GASTROENTEROLOGY 1992;102:818-822

Escherichia coli Enterotoxin (ST,) Binds to Receptors, Stimulates Guanyl Cyclase, and Impairs Absorption in Rat Colon ADAM G. MEZOFF, RALPH A. GIANNELLA, and MITCHELL B. COHEN

MICHAEL

N. EADE,

Division of Pediatric Gastroenterology, Children’s Hospital Research Foundation, Cincinnati, Ohio; Division of Digestive Diseases. Universitv of Cincinnati. and Veterans Administration Hospital, Cincinnati, Ohio; and Department of Physiology, Auckland, New Zealand

To determine the contribution of the colon in Eschediarrichia coli heat-stable enterotoxin-mediated rhea1 disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared. Scatchard analysis suggested a single class of heat-stable enterotoxin receptors with an affinity constant of binding of 10' L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5fold greater in colonocytes than ileocytes (8.32 + 1.33X 10'~s. 2.33IfI 0.28X105 receptors per cell; P = 0.02). Heat-stable enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response. Heatstable enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (-47.8vs. -48.4pL* cm-‘. h-l, respectively) at a dose of 8 nmol/L. At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. At this dose in the ileum, heat-stable enterotoxin induced net secretion. These data are consistent with the concept that heat-stable enterotoxin-induced diarrhea1 disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion. nterotoxigenic Escherichia coli causes diarrhea in humans and animals by the elaboration of large-molecular-weight heat-labile toxins and/or small-molecular-weight heat-stable toxins.’ The heat-stable toxin (STJ produced by E. coli is a polypeptide that binds to an intestinal brush border membrane (BBM) receptor,‘r3 stimulates membrane bound guanyl cyclase,4 and induces intestinal secretion.5 ST, has been thought to primarily affect the small bowel; however, we have previously shown the presence of ST, receptors and ST,-mediated guanyl cyclase activation in human colonic mu-

E

cosa.6,7 In addition, ST, has been shown to increase transepithelial electric potential difference and short-circuit current in Ussing chamber-mounted rabbit cecum and proximal rat co10n.8vgThese data suggest that the colon has the ability to respond to ST,; however, the magnitude of the colonic response, in comparison with the small intestinal response, is unclear. To test the hypothesis that the colon significantly contributes to ST,-mediated diarrhea1 disease, we compared ST, binding, ST,-induced guanyl cyclase activation, and ST,-induced net water flux in the colon and ileum. Materials and Methods Colonocyte

Preparation

Colonocytes were prepared using a modification of the method of Roediger and Truelove.” Two nonfasting, male Sprague-Dawley rats weighing 200-300 g were killed, and one 5-cm segment of proximal colon was removed from each rat. Proximal colon was defined as starting l-2 cm distal to the cecum and measuring 350 mCi/mL) was obtained from Amersham (Arlington Heights, IL). Enzymatic reagents were obtained from Sigma Chemical Co., (St. Louis, MO). All other chemicals used were of reagent grade. Data Presentation

and Statistical

Analysis

Individual points were determined in duplicate in the ST, binding experiments and triplicate in the guanyl cyclase activation experiments. Perfusion studies consisted of 6-10 separate experiments at each dose with one experiment consisting of one loop at one dose in one rat in either the colon or ileum. Data are expressed as means +

0.

0.01

0.1

1 .o

10

100

STa (ntv!) Figure 1. Competitive inhibition of [‘%I]ST, binding to isolated colonocytes and ileocytes. Cell suspensions were incubated for 30 minutes at 37’C with 30 pmol/L [laSI]ST, in the presence of increasing concentrations (0.05-500 nmol/L) of native ST,. Specific binding of [lasI]ST, was calculated as described in Materials and Methods. Binding of [‘“I]ST, was progressively inhibited by increasing doses of native ST, in all cell suspensions of colonocytes and ileocytes. Maximal counts per minute specifically bound per 100 pg of cell protein in colonocytes was more than twice that of ileocytes. Data are means + SE of four separate determinations.

COLONIC RESPONSETO ST, 819

March 1992

of ST, receptors per cell. Colonocytes had a 3.5-fold greater number of ST, receptors per cell than ileocytes (8.32f 1.33X lo5vs.2.33-t0.28X 105,respectively; P = 0.02). ST,-Induced

Guanyl Cyclase Activation

ST, stimulated guanyl cyclase activity in a dose-dependent manner in colonic and ileal membranes. As shown in Figure 2, the maximal ST,stimulated guanyl cyclase activation was approximately 18 pmol cGMP - mg protein-l - min-’ in both colon and ileal membranes. Sensitivity of the colon and ileum to ST,-stimulated guanyl cyclase activation, as measured by dose for 50% of maximal response (ED,,), was also identical (45.0+ 21.0 vs. 49.8+ 7.5nmol/L ST,, respectively). ST,-Induced

Water Flux

Experiments to evaluate the effect of ST, on net water flux in the colon and ileum are shown in Figures 3 and 4, respectively. Water movement was determined in Is-minute study periods. In the first two periods, a toxin-free solution was perfused, and these periods served as the basal or control periods. In the next four periods, ST, was added to the perfusion solution. During the last five periods or recovery phase, the initial toxin-free solution was again perfused. As shown in Figure 3, ST, significantly decreased net water absorption in the colon but did not induce net secretion. As shown in Figure 4, in the

I 20--

?? -•COLON O-O ILEUM

15--

1.0

100

1000

STa ;:h4) Figure 2. Effect of increasing doses of ST, on guanyl cyclase activation in colonic and ileal membranes. Membranes were incubated with increasing doses of ST, for 5 minutes at 32% cGMP generated was measured by radioimmunoassay as described in Materials and Methods. ST, stimulated guanyl cyclase activation in a dose-dependent manner in all colonic and ileal specimens tested. Maximal guanyl cyclase activation and ED, were the same in colon and ileum. Data are means + SE of three separate determinations.

-20

4

-404

:

0

15

: 30

I 45

I 60

: 75

: 90

:

:

:

:

:

105

120

135

150

165

Minutes Figure 3. ST,-induced water flux in continuously perfused segments of colon. A balanced electrolyte solution (see Materials and Methods), containing the nonabsorbable marker [“C]PEG with and without 6 nmol/L ST,, was perfused at a rate of 6.5 mL/min. After an initial 36minute equilibration period, water movement was determined in 1115-minutestudy periods. In the first 2 periods, the basal phase, a toxin-free solution was perfused. In the next 4 periods (stippled), toxin was added to the perfusion solution. In the last 5 periods, the recovery phase, the initial toxin-free solution was again perfused. Data are means + SE of six separate determinations. ANOVA of all 11 perfusion periods showed that the means were significantly different (P = 0.0001). ANOVA between periods 2 (basal), 6 (toxin), and 11 (recovery) showed that the means of these periods were also significantly different (P = 0.0001). There was no difference between the means of the basal and recovery periods.

ileum, the basal level of absorption was less than that in the colon and the addition of ST, to the perfusate did induce net secretion. However, as shown in Table 1,at a dose of 8 nmol/L of ST,, the difference in mean net water flux, between average basal value, and average value during the toxin period was the same in both colon and ileum. Although the effect of ST, on colonic water flux was not significant at a dose of 3 nmol/L, the magnitude of the ST,-induced change was also similar to that observed in the ileum at this dose. Thus, in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. In the ileum, this change induced net secretion. As shown in Table 1, at higher doses of ST,, secretion could also be induced in the colon. In both the colon and ileum, there was rapid onset of ST, action, and maximal toxin effect was shown in the second Is-minute period study period. These effects of ST, on net water flux were also rapidly and completely reversible. The response to toxin and return to baseline during perfusion with toxin-free solution may be even more rapid than depicted in Figures 3 and 4, because the experimental design requires a small amount of residual dead space in the perfusing tubing as well as the volume within the 15-cm loop.

820 MEZOFF ET AL.

GASTROENTEROLOGY Vol. 102, No. 3

140-

I

120.. too--

,________________, Recovery

-2o--401 0

: 15

: 30

I 45

: 60

: 75

: : : I : :' SO 105 120 135 150 165

Minutes Figure 4. ST.-induced water flux in continuously perfused segments of ileum. The experimental design was similar to that used in the colonic experiments (Figure 3); the dose of ST, shown is 8 nmol/L. Data are means + SE of six separate determinations. ANOVA of all 11 perfusion periods showed that the means were significantly different (P = 0.0001). ANOVA between periods 2 (basal), 6 (toxin), and 11 (recovery) showed that the means of these periods were also significantly different (P = 0.0001). There was no difference between the means of the basal and recovery periods.

Discussion Our studies have shown that (a) rat colonocytes harvested from the proximal colon have a 3.5 fold greater number of ST, receptors per cell than enterocytes harvested from the ileum; (b) rat colonocytes and ileocytes have an identical K, for ST, binding (10’ L/mol); (c) ST, stimulates guanyl cyclase activation in proximal colon and distal ileum in a similar dose-dependent manner; and (d) ST, induces alteration in net water transport of a similar degree in proximal colonic and distal ileal intestinal segments when perfused with similar doses of ST,. Although the decrement in net water flux was similar, at lower doses, ST, induced a significant amount of secretion in the ileum and not in the colon. This may have been caused in part by the lower level of absorption in the basal state in the ileum. The capacity of the colon to absorb fluid, however, was greatly diminished. This decrease in fluid absorption represents a significant impairment in the ability of the host to conserve water during ST,-mediated small intestinal secretion. The possibility that the colon is significantly responsive to ST, has been suggested by previous studies. Argenzio and Whipp examined the effect of ST, on the porcine colon and documented both active bicarbonate excretion and abolition of net water transport.” They postulated that this failure of coionic absorption would have a major impact on the associated rapid dehydration observed in porcine di-

arrheal disease caused by ST,. Tantisira et al. have recently shown that ST, induces an increase in electric transepithelial potential difference in both the jejunum and proximal colon of the rata9 We have previously shown that receptors for ST, exist in T,, cells, a human colonic cell line, and that these receptors are coupled to guanyl cyclase activation.‘* We have also previously shown that ST, receptors exist in the human colon6 and that ST,-induces guanyl cyclase activation in both human colon and ileum.’ Thus, considerable data now exist to suggest that the colon is a likely target organ for ST,. We chose to study the proximal colon in our large intestinal experiments because in Ussing chamber studies in rabbits, ST, induced an increased transepithelial electric potential difference and short-circuit current in jejunum, ileum, and cecum, but not in distal co1on.8 Furthermore, other investigators have shown that the responsiveness of the proximal colon to stimuli inducing water and electrolyte movement is different than the responsiveness of the distal co1on.22,23However, we did include the distal colon in our transport studies. Inclusion of the distal colon in these studies has the potential for inclusion of a less responsive distal segment. This would diminish the apparent effect of ST, and cause us to underestimate the magnitude of the proximal colonic response. The ileum was studied because it represents the final processing area for small intestinal contents before contact with the large intestine. ST, has previ-

Table Z. ST,, Effect on Intestinal

Water Flux

Water flux (@a cm-‘. h-‘) ST, dose

(nmol/L)

Basal infusion

Toxin infusion

Net change” (P)

Colon 3 8 19 32 65

82.2-t15.7 98.6f 16.3 52.2+ 9.8 84.8k 12.0 81.2+ 4.2

56.7f 18.2 50.8+ 14.6 -7.5in25.7 6.8z!c 17.2 -54.2I?35.4

-25.5k -47.8f -59.7f -78.0f -135.4f

13.4(NS) 12.2(0.05) 15.4(0.007) 11.7(0.002) 19.9(0.003)

Ileum 1.4 3 8

-3.6f 9.2 -.05f 5.7 27.2f 4.8

-17.6f 5.7 -34.9Ik7.5 -21.2+ 3.9

-14.0f 5.1(0.01) -34.4f 3.9(0.001) -48.4k 3.1(0.002)

NOTE. Data are means f SE of 6-10 separate experiments with one experiment consisting of one loop at one dose in one rat in either the colon or ileum. “The difference in mean net water flux (net change) between average baseline value (electrolyte solution without ST,) and average value during perfusion with toxin (electrolyte solution with ST, during periods five and six as described in Materials and Methods) in colon and ileum is expressed as pL - cm-’ *h-‘.

COLONIC RESPONSE TO ST,

March 1992

ously been shown to bind to ileocytes3 and modulate ileal cGMP levels.24 In addition, we have shown the ileum to be at least as sensitive and responsive to ST,-induced secretion as the jejunum in two different models, i.e., ligated loopsz5 and continuously perfused segments of small intestine.26 Furthermore, the human ileum has been shown to secrete fluid and electrolytes in response to infection with enterotoxigenie E. cohz7 High colonic flow rates resulting from toxin-mediated small bowel secretion may result in relative colonic dysfunction by exceeding the ability of the colon to absorb.‘* However, our data show that independently of flow rate, ST, can impair colonic absorption and can cause net colonic secretion at higher doses. The function of the colon is also affected by cholera toxin, the prototypic enterotoxin.2g’30 Data in humans show that the colon contributes to the clinical expression of cholera, in part, by a diminished absorptive capacity.30 We suggest that the colon may contribute to ST,-mediated diarrhea1 disease in a similar manner. However, in distinct contrast to cholera toxin, the effect of ST, on transport is rapidly reversible by colonic or ileal perfusion with ST,-free buffer at the same flow rate. This rapid recovery from toxin washout does not occur with cholera toxin. The reversibility of ST,-mediated secretion has also been shown in the T, human colonic carcinoma model system.31 In this system, ST, induced sustained, maximal secretion in T,, cells mounted in Ussing chambers within 20-30 minutes. Removal of ST,, by addition of anti-ST, monoclonal antibody 30 minutes after addition of ST,, resulted in levels of cGMP that approached control values within 20-30 minutes. This time course is similar to that observed by us in the perfusion studies. We have also shown that ST, is inactivated in the intestine and that the secretory response is proportional to the rate and degree of inactivation.” Both of these observations support our hypothesis that ST, must remain bound to its receptor for activation of guanyl cyclase and secretion to occur. We have shown that ST, induces an identical dose-dependent activation of guanylate cyclase in the colon and ileum. There is also similar ST,-induced net water flux in the proximal rat colon and ileum. However, whereas the K, of ST, binding is also identical in the proximal colon and ileum, there are more receptors for ST, in the colon than in the ileum. The presence of a greater number of ST, receptors despite the generation of an equal secondary messenger response and an equal change in net water flux suggests the possibility of a different receptor-effector coupling ratio in the colon or the presence of spare receptors. Because our experiments to determine ST, binding and guanylate cyclase activa-

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tion were performed under different conditions, we cannot directly calculate the coupling ratios of ST, binding and ST, activation of guanylate cyclase. Recently, a new plasma membrane form of guanyl cyclase has been shown to function as the heat-stable enterotoxin receptor in rat small intestine.32 In this study, there was no Northern blot analysis of rat colon; therefore, the presence of an identical ST, receptor in rat small intestine and colon was not addressed. In addition, the possibility remains that there are multiple receptors for ST,, including clearance receptors, as have been described for another member of the guanyl cyclase receptor family, i.e., atria1 natriuretic peptide receptor.33 Whereas the mechanism of reduced colonic absorption due to ST, is likely to be related to elevation of intracellular levels of the secondary messenger cGMP, our studies do not address the possibility that the enteric nervous system or cells in the lamina propria could also have a significant influence on ST,-mediated secretion in vivo. In summary, we have shown that the colon contains all the necessary components to respond to ST,, including ST, receptors and ST,-stimulated guanyl cyclase activity. In addition, on the basis of our data we suggest that the colon may significantly contribute to the host’s diarrhea1 response to ST, by a decreased absorptive capacity in the face of increased small intestinal fluid secretion. References 1.

Black RE, Merson MH, Rahman AS, Yunus A, Alim AR, Huq I, Yolken RH, Curlin GT. A two-year study of bacterial, viral, and parasitic agents associated with diarrhea in rural Bangladesh. J Infect Dis 1980;142:660-664. 2. Cohen MB, Moyer MS, Luttrell M, Giannella RA. The immature rat small intestine exhibits an increased sensitivity and response to Escherichia cob heat stable enterotoxin. Pediatr Res 1986;20:555-560, 3. Giannella RA, Luttrell M, Thompson M. Binding of Escherichia coli heat-stable enterotoxin to receptors on rat intestinal cells. Am J Physiol 1983;245:G492-G498. 4. Field M, Graf LH, Laird WJ, Smith PL. Heat stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proc Nat1 Acad Sci USA 1978;75:2800-2804, 5. Giannella RA, Drake KW, Luttrell M. Development of a radioimmunoassay for E. cob heat-stable enterotoxin: comparison with the suckling mouse assay. Infect Immunol1981;33:186192. 6. Cohen MB, Guarino A, Shukla R, Giannella RA. Age-related differences in receptors for Escherichia coli heat-stable enterotoxin in the small and large intestine of children. Gastroenterology 1988;94:367-373. 7. Guarino A, Cohen MB, Giannella RA. Small and large intestinal guanylate cyclase activity in children: effect of age and stimulation by Escherichia coli heat-stable enterotoxin. Pediatr Res 1987;21:551-555. 8. Rao MC, Guandalini S, Smith PL, Field M. Mode of action of

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heat-stable Escherichia coli enterotoxin. Biochim Biophys Acta 1980;632:35-46. 9. Tantisira MH, Jodal M, Lundgren 0. Effects of heat-stable Escherichia coli enterotoxin on intestinal alkaline secretion and transepithelial potential difference in the rat intestines in vivo. Stand J Gastroenterol 1990;25:19-28. 10. Roediger WEW, Truelove SC. Method of preparing isolated colonic epithelial cells (colonocytes) for metabolic studies. Gut 1979;20:484-488, 11. Binder HJ, Stange G, Murer H, Stieger B, Hauri HP. Sodiumproton exchange in colon brush-border membranes. Am J Physiol 1986;251:G382-G390. 12. Weiser MM. Intestinal epithelial cell surface membrane glycoprotein synthesis. 1. An indicator of cellular differentiations. J Biol Chem 1973;248:2536-2541. 13. Staples JS, Asher SE, Giannella RA. Purification and characteristics of heat-stable enterotoxin produced by a strain of E. cob pathogenic for man. J Biol Chem 1980;255:4716-4721. 14.Thompson M, Luttrell M, Overmann G, Giannella RA. Biological and immunological characteristics of ‘*%E. cob heat-stable enterotoxin species purified by HPLC. Anal Biochem 1985;148:26-36. 15. Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 1980;107:220-239. 16.Waldman SA, O’Hanley PD, Falkow S, Schoolnik G, Murad F. A simple, sensitive and specific assay for the heat-stable enterotoxin of Escherichia cob. J Infect Dis 1984;149:83-89. 17. Giannella RA, Serumaga J, Walls D, Drake KW. Effect of clindamycin on intestinal water and glucose transport in the rat. Gastroenterology 1981;80:907-913. 18. Rout WR, Formal SB, Dammin GJ, Giannella RA. Pathophysiology of Salmonella diarrhea in the rhesus monkey: intestinal transport, morphological and bacteriological studies. Gastroenterology 1974;67:59-70. 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. 20. Argenzio RA, Whipp SC. Effect of Escherichia coli heat-stable enterotoxin, cholera toxin, and theophylline on ion transport in porcine colon. J Physiol 1981;320:469-487. K, Giannella RA. 21. Guarino A, Cohen MB, Dharmsathaphorn T, cell receptor binding and guanyl cyclase activation by Escherichia coli heat-stable toxin. Am J Physiol 1987; 253:G775-G780. 22. Foster ES, Budinger ME, Hayslett JP, Binder HJ. Ion transport in proximal colon of the rat. Sodium depletion stimulates neutral sodium chloride absorption. J Clin Invest 1986; 77:228-235. 23. Sellin JH, DeSoigne R. Ion transport in human colon in vitro. Gastroenterology 1987;93:441-448.

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24. Guandalini S, Mrinalini CR, Smith PL, Field M. cGMP modulation of ileal transport: in vitro effects of Escherichia cob heatstable enterotoxin. Am J Physiol 1983;245:G492-G498. 25. Cohen MB, Thompson MR, Giannella RA. Differences in jejunal and ileal response to E. cob enterotoxin: possible mechanisms. Am J Physiol 1989;257:G118-G123. 26. Giannella RA, Walls D, Eade MN. Effect of pure E. coli heatstable enterotoxin on small and large intestinal water and glucose absorption, mucosal histology, and intestinal permeability. In: Kuwahara S, Pierce NF, eds. Advances in research on cholera and related diarrheas. 3. Tokyo: KTK Scientific, 1986:327-332. 27. Banwell JG, Gorbach SL, Pierce NF, Mitra R, Mondal A. Acute undifferentiated human diarrhea in the tropics. II. Alterations in intestinal fluid and electrolyte metabolism. J Clin Invest 1971;50:890-900. 28. Levitan R, Fordtran JS, Burrows BA, Ingelfinger FJ. Water and salt absorption in the human colon. J Clin Invest 1962; 41:1754-1759. 29. Donowitz M, Binder HJ. Effect of enterotoxins of Vibrio cholerae, Escherichia coli, and Shigella dysenteriae type 1 on fluid and electrolyte transport in colon. J Infect Dis 1976;134:135143. 30. Speelman P, Butler T, Kabir I, Ali A, Banwell J. Colonic dysfunction during cholera. Gastroenterology 1986;91:11641170. K. Reversal of E. 31. Giannella RA, Huott P, Dharmsathaphorn cob heat-stable enterotoxin-induced secretion and guanyl cyclase activation by anti-ST, monoclonal antibody (abstr). Gastroenterology 1987;92:A1403. 32. Schulz S, Green CK, Yuen PST, Garbers DL. Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell 1990;63:941-948. 33. Maack T, Suzuki M, Almeida FA, Nussenzweig D, Scarborough RM, McEnroe GA, Lewicki JA. Physiologic role of silent receptors of atria1 natriuretic peptide. Science 1987;238:675678.

Received January 28, 1991. Accepted August 9,199l. Address requests for reprints to: Mitchell B. Cohen, M.D., Division of Gastroenterology, Children’s Hospital Medical Center, 3250 Elland Avenue, Cincinnati, Ohio 45229-2899. Supported by National Institutes of Health grant DK-1908 from the U.S. Public Health Service, American Gastroenterological Association Industry (Glaxo) Scholar Award, and research grant 5393108-01from the Veterans Administration. Presented in part at the North American Society for Pediatric Gastroenterology and Nutrition/European Society for Pediatric Gastroenterology and Nutrition meetings (Amsterdam, Holland May 23-26 1990) and published in abstract form (Pediatr Res 1990;27:541). The authors thank Cynthia Daugherty, M.D., for her assistance.