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Chronic ethanol intake inhibits both the vasoactive intestinal peptide binding and the associated cyclic AMP production in rat enterocytes
Gen. Pharmac. Vol. 23. No. 4, pp. 607-611, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 S5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
CHRONIC ETHANOL INTAKE INHIBITS BOTH THE VASOACTIVE INTESTINAL PEPTIDE BINDING A N D THE ASSOCIATED CYCLIC AMP PRODUCTION IN RAT ENTEROCYTES J. JIMENEZ,J. R. C^Lvo, P. MOLINERO,R. GOBERNA and J. M. GUERRERO* The University of Seville School of Medicine, Department of Medical Biochemistry and Molecular Biology, Avda Sanchez Pizjuan 4, 41009-Sevilla, Spain [Fax 345-490-7048] (Received 14 January 1992)
Abstract--l. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of ~2~I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. Native peptide displaced ~2~I-VIPbinding to enterocytes, exhibiting a ICs0 at about 4 nM native VIP in control and ethanol-treated animals. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-atfinity and the Iow-al~nity binding sites. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.
INTRODUCTION
MATERIALS AND METHODS
A variety of changes in intestinal function have been reported after acute or habitual alcohol consumption (Langman and Bell, 1982). Impaired absorption of vitamins (Halsted et al., 1967; Balaghi and Neal, 1968; T o m a s u d o et al., 1968; Lindenbaum and Lieber, 1969), sugars (Kuo and Shambour, 1978), and amino acids (Israel et al., 1968; Jacobs and Overbold, 1976) has been shown in the presence of ethanol. The chronic consumption of ethanol also induces diminution of sucrase, lactase and alkaline phosphatase activities (Baraona et al., 1974) and a zinc-deficient state (Ahmed and Russell, 1982) associated with diminished total disaccharidase activity and intestinal mucosal protein content (Zarling et al., 1986). Moreover, long-term ethanol ingestion is often associated with diarrhoea. Studies concerning either the role of gastrointestinal hormones in the alcohol consumption-induced modifications o f the enterocyte function or the effect of ethanol consumption on the enterocyte response to gastrointestinal hormones are infrequent. Thus, implication of vasoactive intestinal peptide (VIP), which physiologically causes a striking net secretion of water and electrolytes in the small intestine (Barbezat, 1973), has not been studied, and it could be expected a relationship between the alcohol-induced modifications of the intestinal absorption/secretion and VIP activity. The purpose of this study was to investigate the modifications of VIP binding and the associated cyclic A M P production in enterocytes of rats chronically treated with ethanol.
Chemicals Synthetic rat VIP was purchased from Peninsula Laboratories Europe (Merseyside, England)', bacitracin, bovine serum albumin (BSA) (fraction V), 3-isobutyl-l-methylxanthine (IBMX) and cyclic AMP from Sigma (St Louis, Mo.), carrier-free Natal (IMS 30, 100mCi/ml) and 3Hcyclic AMP (TRK 304, 30 Ci/mmol) from Radiochemical Center (Amersham, England). VIP was radioiodinated by the chloramine T method to a specific activity of about 250 Ci/g (Prieto et al., 1979). Purification of labeled tracer was performed on a Sephadex G-50 column (l x 30cm) eluted with 0.2 M acetic acid containing 0.5% (w/v) BSA and 0.03% (w/v) bacitracin. Ethyl alcohol and all other chemicals were reagent grade.
Male Wistar rats, weighing approx. 200 g, obtained from our animal facility were used. Rats were housed in an air-conditioned room with lighting regulated to provide equal hours of light and dark. During the first week in our laboratory, rats were fed with standard rat chow and drinking water ad libitum. Then, chronic ethanol administration was achieved by adding increasing concentrations of ethanol to the drinking water. Thus, ethanol concentration (v/v) in the water was of 5, 10 and 15%, during the first, second and third week since the beginning of the treatment, respectively. From fourth week up to the 12th week of treatment concentration of ethanol was 20*/,. During the same periods of time, control animals only received water. Animals were killed at 6, 8, 10 or 12 weeks of treatment and small intestines used for obtaining isolated enterocytes. Serum ethanol concentrations were determined by a commercial test (Boehringer Mannheim GmbH, Mannheim, Germany).
*To whom all correspondence should be addressed.
Preparation o f cells Intestinal epithelial cells of villous origin were isolated from jejune-ileum as described Prieto et al. (1979).
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Immediately after decapitation of the rats, the jejune-ileum was removed and flushed free of content with an ice-cold 0.24 M NaCI solution. Gut was everted over a glass rod, filled with 0.34 M NaCI in order to expose the villi, and washed with a dispersing solution containing 2.5raM EDTA and 0.24 M NaCI, pH 7.5; then the villous cells were obtained by gentle hand-shaking for 10 min in 100 ml of the dispersing solution. Cells were sedimented at 2000g for 5rain, washed in 100ml of 0.15 M NaCI, and again sedimented. The addition of 5 ml of 0.15 M NaCI to this pellet was followed by intermittent shaking every 2 min for I0 min and centrifugation; this procedure was repeated three times, then cells were used for binding experiments or cyclic AMP studies. The preparation contained almost exclusively villous cells as attested by the presence of microvilli (brush border) in about 95% of the cells, and was free of nonepithelial cells. Using trypan blue or erythrosin B it was determined that about 95% of the cells were viable. The concentration of the cell DNA (Kissane and Robbins, 1958) and protein (Lowry et al., 1951) was also determined. Binding studies In the standard binding assay, isolated intestinal epithelial cells (5-10 g DNA/ml) were incubated at 15°C in 0.5 ml of 35 mM Tris-HCl buffer (pH 7.5) containing 50 mM NaCI, 1.4% (w/v) BSA, 1 mg/ml bacitracin, and 45 pM t2~-VIP in the absence or presence of increasing concentrations of native pcptide (0-100 nM). Cell bound peptide was separated by centrifugation after 90 rain incubation, and radioactivity associated with the cells was measured in a LKB gamma counter. Results are reported as specific binding; which was calculated by subtracting from total binding the nonspecific binding, i.e. binding in presence of 1M VIP (about 1% of the total radioactivity). Degradation of t2~I-VIP by the cells was tested by its loss of ability to be adsorbed to talc (Jimenez et al., 1989). Cyclic A M P determination Cyclic AMP determination was determined as previously described (Guerrero et al., 1981; Calvo et al., 1986; Segura et al., 1991). VIP-induced cyclic AMP production was studied in a standard solution (0.5 ml final volume) consisting of 35 mM Tris-HCI buffer (pH 7.5), 50 mM NaCI, 1.4% BSA, 1 mg/ml bacitracin, and increasing concentrations of VIP (up to 100 nM). The reaction was initiated by the addition of intestinal epithelial cells (10g DNA/ml) and, after 45 min incubation at 15°C, the reaction was stopped by the addition of 2.5ml methanol, the precipitate was removed by centrifugation, aliquots of the supernatant were evaporated and cyclic AMP was measured by a proteinbinding assay (Gilman, 1970). Statistical methods Binding data were analyzed by the method of Scatchard (1949) using the nonlinear curve-fitting program Ligand (Munson and Robard, 1980). Statistical differences were determined by either paired t-test or analyses of variance, followed by a Student-Newman-Keuls multiple range test.
Table I. Levels of ethanol in serum from rats treated with ethanol for either 0, 6, 8, 10 or 12 weeks. Values represent the mean ± SE of the 8 animals
intake (weeks)
Ethanol
Ethanol concentration (mg/ml)
0 6
0 0.151 ±0.019
8 10 12
0.168 ± 0.018 0.181 ± 0.028 0.250 ± 0.096
et al. RESULTS Characterization o f e t h a n o l - t r e a t m e n t m o d e l
C h r o n i c a d m i n i s t r a t i o n o f e t h a n o l in the drinking water induced the a p p e a r a n c e o f the alcohol in the rat serum (Table 1). F r o m non-detectable levels at the beginning o f the treatment, serum ethanol concentrations progressively increased up to reaching maximal values after 12 weeks. The first point to be m e n t i o n e d in that ethanol intake resulted in a significant decrease o f the rate o f rat p o n d e r a l growth [Fig. I(A)]. After 12 weeks o f treatment, ethanoltreated rats weighed 35% less t h a n control animals. However, total caloric intake o f b o t h groups were similar d u r i n g all t r e a t m e n t [Fig. I(B)]. O u r study shows that e t h a n o l - t r e a t e d animals o b t a i n e d less calories from the s t a n d a r d rat chow t h a n control
600
•
A
Control
~00
~200 1 •
20%
0 I
120
+
I
I
•
Confro[
•
Ethanot trea)ed
I
I
I
I B
8O
~.o , , 15~q
I o
90
•"-
•
20%
I I Total Kcat Kcal in food
I
I
I
I C
3O
0
2
~ 6 T)me ( W e e k s )
8
10
12
Fig. I. Effect of ethanol intake on rat ponderal development (A) or caloric intake (B, C). Animals were treated with 5, 10, I 5, or 20 ethanol in the drinking water during the indicated periods of time. Control animals received only water. During the treatment, animals were weighed weekly (A) and total caloric intake was controlled (B). Moreover, specific caloric intake from either chow or ethanol was also controlled in ethanol-treated animals (C). Result are expressed as the mean (B, C) or the mean + SE of 12 animals.
6O9
Ethanol and VIP receptors Table 2. Ratio betweenprotein and DNA content in intestinalepithelialcellsfrom rats treated with ethanol for either 0, 6, 8, 10 or 12 weeks. Values represent the mean+ SE of 8 animals Ethanol i n t a k e Protein/DNA (weeks) (mg protein/ragDNA) 0 63.3 + 9.4 6 36.6 + 13.6* 8 19.1 +_7.9t 10 20.8 + 5.7t 12 22.8 _+5.3t The significanceof the differenceswithrespectto zero values is given by either *P < 0.05 or tP < 0.001.
7
"w
6
g
,o
2
Q.
animals, and obtained more than 35% of calories from ethanol [Fig. I(C)]. Thus, total caloric intake in ethanol-treated animals, represented as calories obtained from the standard chow plus calories obtained from ethanol, was similar to the caloric intake of control animals, which obtained calories exclusively from the standard rat chow. The different ponderal development between control and ethanol-treated rats did not correlate with morphological changes in the enterocytes when hematoxylin-eosin studies were performed (data not shown). However, protein content of enterocytes was different between both groups. Table 2 shows that enterocyte protein content, expressed as the ratio between and DNA content, exhibited a 50% decrease after 6 weeks of treatment. Moreover, after 8, l0 or 12 weeks of treatment, enterocyte protein content was only a 35% of that found in controls. For that reason, in subsequent experiments, data are referred to the DNA content to avoid errors induced by the effect of ethanol on the enterocyte protein content.
125I-VIP binding studies In the first experiment, ~25I-VIP binding to enterocytes from controls and ethanol-treated animals was studied. As shown in Fig. 2, maximal binding was found in control animals while, in ethanol-treated rats, binding was significantly reduced. Thus, ]25I-VIP
2
t
2
I
0
-I.
1
i
I
l
6
8
10
12
Time (welkl) Fig. 2. Effect of ethanol intake on '2~I-VIP binding to enterocytes. Cells (5-10g DNA/ml) from controls or ethanol-treated animals for 6, 8, or 12 weeks were incubated with ~2sI-VIP at 15°C for 60min. Each point is the mean -t- SE of 8 animals performed in triplicate (*P < 0.05 vs 0 weeks; "*P < 0.05 vs 0 weeks).
0
;rl
0
I
I
'
i
10
9
8
7
-Log VlP concentration(HI Fig. 3. Competitive displacement of ~5I-VIP binding to rat enterocytes by unlabeled VIP. Cells (5-10 g DNA/ml) from controls (0) or ethanol-treated animals for 6 (&), 8 (ll), 10 (©), or 12 (I-q) weeks were incubated with t2~I-VIPand increasing concentration of unlabeled peptide at 15°C for 60 min. Each point is the mean of 8 animals performed in triplicate. In order to clarify the figure, SE were omitted being always lower than 10% of the mean.
binding clearly decreased since 6 weeks of treatment (50% of control), with no further decrease during the following weeks of treatment. Then, stoichiometric experiments were performed in order to investigate whether the ~25I-VIP binding decrease induced by ethanol was due to a diminution in the affinity or the number of binding sites. Figure 3 shows again that, in the absence of native peptide, maximal ~2sI-VIP binding was reached in enterocytes obtained from control animals. However, native peptide displaced ~25I-VIP binding to enterocytes in the same manner in both control and ethanol-treated animals, exhibiting a half-maximal inhibition (IC~) at about 4 nM native VIP in all groups studied. When data were analyzed by the method of Scatchard (1949), using the nonlinear curve-fitting progam Ligand (Munson and Robard, 1980), two different classes of 12~I-VIPbinding sites were found for control and ethanol-treated rats. Table 3 shows that affinities of both the highaffinity and the low-affinity binding sites were similar in all groups. However, the number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. The ethanolinduced reduction of ~25I-VIPbinding sites in enterocytes was observed in both the high-affinity and the low-affinity binding sites. On the other hand, reduction in ~25I-VIP binding to enterocytes was not induced by changes in either the association rate or the degradation of the tracer (data not shown).
Cyclic AMP studies The effect of various concentrations of VIP in promoting a cyclic AMP rise in enterocytes from control or ethanol-treated rats is shown in Fig. 4. After 6 weeks of treatment, cyclic AMP production
J. JIMENEZ et al.
610
Table 3. Characteristics of VIP receptors in intestinal epithelial cells from control and ethanol treated rats
Control Ke (nM) BC (pg/mg DNA)
High affinity binding sites
Low affinity binding sites
1.1 +_0.2 19.2 _+ 3.5
79 + 13 581 + 92
Ethanol-treatment (6 weeks) K~ (nM) BS (pg/mg DNA)
0.8 +_0.2 5.6 + 1.8+
93 + 63 766 4. 97
l.I +_.0.7 7.1 4-_3.6'1"
93 _ 37 343 + 209
0.8 = 0.3 6.1 + 0.6t
75 + 26 329 4- 112"
I.I 4- 0.3 9.7 + 2.2?
95 + 33 296 4- 93*
Ethanol-treatment (8 weeks) Kd (nM) BC (pg/mg DNA)
Ethanol-treatment (10 weeks) Kd (nM) BC (pg/mg DNA)
Ethanol-treatment (12 weeks) Kn (nM) BS (pg/mg DNA)
Values are calculated from Scatchard plot and represent mean + SE of 6 experiments. The significant of the differences between control animals and all weeks of ethanol treatment studied is given by *P < 0.05 and t P < 0.01. Ka = dissociation constant; BC binding capacity. =
stimulated by VIP was similar to that obtained in control animals. However, after 8 weeks of treatment, a significant decrease in cyclic AMP production was observed. Finally, enterocytes from animals treated for 10 or 12 weeks exhibited the lowest cyclic AMP values. The fact that the half-maximal stimulation (EDs0) was elicited at about 1 nM VIP in all groups, and maximal stimulation was obtained at 0.1 #M VIP, indicated no changes in the sensitivity of adenylate cyclase system to the peptide.
4 &
o
.d . {; 0
I
10
I
l
9
8
7
-Log VIP concentration IM) Fig. 4. Effect of increasing concentrations of VIP on cyclic AMP production in rat enterocytes. Cells (5-10 g DNA/ml) from controls (O) or ethanol-treated animals for 6 (&), 8(m), 10 (O), or 12 (l'q) weeks were incubated with increasing concentration of VIP at 15°C for 45 rain. Each point is the mean of 8 animals performed in triplicate. In order to clarify the figure, SE were omitted being always lower than 10% of the mean.
DISCUSSION
In the present study, we reported for the first time the effect of chronic intake of ethanol on the VIP binding to rat intestinal epithelial cells and the cyclic AMP production stimulated by the peptide. This study was carried out with an experimental model of alcoholism very similar to that used in previous reports (Chariot et al., 1977; Lamb et al., 1979; Roze et al., 1979; Jimenez et al., 1989). The results showed that chronic intake of ethyl alcohol induced significant changes in VIP binding and cyclic AMP production in these cells. It has long been known, that digestion and absorption are compromised by ethanol consumption. This functional impairment is accompanied by structural alteration in the cells of the small intestine (Worthington et al., 1978). The loss of gut integrity significantly reduces the nutritional status by promoting malabsorption. In fact, we found a decrease in the rat ponderal growth during ethanol treatment. Moreover, after 8 weeks of ethanol intake the protein content of enterocytes decreased up to a 30% of the control. Most of the studies of changes in protein content have been performed on the liver, where it has been shown a reduction in mitochondrial membane proteins (Taraschi and Rubin, 1985). Thus, chronic intoxicated rats exhibit a 50% decrease in mitochondrial cytochrome oxidase and cytochrome b. Apparently, one of the proteins affected in the enterocyte membrane is the VIP binding site. As indicated by binding experiments, the specific binding of ~'sI-VIP binding to enterocytes of alcoholic rats was significantly lower than in control rats. The Scatchard plot of binding data, showed that ethanol elicited a decrease in binding capacity of both the high and low binding sites. No changes in the affinities of binding sites were observed. It is important to point out that the characteristics of VIP binding sites in intestinal epithelial cells reported in this paper for control rats correlated well with those previously reported by Prieto et al. (1979). We also show that cyclic AMP production stimulated by VIP is clearly diminished in ethanol treated rats rising from the suggestion that the decrease in VIP binding sites is reflected in a decrease in cyclic AMP production. We have previously reported that ethanol affects VIP binding sites in rat spleen lymphoid cells, speculating that VIP could be involved in the depressed lymphocytes seen in chronic ethanol intake (Jimenez et al., 1989). In acinar cells from guinea-pig pancreas, ethanol also modify the in vitro effect of VIP. Thus, ethanol inhibited the increase in the amylase secretion caused by VIP, although ethanol did not alter binding of VIP to the cells and it potentiated the cyclic AMP production stimulated by the peptide (Uhlemann et al., 1979). For other peptides, such as somatostatin, it has been described that, while acute ethanol administration resulted in an increase in the number of somatostatin binding sites, chronic administration of ethanol caused a decrease in the number of somatostatin binding sites in frontoparietal cortex suggesting a possible role for the somatostatin in the nervous system during alcoholism (Barrios et al., 1990). Now, we suggest that the impairment of VIP binding sites seen during alcoholism can participate
Ethanol and VIP receptors in the malabsorption syndrome that always accompanies ethanol intake. We do not demonstrate whether the decrease of VIP binding sites is due to either a direct effect of ethanol, an effect of the reduced nutritional status, or any other unknown cause. However, it is conceivable that the changes in the enterocyte VIP binding sites after chronic ethanol consumption may be due to the derangement in receptor biosynthesis. Various studies have led to the conclusion that chronic ethanol consumption interferes with genetic code transcription and protein synthesis (Schenker, 1982). Many proteins of membranes are disrupted by ethanol and the cellular adaptation that it provokes (Sun and Sun, 1985). In conclusion, our study shows for the first time the effects o f chronic intake of ethanol on the VIP binding to rat intestinal epithelial cells. This result contributes to one better definition of the role of VIP in the regulation of enterocyte function in physiological and pathological conditions. Acknowledgement--This research was supported by a Grant from el Fondo de Investigaciones Sanitarias de la Seguridad Social (90/0688).
REFERENCES Ahmed S. B. and Russell R. M. (1982) The effect of ethanol feeding on zinc balance and tissue zinc levels in rats maintained on zinc-deficient diets. J. Lab. clin. Med. I00, 211-217. Balaghi M. and Neal R. A. (1968) Effect of chronic ethanol administration on thiamine metabolism in the rat. J. Nutr. 107, 2144-2152. Baraona E., Pirola R. C. and Lieber C. S. (1974) Small intestinal damage and changes in cell population by ethanol ingestion in the rat. Gastroenterology 66, 226-234. Barbezat G. O. (1973) Stimulation of intestinal secretion by polypeptide hormones. Scand. J. Gastroent. 8 (Suppl. 22), 1-21. Barrios V., Rodriguez-Sanchez M. N. and Arilla E. (1990) Effects of acute and chronic ethanol administration and its withdrawal on the level and binding of somatostatin in rat brain. Clin. Sci. 79, 451-456. Calvo J. R., Guerrero J. M., Molinero P., Blasco R. and Goberna R. (1986) Interaction of vasoactive intestinal peptide (VIP) with human peripheral blood lymphocytes: specific binding and cyclic AMP production. Gen. Pharmac. 17, 185-189. Chariot J., Roze C., De La Tour J., Souchard M., Hollande E., Vaille C. and Debray C. (1977) Effects of chronic ethanol consumption on pancreatic response to central vagal stimulation by 2-deoxy-D-giucos¢ in the rat. Digestion 15, 425-437. Gilman A. C. (1970) A protein binding assay for adenosine 3'-5'-cyclic monophosphate. Proc. ham. Acad. Sci. U.S.A. 67, 305-312. Guerrero J. M., Prieto J. C., Elorza F. L., Ramirez R. and Gobcrna R. (1981) Interaction of vasoactiv¢ intestinal peptide with human blood mononuclear cells. Molec. cell. Endocr. 21, 151-160. Halsted C. H., Griggs R. C. and Harris J. W. (1967) The effect of alcoholism on the absorption of folic (H3-PGA) evaluated by plasma levels and urine excretion. J. Lab. clin. Med. 69, 116-131.
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Israel Y., Salazar !. and Roscnmann E. (1968) Inhibitory effects of alcohol on intestinal amino acid transport in vivo and/n vitro. J. Nutr. 96, 499-504. Jacobs F. A. and Overbold C. (1976) Inhibition of leucin¢ absorption by ethanol. Fedn. Proc. 35, 463. Jimenez J., Gobcrna R., Mesa J. C., Morales M. F. and Calvo J. R. (1989) Effect of chronic intake of ethanol on the binding of vasoactive intestinal peptide to rat spleen lymphoid cells~ Gen. Pharmac. 20, 659-662. Kissane J. M. and Robbins E. (1958) The fluorimetric measurement of DNA in animal tissues with special references to the central nervous systems. J. biol. Chem. 233, 184--188. Kuo Y. J. and Shambour L. L. (1978) Effects of ethanol on sodium, 3-O-methylglucose and l-alanine transport in the jejunum. Am. J. dig. Dis. 23, 51-56. Lamb R. G., Wood C. K. and Fallon H. J. (1979) The effect of acute and chronic ethanol intake on hepatic glycerolipid biosynthesis in the hamster. J. clin. Invest. 63, 14-20. Langman J. S. and Bell G. D. (1982) Alcohol and the gastrointestinal tract. Br. med. Bull. 82, 71-75. Lindenbaum J. and Lieber C. S. (1969) Alcohol induced malabsorption of vitamin BI2 in man. Nature (Lond.) 244, 806. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Munson P. J. and Robard D. (1980) Ligand: a versatile computerized approach for characterization of ligand binding systems. Analyt. Biochem. 107, 220--239. Prieto J. C., Laburthe M. and Rosselin G. (1979) Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cell from rat. I. Characterization, quantitative aspects and structural requirements of binding sites. Fur. J. Biochem. 96, 229-239. Roze C., Chariot J., De La Tour J. and Camilleri J. P. (1979) Effects of long term alcohol intake (3-18 months) on the rat pancreas. Gastroent. clin. Biol. 3, 657-666. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660--672. Schenker S. (1982) Medical Disorders o f Alcoholism: Pathogenesis and Treatment, pp. 480-525. Saunders, Philadelphia, Pa. Segura J. J., Guerrero J. M., Goberna R. and Cairo J. R. (1991 ) Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages. Regul. Pept. 33, 133-143. Sun G. Y. and Sun A. Y. (1985) Ethanol and membrane lipids. Alcohol. clin. Exp. Res. 9, 164-180. Taraschi T. F. and Rubin E. (1985) Effects of ethanol on the chemical and structural properties of biologic membranes. Lab. Invest. 52, 120-131. Tomasudo P. A., Kater R. M. H. and Iber F. L. (1968) Impairment of thiamine absorption in alcoholism. Am. J. clin. Nutr. 21, 1341-1344. Uhlemann E. R., Robberecht P. and Gardner J. D. (1979) Effects of alcohols on the actions of VIP and secretion on acinar cells from guinea pig pancreas. Gastroenterology 76, 917-925. Worthington B. S., Meserole L. and Syrotuck J. A. (1978) Effect of daily ethanol ingestion on intestinal permeability to macromolecules. Dig. Dis. 23, 23-32. Zarling E. J., Mobarhan S. and Donahue P. E. (1986) Effect on moderate prolonged ethanol ingestion on intestinal disaccharidase activity and histology. J. Lab. clin. Med. 108, 7-10.
0306-3623/92 S5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
CHRONIC ETHANOL INTAKE INHIBITS BOTH THE VASOACTIVE INTESTINAL PEPTIDE BINDING A N D THE ASSOCIATED CYCLIC AMP PRODUCTION IN RAT ENTEROCYTES J. JIMENEZ,J. R. C^Lvo, P. MOLINERO,R. GOBERNA and J. M. GUERRERO* The University of Seville School of Medicine, Department of Medical Biochemistry and Molecular Biology, Avda Sanchez Pizjuan 4, 41009-Sevilla, Spain [Fax 345-490-7048] (Received 14 January 1992)
Abstract--l. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of ~2~I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. Native peptide displaced ~2~I-VIPbinding to enterocytes, exhibiting a ICs0 at about 4 nM native VIP in control and ethanol-treated animals. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-atfinity and the Iow-al~nity binding sites. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.
INTRODUCTION
MATERIALS AND METHODS
A variety of changes in intestinal function have been reported after acute or habitual alcohol consumption (Langman and Bell, 1982). Impaired absorption of vitamins (Halsted et al., 1967; Balaghi and Neal, 1968; T o m a s u d o et al., 1968; Lindenbaum and Lieber, 1969), sugars (Kuo and Shambour, 1978), and amino acids (Israel et al., 1968; Jacobs and Overbold, 1976) has been shown in the presence of ethanol. The chronic consumption of ethanol also induces diminution of sucrase, lactase and alkaline phosphatase activities (Baraona et al., 1974) and a zinc-deficient state (Ahmed and Russell, 1982) associated with diminished total disaccharidase activity and intestinal mucosal protein content (Zarling et al., 1986). Moreover, long-term ethanol ingestion is often associated with diarrhoea. Studies concerning either the role of gastrointestinal hormones in the alcohol consumption-induced modifications o f the enterocyte function or the effect of ethanol consumption on the enterocyte response to gastrointestinal hormones are infrequent. Thus, implication of vasoactive intestinal peptide (VIP), which physiologically causes a striking net secretion of water and electrolytes in the small intestine (Barbezat, 1973), has not been studied, and it could be expected a relationship between the alcohol-induced modifications of the intestinal absorption/secretion and VIP activity. The purpose of this study was to investigate the modifications of VIP binding and the associated cyclic A M P production in enterocytes of rats chronically treated with ethanol.
Chemicals Synthetic rat VIP was purchased from Peninsula Laboratories Europe (Merseyside, England)', bacitracin, bovine serum albumin (BSA) (fraction V), 3-isobutyl-l-methylxanthine (IBMX) and cyclic AMP from Sigma (St Louis, Mo.), carrier-free Natal (IMS 30, 100mCi/ml) and 3Hcyclic AMP (TRK 304, 30 Ci/mmol) from Radiochemical Center (Amersham, England). VIP was radioiodinated by the chloramine T method to a specific activity of about 250 Ci/g (Prieto et al., 1979). Purification of labeled tracer was performed on a Sephadex G-50 column (l x 30cm) eluted with 0.2 M acetic acid containing 0.5% (w/v) BSA and 0.03% (w/v) bacitracin. Ethyl alcohol and all other chemicals were reagent grade.
Male Wistar rats, weighing approx. 200 g, obtained from our animal facility were used. Rats were housed in an air-conditioned room with lighting regulated to provide equal hours of light and dark. During the first week in our laboratory, rats were fed with standard rat chow and drinking water ad libitum. Then, chronic ethanol administration was achieved by adding increasing concentrations of ethanol to the drinking water. Thus, ethanol concentration (v/v) in the water was of 5, 10 and 15%, during the first, second and third week since the beginning of the treatment, respectively. From fourth week up to the 12th week of treatment concentration of ethanol was 20*/,. During the same periods of time, control animals only received water. Animals were killed at 6, 8, 10 or 12 weeks of treatment and small intestines used for obtaining isolated enterocytes. Serum ethanol concentrations were determined by a commercial test (Boehringer Mannheim GmbH, Mannheim, Germany).
*To whom all correspondence should be addressed.
Preparation o f cells Intestinal epithelial cells of villous origin were isolated from jejune-ileum as described Prieto et al. (1979).
607
Animals
608
J.
JIMENEZ
Immediately after decapitation of the rats, the jejune-ileum was removed and flushed free of content with an ice-cold 0.24 M NaCI solution. Gut was everted over a glass rod, filled with 0.34 M NaCI in order to expose the villi, and washed with a dispersing solution containing 2.5raM EDTA and 0.24 M NaCI, pH 7.5; then the villous cells were obtained by gentle hand-shaking for 10 min in 100 ml of the dispersing solution. Cells were sedimented at 2000g for 5rain, washed in 100ml of 0.15 M NaCI, and again sedimented. The addition of 5 ml of 0.15 M NaCI to this pellet was followed by intermittent shaking every 2 min for I0 min and centrifugation; this procedure was repeated three times, then cells were used for binding experiments or cyclic AMP studies. The preparation contained almost exclusively villous cells as attested by the presence of microvilli (brush border) in about 95% of the cells, and was free of nonepithelial cells. Using trypan blue or erythrosin B it was determined that about 95% of the cells were viable. The concentration of the cell DNA (Kissane and Robbins, 1958) and protein (Lowry et al., 1951) was also determined. Binding studies In the standard binding assay, isolated intestinal epithelial cells (5-10 g DNA/ml) were incubated at 15°C in 0.5 ml of 35 mM Tris-HCl buffer (pH 7.5) containing 50 mM NaCI, 1.4% (w/v) BSA, 1 mg/ml bacitracin, and 45 pM t2~-VIP in the absence or presence of increasing concentrations of native pcptide (0-100 nM). Cell bound peptide was separated by centrifugation after 90 rain incubation, and radioactivity associated with the cells was measured in a LKB gamma counter. Results are reported as specific binding; which was calculated by subtracting from total binding the nonspecific binding, i.e. binding in presence of 1M VIP (about 1% of the total radioactivity). Degradation of t2~I-VIP by the cells was tested by its loss of ability to be adsorbed to talc (Jimenez et al., 1989). Cyclic A M P determination Cyclic AMP determination was determined as previously described (Guerrero et al., 1981; Calvo et al., 1986; Segura et al., 1991). VIP-induced cyclic AMP production was studied in a standard solution (0.5 ml final volume) consisting of 35 mM Tris-HCI buffer (pH 7.5), 50 mM NaCI, 1.4% BSA, 1 mg/ml bacitracin, and increasing concentrations of VIP (up to 100 nM). The reaction was initiated by the addition of intestinal epithelial cells (10g DNA/ml) and, after 45 min incubation at 15°C, the reaction was stopped by the addition of 2.5ml methanol, the precipitate was removed by centrifugation, aliquots of the supernatant were evaporated and cyclic AMP was measured by a proteinbinding assay (Gilman, 1970). Statistical methods Binding data were analyzed by the method of Scatchard (1949) using the nonlinear curve-fitting program Ligand (Munson and Robard, 1980). Statistical differences were determined by either paired t-test or analyses of variance, followed by a Student-Newman-Keuls multiple range test.
Table I. Levels of ethanol in serum from rats treated with ethanol for either 0, 6, 8, 10 or 12 weeks. Values represent the mean ± SE of the 8 animals
intake (weeks)
Ethanol
Ethanol concentration (mg/ml)
0 6
0 0.151 ±0.019
8 10 12
0.168 ± 0.018 0.181 ± 0.028 0.250 ± 0.096
et al. RESULTS Characterization o f e t h a n o l - t r e a t m e n t m o d e l
C h r o n i c a d m i n i s t r a t i o n o f e t h a n o l in the drinking water induced the a p p e a r a n c e o f the alcohol in the rat serum (Table 1). F r o m non-detectable levels at the beginning o f the treatment, serum ethanol concentrations progressively increased up to reaching maximal values after 12 weeks. The first point to be m e n t i o n e d in that ethanol intake resulted in a significant decrease o f the rate o f rat p o n d e r a l growth [Fig. I(A)]. After 12 weeks o f treatment, ethanoltreated rats weighed 35% less t h a n control animals. However, total caloric intake o f b o t h groups were similar d u r i n g all t r e a t m e n t [Fig. I(B)]. O u r study shows that e t h a n o l - t r e a t e d animals o b t a i n e d less calories from the s t a n d a r d rat chow t h a n control
600
•
A
Control
~00
~200 1 •
20%
0 I
120
+
I
I
•
Confro[
•
Ethanot trea)ed
I
I
I
I B
8O
~.o , , 15~q
I o
90
•"-
•
20%
I I Total Kcat Kcal in food
I
I
I
I C
3O
0
2
~ 6 T)me ( W e e k s )
8
10
12
Fig. I. Effect of ethanol intake on rat ponderal development (A) or caloric intake (B, C). Animals were treated with 5, 10, I 5, or 20 ethanol in the drinking water during the indicated periods of time. Control animals received only water. During the treatment, animals were weighed weekly (A) and total caloric intake was controlled (B). Moreover, specific caloric intake from either chow or ethanol was also controlled in ethanol-treated animals (C). Result are expressed as the mean (B, C) or the mean + SE of 12 animals.
6O9
Ethanol and VIP receptors Table 2. Ratio betweenprotein and DNA content in intestinalepithelialcellsfrom rats treated with ethanol for either 0, 6, 8, 10 or 12 weeks. Values represent the mean+ SE of 8 animals Ethanol i n t a k e Protein/DNA (weeks) (mg protein/ragDNA) 0 63.3 + 9.4 6 36.6 + 13.6* 8 19.1 +_7.9t 10 20.8 + 5.7t 12 22.8 _+5.3t The significanceof the differenceswithrespectto zero values is given by either *P < 0.05 or tP < 0.001.
7
"w
6
g
,o
2
Q.
animals, and obtained more than 35% of calories from ethanol [Fig. I(C)]. Thus, total caloric intake in ethanol-treated animals, represented as calories obtained from the standard chow plus calories obtained from ethanol, was similar to the caloric intake of control animals, which obtained calories exclusively from the standard rat chow. The different ponderal development between control and ethanol-treated rats did not correlate with morphological changes in the enterocytes when hematoxylin-eosin studies were performed (data not shown). However, protein content of enterocytes was different between both groups. Table 2 shows that enterocyte protein content, expressed as the ratio between and DNA content, exhibited a 50% decrease after 6 weeks of treatment. Moreover, after 8, l0 or 12 weeks of treatment, enterocyte protein content was only a 35% of that found in controls. For that reason, in subsequent experiments, data are referred to the DNA content to avoid errors induced by the effect of ethanol on the enterocyte protein content.
125I-VIP binding studies In the first experiment, ~25I-VIP binding to enterocytes from controls and ethanol-treated animals was studied. As shown in Fig. 2, maximal binding was found in control animals while, in ethanol-treated rats, binding was significantly reduced. Thus, ]25I-VIP
2
t
2
I
0
-I.
1
i
I
l
6
8
10
12
Time (welkl) Fig. 2. Effect of ethanol intake on '2~I-VIP binding to enterocytes. Cells (5-10g DNA/ml) from controls or ethanol-treated animals for 6, 8, or 12 weeks were incubated with ~2sI-VIP at 15°C for 60min. Each point is the mean -t- SE of 8 animals performed in triplicate (*P < 0.05 vs 0 weeks; "*P < 0.05 vs 0 weeks).
0
;rl
0
I
I
'
i
10
9
8
7
-Log VlP concentration(HI Fig. 3. Competitive displacement of ~5I-VIP binding to rat enterocytes by unlabeled VIP. Cells (5-10 g DNA/ml) from controls (0) or ethanol-treated animals for 6 (&), 8 (ll), 10 (©), or 12 (I-q) weeks were incubated with t2~I-VIPand increasing concentration of unlabeled peptide at 15°C for 60 min. Each point is the mean of 8 animals performed in triplicate. In order to clarify the figure, SE were omitted being always lower than 10% of the mean.
binding clearly decreased since 6 weeks of treatment (50% of control), with no further decrease during the following weeks of treatment. Then, stoichiometric experiments were performed in order to investigate whether the ~25I-VIP binding decrease induced by ethanol was due to a diminution in the affinity or the number of binding sites. Figure 3 shows again that, in the absence of native peptide, maximal ~2sI-VIP binding was reached in enterocytes obtained from control animals. However, native peptide displaced ~25I-VIP binding to enterocytes in the same manner in both control and ethanol-treated animals, exhibiting a half-maximal inhibition (IC~) at about 4 nM native VIP in all groups studied. When data were analyzed by the method of Scatchard (1949), using the nonlinear curve-fitting progam Ligand (Munson and Robard, 1980), two different classes of 12~I-VIPbinding sites were found for control and ethanol-treated rats. Table 3 shows that affinities of both the highaffinity and the low-affinity binding sites were similar in all groups. However, the number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. The ethanolinduced reduction of ~25I-VIPbinding sites in enterocytes was observed in both the high-affinity and the low-affinity binding sites. On the other hand, reduction in ~25I-VIP binding to enterocytes was not induced by changes in either the association rate or the degradation of the tracer (data not shown).
Cyclic AMP studies The effect of various concentrations of VIP in promoting a cyclic AMP rise in enterocytes from control or ethanol-treated rats is shown in Fig. 4. After 6 weeks of treatment, cyclic AMP production
J. JIMENEZ et al.
610
Table 3. Characteristics of VIP receptors in intestinal epithelial cells from control and ethanol treated rats
Control Ke (nM) BC (pg/mg DNA)
High affinity binding sites
Low affinity binding sites
1.1 +_0.2 19.2 _+ 3.5
79 + 13 581 + 92
Ethanol-treatment (6 weeks) K~ (nM) BS (pg/mg DNA)
0.8 +_0.2 5.6 + 1.8+
93 + 63 766 4. 97
l.I +_.0.7 7.1 4-_3.6'1"
93 _ 37 343 + 209
0.8 = 0.3 6.1 + 0.6t
75 + 26 329 4- 112"
I.I 4- 0.3 9.7 + 2.2?
95 + 33 296 4- 93*
Ethanol-treatment (8 weeks) Kd (nM) BC (pg/mg DNA)
Ethanol-treatment (10 weeks) Kd (nM) BC (pg/mg DNA)
Ethanol-treatment (12 weeks) Kn (nM) BS (pg/mg DNA)
Values are calculated from Scatchard plot and represent mean + SE of 6 experiments. The significant of the differences between control animals and all weeks of ethanol treatment studied is given by *P < 0.05 and t P < 0.01. Ka = dissociation constant; BC binding capacity. =
stimulated by VIP was similar to that obtained in control animals. However, after 8 weeks of treatment, a significant decrease in cyclic AMP production was observed. Finally, enterocytes from animals treated for 10 or 12 weeks exhibited the lowest cyclic AMP values. The fact that the half-maximal stimulation (EDs0) was elicited at about 1 nM VIP in all groups, and maximal stimulation was obtained at 0.1 #M VIP, indicated no changes in the sensitivity of adenylate cyclase system to the peptide.
4 &
o
.d . {; 0
I
10
I
l
9
8
7
-Log VIP concentration IM) Fig. 4. Effect of increasing concentrations of VIP on cyclic AMP production in rat enterocytes. Cells (5-10 g DNA/ml) from controls (O) or ethanol-treated animals for 6 (&), 8(m), 10 (O), or 12 (l'q) weeks were incubated with increasing concentration of VIP at 15°C for 45 rain. Each point is the mean of 8 animals performed in triplicate. In order to clarify the figure, SE were omitted being always lower than 10% of the mean.
DISCUSSION
In the present study, we reported for the first time the effect of chronic intake of ethanol on the VIP binding to rat intestinal epithelial cells and the cyclic AMP production stimulated by the peptide. This study was carried out with an experimental model of alcoholism very similar to that used in previous reports (Chariot et al., 1977; Lamb et al., 1979; Roze et al., 1979; Jimenez et al., 1989). The results showed that chronic intake of ethyl alcohol induced significant changes in VIP binding and cyclic AMP production in these cells. It has long been known, that digestion and absorption are compromised by ethanol consumption. This functional impairment is accompanied by structural alteration in the cells of the small intestine (Worthington et al., 1978). The loss of gut integrity significantly reduces the nutritional status by promoting malabsorption. In fact, we found a decrease in the rat ponderal growth during ethanol treatment. Moreover, after 8 weeks of ethanol intake the protein content of enterocytes decreased up to a 30% of the control. Most of the studies of changes in protein content have been performed on the liver, where it has been shown a reduction in mitochondrial membane proteins (Taraschi and Rubin, 1985). Thus, chronic intoxicated rats exhibit a 50% decrease in mitochondrial cytochrome oxidase and cytochrome b. Apparently, one of the proteins affected in the enterocyte membrane is the VIP binding site. As indicated by binding experiments, the specific binding of ~'sI-VIP binding to enterocytes of alcoholic rats was significantly lower than in control rats. The Scatchard plot of binding data, showed that ethanol elicited a decrease in binding capacity of both the high and low binding sites. No changes in the affinities of binding sites were observed. It is important to point out that the characteristics of VIP binding sites in intestinal epithelial cells reported in this paper for control rats correlated well with those previously reported by Prieto et al. (1979). We also show that cyclic AMP production stimulated by VIP is clearly diminished in ethanol treated rats rising from the suggestion that the decrease in VIP binding sites is reflected in a decrease in cyclic AMP production. We have previously reported that ethanol affects VIP binding sites in rat spleen lymphoid cells, speculating that VIP could be involved in the depressed lymphocytes seen in chronic ethanol intake (Jimenez et al., 1989). In acinar cells from guinea-pig pancreas, ethanol also modify the in vitro effect of VIP. Thus, ethanol inhibited the increase in the amylase secretion caused by VIP, although ethanol did not alter binding of VIP to the cells and it potentiated the cyclic AMP production stimulated by the peptide (Uhlemann et al., 1979). For other peptides, such as somatostatin, it has been described that, while acute ethanol administration resulted in an increase in the number of somatostatin binding sites, chronic administration of ethanol caused a decrease in the number of somatostatin binding sites in frontoparietal cortex suggesting a possible role for the somatostatin in the nervous system during alcoholism (Barrios et al., 1990). Now, we suggest that the impairment of VIP binding sites seen during alcoholism can participate
Ethanol and VIP receptors in the malabsorption syndrome that always accompanies ethanol intake. We do not demonstrate whether the decrease of VIP binding sites is due to either a direct effect of ethanol, an effect of the reduced nutritional status, or any other unknown cause. However, it is conceivable that the changes in the enterocyte VIP binding sites after chronic ethanol consumption may be due to the derangement in receptor biosynthesis. Various studies have led to the conclusion that chronic ethanol consumption interferes with genetic code transcription and protein synthesis (Schenker, 1982). Many proteins of membranes are disrupted by ethanol and the cellular adaptation that it provokes (Sun and Sun, 1985). In conclusion, our study shows for the first time the effects o f chronic intake of ethanol on the VIP binding to rat intestinal epithelial cells. This result contributes to one better definition of the role of VIP in the regulation of enterocyte function in physiological and pathological conditions. Acknowledgement--This research was supported by a Grant from el Fondo de Investigaciones Sanitarias de la Seguridad Social (90/0688).
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