-O-MG, and L-alanine. Ethanol at 5.4% potentiated the effects on PD, I~, and the permeability for electrolytes and organic substances. These effects of ethanol could not be fully explained by an osmotic action.

Ethanol has been reported to inhibit the absorption of amino acids and glucose (1-4) when administered into the intestinal lumen of the rat. The absorption of these organic substances is dependent to a considerable extent on the active transport of sodium across tJhe intestine (5, 6). The mechanism of inhibition of glucose and amino acid transport which are linked with Na + transport has not been completely documented. Although Dinda et al (7) found that 2.6% (v/v) ethanol inhibited glucose transport and mucosal (M) to serosal (S) Na + flux but did not affect net Na + flux in the hamster jejunum, it was still uncertain whether ethanol inhibited the active absorption of Na +, since neither M-to-S Na + flux nor net Na + flux could represent the active transport of Na + due to the presence of electrochemical gradients across the mucosa in their studies. Net absorption of Na + is evident when o-glucose or 3-O-MG is present in the bathing bicarbonate From the Department of Physiology, The University of Texas Medical School at Houston, Houston, Texas 77030. This research was supported by funds from NIAAA grant 2R01 AA-00194-05. Dr. L.L. Shanbour is recipient of Research Scientist Development Award 5K02-AA-70463-04. Address for reprint requests: Dr. L.L. Shanbour, Department of Physiology, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77025.

Ringer solution (8) in the isolated rabbit jejunal mucosa. This in vitro technique was used in the present studies to determine the effects of ethanol on the fluxes of Na +, CI-, and two actively transported but not metabolized organic solutes, 3-O-MG and L-alanine (5). The advantages of these studies are: (1) The net flux represents the active transport of the electrolyte or organic substance since the electrochemical gradients across the mucosa are maintained at zero. (2) The changes in unidirectional fluxes represent changes in permeability. (3) The changes in the electrophysiological parameters, including electrical potential difference (PD) and short-circuit current (Isc) can also be simultaneously detected. It is well established that the effects of ethanol on intestinal function are dose dependent (1, 3, 7, 9, 10). Israel et al (4) have found that the ethanol concentration is about 1-3% in the human upper jejunum during moderate drinking. However, the concentration of ethanol in the jejunal lumen could increase to over 6% with prolonged alcohol consumption (11). Several concentrations of ethanol, 1.8, 3.0, and 5.4% were selected in this study because they would approximate the concentration present in the jejunal lumen when drinking table wine, one martini, and common spirits, respectively.

Digestive Diseases, Vol. 23, No. 1 (January 1978)

0002-9211/78/0100-0051505.00/1 9 1978DigestiveDiseaseSystems,Inc.

51

KUO AND SHANBOUR MATERIALS

AND METHODS

New Zealand white rabbits (Oryctolagus cunicutus), weighing 1.5-3.0 kg, were anesthetized with intravenous administration of nembutal (approximately 25-50 mg/kg). The abdomen was opened and the jejunum just distal to the ligament of Treitz was excised. After dissecting off the serosa and muscularis (12), paired adjacent segments of the mucosae were mounted in Ussing type flux chambers with an aperture of 1 cm2 between the two compartments. The mucosal and serosal surfaces of the tissues were bathed with 10 ml of identical ionic solutions of bicarbonate Ringer maintained at 37~ C with a thermoregulator. The solutions were gassed with 95% 02-5% CO2 and had the following ionic composition, in millimoles per liter: Na +, 141; K +, 10; Mg2+, 1.1; Ca2+, 1.25; CI-, 127; HCO3-, 25; H2PO4-, 0.3; HPO4 2-, 1.6. Control tissues were incubated in bicarbonate Ringer with 10 mM of glucose (J.T. Baker Chemical Co.) or 3-O-MG or u-alanine (Sigma Chemical Co.), and experimental tissues were similarly incubated with the exception that the bicarbonate Ringer solution contained 1.8, 3.0, or 5.4% (v/v) ethanol. All tissues were short-circuited throughout the course of the experiment, but the current was interrupted periodically to determine the spontaneous transmembrane PD. Potential difference and Isc across the tissue were determined as previously described (13). After PD and Isc stabilized, a 50-X aliquot of radioisotopic 2~Na, a~CI, [14C]-3-O-MG, or [~4C]L-alanine (New England Nuclear, approximately 3 ~Ci) was added to the opposite sides of paired mucosae. A 1-ml sample was removed from the unlabeled half chambers and replaced with the same fresh and prewarmed solutions at 20, 35, and 50 min after the addition of the isotopes. The I~c and PD of paired tissues were not significantly different. Unidirectional fluxes of Na +, CI-, 3-O-MG, or u-alanine were measured as the mean value of two consecutive 15-rain periods (14) in one direction from one chamber and in the opposite direction from the other chamber. The 2ZNa, 36C1, [~4C]3-O-MG, and ["C]L-alanine were counted in a liquid scintillation detector (Nuclear Chicago). The concentration of chloride was determined by a chloridometer (Buchler) and that of sodium by a flame photometer (Brinkman). The effect of hyperosmolarity on the electrophysiology of the isolated rabbit jejunal mucosa was determined by using 940 mM mannitol in the bicarbonate Ringer solution. The first 30 min served as control, then the bicarbonate Ringer solution of both mucosal and serosal sides was replaced with fresh bicarbonate Ringer (control group), 5.4% ethanol (930 mOsm/liter, Precision Osmette osmometer), or 940 mM mannitol, respectively. Potential difference and I~ were recorded every 5 or 15 min. The values reported are means -+ standard errors. Differences were considered significant if the P value calculated from the Student's t test was less than 0.05. RESULTS

Figure 1 illustrates the effects of various concentrations of ethanol on PD, Ise, and N a + fluxes in the jejunum. The PD across the jejunum was 5.9 -- 0.3

52

(8)

6

1

s ~ ( 8 )

m

i~

.

~(4)

o

-2

>

g Q 0.

3

2-

)

-1 I

1 I

30. o" DA

2o

I

I

I

M--~S S--~M

x

_= u.

+

10

ETHANOL (%, v/v) Fig 1. Effects of various concentrations of ethanol on PD, Ise, and Na + fluxes in the rabbit jejunum. Each point is the mean of several animals (shown in parenthesis). The vertical lines represent

standard errors of means. Asterisks (*) show statistical (P < 0.05) significant difference compared to the control (no ethanol) with unpaired t test.

mV, the mucosal side negative. This value was slightly higher than reported by F r o m m (8), which was probably due to the different procedure employed; ie, removing the jejunum when the rabbit was anesthetized rather than after the animal was killed. Ethanol, at a concentration of 1.8%, did not significantly alter either PD or Isc. H o w e v e r , both PD and I~c were significantly decreased when the concentration of ethanol was increased to 3.0 or 5.4%. The unidirectional flux of N a + from m u c o s a (M) to serosa (S) was significantly greater than the unidirectional S-to-M flux of N a +, resulting in a net flux which represented active transport of N a + (A N a +) from M to S during short-circuited conditions. There was no difference between the net flux of N a + and the Isc. Sodium fluxes were not significantly altered in the presence of 1.8% ethanol; but there was a marked decrease in net flux of N a + with 3% ethanol. Ethanol at 5.4%, in addition to inhibiting a c t i v e N a + t r a n s p o r t , also i n c r e a s e d uniDigestive Diseases, Vol. 23, No. 1 (January 1978)

ETHANOL AND INTESTINAL TRANSPORT directional M-to-S and S-to-M fluxes, indicating increased mucosal permeability to Na § Net flux of C1- across the jejunum was not apparent although stripped rather than unstripped mucosae were used (Figure 2). With 1.8% ethanol in the bicarbonate Ringer, unidirectional C1- fluxes were not significantly different from the control values. In contrast, 3.0% ethanol produced an increase in the unidirectional flux of C1- in both directions. Since the conditions were essentially the same as the Na § flux studies, PD and Ise (not shown) exhibited the, same changes as illustrated in Figure 1. Figure 3 shows the effects of various concentrations of ethanol on the rates of 3-O-methyl glucose (3-O-MG) unidirectional and net fluxes and the correlated electrophysiological changes. This form of glucose is a specific monosaccharide which is not metabolized but is transported actively through the small intestine (5). In the control preparation, PD was lower than the values in Na + and C1- flux studies in which o-glucose rather than 3-O-MG was added as art actively transported organic substrate. Net flux of 3-O-MG (A 3-O-MG) represents active absorption across the jejunum. Significant decreases in PD and I~c were observed with 1.8, 3.0, and 5.4% ethanol in the bathing solutions. There was significant reduction in net flux of 3-O-MG in 3.0 and 5.4% ethanol due to the increase in the unidirectional flux of 3-O-MG from S to M and slight decrease in the unidirectional flux of 3-O-MG from M to S. The increase in the unidirectional S-to-M flux of 3-O-MG also indicated that the permeability of the jejunum to 3-O-MG increased.

"C" E

~

o

LU

&

S. . . ~ - x -

30

M--~S

20

x

"

lo

L-

i

i

I

1

2

5

ETHANOL (%, v/v)

Fig 2. Effects o f 1.8% and 3.0% ethanol on C1- fluxes in the isolated rabbit jejunal m u c o s a . M e a n s _+ SEM for the n u m b e r of animals s h o w n in p a r e n t h e s e s . A s t e r i s k s (*) s h o w statistical (p < 0.05) significant difference c o m p a r e d to the control with unpaired t test. Digestive Diseases, Vol. 23, No. 1 (January 1978)

-3

C6) 4:

~-

-2

3-

(5)

o..r" 2 -

3

*

~

7

)

-1

1"~"

0j ~

,

,

m t~

i

i

I

0

i

i . LI.

i

? m

0

~ 1

~ i [ 2 3 4 ETHANOL (%, v/v)

~ i 5

I 6

Fig

3. Effects of various concentrations of ethanol on PD, I~c, and 3-O-methyl glucose fluxes in the jejunal m u c o s a . M e a n s _+ SEM for the n u m b e r of animals s h o w n in p a r e n t h e s e s . Asterisks (*) s h o w statistical (P < 0.05) significant difference compared to the control with unpaired t test.

Similar results were obtained in L-alanine flux studies in which L-alanine was used as an organic substrate in the bathing bicarbonate Ringer solution (Figure 4). Potential difference, Isc, and net L-alanine flux were partially depressed in 3% ethanol and completely suppressed in 5.4% ethanol. The permeability of the jejunal mucosa for L-alanine was increased as demonstrated by the threefold increase in the unidirectional S-to-M flux of L-alanine in 3% ethanol and sixfold increase in 5.4% ethanol. The electrophysiological responses of the jejunal mucosa to 5.4% ethanol and 940 mM mannitol are illustrated in Figure 5. Potential difference and Isc decreased to zero within 1 min and remained at zero when the bicarbonate Ringer solution was replaced with hyperosmotic mannitol solution in both mucosal and serosal sides. However, PD and Ise decreased progressively in 5.4% ethanol. In the above studies, ethanol was present in both mucosal and serosal solutions to prevent an osmotic gradient. In order to determine whether ethanol exhibited any polarity in its action on intestinal transport, studies were conducted in which ethanol was only present in either mucosal or serosal solutions. When present in only the mucosal solution, the effects were essentially the same as when ethanol was present on both sides. However, when only the se-

53

KUO AND SHANBOUR

gll ~g

0t

8

~0

S--~-M

2

0

~

~

~

~,

ETHANOL (%, v/v) Fig 4. Effects of various concentrations of ethanol on PD, Isc, and L-alanine fluxes in the jejunal m u c o s a . M e a n s -+ sE~ for the n u m b e r of animals s h o w n in p a r e n t h e s e s . Asterisks (*) s h o w statistical (P < 0.05) significant difference compared to the control with unpaired t test.

ster jejunum could reflect a species difference and, in addition, the net Na + flux in their studies could not precisely represent the active absorption of Na + because electrochemical gradients existed across the mucosa. Inhibition of Na +, 3-O-MG, and L-alanine absorption will produce intestinal fluid loss which may be a major factor contributing to the diarrhea which frequently occurs with consumption of alcohol (15). The ethanol-treated jejunum also showed greater fluxes of Na +, CI-, 3-O-MG, and L-alanine in the Sto-M direction than the controls, indicating an increase in permeability of the tissue. These effects were concentration dependent and could be the reason for, or the result of, cellular damage. Chang et al (1) reported an increase in phenylalanine permeability associated with tissue damage in the rat small intestine treated with 3% ethanol. They also suggested that both the duration of exposure and concentration of ethanol may influence the extent of damage to the cellular membrane. Baraona et al (9) also found that the severity of the rat jejunal erosions varied with the concentrations of ethanol used. Whether the increase in permeability is asso-

rosal solution contained ethanol, the measured parameters were not significantly altered from control values. This is further evidence for a primary action of ethanol on active transport rather than an increase in permeability or membrane toxicity. In addition, at the end of the experiments when ethanol was washed out of the chambers and replaced with fresh solutions, the measured parameters did not further decrease, although they did not return to control values.

~I 1

0

? V

H H ~

Control

5.4% Ethanol 940 mM Mannitol

t

9

9

60

70

DISCUSSION The administration of 3.0 or 5.4% ethanol to the isolated rabbit jejunal mucosa produced a sustained inhibition in absorption of 3-O-MG and L-alanine. There was also a significant decrease in net Na + flux corresponding to decreases in PD and Isc. Since there is no active absorption of Na § when the jejunal mucosa is bathed in bicarbonate Ringer solution without the addition of actively transported organic solutes, the active transport of Na + is glucose- or amino acid-dependent (8). The diminution of Na § transport by ethanol is thus linked to the inhibition of absorption of glucose or amino acids across the jejunum. The observations of Dinda et al (7) that 2.6% ethanol did not alter net Na + flux in the ham-

54

o kU

2

&

15

30

45

Time (rain.) Fig 5. C o m p a r i s o n b e t w e e n the effects of bicarbonate Ringer, 5.4% ethanol, and 940 m M mannitol of PD and Iso in the isolated rabbit jejunal m u c o s a . Ethanol and mannitol are added at time 30 m i n . E a c h p o i n t is t h e m e a n o f v a l u e s for 2 - 3 a n i m a l s . 9 9 bicarbonate Ringer control, N = 2; 9 II, bicarbonate Ringer and 5.4% ethanol, N = 2; 9 9 bicarbonate Ringer and 940 m M mannitol, N = 3. Digestive Diseases, Vol. 23, No. 1 (January 1978)

ETHANOL AND INTESTINAL TRANSPORT ciated with inhibition of active transport is not fully explained, but from the present experiments, as well as previous studies (16-18), it seems likely that the agents which produce damage to the gastrointestinal m u c o s a (ethanol, aspirin, and bile salts) initially inhibit ion transport which is followed b y an increase in mucosal permeability with treatment for longer duration or with higher concentrations. Other reports (19) have noted that the disturbances in permeability m a y be due to insufficient energy supply for active transport b e c a u s e the m e m b r a n e permeability m a y be increased under anaerobic conditions, with lack of glucose, under conditions of low t e m p e r a t u r e , or by the addition of iodoacetate, dinitrophenol, or cyanide. T h e progressive increase in permeability occurring with the inhibition of active transport would further contribute to intestinal fluid loss. In order to evaluate the direct effects of ethanol on m e m b r a n e transport, the h y p e r o s m o t i c properties of an ethanol solution h a v e to be considered. When mannitol solution, equivalent in osmolality to 5.4% ethanol in bicarbonate Ringer solution, was present in both luminal and serosal sides of the jejunal m u c o s a , PD and I~e decreased to zero immediately rather than progressively as o b s e r v e d with 5.4% ethanol. T h e s e results d e m o n s t r a t e that the effects of ethanol on intestinal function are not entirely a direct c o n s e q u e n c e of its hyperosmolality. T h e r e are s o m e differences in properties b e t w e e n ethanol and mannitol, such as accessibility to the intracellular space. H o w e v e r , mannitol will app r o x i m a t e the osmotic conditions of ethanol as well as any other selected substance. Ethanol has a distinct polarity with respect to its application. Administration of ethanol on the luminal side of the jejunal m u c o s a produced the s a m e results as when present on both sides. H o w e v e r , when ethanol was present on the serosal side only, the PD, Isc, and N a § fluxes were not significantly different f r o m controls. T h e s e results are consistent with studies in the r a b b i t gastric m u c o s a in vitro (20) and canine s t o m a c h in v i v o (21) in which 95% ethanol was infused at 0.5-1.5 ml/min into the splenic artery to a p p r o x i m a t e a 10-30% ethanol concentration on the blood side of the stomach. The PD across the s t o m a c h remained unchanged compared to the p r o m p t and p r o n o u n c e d decrease in PD when 10-20% ethanol was instilled into the lumen. The question has b e e n debated as to w h e t h e r ethanol has direct effects on other tissues, such as the liver, or if the effects are due to inadequate dietary Digestive Diseases, Vol. 23, No. 1 (January 1978)

intake. Lieber et al (22) h a v e reported that longt e r m administration of ethanol given with balanced liquid diets in the b a b o o n still produced fatty liver and suggested that ethanol has direct toxic effects on the liver, independent of nutritional factors. H o w e v e r , the present studies suggest that ethanol, by inhibiting intestinal absorption of N a § 3-O-MG, and L-atanine and enhancing possible fluid loss through increased permeability, m a y produce an effective nutritional deficiency e v e n with adequate dietary intake.

REFERENCES 1. 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 135:1000-1007, 1967 2. Ghirardi P, Marzo A, Sardini D, Marchetti G: Changes in

intestinal absorption of glucose in rats treated with ethanol. Experientia 27:61-62, 1971 3. Israel Y, Salazar I, Rosenmann E: Inhibitory effect of alcohol on intestinal amino acid transport in vivo and in vitro. J Nutr 96:49%504, !968 4. Israel Y, Valenzuela JE, Salazar I, Ugarte G: Alcohol and amino acid transport in the human small intestine. J Nutr 98~222-224, 1969 5. Schultz SG, Fuisz RE, Curran PF: Amino acid and sugar transport in rabbit ileum. J Gen Physiol 49:849-866, 1966 6. Schultz SG, Curran PF: Coupled transport of sodium and organic solutes. Physiol Re 50:637-718, 1970 7. Dinda PK, Beck IT, Beck M, McElligott TF: Effect of ethanol on sodium-dependent glucose transport in the small intestine of the hamster. Gastroenterology 68:1517-1526, 1975 8. Fromm D: Na and C1 transpo~ across isolated proximal small intestine of the rabbit. Am J Physio1224:110-116, 1973 9. Baraona E, Pirola RC, Lieber CS; Small intestinal damage and changes in cell population produced by ethanol ingestion in the rat. GaStroenterology 66:226-234, !974 10. Lindenbaum J, Lieber CS: Effects of chronic ethanol administration on intestinal absorption in man in the absence of nutritional deficiency. Ann NY Acad Sci 252:228-234, 1975 11. Ha!sted CH, Robles EA, Mezey E: Distribution of ethanol in the human gastrointestinal tract. Am J Clin Nutr 26:831-8341 1973 12. Field M, Fromm D, McColl I: Ion transport in rabbit ileal mucosa. I. Na and C1 fluxes and short-circuit current. Am J Physiol 220:1388-1396, 1971 13, Shanbour LL: An automatic voltage-clamp system for in vivo or in vitro studies. Am J Dig Dis 19:367-371, 1974 14. Schultz SG, Zalusky R: Ion transport in isolated rabbit ileum. I. Short-circuit current and Na fluxes. J Gen Physiol 47:567-584, 1964 15. Mezey E: Intestinal function in chronic alcoholism. Ann NY Acad Sci 252:215-227, 1975 16. Kuo Y-J, Shanbour LL, Sernka TJ: Effect of ethanol on permeabi.lity and ion transport in the isolated dog stomach. Am J Dig Dis 19:818-824, 1974 17. Kuo y-J, Shanbour LL: Mechanism of action of aspirin on canine gastric mucosa. Am J Physiol 230:762-767, 1976 55

KUO AND SHANBOUR 18. Kuo Y-J, Shanbour LL: Inhibition of ion transport by bile salts in the canine gastric mucosa. Am J Physiol 231:14331437 19. Hess B: Enzymes in Blood Plasma. New York, Academic Press, 1963, pp 48-55 20. Fromm D, Robertson R: Effects of alcohol on ion transport

56

by isolated gastric and esophageal mucosa. Gastroenterology 70:220-225, 1976 21. Sernka TJ, Gilleland CW, Shanbour LL: Effects of ethanol on active transport in the dog stomach. Am J Physiol 226:397--400, 1974 22. Lieber CS: The metabolism of alcohol. Sci Am 234:25-33, 1976

Digestive Diseases, Vol. 23, No. I (January 1978)