disease both in humans and in domestic animals ... sorption of salt and water into uncompensated ... 149) isolated from an outbreak of fatal watery diarrhea.
INFECTION AND IMMUNITY, June 1979, p. 900-905 0019-9567/79/06-0900/06$02.00/0
Vol. 24, No. 3
Chlorpromazine Reverses Diarrhea in Piglets Caused by Enterotoxigenic Escherichia coli IVAR LONNROTH,' * BIRGITTA ANDRAN,2 STEFAN LANGE,' KJELL MARTINSSON,2 AND JAN
HOLMGREN'
College of Veterinary Medicine, Swedish University ofAgricultural Science, S-750 07 Uppsala,2 and Institute of Medical Microbiology, University of Goteborg, S-413 46 Goteborg,' Sweden Received for publication 15 March 1979
The effect of chlorpromazine (CPZ) on diarrhea caused by enterotoxigenic Escherichia coli was tested in piglets since CPZ has been shown to be a potent antagonist to enterotoxins in vitro in a cell system and in vivo in a mouse model. Experimental diarrhea was induced in three litters of newborn piglets which were infected by mouth with 2 x 109 E. coli bacteria, which produce heat-labile (LT) and heat-stable (ST) enterotoxins. Treatment with CPZ given intramuscularly 1 h after the onset of diarrhea reversed fluid secretion in small intestine as well as dehydration, as judged by clinical criteria. A dose of 5 mg of CPZ per kg of body weight completely normalized the intestinal-fluid content measured 4 h after diarrhea developed, whereas 1 to 2 mg of CPZ per kg of body weight was somewhat less effective but still caused significant reduction of fluid (P < 0.001). Studies with radioactive [35S]CPZ showed preferential and dose-dependent uptake of 35S in the intestinal mucosa, the radioactivity being evenly distributed in the membranes of both crypt and villus cells. The enzyme adenylate cyclase, which probably mediates the cellular effects of LT, was shown to have two- to threefold higher activity in the infected than in the uninfected animals. This activation was reduced about 50% by the CPZ treatment (2 mg/kg of body weight). In a preliminary field trial the effect of CPZ was tested in a spontaneous outbreak of diarrhea in piglets due to enterotoxinogenic E. coli. The animals were treated either with oral electrolyte solution and standard antimicrobial agents only (controls) or with 1 mg of CPZ per kg of body weight intramuscularly in addition to this treatment. The mean duration of diarrhea in CPZ-treated animals was significantly shorter, 4.1 h (n = 23), than that in controls, 7.2 h (P < 0.05).
Enterotoxin-producing bacteria of various species have in recent years attracted increasing attention as an important cause of diarrheal disease both in humans and in domestic animals (3, 13). The severe, often lethal pathophysiological disturbance in these diseases result from the action of the enterotoxins on the small intestinal epithelium shifting the normal state of net absorption of salt and water into uncompensated net secretion (16). In the case of the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli this effect on intestinal transport processes is associated with and probably controlled by a toxin-induced increase in intraepithelial cyclic adenosine 5'-monophosphate (cAMP) (5, 9), whereas the low-molecular heat-stable enterotoxin(s) seems to act through cyclic guanosine 5'-monophosphate (6, 8). We have concentrated on the problem of identifying pharmacological agents which could re-
duce diarrhea in these diseases by interfering with the secretary process in the small intestine. Such agents could ideally eliminate the need for intravenous fluid replacement therapy by reducing diarrheal fluid loss to a level manageable with oral intake of sugar-electrolyte solution. We recently found that the phenothiazine chlorpromazine (CPZ) is a potent antagonist of cholera toxin-induced cAMP production in an in vitro cell test (11). Subsequent studies in a mouse model showed that this drug also very effectively inhibits the small intestinal secretion induced by cholera toxin, E. coli heat-labile enterotoxin (LT), prostaglandin El, or dibutyrylcAMP (7, 11). It is important to know, before considering testing of CPZ in humans with diarrhea, that the drug has an antisecretory effect in species other than mice, ideally in an animal naturally susceptible to enterotoxic diarrheal diseases. In the present study we have therefore 900
VOL. 24, 1979
EFFECT OF CPZ ON DIARRHEA IN PIGLETS
901
examined whether CPZ would reduce intestinal 3n a sample of the suspension (12) after hydrolysis in secretion and improve the clinical condition of 3 M NaOH at 100°C for 2 h. Adenylate cyclase activity in the mucosal mempiglets with diarrhea due to enterotoxinogenic brane preparation was estimated by a modification of E. coli. the method described by Krishna et al. (10). A sample MATERIALS AND METHODS Experimental infection. The E. coli bacteria used for infection were of a strain (BD 3027/76; 0 group 149) isolated from an outbreak of fatal watery diarrhea among a herd of piglets in Sweden. This strain gave rise to fluid accumulation in ligated intestinal loops of piglets (0. Soderlind, personal communication). The bacteria were cultivated at 370C for 10 h in a tryptoneyeast extract medium containing, per liter: tryptone (Difco), 10 g; yeast extract (Difco), 5 g; NH4C1, 2.5 g, Na2HPO4. 12 H20, 15 g; KH2PO4, 6 g; Na2SO4. 1OH20, 0.5 g; MgSO4, 2 mg; MnSO4, 1 mg; FeSO4, 0.6 mg; citric acid, 0.6 mg. The cell density was then adjusted to 2 x 109 bacteria per ml. Three litters with 10 to 11 newborn piglets in each (Swedish "Lant" race) were used in three consecutive similar experiments. To avoid suckling, the piglets were immediately separated from the sows. One to 2 h after birth two-thirds of the animals were given 4 ml of the E. coli culture together with an equal volume of 0.6 M NaHCO3 solution by mouth, using a plastic pipette introduced towards the pharynx wall. Approximately one-third of the animals were kept uninfected to serve as controls. The infected animals developed watery diarrhea after 3 to 4 h. When the diarrhea had been manifest for 1 h, half of the sick animals were injected in the thigh muscle with CPZ (Hibernal, AB Leo, Helsingborg, Sweden) or [nS]CPZ (4 Ci/mol; The Radiochemical Centre, Amersham, England) in doses between 1 and 5 mg/kg of body weight, and the remaining ones were left untreated. About 4 h later the animals were killed by a skull blow. The small intestine was then carefully freed from the mesenteric tissue, and the entire free-dissected small bowel with its fluid content was weighed. The fluid content was estimated as the difference in relative intestinal weight (grams per meter) between the test piglets and uninfected control animals within the same litter. It was decided in advance, based on a randomized procedure, which treatment every piglet would get so that each CPZ-treated infected animal had one untreated, infected control and one uninfected control. In one experiment (no. 1) there was also a CPZtreated, uninfected control for each treated, infected animal. Assay of adenylate cyclase. The middle third of the small bowel was cut open and washed with icecold phosphate-buffered isotonic saline, pH 7.5, after which the mucosa was carefully scraped off the muscularis with a scalpel. After suspension of the tissue in a buffer containing 75 mM tris(hydroxymethyl)aminomethane-hydrochloride 12 mM MgCl2-1 mM dithiothreitol, pH 7.5 (50 mg [wet weight]/ml), the mucosal preparation was homogenized at 0°C in a glass homogenizer. Membranes were recovered, washed once by centrifugation (800 x g, 5 min), and resuspended in the same buffer. The amount of membrane-bound protein was then determined according to Lowry et al.
of the suspended intestinal membranes was centrifuged (800 x g, 5 min) and resuspended to the same volume in a buffer, pH 7.5, containing 40 mM tris(hydroxymethyl)aminomethane-hydrochloride, 3.3 mM dithiothreitol, 5 mM phospho(enol)pyruvate, 0.1% (wt/vol) bovine serum albumin, 0.1% (wt/vol) myokinase (Sigma grade III), and 0.004% (wt/vol) pyruvate kinase (Sigma type II). To 100 p1l of this mixture (containing 300 to 600 ,g of membrane protein) was added 10 pJ of 2 mM [a-3P]adenosine 5'triphosphate (25 Ci/mol), and in some experiments 10 p1 of 0.1 M potassium fluoride was also added. Incubation was performed at 30°C for 10 min, and the reaction was stopped by the addition of 0.65 ml of 0.2 mM adenosine 5'-triphosphate, 0.15 mM [3H]cAMP (2,000 cpm), and 0.25 ml of 20% trichloroacetic acid. The formed [32P]cAMP together with added carrier [3H]cAMP was isolated by ion-exchange chromatography on a 5- by 60-mm column of AG 5OW x 2, 200 to 400 mesh (Bio-Rad). The neutral fraction (2.5 ml) containing cAMP was transferred directly into a small column, where it was absorbed on 0.5 g of neutral alumina (type WN3, Sigma), after which the nucleotide was eluted again with 2 ml of 0.1 M imidazole solution (14). Double isotope counting was performed on the final supernatant, and the values of formed [32P]cAMP were corrected for loss during isolation by estimation of the recovery of [3H]cAMP. Tissue incorporation of [35S]CPZ. Mucosal membrane preparations (see above) from animals injected with [3S]CPZ were dissolved in Soluene 350 (Packard Instrument Co., Downers Grove, Ill.), and the radioactivity was measured in a liquid scintillator. Correction for quenching of radiation by dissolved tissue was done by use of an internal 5S standard. The localization of radiolabel in the intestinal epithelium was studied microscopically by autoradiography on intestinal tissue sections. The specimens were taken from the mucosa adjacent to thdportion used for the membrane preparations and kept frozen in liquid nitrogen until sectioned as described earlier (7). Field study. In a swine herd with 40 sows, all litters developed watery diarrhea about 1 day after birth; the diarrhea was lethal if not treated. Postmortem examination showed signs of an acute hemorrhagic gastroenteritis. Beta-hemolytic E. coli of the commonly enterotoxigenic 0 group 149 were isolated in large numbers from the small intestinal fluid. Since the organisms were sensitive to trimethoprim-sulfonamide, all animals were given 200 mg of sulfadoxin plus 40 mg of trimethoprim intramuscularly as soon as diarrhea appeared, and, in addition, an electrolyte solution consisting of 80 mM glucose-64 mM NaCl-35 mM NaHCO3-6 mM KCl was made available as the only drinking fluid. Five litters of diseased animals were split into two equal-sized groups (test and control), every second piglet being given 1 mg of CPZ per kg of body weight injected intramuscularly simultaneously with the antimicrobial agent. Persistance of
902
LONNROTH ET AL.
INFECT. IMMUN.
diarrhea was examined 4, 8, 12, and 24 h after the treatment with CPZ.
reversed the intestinal net secretion of fluid but also had a marked sedative effect on the animals, Enterotoxin assays. Bacterial cultures and diar- whereas a dose of 1 mg/kg, which caused little rheal fluid collected from the small intestine of in- if any sedation, was somewhat less effective. The fected piglets were examined for LT by the ganglioside-enzyme-linked immunosorbent assay (GMI- effect of CPZ was then confirmed in two consecELISA) (17) and rabbit skin (2) tests and for heat- utive experiments in which an intermediate dose stable enterotoxin (ST) by the suckling mouse assay of CPZ (2 mg/kg) was given (Fig. 1). Statistically, the difference in fluid accumulation be(4). tween the CPZ-treated piglets and their unRESULTS treated matched controls was highly significant Effect on experimental E. coli diarrhea. (P < 0.001, Student's t test). Also, clinically the Newborn piglets given enterotoxigenic E. coli dehydration as judged by subjective criteria was perorally together with sodium bicarbonate con- less severe in the CPZ-treated than in the unsistently developed a watery diarrhea within 4 h treated animals at the end of the experiments. Enterotoxin determination. When culafter infection. Other symptoms of the infected animals progressing with time after onset of tured in vivo, the E. coli strain used for infection diarrhea were a yellow-greyish change in skin of the piglets produced a heat-labile factor which color, decrease in turgor, and sunken eyes. Fur- gave a positive GMl-ELISA reaction and charthermore, as a quantitative objective measure of acteristic bluing and induration in the rabbit pathophysiological change, these animals skin test, indicating its LT nature. The filtrate showed fluid distension of their intestines when also contained detectable ST activity as tested autopsied. This increased the weight of the small in suckling mice (Table 1). Fluid collected from intestine by 67 ± 13% (mean ± standard error the small bowel of two infected piglets with for nine piglets) in comparison with the unin- diarrhea demonstrated significant LT but not ST activity (Table 1). fected littermate controls. Studies with [36S]CPZ. The amount of raTreatment with 1 to 5 mg of CPZ per kg of body weight changed this pattern significantly dioactive 5S incorporated into the intestinal mucosal membranes was directly proportional to as studied in three experiments (Fig. 1). An initial experiment showed that an intramuscular the injected dose of [35S]CPZ (Fig. 2a). Provided injection of 5 mg of CPZ per kg completely that most of this 35S was still in the form of unmetabolized [35S]CPZ, the results indicate a >100-fold concentration of CPZ in intestinal membrane as compared to the initial body concentrations. Autoradiography on sections of small bowel 2
8-
4
TABLE 1. Assays of LT and ST enterotoxins in culture filtrate of E. coli 3027/76 and in small intestinal fluid ofpiglets developing diarrhea after infection with this straina
~~~~~~3
4~~~~~~~~~~
Enterotoxin in:
testb
3
2
2
0
Exp.1
Exp.2
GMILT
Exp.3
FIG. 1. Inhibition ofexperimental E. coli diarrhea by CPZ. The fluid accumulated in the small intestine is shown in infected animals which were untreated (5) or treated with 1 (O), 2 (MM), and 5 (E) mg of CPZ per kg of body weight. Mean ± standard error and number of infected animals are given. Uninfected control animals were used in each experiment (litter of piglets) for calculation of basal intestinal weight (in grams per meter).
Skin PF LT test'
Infant mouse ST test
Culture filtrate 1.1 4 Heated filtrate (80'C, 0.1 0.132c 20 min) 4.2 6 Intestinal fluid, piglet 1 Heated fluid 0.0 0.066 8 Intestinal fluid, piglet 2 8.5 0.0 0.061 Heated fluid Appropriate positive and negative control materials tested simultaneously gave the expected results. b Absorbance change above background in 100 min; positive test requires value above 0.2. GM1-ELISA, Ganglioside-enzyme-linked immunosorbent assay. 'Diameter of bluing zone. PF, permeability factor in milliliters. d Weight ratio intestine/whole animal; positive test requires value above 0.085. a
903
EFFECT OF CPZ ON DIARRHEA IN PIGLETS
VOL. 24, 1979
b
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FIG. 2. Uptake of radiolabel in mucosal membranes of small intestine after intramuscular injection of [35S]CPZ. The amount of 35S bound to mucosal membrane is shown in (a), and (b) illustrates the autoradiographic distribution of radiolabel in a section of small intestine (1 = labeled plasma membrane; 2 = intracellular label; LP = lamina propria; E = epithelial cell [limits indicated with small arrows], C = crypt). b.w., Body weight.
showed a similar distribution of radiolabel in crypt and villus cells. Specific staining was evident in plasma membranes as well as intracellularly (Fig. 2b). Effects on adenylate cyclase. The adenylate cyclase activity in the intestinal mucosa was assayed on specimens from piglets in each of the three test groups in experiment 2. The basal enzymatic activity in the uninfected control animals was about 7 pmol of cAMP formed per mg of membrane protein per min (Table 2). The infected animals with diarrhea, which did not receive CPZ treatment, had two- to threefold higher adenylate cyclase activity. The CPZtreated animals, on the other hand, had distinctly lower enzyme activity, although it was still above the basal activity of the control animals. Effects in a preliminary field trial. CPZ treatment was tested also in a spontaneous outbreak of severe watery diarrhea among newborn piglets. The apparent causative agent, an E. coli 0 149 strain isolated from the intestinal fluid, was found to produce LT and ST as based on
TABLE 2. Effect of CPZ on intestinal adenylate
cyclase
Animalsa
Small intestinal fluid (g/
m)b
Infected Untreated 1 2
Adenylate cyclase (pmol of cAMP mg
of protein-'
min'-)c
6.7 4.2
15.8 ± 0.5 15.2 ± 1.4
0.1 0.0
11.8 + 1.2 9.3 ± 0.6
-0.2 0.2
6.0 + 0.3 8.4 ± 0.3
CPZ treated 1
2 Controls 1 2
aPiglets were from experiment 2. increase in comparison with the mean value for all uninfected controls in experiment 2. e Mean ± standard error of triplicate determinations of washed mucosal membrane.
bWeight
examination of culture filtrate with the GM1ELISA and infant mouse tests. The effect of CPZ was evaluated by comparing the duration of diarrhea in piglets treated either
904
LONNROTH ET AL.
INFECT. IMMUN.
with standard antibiotics and oral electrolyte solution only (controls) or with 1 mg of CPZ per kg of body weight intramuscularly in addition to this treatment. The results in Table 3 show that the mean duration of diarrhea in the CPZtreated group was 4.1 h and that in the control group was 7.2 h, the difference between the groups being statistically significant (P < 0.05, Student's t test).
DISCUSSION The results of this study indicate that CPZ can stop diarrheal secretion in piglets caused by experimental or natural infection with enterotoxinogenic E. coli. The doses of CPZ, kilogram of body weight found to be effective in the piglets are similar to those previously shown to reverse intestinal fluid loss in mice in response to LT as well as other agents stimulating secretion by increasing intestinal cAMP (cholera toxin, prostaglandin El, and dibutyryl-cAMP [7]). The effect of CPZ on enterotoxic diarrhea in piglets was best documented in the experiments in which newborn piglets were intentionally infected with a diarrheagenic E. coli strain shown to produce LT and ST when cultured in vitro. After diarrhea had been manifest for 1 h, CPZ was given to every second animal, and the treated piglet was compared with the untreated paired control as well as with another paired uninfected piglet within the litter. In the infected, untreated animals there was a clear progression of dehydration during the observed course of diarrheal illness associated with a marked increase in intestinal-fluid content at the time of sacrifice. The weight increase of the small intestine due to this fluid accumulation was about 2% of the body weight. Since the transit time in the gut is probably shorter than 1 h, this fluid loss from the small intestine explains the lethal course of this experimental infection in piglets, which in untreated animals usually causes death 10 to 15 h after diarrhea has started (B. Andren, submitted for publication). In accordance with the in vitro bacteriological findings, LT activity could be demonstrated in the small intestinal fluid of the inTABLE 3. Preliminary field trial: effect of CPZ on duration of E. coli diarrheal disease Amaa No. Aniimalsa teste tested
No. with diarrhea at: 4h
8h
12h 24h
4 1 26 16 10 Controls 3 9 0 0 CPZ-treated (1 mg/kg, 23 i.m.) All piglets were given oral glucose-electrolyte solution and an intramuscular (i.m.) injection of trimetha
oprim-sulfamethoxazole.
fected piglets, and enzymatic studies revealed a two- to threefold increase in mucosal adenylate cyclase activity. These findings support the conclusion that the dehydrating diarrhea was due to an effect of LT on small intestinal cAMP. The infant mouse test was used to assess the production of ST by the challenge E. coli strain. No ST activity could be demonstrated with this method in the diarrheal fluid. This does not, however, exclude ST production in subdetectable concentrations. Furthermore, it was recently reported that porcine E. coli strains may produce an ST which is undetectable in suckling mice (1). The treatment with CPZ in doses of 1 to 5 mg/kg of body weight reduced fluid accumulation in the small intestine almost to the base line level, the higher dose tending to be more effective than the lower ones. However, the side effect of sedation was so pronounced with the 5-mg/kg dose that it might have affected suckling and other fluid intake (e.g., oral sugar-electrolyte solution) adversely. This does not seem to pose problems with doses of 1 to 2 mg of CPZ per kg, which still had a strong antisecretory effect. Clinically there were also clear indications that the CPZ treatment interrupted the progression of dehydration seen in the untreated, infected controls. Earlier studies in mice challenged with cholera toxin (7) indicated that the intestinal epithelium probably is the main target tissue for the antisecretory action of CPZ. Thus, there was a close relationship between the antisecretory activity and the amount of radioactive [35S]CPZ incorporated into the intestinal mucosa membranes after treatment (I. Lonnroth, submitted for publication). Proportionally more (about twice as much) of the injected CPZ radioactivity became localized to the intestinal membranes in piglets than in mice. The autoradiographic pattern of the incorporated [35S]CPZ showed an even distribution in crypt and villus cells. LT, in analogy with cholera toxin, probably causes diarrhea by stimulating active secretion by crypt cells and inhibiting absorption by villus cells (5). The cellular distribution of radioactive CPZ suggests that CPZ might interfere with both of these processes. Like cholera toxin, E. coli LT affects both the absorption and the secretion by stimulating mucosal adenylate cyclase (5). In mice, treatment with CPZ completely normalized the increased adenylate cyclase activity of cholera toxin-stimulated intestinal epithelium (7). In piglets, however, a dose of CPZ of 2 mg/ kg of body weight, which abolished the small intestinal fluid accumulation completely, did reduce adenylate cyclase activity too, but not to
EFFECT OF CPZ ON DIARRHEA IN PIGLETS
VOL. 24, 1979
the "basal" activity of control animals. Since adenylate cyclase is a key enzyme in the chain of events leading to intestinal secretion, its inhibition by CPZ in all probability is of great significance. However, in the CPZ-treated piglets studied it seems likely that the remaining stimulation of adenylate cyclase per se would have been sufficient to maintain a net secretary state in the intestinal mucosa. Effects additional to those on adenylate cyclase must therefore be considered for the CPZ incorporated in the intestinal mucosa. CPZ is known to affect cell membrane structure and function in several respects, e.g., by causing a volume expansion of the lipid bilayer, stabilizing the membrane to inducing a release of Ca2" (15). Such "unspecific" effects could obviously lead to interference with numerous membrane enzymes and other components involved in electrolyte transport processes.
The effect of CPZ on E. coli diarrhea was also indicated in a preliminary field trial, in which the addition of CPZ to the ordinary antibioticoral electrolyte treatment was found to shorten the duration of diarrhea significantly. The promising results obtained with CPZ, first in mice and now in piglets, certainly warrant studies of whether CPZ will also reduce fluid loss in human adults and children with cholera and E. coli diarrhea. It is also important to clarify more directly than in the present study whether the secretary mechanisms stimulated by E. coli ST are also amenable to treatment with CPZ, since E. coli diarrhea both in humans and in domestic animals usually is caused by strains producing ST as well as LT. ACKNOWLEDGMENTS Erva Dr. Olsson, Uppsala, is gratefully acknowledged for assaying ST production by the E. coli strains, and Gudrun Andersson for technical assistance. This study was financially supported by the Swedish Medical Research Council (grant 16X-3382), the Swedish Research Council for Forestry and Agriculture (grant 5092-B3608), and the Medical Faculty of the University of G6teborg (grant
B321a, 485/78). LITERATURE CITED 1. Burgess, M. N., R. J. Bywater, C. M. Cowley, N. A.
905
Mullan, and R. M. Newsome. 1978. Biological evaluation of a methanol-soluble heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves. Infect. Immun. 21:526-531. 2. Craig, J. P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature (London) 207: 614-616. 3. Craig, J. P. 1972. The enterotoxic enteropathies, p. 129155. In H. Smith and J. H. Pearce (ed.), Microbial pathogenicity in man and animals. Cambridge University Press, Cambridge. 4. Dean, A. G., Y.-C. Ching, R. G. Williams, and L B. Harden. 1972. Test for Escherichia coil enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 5. Field, M. 1976. Regulation of active ion transport in the small intestine, p. 109-122. In K. Elliott and J. Knight (ed.), Acute diarrhea in childhood. Ciba Foundation Symposium 42. Elsevier, Amsterdam. 6. Field, M., L H. Graf, N. J. Laird, and P. L. Smith. 1978. Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proc. Natl. Acad. Sci. U.S.A. 75:2800-2804. 7. Holmgren, J., S. Lange, and I. Linnroth. 1978. Reversal of cyclic AMP-mediated intestinal secretion in mice by chlorpromazine. Gastroenterology 75:1103-1108. 8. Hughes, J. M., F. Murad, B. Chang, and R. L. Guerrant. 1978. Role of cyclic GMP in the action of heatstable enterotoxin of Escherichia coli. Nature (London) 271:755-756. 9. Kimberg, D. V. 1974. Nucleotides and their role in gastrointestinal secretion. Gastroenterology 67:1023-1064. 10. Krishna, G., B. Weiss, and B. B. Brodie. 1968. A simple, sensitive method for the assay of adenyl cyclase. J. Pharmacol. Exp. Ther. 163:379-385. 11. LMnnroth, I., J. Holmgren, and S. Lange. 1977. Chlorpromazine inhibits toxin-induced intestinal hypersecretion. Med. Biol. (Helsinki) 55:126-129. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Sack, R. B. 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29:333-354. 14. Salomon, Y., C. Londos, and M. Rodbell. 1974. A highly sensitive adenylate cyclase assay. Anal. Biochem.
58:541-548. 15. Seeman, P. 1972. The membrane actions of anaesthetics and tranquilizers. Pharmacol. Rev. 24:583-653. 16. Sharp, G. W. G. 1973. Action of cholera toxin in fluid and electrolyte movement in the small intestine. Annu. Rev. Med. 24:19-29. 17. Svennerholm, A.-M., and J. Holmgren. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GMrELISA) procedure. Curr. Microbiol. 1:19-23.
