INFECTION AND IMMUNITY, Aug. 1978, p. 373-380 0019-9567/78/0021-0373$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 21, No. 2
Printed in U.S.A.
Cholera Toxin Effects on Fluid Secretion, Adenylate Cyclase, and Cyclic AMP in Porcine Small Intestine G. W. FORSYTH,* D. L. HAMILTON, K. E. GOERTZ, AND M. R. JOHNSON Department of Veterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Received for publication 2 March 1978
The effects of cholera toxin on mucosal cyclic nucleotide concentrations and on net fluid secretion in the porcine small intestine are reported. Cholera toxin causes net secretion of fluid into the small intestine of weanling pigs, and secretary rates are dependent on the dose of the toxin placed in intestinal loops. Intestinal secretion due to cholera toxin exposure was not consistently accompanied by elevated concentrations of mucosal cyclic AMP or cyclic GMP. Net fluid fluxes in individual loops did not correlate with mucosal cyclic AMP concentration in the same loop. Jejunal adenylate cyclase was activated to a lesser extent in pigs, compared with rabbits, after in vivo treatment with cholera toxin. In vitro activation in cell-free homogenates was similar for both species. Papaverine was similar to cholera toxin in causing fluid secretion without cyclic AMP accumulations, but 3-isobutyl-l-methyl xanthine significantly increased cyclic AMP concentration and induced fluid secretion in pigs. Weanling pigs appeared to differ from rabbits in having a secretary response to cholera toxin which was independent of elevations in total mucosal cyclic AMP concentration. Active secretion of electrolytes and water into the gut lumen is a major feature of diarrheal disease caused by enterotoxigenic bacteria. Intestinal colonization by Vibrio cholerae or by certain strains of Escherichia coli may cause a severe diarrhea through inhibition of Na' and Cl- absorptive processes and by inducing active ion secretion (1, 2, 7). Cyclic 3',5'-AMP (cAMP) concentrations in intestinal mucosa are elevated in dogs, rabbits, and humans after exposure to cholera toxin (CT) or to the E. coli heat-labile enterotoxin (4, 15, 20). Phosphodiesterase inhibitors such as theophylline and caffeine induce fluid secretion into the intestinal lumen (1, 19, 25), and in vitro addition of cAMP to rabbit leal mucosa causes changes in ion flux and short circuit current which mimic the action of CT or theophylline (5, 19). These observations support a model of fluid secretion mediated by increased intracellular cAMP concentration resulting from adenylate cyclase activation. However, cAMP-independent secretary processes may occur. Expansion of fluid volume by rapid saline infusion or intraluminal addition of the heat-stable enterotoxin of E. coli are believed to cause net fluid secretion into the gut without activation of adenylate cyclase or increases in mucosal cAMP concentration (3, 12; D. L. Hamilton, W. E. Roe, and N. 0. Nielsen, Can. J. Comp. Med., in press). Recent reports also suggest that some of the secretary effects of
CT are not related to cAMP. Epinephrine was reported to decrease CT-induced cAMP concentrations without affecting secretion, and application of CT to the serosal side of stripped rabbit ileal mucosa elevated cAMP concentration without the expected secretary response (8, 24). Porcine intestinal mucosal cAMP concentrations were not increased by CT doses which caused fluid secretion (Hamilton et al., Can. J. Comp. Med., in press). The role of cAMP in regulation of intestinal secretion needs some clarification in light of these conflicting observations. This study was designed to examine the role of cAMP and adenylate cyclase in intestinal fluid secretion induced by CT in the pig small intestine.
MATERIAlS AND MERHODS Animals and surgical procedure. Cross-bred, weanling pigs (5 to 8 weeks old), weighing between 5 and 7 kg, and New Zealand white rabbits (8 to 12 weeks old) were used in these studies. Flux studies and intestinal sampling were carried out under general anesthesia induced and maintained by halothane. Subjects were positioned in dorsal recumbency, and the abdomen was entered by midline incision. Isolated loops of proximal jejunum (20 cm in length in pigs or 10 cm in rabbits, as measured in situ) were prepared aborally from the Treitz ligament. One end of the loop was ligated, and a lucite cannula was inserted in the other end for sample introduction and removal. For studies in the ileum, loops were placed orad to the
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leal cecal ligament, approximately 40 cm proximal to the ileal-cecal valve. Flux studies. Net fluid flux was measured after incubation with CT by draining the loops and introducing the appropriate loop fluid and ['4C]polyethylene glycol 4000 (0.5 g/liter) as a dilution marker. Proximal jejunal fluid contained 140 mM Na', 5.0 mM K+, 120 mM C1-, and 20 mM HC03-. Chloride and bicarbonate concentrations were changed to 40 mM and 105 mM, respectively, for ileal studies (14). For acute flux studies, 3-methyl isobutyl xanthine (MIX) or papaverine was dissolved in and administered with the loop fluid. Net fluid volume changes were calculated from changes in ['4C]polyethylene glycol concentrations by published procedures (14). Adenylate cyclase and cyclic nucleotides. At the completion of flux measurements, intestinal loops were removed and immediately chilled on ice, the serosal and muscularis layers were stripped off, and mucosal samples were obtained. For cyclic nucleotide determinations, mucosal samples of 100 to 200 mg (wet weight) were homogenized in 5 ml of 6% trichloroacetic acid at 4°C within 40 s of loop removal. Protein concentration of the trichloroacetic acid suspension was determined by the method of Zak and Cohen (27). Trichloroacetic acid was removed from a 2-ml volume of the supernatant solution resulting from centrifugation at 10,000 x g by three washings with diethyl ether, and the trichloroacetic acid-free extract was dried in a forced-air oven and reconstituted to 500-1d volume with tris(hydroxymethyl)aminomethane (Tris)-hydrocloride. cAMP was measured by a competitive protein binding technique (with a cAMP assay kit; Amersham/Searle, Arlington Heights, Ill.); cGMP was measured by a commercial radioimmunoassay procedure (with a cGMP RIA kit; Amersham/Searle). With this assay system, the recovery of cAMP (4 or 8 pmol) added to pig jejunal trichloroacetic acid homogenates averaged 102 ± 22%. Adenylate cyclase activity was assayed in mucosal samples homogenized in 60 volumes of 10 mM N'-2hydroxyethyl piperazine-N-2-ethanesulfonic acid (HEPES; pH 8.3) with 1.0 mM disodium ethylenediaminetetraacetic acid (EDTA). The assay system contained 2 mM ATP, an ATP regenerating system, and other standard ingredients (10). Linearity with time and protein concentration have been established, and cAMP production was measured by competitive protein binding (9). In vitro adenylate cyclase activation by CT. Samples of intestinal mucosa were homogenized in 10 volumes of HEPES-EDTA buffer and incubated with a range of concentrations of nicotinamide adenine dinucleotide (NAD) and CT for 10 min at 24°C. Incubated homogenates were centrifuged for 10 min at 4°C, and the supernatant solution was discarded. Pellets were suspended in 20 volumes of HEPES-EDTA buffer (based on original wet weight of tissue) and assayed for adenylate cyclase activity. Activation was measured in comparison with appropriate control samples, with NAD and CT omitted during the incubation. Neutralization of CT by gangliosides. A mixed ganglioside preparation (from bovine brain; type II, Sigma Chemical Co.) was preincubated with CT for 30 min at 24°C before CT injection into ligated jejunal
INFECT. IMMUN. loops in rabbits or pigs. One part of ganglioside was added to 10 parts of crude CT (wt/wt) for the preincubation. In these neutralization experiments, rabbit loops were injected with 5 mg of CT with or without gangliosides, and pig loops received 50 mg of CT with or without added ganglioside. Control and experimental loops were incubated for 3 h in situ before fluid flux rates and mucosal adenylate cyclase were measured. CT. Crude CT was obtained as a lyophilized broth filtrate from the National Institute of Allergy and Infectious Diseases (Wyeth lot no. 002). Purified CT was purchased from Schwarz/Mann, Orangeburg, N.Y. CT doses were dissolved in 10 ml of 0.9% NaCl for use in pig loops, or in 4 ml for rabbit loops. Statistics. Significant differences in the dose response study were determined by analysis of variance, and significantly different treatment means were detected by a multiple range test.
RESULTS The potency of several concentrations of a crude preparation of CT was assessed by measuring in situ fluid secretion into ligated intestinal loops after incubation with the toxin. Fluid secretion was recorded by two independent techniques. The amount of fluid collected from a loop at the end of a 3-h incubation period with CT gave an estimate of total secretary capacity in this time period. The actual secretion rate 3 h after CT addition was estimated after removal of the accumulated fluid by changes in marker polyethylene glycol concentration during a 10min flux study. The effect of different amounts of CT on these secretion measurements is shown in Fig. 1. Both of the indexes increased with CT dose over a range of 3 to 1,000 mg of crude CT per intestinal loop, and all values measured after CT treatment were significantly higher than in control loops (P < 0.05). There was no clear evidence for saturation ofthe secretary response, even with 1 g of crude CT per loop. The activity of intestinal mucosal adenylate cyclase and the mucosal cell concentrations of cAMP were measured, along with rates of fluid secretion. Changes in these parameters in the same study are shown in Fig. 2. Significant increases in adenylate cyclase activity (P < 0.05) occurred with 3 to 30 mg of CT per loop. Adenylate cyclase activity decreased with doses of CT above 30 mg per loop and declined to basal values with CT concentrations which produced the maximum secretary response. cAMP concentration in intestinal mucosa was significantly elevated above control values at CT doses of 100 and 300 mg per loop (P < 0.05) but then decreased as CT dose and fluid secretion increased. Any potential masking of significant effects by variations in basal levels of adenylate cyclase or cAMP among pigs was investigated in the fol-
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CHOLERA TOXIN IN PORCINE SMALL INTESTINE
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FIG. 1. Secretory responses to crude CT in ligated pigjejunal loops. Responses were measured as the total fluid accumulated in closed jejunal loops for a 3-h period after introduction of CT. Net fluid volume changes (flux rate) were measured by the change in concentration ofpolyethylene glycol during a 10-min study carried out at the end of the 3-h incubation with CT. Each point is the average of observations on eight separate loops, with standard deviation indicated. Proximal Jejunum I 156
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FIG. 2. CT effects onjejunal adenylate cyclase and cAMP in pigs. Ligatedjejunal loops were incubated for 3 h with the indicated levels of CT. Then cAMP content and adenylate cyclase activity were determined in jejunal mucosal samples which were processed immediately after removal ofintestinal tissue from anesthetized pigs. Each point is the average of duplicate determinations on eight separate loops, with standard deviation indicated. (*) Significant differences from control values (P < 0.05).
lowing manner. Correlation of independent variables from individual intestinal loops was studied to measure the relationships of fluid content, fluid flux rate, cAMP concentration, and adenylate cyclase activity within loops. In addition, regression equations were developed to describe the dependence of each parameter on the independent variable, the CT dose (Table 1). The regression equations indicate that loop fluid content and flux rate were much more dependent on CT dose than were adenylate cyclase activity or mucosal cAMP concentration. Based on the values for the correlation coefficients, 56% of the variation in fluid per loop was dependent on flux rate changes, whereas only 23% could be attributed to cAMP concentration changes. Less than 2% of the variation in flux rate changes was
determined by changes in adenylate cyclase activity or cAMP concentration. Changes in mucosal cyclic nucleotide concentration induced by an intermediate level of CT were investigated in the pig jejunum. A 100-mg amount of CT was used in four ligated 20-cm loops in each of four pigs, with a similar number of control loops. Each loop was sampled twice for cAMP concentration and four times for assay of adenylate cyclase activity. The mucosal cAMP concentration was not significantly increased after 3 h of incubation with CT (Table 2). Cyclic 3',5'-guanosine monophosphate concentration was reduced slightly, but not significantly, and adenylate cyclase activity increased by 50% (P 0.05; n = The role of CT receptors in adenylate cyclase 24. ePicomoles per milligram of protein per 20 min; P activation was compared in pigs and rabbits. Preincubation of CT with ganglioside neutral< 0.001; n = 64. ized secretary and adenylate cyclase effects of using purified CT, because the crude toxin con- CT in both species (Table 4). The binding of CT tains large amounts of nontoxin material which to cell membrane receptors appeared to be an might influence secretion by osmotic or enzy- obligatory step for fluid secretion in rabbits and matic means. The 20-cm jejunal loops had a pigs. The extent of potential activation of rabbit linear secretary response to the logio of CT dose from 2.5 to 250 ,ug of CT per loop. Net flux, and pig jejunal adenylate cyclase by CT was cAMP, and adenylate cyclase data are shown in estimated in a cell-free activation system. The Table 3. Correlation coefficients were calculated response of adenylate cyclase activity of cell-free as before, comparing secretion rates, cAMP con- homogenates to in vitro activation is shown in centration, and adenylate cyclase activity within Table 5. This system, which is reported to be each loop to look for any association between free of receptor requirements (10), gave similar secretary rate and changes in the adenylate cy- degrees of activation for rabbit and pig adenylate clase system (Table 3). As found with the crude cyclase. Enzyme activity doubled in both species CT, secretary effects were not associated with after a 10-min preincubation with 5 mM NAD increases in total mucosal cAMP concentration. and 1 mg of crude CT per ml. Therefore, the pig CT effects on unidirectional ion flux are enzyme did not show any unique inherent limits known to vary depending on the region of the to CT-induced activation in this system. Other intestinal secretagogues were investigut chosen for study. Ileal effects were also investigated because the pig jejunal response is gated in a search for differing effects on the increased unidirectional Na' secretion, whereas jejunal adenylate cyclase-cAMP system in pigs. decreased unidirectional Na+ absorption has A comparison of the responses of pig jejunal TABLE 2. Mucosal cyclic nucleotide concentrations and adenylate cyclase activity after exposure ofpig jejunal loops to crude CTa Concn of:'
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TABLE 3. Pig jejunal loop responses to incubation with purified CT' Dose of purified CT (ag) Determination
Fluid (ml/20-cm loop)b Net fluid flux rate (ll/loop per
0 1.4 ± 1.7
2.5 3.2 ± 2.6
0
35 ± 33
250 7.5 ± 2.2 107 ± 43
25 5.1 ± 2.0 61 ± 14
p
< 0.001 < 0.001
min)c
0.82 21.2 ± 7.9 22.7 ± 10.8 21.5 ± 9.0 Mucosal cAMP (pmol/mg of pro- 19.8 ± 7.1 tein) 0.008 99± 33 125± 35 120 ±46 Adenylate cyclase (pmol/mg of 75± 34 protein per 20 min) a Correlation coefficients: log CT dose versus fluid per loop, 0.76; log CT dose versus net fluid flux rate, 0.69; ldg CT dose versus mucosal cAMP, 0.02; log CT dose versus adenylate cyclase, 0.50; fluid flux versus mucosal cAMP, 0.10; fluid flux versus adenylate cyclase, 0.22. Means not over a common line are significantly different (Pb< 0.01). Values indicate means of observations from nine separate loops ± standard deviation. c Values indicate difference between flux rates in control and CT loops. x
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FIG. 3. Secretory responses to crude CT in ligated pig ileal loops. Incubation with CT and fluid measurements were as described in the legend to Fig. 1. Points are averages of observations on six separate loops, with standard deviation indicated. (*) Significant increases above control values (P < 0.05). Distal Ileum
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loops to 20-min exposure to papaverine and to 3-isobutyl-1-methyl xanthine (MIX) is reported in Table 6. Fluid secretion was induced by 1 and
10 mM papaverine with no detectable accompanying increase in cAMP concentration in intestinal mucosa. MIX was more potent than
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TABLE 4. Neutralization of CT-induced fluid secretion by preincubation with ganglioside Jejunal loop response in:
Rabbits"
Pigs-
Type of incubation Fluid flux'
Adenylate cyclased 120 ± 46 108 ± 6
Fluid flux'
Adenylate cyclased 135 ± 47 88 ± 16
-14 ± 53 12 ± 53 Control -14 ± 76 -24 ± 29 Ganglioside plus CT 194±36 CT 146±54 88±16 215±15 indicated. added where aJejunal loops (20 cm each) with 50 mg of crude CT and 5 mg of ganglioside b Jejunal loops (10 cm each) with 5 mg of crude CT and 0.5 mg of ganglioside added where indicated. 'Values indicate microliters of fluid entering the loop per minute; mean of four observations ± standard deviation. d Values indicate picomoles per minute per milligram of protein; mean of duplicate determinations from two loops in two animals (n = 8) ± standard deviation. TABLE 5. Activation of adenylate cyclase by CT in
cell-free jejunal homogenates Adenylate cyclase activity (% control activity) in:' Concn of crude CT
(Ug/ml)
PiRabt Pigs Rabbits 0.5 mM 5.0 mM 0.5 mM 5.0 mM NAD NAD NAD NAD
104 ± 40 121 ± 27 110 ± 95 120 ± 55 103±32 131 ±31 85±42 111 ±35 107 ± 30 183 ± 17 92 ± 32 162 ± 85 117 ± 36 209 ± 55 136 ± 94 232 ± 74 a Values indicate average ± standard deviation of duplicate determinations from four separate incubations. TABLE 6. Fluid secretion into ligatedpigjejunal loops mediated by papaverine and by MIX Fluid flux' Inhibitor cAMPb 20 100 500 1,000
Papaverine 0mM 1mM 10mM
0 40±27 58±35
16±6 17±7 16±5
MIX 0 0mM 19±5 1mM 112±26 22±4c 10 mM 173 ± 37 25 ± 6d a Values indicate (in micromoles per 20-cm loop) difference in fluid movement between control loops and loops treated with the indicated concentration of phosphodiesterase inhibitor ± standard deviation. Positive values represent secretion. b Values indicate picomoles per milligram of mucosal protein after 20-min exposure to phosphodiesterase inhibitor ± standard deviation. Positive values represent secretion. Significantly greater than control (P < 0.05). d Significantly greater than control (P < 0.01).
papaverine in causing fluid secretion. MIX also caused a significant increase in mucosal cAMP concentration, which appeared to correlate with the fluid secretary response. Both MIX and papaverine are cyclic nucleotide phosphodies-
terase inhibitors in pig jejunal mucosa (G. W. Forsyth, unpublished data).
DISCUSSION Activation of adenylate cyclase is thought to play a causal role in intestinal secretary responses because fluid secretion mediated by CT, by prostaglandins, and by vasoactive intestinal peptide is associated with increased adenylate cyclase activity (16, 21, 22, 23). The effects of cyclic nucleotide phosphodiesterase inhibitors (14, 19, 25) and in vitro addition of cAMP (5, 19) support the proposed role of cAMP as an intracellular regulator of ion transport in intestinal mucosa. This model is strongly supported by evidence obtained from several species of experimental animals. Some criteria for a test of this model in another species, domestic pigs, are as follows. (i) CT levels sufficient to cause a secretory response should significantly elevate mucosal cAMP concentrations, and (ii) dose response relations should be orderly for the change in cAMP concentration and for the secretary response to CT (17). In contrast to investigations reported with dogs (13), these two criteria were not satisfied in this study by using pig small intestine. Doses of CT sufficient to induce secretion did not cause consistent increases in cAMP concentration, and secretary rates did not correlate well with mucosal cAMP concentrations. Intermediate dose levels of CT caused a significant activation of adenylate cyclase, whereas cAMP concentration increased significantly at only two dose levels in the jejunum. An involvement of cAMP in porcine intestinal secretion could be supported by a careful choice of CT dose, but low or high CT doses did not significantly affect cAMP concentration. This low correlation between cAMP concentration and secretory rates is difficult to reconcile with a regulatory role for cAMP in this process. The validity of the cAMP assay used for intestinal mucosa was supported by our findings of increased
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CHOLERA TOXIN IN PORCINE SMALL INTESTINE
cAMP after CT treatment of the rabbit ileum (Hamilton et al., Can. J. Comp. Med., in press) and by similar increases reported in this study after MIX incubation in the pig jejunum. The effects of CT in the pig jejunum occur mainly through increased unidirectional Na' secretion (14). Field has suggested that active ion secretion occurs mainly in the mucosal crypt cells and that altered cAMP concentrations in a small subpopulation of mucosal cells may not be detectable (6). The main leal response to CT is decreased unidirectional Na' absorption in the pig (14), and Na' absorption is a property of the major mucosal cell type, the villus cell (6). Therefore, CT effects associated with increased cAMP concentration could be easier to detect in the ileum than in the jejunum. CT did not significantly elevate ileal mucosal cAMP concentration in the pig even in the presence of a copious secretary response. Fluid secretion caused by cAMP accumulation in a minor population of secretary cells may be possible, but reported adenylate cyclase activation in the major villus cell population in other species (26) reduces the likelihood of selective activation in pigs. Neutralization of CT effects by preincubation with ganglioside suggests the existence of a binding requirement for CT activity which may not relate to activation of adenylate cyclase. The low activation of adenylate cyclase observed in pigs relative to that in rabbits must account in part for the absence of mucosal cAMP accumulations induced by CT in pigs. Cyclic nucleotide phosphodiesterase activity has not been studied in pig intestinal mucosa. The diminished response of porcine intestinal adenylate cyclase to activation by CT is probably not due to unusual enzyme properties, since the pig and rabbit enzyme properties are quite similar (9). The similar extent of in vitro activation of pig and rabbit jejunal adenylate cyclase by CT and NAD indicates regulatory similarities for the two enzymes. Differences in the extent of in vivo and in vitro activation of the porcine enzyme could be due to limitations at the stage of receptor binding or at some stage in the transduction process. Preparations containing E. coli heat-stable enterotoxin activity cause fluid secretion into ligated jejunal loops in pigs without obvious increases in vascular permeability or ultrastructural changes (18). A cAMP-independent secretory system must exist in species susceptible to heat-stable E. coli enterotoxin. The secretary responses elicited by papaverine also provide evidence for the existence of such a system. Papaverine has been reported to be a phosphodiesterase inhibitor (11), but it did not cause
379
cAMP accumulation in pig jejunal mucosa. The secretory effects of MIX relate directly to mucosal cAMP concentration, but the causal nature of this relationship is not definitely established. Sheerin and Field have recently reported that mucosal or serosal application of CT increases intracellular cAMP concentrations, but the strongest secretary response requires exposure to the mucosal surface of rabbit ileum to CT (24). Intestinal secretary responses to CT observed in this study may involve factors other than total mucosal cAMP concentration. ACKNOWLEDGMENTS Financial support by the Alberta Agricultural Research Trust, the Alberta Hog Producers Marketing Board, and the National Research Council is gratefully acknowledged.
LITERATURE CITED 1. Al-Awqati, Q., J. L Cameron, and W. B. Greenough Im. 1973. Electrolyte transport in human ileum: effect of purified cholera exotoxin. Am. J. Physiol. 224:818-823. 2. Al-Awqati, Q., C. K. Wallace, and W. B. Greenough III. 1972. Stimulation of intestinal secretion in vitro by culture filtrates of Escherichia coli. J. Infect. Dis.
125:300-303. 3. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974. Detection of heat-labile Escherichia coli enterotoxins with the use of adrenal cells in culture. Science 183:334-336. 4. Evans, D. J., L C. Chen, G. T. Curlin, and D. G. Evans. 1972. Stimulation of adenyl cyclase by Escherichia coli enterotoxin. Nature (London) New Biol. 236:137-138. 5. Field, M. 1971. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3',5'-AMP. Am. J. Physiol. 221: 992-997. 6. Field, M. 1976. Regulation of ion transport in the small intestine, p. 109-122. In Acute diarrhoea in childhood. CIBA Foundation Symposium no. 42. Elsevier-North Holland Pub. Co., New York. 7. Field, M, D. Fromm, Q. Al-Awqati, and W. B. Greenough HI. 1972. Effect of cholera toxin on ion transport across isolated ileal mucosa. J. Clin. Invest. 51:796-804. 8. Field, M., H. E. Sheerin, A. Henderson, and P. L Smith. 1975. Catecholamine effects on cyclic AMP levels and on ion secretion in rabbit ileal mucosa. Am. J. Physiol. 229:86-92. 9. Forsyth, G. W., D. L Hamilton, K. E. Goertz, and L. W. Oliphant. 1978. Some comparative properties and localization of porcine jejunal adenylate cyclase. Can. J. Biochem. 6:280-286. 10. Gill, D. MK 1976. Multiple roles of erythrocyte supernatant in the activation of adenylate cyclase by Vibrio cholerae toxin in vitro. J. Infect. Dis. 133:S55S63. 11. Goldberg, N. D., W. D. Lust, R. F. O'Dea, S. Wei, and A. G. O'Toole. 1970. A role of cyclic nucleotides in brain metabolism. Adv. Biochem. Psychopharmacol. 3:67-87. 12. Guerrant, R. L,. and C. C. J. Carpenter. 1975. Diarrheagenic effect of volume expansion: intestinal fluid secretion without mucosal adenyl cyclase stimulation. Johns Hopkins Med. J. 136:209-211. 13. Guerrant, R. L, L C. Chen, and G. W. G. Sharp. 1972. Intestinal adenyl-cyclase activity in canine cholera: correlation with fluid accumulation. J. Infect. Dis. 125: 377-381.
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14. Hamilton, D. L., W. E. Roe, and N. 0. Nielsen. 1977. Effect of heat stable and heat labile Escherichia coli enterotoxins, cholera toxin and theophylline on unidirectional sodium and chloride fluxes in the proximal and distal jejunum of weanling swine. Can. J. Comp. Med. 41:306-317. 15. Kantor, H. S., P. Tao, and S. L Gorbach. 1974. Stimulation of intestinal adenyl cyclase by Escherichia coli enterotoxin: comparison of strains from an infant and an adult with diarrhea. J. Infect. Dis. 129:1-9. 16. Kimberg, D. V., M. Field, J. Johnson, A. Henderson, and E. Gershon. 1971. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J. Clin. Invest. O:1218-1230. 17. Huddle, G. W., and J. G. Hardmen. 1971. Cyclic adenosine monophosphate as a mediator of hormone action. N. Engl. J. Med. 285:560-566. 18. Moon, H. W., S. C. Whipp and A. L Baetz. 1971. Comparative effects of enterotoxins from Escherichia coli and Vibrio chokrae on rabbit and swine small intestine. Lab. Invest. 25:133-140. 19. Powell, D. W., R. K. Farris, and S. T. Carbonetto. 1974. Theophylline, cyclic AMP, choleragen, and electrolyte transport by rabbit ileum. Am. J. Physiol. 227:1428-1435. 20. Schafer, D. E., W. D. Lust, B. Sircar, and N. D. Goldberg. 1970. Elevated concentrations of adenosine
3',5'-cyclic monophosphate in intestinal mucosa after treatment with cholera toxin. Proc. Natl. Acad. Sci. U.S.A. 67:851-856. 21. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, K. Field, and S. L Said. 1974. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J. Clin. Invest.
54:536-544. 22. Sharp, G. W. G., and S. Hynie. 1971. Stimulation of intestinal adqnyl cyclase by cholera toxin. Nature (London) 229:226-268. 23. Sharp, G. W. G., S. Hynie, H. Ebel, D. K. Parkinson, and P. Witkum. 1973. Properties of adenylate cyclase in mucosal cells of the rabbit ileum and the effect of cholera toxin. Biochim. Biophys. Acta 309:339-348. 24. Sheerin, H. E., and M. Field. 1977. leal mucosal cyclic AMP and CL secretion: serosal vs mucosal addition of cholera toxin. Am. J. Physiol. 232:E210-E215. 25. Wald, A., C. Back, and T. M. Bayless. 1976. Effect of caffeine on the human small intestine. Gastroenterology 71:738-742. 26. Weiser, M. M., and H. Quill. 1975. Intestinal villus and crypt cell responses to cholera toxin. Gastroenterology 69:479-482. 27. Zak, B., and J. Cohen. 1961. Automatic analysis of tissue culture proteins with stable Folin reagents. Clin. Chim. Acta 6:665-670.
Vol. 21, No. 2
Printed in U.S.A.
Cholera Toxin Effects on Fluid Secretion, Adenylate Cyclase, and Cyclic AMP in Porcine Small Intestine G. W. FORSYTH,* D. L. HAMILTON, K. E. GOERTZ, AND M. R. JOHNSON Department of Veterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Received for publication 2 March 1978
The effects of cholera toxin on mucosal cyclic nucleotide concentrations and on net fluid secretion in the porcine small intestine are reported. Cholera toxin causes net secretion of fluid into the small intestine of weanling pigs, and secretary rates are dependent on the dose of the toxin placed in intestinal loops. Intestinal secretion due to cholera toxin exposure was not consistently accompanied by elevated concentrations of mucosal cyclic AMP or cyclic GMP. Net fluid fluxes in individual loops did not correlate with mucosal cyclic AMP concentration in the same loop. Jejunal adenylate cyclase was activated to a lesser extent in pigs, compared with rabbits, after in vivo treatment with cholera toxin. In vitro activation in cell-free homogenates was similar for both species. Papaverine was similar to cholera toxin in causing fluid secretion without cyclic AMP accumulations, but 3-isobutyl-l-methyl xanthine significantly increased cyclic AMP concentration and induced fluid secretion in pigs. Weanling pigs appeared to differ from rabbits in having a secretary response to cholera toxin which was independent of elevations in total mucosal cyclic AMP concentration. Active secretion of electrolytes and water into the gut lumen is a major feature of diarrheal disease caused by enterotoxigenic bacteria. Intestinal colonization by Vibrio cholerae or by certain strains of Escherichia coli may cause a severe diarrhea through inhibition of Na' and Cl- absorptive processes and by inducing active ion secretion (1, 2, 7). Cyclic 3',5'-AMP (cAMP) concentrations in intestinal mucosa are elevated in dogs, rabbits, and humans after exposure to cholera toxin (CT) or to the E. coli heat-labile enterotoxin (4, 15, 20). Phosphodiesterase inhibitors such as theophylline and caffeine induce fluid secretion into the intestinal lumen (1, 19, 25), and in vitro addition of cAMP to rabbit leal mucosa causes changes in ion flux and short circuit current which mimic the action of CT or theophylline (5, 19). These observations support a model of fluid secretion mediated by increased intracellular cAMP concentration resulting from adenylate cyclase activation. However, cAMP-independent secretary processes may occur. Expansion of fluid volume by rapid saline infusion or intraluminal addition of the heat-stable enterotoxin of E. coli are believed to cause net fluid secretion into the gut without activation of adenylate cyclase or increases in mucosal cAMP concentration (3, 12; D. L. Hamilton, W. E. Roe, and N. 0. Nielsen, Can. J. Comp. Med., in press). Recent reports also suggest that some of the secretary effects of
CT are not related to cAMP. Epinephrine was reported to decrease CT-induced cAMP concentrations without affecting secretion, and application of CT to the serosal side of stripped rabbit ileal mucosa elevated cAMP concentration without the expected secretary response (8, 24). Porcine intestinal mucosal cAMP concentrations were not increased by CT doses which caused fluid secretion (Hamilton et al., Can. J. Comp. Med., in press). The role of cAMP in regulation of intestinal secretion needs some clarification in light of these conflicting observations. This study was designed to examine the role of cAMP and adenylate cyclase in intestinal fluid secretion induced by CT in the pig small intestine.
MATERIAlS AND MERHODS Animals and surgical procedure. Cross-bred, weanling pigs (5 to 8 weeks old), weighing between 5 and 7 kg, and New Zealand white rabbits (8 to 12 weeks old) were used in these studies. Flux studies and intestinal sampling were carried out under general anesthesia induced and maintained by halothane. Subjects were positioned in dorsal recumbency, and the abdomen was entered by midline incision. Isolated loops of proximal jejunum (20 cm in length in pigs or 10 cm in rabbits, as measured in situ) were prepared aborally from the Treitz ligament. One end of the loop was ligated, and a lucite cannula was inserted in the other end for sample introduction and removal. For studies in the ileum, loops were placed orad to the
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leal cecal ligament, approximately 40 cm proximal to the ileal-cecal valve. Flux studies. Net fluid flux was measured after incubation with CT by draining the loops and introducing the appropriate loop fluid and ['4C]polyethylene glycol 4000 (0.5 g/liter) as a dilution marker. Proximal jejunal fluid contained 140 mM Na', 5.0 mM K+, 120 mM C1-, and 20 mM HC03-. Chloride and bicarbonate concentrations were changed to 40 mM and 105 mM, respectively, for ileal studies (14). For acute flux studies, 3-methyl isobutyl xanthine (MIX) or papaverine was dissolved in and administered with the loop fluid. Net fluid volume changes were calculated from changes in ['4C]polyethylene glycol concentrations by published procedures (14). Adenylate cyclase and cyclic nucleotides. At the completion of flux measurements, intestinal loops were removed and immediately chilled on ice, the serosal and muscularis layers were stripped off, and mucosal samples were obtained. For cyclic nucleotide determinations, mucosal samples of 100 to 200 mg (wet weight) were homogenized in 5 ml of 6% trichloroacetic acid at 4°C within 40 s of loop removal. Protein concentration of the trichloroacetic acid suspension was determined by the method of Zak and Cohen (27). Trichloroacetic acid was removed from a 2-ml volume of the supernatant solution resulting from centrifugation at 10,000 x g by three washings with diethyl ether, and the trichloroacetic acid-free extract was dried in a forced-air oven and reconstituted to 500-1d volume with tris(hydroxymethyl)aminomethane (Tris)-hydrocloride. cAMP was measured by a competitive protein binding technique (with a cAMP assay kit; Amersham/Searle, Arlington Heights, Ill.); cGMP was measured by a commercial radioimmunoassay procedure (with a cGMP RIA kit; Amersham/Searle). With this assay system, the recovery of cAMP (4 or 8 pmol) added to pig jejunal trichloroacetic acid homogenates averaged 102 ± 22%. Adenylate cyclase activity was assayed in mucosal samples homogenized in 60 volumes of 10 mM N'-2hydroxyethyl piperazine-N-2-ethanesulfonic acid (HEPES; pH 8.3) with 1.0 mM disodium ethylenediaminetetraacetic acid (EDTA). The assay system contained 2 mM ATP, an ATP regenerating system, and other standard ingredients (10). Linearity with time and protein concentration have been established, and cAMP production was measured by competitive protein binding (9). In vitro adenylate cyclase activation by CT. Samples of intestinal mucosa were homogenized in 10 volumes of HEPES-EDTA buffer and incubated with a range of concentrations of nicotinamide adenine dinucleotide (NAD) and CT for 10 min at 24°C. Incubated homogenates were centrifuged for 10 min at 4°C, and the supernatant solution was discarded. Pellets were suspended in 20 volumes of HEPES-EDTA buffer (based on original wet weight of tissue) and assayed for adenylate cyclase activity. Activation was measured in comparison with appropriate control samples, with NAD and CT omitted during the incubation. Neutralization of CT by gangliosides. A mixed ganglioside preparation (from bovine brain; type II, Sigma Chemical Co.) was preincubated with CT for 30 min at 24°C before CT injection into ligated jejunal
INFECT. IMMUN. loops in rabbits or pigs. One part of ganglioside was added to 10 parts of crude CT (wt/wt) for the preincubation. In these neutralization experiments, rabbit loops were injected with 5 mg of CT with or without gangliosides, and pig loops received 50 mg of CT with or without added ganglioside. Control and experimental loops were incubated for 3 h in situ before fluid flux rates and mucosal adenylate cyclase were measured. CT. Crude CT was obtained as a lyophilized broth filtrate from the National Institute of Allergy and Infectious Diseases (Wyeth lot no. 002). Purified CT was purchased from Schwarz/Mann, Orangeburg, N.Y. CT doses were dissolved in 10 ml of 0.9% NaCl for use in pig loops, or in 4 ml for rabbit loops. Statistics. Significant differences in the dose response study were determined by analysis of variance, and significantly different treatment means were detected by a multiple range test.
RESULTS The potency of several concentrations of a crude preparation of CT was assessed by measuring in situ fluid secretion into ligated intestinal loops after incubation with the toxin. Fluid secretion was recorded by two independent techniques. The amount of fluid collected from a loop at the end of a 3-h incubation period with CT gave an estimate of total secretary capacity in this time period. The actual secretion rate 3 h after CT addition was estimated after removal of the accumulated fluid by changes in marker polyethylene glycol concentration during a 10min flux study. The effect of different amounts of CT on these secretion measurements is shown in Fig. 1. Both of the indexes increased with CT dose over a range of 3 to 1,000 mg of crude CT per intestinal loop, and all values measured after CT treatment were significantly higher than in control loops (P < 0.05). There was no clear evidence for saturation ofthe secretary response, even with 1 g of crude CT per loop. The activity of intestinal mucosal adenylate cyclase and the mucosal cell concentrations of cAMP were measured, along with rates of fluid secretion. Changes in these parameters in the same study are shown in Fig. 2. Significant increases in adenylate cyclase activity (P < 0.05) occurred with 3 to 30 mg of CT per loop. Adenylate cyclase activity decreased with doses of CT above 30 mg per loop and declined to basal values with CT concentrations which produced the maximum secretary response. cAMP concentration in intestinal mucosa was significantly elevated above control values at CT doses of 100 and 300 mg per loop (P < 0.05) but then decreased as CT dose and fluid secretion increased. Any potential masking of significant effects by variations in basal levels of adenylate cyclase or cAMP among pigs was investigated in the fol-
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FIG. 1. Secretory responses to crude CT in ligated pigjejunal loops. Responses were measured as the total fluid accumulated in closed jejunal loops for a 3-h period after introduction of CT. Net fluid volume changes (flux rate) were measured by the change in concentration ofpolyethylene glycol during a 10-min study carried out at the end of the 3-h incubation with CT. Each point is the average of observations on eight separate loops, with standard deviation indicated. Proximal Jejunum I 156
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FIG. 2. CT effects onjejunal adenylate cyclase and cAMP in pigs. Ligatedjejunal loops were incubated for 3 h with the indicated levels of CT. Then cAMP content and adenylate cyclase activity were determined in jejunal mucosal samples which were processed immediately after removal ofintestinal tissue from anesthetized pigs. Each point is the average of duplicate determinations on eight separate loops, with standard deviation indicated. (*) Significant differences from control values (P < 0.05).
lowing manner. Correlation of independent variables from individual intestinal loops was studied to measure the relationships of fluid content, fluid flux rate, cAMP concentration, and adenylate cyclase activity within loops. In addition, regression equations were developed to describe the dependence of each parameter on the independent variable, the CT dose (Table 1). The regression equations indicate that loop fluid content and flux rate were much more dependent on CT dose than were adenylate cyclase activity or mucosal cAMP concentration. Based on the values for the correlation coefficients, 56% of the variation in fluid per loop was dependent on flux rate changes, whereas only 23% could be attributed to cAMP concentration changes. Less than 2% of the variation in flux rate changes was
determined by changes in adenylate cyclase activity or cAMP concentration. Changes in mucosal cyclic nucleotide concentration induced by an intermediate level of CT were investigated in the pig jejunum. A 100-mg amount of CT was used in four ligated 20-cm loops in each of four pigs, with a similar number of control loops. Each loop was sampled twice for cAMP concentration and four times for assay of adenylate cyclase activity. The mucosal cAMP concentration was not significantly increased after 3 h of incubation with CT (Table 2). Cyclic 3',5'-guanosine monophosphate concentration was reduced slightly, but not significantly, and adenylate cyclase activity increased by 50% (P 0.05; n = The role of CT receptors in adenylate cyclase 24. ePicomoles per milligram of protein per 20 min; P activation was compared in pigs and rabbits. Preincubation of CT with ganglioside neutral< 0.001; n = 64. ized secretary and adenylate cyclase effects of using purified CT, because the crude toxin con- CT in both species (Table 4). The binding of CT tains large amounts of nontoxin material which to cell membrane receptors appeared to be an might influence secretion by osmotic or enzy- obligatory step for fluid secretion in rabbits and matic means. The 20-cm jejunal loops had a pigs. The extent of potential activation of rabbit linear secretary response to the logio of CT dose from 2.5 to 250 ,ug of CT per loop. Net flux, and pig jejunal adenylate cyclase by CT was cAMP, and adenylate cyclase data are shown in estimated in a cell-free activation system. The Table 3. Correlation coefficients were calculated response of adenylate cyclase activity of cell-free as before, comparing secretion rates, cAMP con- homogenates to in vitro activation is shown in centration, and adenylate cyclase activity within Table 5. This system, which is reported to be each loop to look for any association between free of receptor requirements (10), gave similar secretary rate and changes in the adenylate cy- degrees of activation for rabbit and pig adenylate clase system (Table 3). As found with the crude cyclase. Enzyme activity doubled in both species CT, secretary effects were not associated with after a 10-min preincubation with 5 mM NAD increases in total mucosal cAMP concentration. and 1 mg of crude CT per ml. Therefore, the pig CT effects on unidirectional ion flux are enzyme did not show any unique inherent limits known to vary depending on the region of the to CT-induced activation in this system. Other intestinal secretagogues were investigut chosen for study. Ileal effects were also investigated because the pig jejunal response is gated in a search for differing effects on the increased unidirectional Na' secretion, whereas jejunal adenylate cyclase-cAMP system in pigs. decreased unidirectional Na+ absorption has A comparison of the responses of pig jejunal TABLE 2. Mucosal cyclic nucleotide concentrations and adenylate cyclase activity after exposure ofpig jejunal loops to crude CTa Concn of:'
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VOL. 21, 1978
TABLE 3. Pig jejunal loop responses to incubation with purified CT' Dose of purified CT (ag) Determination
Fluid (ml/20-cm loop)b Net fluid flux rate (ll/loop per
0 1.4 ± 1.7
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0
35 ± 33
250 7.5 ± 2.2 107 ± 43
25 5.1 ± 2.0 61 ± 14
p
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0.82 21.2 ± 7.9 22.7 ± 10.8 21.5 ± 9.0 Mucosal cAMP (pmol/mg of pro- 19.8 ± 7.1 tein) 0.008 99± 33 125± 35 120 ±46 Adenylate cyclase (pmol/mg of 75± 34 protein per 20 min) a Correlation coefficients: log CT dose versus fluid per loop, 0.76; log CT dose versus net fluid flux rate, 0.69; ldg CT dose versus mucosal cAMP, 0.02; log CT dose versus adenylate cyclase, 0.50; fluid flux versus mucosal cAMP, 0.10; fluid flux versus adenylate cyclase, 0.22. Means not over a common line are significantly different (Pb< 0.01). Values indicate means of observations from nine separate loops ± standard deviation. c Values indicate difference between flux rates in control and CT loops. x
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FIG. 3. Secretory responses to crude CT in ligated pig ileal loops. Incubation with CT and fluid measurements were as described in the legend to Fig. 1. Points are averages of observations on six separate loops, with standard deviation indicated. (*) Significant increases above control values (P < 0.05). Distal Ileum
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loops to 20-min exposure to papaverine and to 3-isobutyl-1-methyl xanthine (MIX) is reported in Table 6. Fluid secretion was induced by 1 and
10 mM papaverine with no detectable accompanying increase in cAMP concentration in intestinal mucosa. MIX was more potent than
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TABLE 4. Neutralization of CT-induced fluid secretion by preincubation with ganglioside Jejunal loop response in:
Rabbits"
Pigs-
Type of incubation Fluid flux'
Adenylate cyclased 120 ± 46 108 ± 6
Fluid flux'
Adenylate cyclased 135 ± 47 88 ± 16
-14 ± 53 12 ± 53 Control -14 ± 76 -24 ± 29 Ganglioside plus CT 194±36 CT 146±54 88±16 215±15 indicated. added where aJejunal loops (20 cm each) with 50 mg of crude CT and 5 mg of ganglioside b Jejunal loops (10 cm each) with 5 mg of crude CT and 0.5 mg of ganglioside added where indicated. 'Values indicate microliters of fluid entering the loop per minute; mean of four observations ± standard deviation. d Values indicate picomoles per minute per milligram of protein; mean of duplicate determinations from two loops in two animals (n = 8) ± standard deviation. TABLE 5. Activation of adenylate cyclase by CT in
cell-free jejunal homogenates Adenylate cyclase activity (% control activity) in:' Concn of crude CT
(Ug/ml)
PiRabt Pigs Rabbits 0.5 mM 5.0 mM 0.5 mM 5.0 mM NAD NAD NAD NAD
104 ± 40 121 ± 27 110 ± 95 120 ± 55 103±32 131 ±31 85±42 111 ±35 107 ± 30 183 ± 17 92 ± 32 162 ± 85 117 ± 36 209 ± 55 136 ± 94 232 ± 74 a Values indicate average ± standard deviation of duplicate determinations from four separate incubations. TABLE 6. Fluid secretion into ligatedpigjejunal loops mediated by papaverine and by MIX Fluid flux' Inhibitor cAMPb 20 100 500 1,000
Papaverine 0mM 1mM 10mM
0 40±27 58±35
16±6 17±7 16±5
MIX 0 0mM 19±5 1mM 112±26 22±4c 10 mM 173 ± 37 25 ± 6d a Values indicate (in micromoles per 20-cm loop) difference in fluid movement between control loops and loops treated with the indicated concentration of phosphodiesterase inhibitor ± standard deviation. Positive values represent secretion. b Values indicate picomoles per milligram of mucosal protein after 20-min exposure to phosphodiesterase inhibitor ± standard deviation. Positive values represent secretion. Significantly greater than control (P < 0.05). d Significantly greater than control (P < 0.01).
papaverine in causing fluid secretion. MIX also caused a significant increase in mucosal cAMP concentration, which appeared to correlate with the fluid secretary response. Both MIX and papaverine are cyclic nucleotide phosphodies-
terase inhibitors in pig jejunal mucosa (G. W. Forsyth, unpublished data).
DISCUSSION Activation of adenylate cyclase is thought to play a causal role in intestinal secretary responses because fluid secretion mediated by CT, by prostaglandins, and by vasoactive intestinal peptide is associated with increased adenylate cyclase activity (16, 21, 22, 23). The effects of cyclic nucleotide phosphodiesterase inhibitors (14, 19, 25) and in vitro addition of cAMP (5, 19) support the proposed role of cAMP as an intracellular regulator of ion transport in intestinal mucosa. This model is strongly supported by evidence obtained from several species of experimental animals. Some criteria for a test of this model in another species, domestic pigs, are as follows. (i) CT levels sufficient to cause a secretory response should significantly elevate mucosal cAMP concentrations, and (ii) dose response relations should be orderly for the change in cAMP concentration and for the secretary response to CT (17). In contrast to investigations reported with dogs (13), these two criteria were not satisfied in this study by using pig small intestine. Doses of CT sufficient to induce secretion did not cause consistent increases in cAMP concentration, and secretary rates did not correlate well with mucosal cAMP concentrations. Intermediate dose levels of CT caused a significant activation of adenylate cyclase, whereas cAMP concentration increased significantly at only two dose levels in the jejunum. An involvement of cAMP in porcine intestinal secretion could be supported by a careful choice of CT dose, but low or high CT doses did not significantly affect cAMP concentration. This low correlation between cAMP concentration and secretory rates is difficult to reconcile with a regulatory role for cAMP in this process. The validity of the cAMP assay used for intestinal mucosa was supported by our findings of increased
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CHOLERA TOXIN IN PORCINE SMALL INTESTINE
cAMP after CT treatment of the rabbit ileum (Hamilton et al., Can. J. Comp. Med., in press) and by similar increases reported in this study after MIX incubation in the pig jejunum. The effects of CT in the pig jejunum occur mainly through increased unidirectional Na' secretion (14). Field has suggested that active ion secretion occurs mainly in the mucosal crypt cells and that altered cAMP concentrations in a small subpopulation of mucosal cells may not be detectable (6). The main leal response to CT is decreased unidirectional Na' absorption in the pig (14), and Na' absorption is a property of the major mucosal cell type, the villus cell (6). Therefore, CT effects associated with increased cAMP concentration could be easier to detect in the ileum than in the jejunum. CT did not significantly elevate ileal mucosal cAMP concentration in the pig even in the presence of a copious secretary response. Fluid secretion caused by cAMP accumulation in a minor population of secretary cells may be possible, but reported adenylate cyclase activation in the major villus cell population in other species (26) reduces the likelihood of selective activation in pigs. Neutralization of CT effects by preincubation with ganglioside suggests the existence of a binding requirement for CT activity which may not relate to activation of adenylate cyclase. The low activation of adenylate cyclase observed in pigs relative to that in rabbits must account in part for the absence of mucosal cAMP accumulations induced by CT in pigs. Cyclic nucleotide phosphodiesterase activity has not been studied in pig intestinal mucosa. The diminished response of porcine intestinal adenylate cyclase to activation by CT is probably not due to unusual enzyme properties, since the pig and rabbit enzyme properties are quite similar (9). The similar extent of in vitro activation of pig and rabbit jejunal adenylate cyclase by CT and NAD indicates regulatory similarities for the two enzymes. Differences in the extent of in vivo and in vitro activation of the porcine enzyme could be due to limitations at the stage of receptor binding or at some stage in the transduction process. Preparations containing E. coli heat-stable enterotoxin activity cause fluid secretion into ligated jejunal loops in pigs without obvious increases in vascular permeability or ultrastructural changes (18). A cAMP-independent secretory system must exist in species susceptible to heat-stable E. coli enterotoxin. The secretary responses elicited by papaverine also provide evidence for the existence of such a system. Papaverine has been reported to be a phosphodiesterase inhibitor (11), but it did not cause
379
cAMP accumulation in pig jejunal mucosa. The secretory effects of MIX relate directly to mucosal cAMP concentration, but the causal nature of this relationship is not definitely established. Sheerin and Field have recently reported that mucosal or serosal application of CT increases intracellular cAMP concentrations, but the strongest secretary response requires exposure to the mucosal surface of rabbit ileum to CT (24). Intestinal secretary responses to CT observed in this study may involve factors other than total mucosal cAMP concentration. ACKNOWLEDGMENTS Financial support by the Alberta Agricultural Research Trust, the Alberta Hog Producers Marketing Board, and the National Research Council is gratefully acknowledged.
LITERATURE CITED 1. Al-Awqati, Q., J. L Cameron, and W. B. Greenough Im. 1973. Electrolyte transport in human ileum: effect of purified cholera exotoxin. Am. J. Physiol. 224:818-823. 2. Al-Awqati, Q., C. K. Wallace, and W. B. Greenough III. 1972. Stimulation of intestinal secretion in vitro by culture filtrates of Escherichia coli. J. Infect. Dis.
125:300-303. 3. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974. Detection of heat-labile Escherichia coli enterotoxins with the use of adrenal cells in culture. Science 183:334-336. 4. Evans, D. J., L C. Chen, G. T. Curlin, and D. G. Evans. 1972. Stimulation of adenyl cyclase by Escherichia coli enterotoxin. Nature (London) New Biol. 236:137-138. 5. Field, M. 1971. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3',5'-AMP. Am. J. Physiol. 221: 992-997. 6. Field, M. 1976. Regulation of ion transport in the small intestine, p. 109-122. In Acute diarrhoea in childhood. CIBA Foundation Symposium no. 42. Elsevier-North Holland Pub. Co., New York. 7. Field, M, D. Fromm, Q. Al-Awqati, and W. B. Greenough HI. 1972. Effect of cholera toxin on ion transport across isolated ileal mucosa. J. Clin. Invest. 51:796-804. 8. Field, M., H. E. Sheerin, A. Henderson, and P. L Smith. 1975. Catecholamine effects on cyclic AMP levels and on ion secretion in rabbit ileal mucosa. Am. J. Physiol. 229:86-92. 9. Forsyth, G. W., D. L Hamilton, K. E. Goertz, and L. W. Oliphant. 1978. Some comparative properties and localization of porcine jejunal adenylate cyclase. Can. J. Biochem. 6:280-286. 10. Gill, D. MK 1976. Multiple roles of erythrocyte supernatant in the activation of adenylate cyclase by Vibrio cholerae toxin in vitro. J. Infect. Dis. 133:S55S63. 11. Goldberg, N. D., W. D. Lust, R. F. O'Dea, S. Wei, and A. G. O'Toole. 1970. A role of cyclic nucleotides in brain metabolism. Adv. Biochem. Psychopharmacol. 3:67-87. 12. Guerrant, R. L,. and C. C. J. Carpenter. 1975. Diarrheagenic effect of volume expansion: intestinal fluid secretion without mucosal adenyl cyclase stimulation. Johns Hopkins Med. J. 136:209-211. 13. Guerrant, R. L, L C. Chen, and G. W. G. Sharp. 1972. Intestinal adenyl-cyclase activity in canine cholera: correlation with fluid accumulation. J. Infect. Dis. 125: 377-381.
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14. Hamilton, D. L., W. E. Roe, and N. 0. Nielsen. 1977. Effect of heat stable and heat labile Escherichia coli enterotoxins, cholera toxin and theophylline on unidirectional sodium and chloride fluxes in the proximal and distal jejunum of weanling swine. Can. J. Comp. Med. 41:306-317. 15. Kantor, H. S., P. Tao, and S. L Gorbach. 1974. Stimulation of intestinal adenyl cyclase by Escherichia coli enterotoxin: comparison of strains from an infant and an adult with diarrhea. J. Infect. Dis. 129:1-9. 16. Kimberg, D. V., M. Field, J. Johnson, A. Henderson, and E. Gershon. 1971. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J. Clin. Invest. O:1218-1230. 17. Huddle, G. W., and J. G. Hardmen. 1971. Cyclic adenosine monophosphate as a mediator of hormone action. N. Engl. J. Med. 285:560-566. 18. Moon, H. W., S. C. Whipp and A. L Baetz. 1971. Comparative effects of enterotoxins from Escherichia coli and Vibrio chokrae on rabbit and swine small intestine. Lab. Invest. 25:133-140. 19. Powell, D. W., R. K. Farris, and S. T. Carbonetto. 1974. Theophylline, cyclic AMP, choleragen, and electrolyte transport by rabbit ileum. Am. J. Physiol. 227:1428-1435. 20. Schafer, D. E., W. D. Lust, B. Sircar, and N. D. Goldberg. 1970. Elevated concentrations of adenosine
3',5'-cyclic monophosphate in intestinal mucosa after treatment with cholera toxin. Proc. Natl. Acad. Sci. U.S.A. 67:851-856. 21. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, K. Field, and S. L Said. 1974. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J. Clin. Invest.
54:536-544. 22. Sharp, G. W. G., and S. Hynie. 1971. Stimulation of intestinal adqnyl cyclase by cholera toxin. Nature (London) 229:226-268. 23. Sharp, G. W. G., S. Hynie, H. Ebel, D. K. Parkinson, and P. Witkum. 1973. Properties of adenylate cyclase in mucosal cells of the rabbit ileum and the effect of cholera toxin. Biochim. Biophys. Acta 309:339-348. 24. Sheerin, H. E., and M. Field. 1977. leal mucosal cyclic AMP and CL secretion: serosal vs mucosal addition of cholera toxin. Am. J. Physiol. 232:E210-E215. 25. Wald, A., C. Back, and T. M. Bayless. 1976. Effect of caffeine on the human small intestine. Gastroenterology 71:738-742. 26. Weiser, M. M., and H. Quill. 1975. Intestinal villus and crypt cell responses to cholera toxin. Gastroenterology 69:479-482. 27. Zak, B., and J. Cohen. 1961. Automatic analysis of tissue culture proteins with stable Folin reagents. Clin. Chim. Acta 6:665-670.
