Mechanisms Escherichia Michael
Field,3
of action co/i
of cholera 2
M.D. ABSTRACT
Current
including
how
properties
of
separate probably
in
transfer
triphosphatase, basis
for
both
heat-labile
processes is inhibited
cells.
Cholera
mucosal
and
this
stimulates
its enterotoxicity.
other
with
diar-
How the small intestine secretes: the relation between diarrhea in a patient with cholera and active secretion in an Ussing chamber.
The
American
cholera can 1 liter/hr.
JournalofC/inica/
Nutrition
have The
massive excreted
32: JANUARY
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/1/189/4666372 by University of Glasgow user on 26 April 2018
by E.
co/i
toxins
from
NAD
guanylate
32:
189-196,
secretion their
results represses cyclase,
and
enterotoxins.
Two
secretory from
the
effect A,
by
subunitguanosine
adenylate which
1)
is stimulated,
to a membrane-bound
normally
mucosal
exert
intestine,
structures
monophosphate:
anion
stimulation
the
coli
3’,5’-adenosine
2) active
which
in small on
Escherichia
cyclic
This
Nuir.
and
and
ribose
enzyme,
J. C/in.
rheal disease-one heat labile (1, 2) and the other heat stable (1, 3, 4). The former is immunologically cross-reactive with cholera toxin (5) and, like cholera toxin, stimulates adenylate cyclase (6). The latter is not neutralized by cholera antitoxin (7) and heretofore there has been little insight into its mode of action. The purpose of this report is 2-fold: first, summarize current information on intestinal water and electrolyte transport in order to provide a conceptual framework for understanding how enterotoxins, in particubar, and enteric infection, in general, can stimulate intestinal secretion. Second, summarize current information on cholera toxin and the E. coli enterotoxins, including some of our own recent studies with the heat-stable enterotoxin of E. coli.
A patient with diarrhea exceeding
cyclase.
ofsecretion
cells
heat-labile
intestinal
Am.
mammals
altered
diphosphate
mechanism
monophosphate heat-stable
in villus
Certain entenc bacteria excrete substances that interact with the intestinal epitheium and cause the transport of water and electrobytes to reverse from absorption to secretion. Cholera is a classic example of this process and has been extensively studied. Two enterotoxins have been identified among the extracellular products of Escherichia coli isolated humans
are
and
inhibiting
enterotoxin
and
adenylate
of adenosine
on the
3’, 5’-adenosine
transport
thereby
E. co/i
is reviewed
cyclic
of NaCl
ion crypt
by
and
intestinal
catalyzed stable
cholera
absorption
stimulating
information
it is altered
active
coupled
from
and
cyclase. appears
Heatto be the
1979.
fluid is nearly protein-free and has an dcctrobyte composition similar to that of serum except that the chloride concentration is lower and the bicarbonate concentration higher. Dehydration, metabolic acidosis and their consequences may develop but, if water and electrolyte losses are adequately replaced, no fever or other evidence of tissue necrosis or systemic involvement appears and recovery is universal. The diarrhea originates in the small intestine, all of which is usually involved. There is neither epitheial invasion by bacteria nor evidence of damage to epitheial cells, even at the ultrastructural bevel. There is also no impairment of absorptive processes for nutrients such as sugars and amino acids. This diarrheal process can be reproduced in animals by innoculating the small intestine with either cholera organisms, a bacteria-free culture filtrate or the purified toxin (see below). At about the time that these cardinal features of clinical cholera gained general acceptance, the effects of cyclic 3’, 5’-adenosine monophosphate (cAMP) on an in vitro preparation of small intestine (8) were demonstrated, thereby setting the stage for much of the subsequent research into the cellular and molecular changes through which Vibrio I
From
the
Departments
of Medicine
logical cago,
and Physiological Sciences, Chicago, Illinois 60637. 2 Supported by National Institute AI-09029 and AM-2l345. 3
1979,
and
Pharmaco-
University of
Health
of
Chi-
Grants
Professor. pp.
189-196.
Printed
in U.S.A.
189
FIELD
190
brane couples the transmembrane movement of glucose to that of Na; the Na that enters with glucose is then pumped out of the cell into the lateral space; in the process adenosine triphosphate is hydrolyzed by Na, K-activated adenosine triphosphatase located in the basolateral membrane. The driving force for gl ucose absorption, therefore is the electrochemical potential difference for Na across the brush-border membrane. This difference, which consists of both a difference in Na’ concentration (intracellular Na” is low) and in electric potential (the cell interior is dcctronegative), favors the influx and restricts the efflux of Na and also glucose, whose movement is coupled to that of Na. Once glucose accumulates in the absorptive cell, it diffuses across the basolateral border by a process that may be carrier-mediated but is not coupled to Na’. A corollary of the Na’dependence ofglucose absorption is the stimulation of Na’ (and secondarily of C1 and water) absorption by luminal glucose. Absorption of neutral amino acids is also coupled to that of Na (1 1). As shown in Figure 1, the addition of theophylline, which inhibits enzymatic degradation of cAMP, to the serosal side of the ileal mucosa, causes a rapid and sustained increase in PD and 5CC. The same result can be obtained by adding cAMP itself. Figure 1 also illustrates the changes in net fluxes that develop under short-circuit conditions: the
cho/erae enterotoxin exerts its secretory effect. I will briefly describe the experimental preparation and some of its ion transport properties so that subsequent comments on underlying mechanisms will make sense: rabbit ileum can be stripped of muscle and mounted in an “Ussing” chamber (9) as a flat sheet separating two identical, oxygenated, bicarbonate-buffered Ringer solutions. Under basal conditions, 1 to 4 mV electric potential difference (PD) across the ileab mucosa (serosa-positive) can be measured (10). For ion flux measurements, this PD is usually nuffifled (short-circuited) by passing current from an external source through the tissue. When the epitheium is short-circuited, all net ion fluxes can be attributed to active transport. A net flux is determined as the difference between opposing unidirectional fluxes measured with radioisotopes. The unidirectional fluxes are often larger than the net fluxes because thejunctions between cells are highly permeable to monovalent ions. Normally the ileum generates net absorptive fluxes of Na’ and Cl and a larger net secretory flux of HCO (10). The PD and short-circuit current (5CC) are due mainly to the latter. If glucose is added to the buminal side of the tissue, the PD and SCC increase very quickly due to stimulation of active Na’ absorption (I 1). The relation between glucose and Na transports can be explained as follows (1 1): a carrier in the brush border mem-
6
C”
E
4
C.) #{188}.
-4--
C..)
2
+Theo
(I)
30
0
FIG. (J,),
and
theophylline.
I. Effects residual From
60
90
minutes of theophylline ion flux (Jet) Reference
on SSC corresponds
and
net ion fluxes. to the
12.
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/1/189/4666372 by University of Glasgow user on 26 April 2018
actual
time
The
inset
period
showing over
which
net Na flux fluxes
were
(J,) , net measured.
Cl flux Theo,
CHOLERA
AND
ESCHERICHIA
-5 -5
2
HOURS
FIG. vitro.
2. Effects
From
AFTER
ofcholera Reference 13.
TOXIN
toxin
AOOED
on transmural
PD
in
direction of net C1 transport reverses and, in the absence of luminal glucose, net absorption of Na” disappears. Under some circumstances, net Na secretion develops. The residual ion flux (that portion of the 5CC not accounted for by Na” and Cb), which is a measure of HCO secretion (10), either remains the same or increases. In general, the increase in 5CC represents the difference between the change in net anion flux (Cl and HCO) and the change in net Na flux. Since these changes have all been measured under short-circuit conditions, there appears to be a change in the active transport of each of these ions. The sum of these effects constitutes a large driving force for the secretion not only of electrolytes but also of water, which follows the ion movements passively. The action of cAMP and theophylline is closely mimicked by that of cholera toxin ( 10). The time course is different (Fig. 2), since the activation of adenylate cyclase by cholera toxin is slow, but the end result is the same. Indeed, theophylline, added 4 hr after cholera toxin, evokes little further change in PD. A detailed consideration of the cellular mechanisms responsible for these alterations of ion transport is beyond the scope of this presentation and, indeed, these mechanisms are as yet only very imperfectly understood. I would nonetheless like to briefly and, of necessity, superficially state my current understanding of these mechanisms since the way they are viewed affects our thinking not only about enterotoxic diarrheas but also about diarrheas associated with invasive and destructive enteric organisms. Normally, a
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COLI
ENTEROTOXINS
191
major part of salt and water absorption from the small intestine appears to be a consequence of the presence in the brush-border membrane ofa channel or carrier that accepts and transbocates the ion pair, NaC1 (14). In the absence of Na’, Cl uptake is diminished and, in the absence of Cl, Na uptake is diminished by the same amount. The Na that enters the cell along with C1, is then actively extruded across the basolateral membrane by the ouabain-inhibitable Na’s pump. The efflux of C1 from cell to lumen over the coupled pathway is thus restricted by the bow intracellular Na concentration and therefore C1 accumulates in the intestinal epithelial cell above ebectrochemical equilibrium and diffuses out of the cell across the basolateral membrane (the latter is presumably far more permeable to free C1 than is the brush border). This mechanism for Cl absorption is directly analogous to that for glucose absorption (see above) and is another example of how the intestinal epitheial cell uses the potential energy in the Na gradient for the transport of a variety of other solutes. Coupled uptake of NaCl across the brush border is inhibited by cAMP, which thereby inhibits “active” Cl absorption and also a large portion of active Na absorption (14). Indeed, in the absence of nutrients which specifically stimulate Na absorption, the majority of salt and water absorption in the small intestine probably occurs by this cAMP-inhibitable mechanism. It is important to bear in mind that this action of cAMP appears to be specific for coupled NaCl transbocation across the brush border and does not constitute a general inhibition of all absorptive processes. The capacity of the Na pump to respond to an increase in Na influx is not impaired and the Na-coupled absorptive processes for sugars and neutral amino acids are also not impaired. Fluid losses of cholera patients can be replaced orally if glucose is added to the replacement solution (15). Although cAMP plays shift
from
the antiabsorptive a significant role absorption
to
secretion,
effect of in the overall it cannot
by itself explain net secretion. Either an underlying secretory process is unmasked through the inhibition of coupled NaC1 absorption or cAMP must have a second effect, directly stimulating active secretion. That the latter alternative is the correct one has been
FIELD
192
demonstrated with pharmacologic agents (acetazobomide, furosemide) which, when placed on the luminab side of the intestine, inhibit coupled NaCl absorption without stimulating secretion ((16); M. Field, unpublished data). Figure 3 summarizes these separate actions of cAMP. The antiabsorptive action is exerted on villus cells, (these are the only cells accessible to measurements of mitial rates of Na’ and Cl uptake, which were made over 45 sec (14)). There is considerable indirect, but nonetheless compelling evidence that the secretory action of cAMP is exerted mainly or exclusively on cells in the crypts of Lieberkuhn (10). The crypt portion of the epitheium, in addition to being the site for cell renewal, also appears to function as an exocrine gland. cAMP, which is an intracelbular mediator for the stimulation by secretin of pancreatic NaHCO3 and water secretion (17), appears also to be an intracellular mediator of secretion in the small intestine, responding to both intraluminal stimuli (i.e., cholera toxin) and hormonal stimuli (i.e., vasoactive intestinal peptide ( 1 8)). Just as the “resting” pancreas still maintains a low rate of secretion, the intestinal crypts, in the absence of extraordinary stimuli such as cholera toxin, may do the same. This “basal” secretion is normally more than offset by the absorptive activities of the villus cells and is therefore difficult to quantitate. If villus cells are damaged or destroyed, however, some LUMEN
VILLUS
BLOOD
CELL
CRYPT
CRYPT
FIG. transport adenosine
CELL
LUMEN
3. Postulated separate actions of cAMP on ion by small intestine. See text for details. ATP, triphoSphate; ADP, adenosine diphoSphate.
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degree of underlying secretion may be unmasked. In viral enteritis, for example, viruses seem to preferentially invade villus cells and crypt cells are often spared (19, 20). Indeed, partial villus atrophy and associated malabsorption of nutrients has been described (19). The watery diarrhea sometimes observed in viral enteritis could therefore be due to the unmasking of crypt cell secretion (20). The mechanism for active anion secretion appears to be fundamentally the same in intestinal crypts as in a variety of other secretory epitheia and is not well understood. A model for active Cl secretion that is closely rebated to the model for coupled NaC1 absorption has recently been proposed (10). Molecular mechanism and heat-labile E. coil
of action enterotoxins
of cholera
Cholera toxin exerts its secretory effect by stimulating the activity of intestinal mucosal adenylate cyclase, thereby increasing the concentration of cAMP in intestinal epitheiab cells (10). The time course of these changes is the same as that of the resulting alterations of ion transport (10). The structure, cell membrane receptor, and intracellular action of cholera toxin have recently been reviewed ( 10, 2 1). It is a protein of 84,000 MW consisting of three peptide chains, A1, A2, and B which weigh about 24,000, 5,000 and 1,000 daltons, respectively. Each toxin molecule contains one each of the A chains and five B chains. A1 and A2 are connected by a single disulfide bond. The B chains can be isolated as a single subunit, which is known as “choberagenoid”. Choleragenoid does not stimulate adenybate cyclase but binds to cell membrane receptors in a manner indistinguishable from that ofthe full toxin molecule. The cell receptor for cholera toxin and choleragenoid is the monosiaboganglioside, Gmi. The bond between Gmi and B subunits is very tight, dissociation constants in the range of bO having been reported. The A subunit (A1 + A2) is nontoxic when added to intact cells but highly active when added to lysates. This toxicity resides in the A1 peptide: by itself A2 is nontoxic, and it does not enhance the toxicity of A1 in lysates. Adenylate cyclase stimulation by cholera toxin therefore involves attachment of the B subunits to cell surface receptors, followed by
CHOLERA
insertion through
AND
ESCHERICHIA
and possibly where it interacts with adenylate cyclase. This reorientation of the A1 peptide in the cell membrane is temperature-dependent and slow. At least 15 miii transpires before detectable stimulation ofthe intestinal cyclase develops whereas addition of A1 to a lysate produces measurable enzyme activation within seconds. As shown by Gill (21), adenylate cyclase activation by the A1 peptide of cholera toxin requires NAD and is greatly facilitated by one or more cytosol macromolecules. Like the active fragment of diptheria toxin, the A1 peptide of cholera toxin splits NAD into adenosine diphosphoribose and nicotinamide, the former being covalently bonded to a receptor protein in the plasma membrane. As shown by Cassel and Selinger (22), the receptor protein is a guanosine triphosphatase, or part of a guanosine triphosphatase, which regulates the activity of adenylate cyclase by converting this enzyme from its active, guanosine triphosphate-bound form to an inactive form. A1 peptide inactivates this guanosine triphosphatase activity. These reactions are summarized in Figure 4. Some E. co/i produce a heat-labile enterotoxin (LT) which is similar to and possibly identical with cholera toxin. This toxin can be neutralized with cholera antitoxin and also with anticholeragenoid (23). It contains a 24,000 dalton subunit which is an ADPR transferase (D. M. Gill, personal communication). It is possible, as suggested by Rappaport et al. (24), that E. coli secrete LT in the form of a larger protein that is then activated by bacterial and possibly mammaian proteases. The genetic information for LT production is contained in a plasmid (25) and therefore toxicity can be transmitted from one strain of E. coli to another. Purification heat-stable
COLI
of the A1 peptide into the plasma membrane,
and biological actions of the enterotoxin (ST) of E. coil
In addition to LT, ST has been isolated from diarrheagenic strains of E. coli (1, 3, 4). Toxigenic E. co/i can produce both LT and ST, LT alone, or ST alone. As is the case with LT, the genetic information for ST production has been found in a pbasmid and a single plasmid can carry genes for both LT and ST production (25).
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193
ENTEROTOXINS
GTP
(AlP
cAMP
GDP NA
NAD
FIG. 4. Postulated A1 subunit of cholera guanosine
GDP,
triphosphate;
adenosine
molecular mode of action toxin. See text for details. ATP,
adenosine
of the GTP,
triphosphate;
diphosphate.
Purification and composition of ST has recently been described by Alderete and Robertson (26). ST appears to be a peptide of 4400 to 5 100 dabtons. Amino acid analysis reveals a high proportion of cystine and three tyrosines. A single N-terminal residue, glycine, was observed. The purified toxin is stable to heating (100 C, 30 mm) and does not lose biological activity after treatment with pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, or phospholipase C. Organic
solvents
(acetone,
phenol,
chloroform,
methanol) have no effect on its biological activity. The purified toxin is stable to acid (pH 1.0) but loses biological activity in alkali (pH 9.0). Neither lipopolysaccharide nor lipid could be detected in the purified material, but a positive carbohydrate reaction was obtained. Periodate oxidation did not, however, diminish the biological activity of ST. ST is a poor antigen but neutralization of ST activity was observed with serum from rabbits immunized with ST. The purification technique of Alderete and Robertson (26) used the following sequence: ultrafiltration through PM-bO and UM-2 Diaflo membranes, acetone fractionation, chloroform-methanol extraction, preparative gel electrophoresis, ion-exchange chromatography on DEAE agarose, and gel filtration on Bio-GEL P-l0. An alternative procedure for partial purification developed by W. J. Laird and D. M. Gill (unpublished data) was used to prepare ST for our own in vitro studies: chromatography on Amberlite XAD-2, elution with 99% methanol and 1% acetic acid,
FIELD
194
gel filtration on Sephadex LH-20, and elution with water. One ng of the purified ST of Alderete and Robertson and 10 ng of the Laird and Gill preparation elicit a half-maximal secretory response in a suckling mouse (one mouse unit). We have examined the in vitro effects of ST on ion transport and cyclic nucleotide metabolism (27). Addition of ST to the luminal side of rabbit ileal mucosa mounted in an Ussing Chamber produced a rapid increase in PD and 5CC that was slowly reversed by removing ST from the buminal bathing solution (Fig. 5, upper panel). Electrical responses could be obtained with as little as 0. 1 mouse unit/ml, which, according to Alderete and Robertson (26), is equivalent to 0. 1 ng/mb. Increasing concentrations of ST caused corresponding increases in PD (Lower panel). Addition of theophylli.ne after 60 mouse units of ST per ml produced a substantial further increase in PD that was, however, smaller than that produced by theophylline in the absence ofST. These electrical changes are associated with changes in unidirectional and net Na” and Cl fluxes under short-circuit conditions that are qualitatively identical with, although 25 to 50% smaller in magnitude than, those produced by theo-
U) 0 >
Minutes
FIG.
5. Effect of ST on transepithelial PD. The upand lower panels give results for separate tissues from the same animal. ST was added on the mucosal side. The specified increments in concentration are in mouse units per milliliter. Theophylline (THEO) was added to the serosal side. Reversibility was examined by replacing the solution in the mucosal-side reservoir several times, each change representing a 1: 10 dilution of the prior concentration. From Reference 27.
per
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phylline or cAMP ((27) and S. Guandalini and M. Field, unpublished data). ST did not alter cAMP concentration but produced a rapid and persistent elevation of cyclic guanosine triphosphate (cGMP) concentration (Fig. 6). cGMP concentration increased linearly with the square root of ST concentration over the entire range of concentrations tested (up to 266 mouse units/mb, which produced a 46-fold elevation). Furthermore, at the low end of their dose-response curves, electrical and cGMP responses showed similar sensitivities to ST. For cxample, 0.3 mouse unit/mi, which produced about one-third of the maximal electrical response, caused about a 4-fold elevation of cGMP concentration. Theophylline produced about a 3-fold elevation of cGMP concentration in both ST-treated and control intestine (Fig. 6), suggesting that ST does not affect cGMP phosphodiesterase activity. In order to ascertain that ST does indeed stimulate guanybate cyclase, we determined the activity of this enzyme is a crude membrane fraction from epitheiai cells isolated from rabbit ileum. As shown in Figure 7, addition of ST to the in vitro assay stimulated enzyme activity, the relative stimulation being more marked at low (0. 1 mM) than at high (1 mM) Mn2 concentration. The sensitivity of this membrane assay to ST was considerably lower than that of the intact cell assays, although, because of the small assay volume required, the total amounts of toxin added were similar. ST-containing E. coli culture filtrates also stimulate cGMP accumulation in mouse and rabbit small intestine in vivo, as recently reported by Hughes et al. (28). These investigations also showed that intraluminal 8-bromo-cGMP stimulated fluid secretion without increasing cAMP concentration. Preliminary reports from several laboratories suggest that, unlike cholera toxin and LT, ST stimulation of a nucleotide cyclase may be specific
for
intestine
(including
colon).
Guan-
ybate cyclase stimulation by ST could not be demonstrated in a variety of other tissues. Thus the intestine may have a unique receptor, a unique guanylate cyclase, or an enzyme that is needed to activate the toxin. These studies demonstrate another target enzyme for bacterial enterotoxins. That the ST-induced increments in intestinal mucosal
CHOLERA
AND
ESCHERICHIA
COLI for
cAMP
2.
C
4)
4,
0
0
3.
0.
E
0.
I
4.
6 35
5
FIG. and
cGMP
6. Effect
Minutes of heat-stable
concentrations
in
5.
enterotoxin rabbit
ileal
on cAMP mucosa.
ST
added at time zero; theophylline (Theo) added at 30 min. ST concentrations in mouse units per milliliter are shown. Dashed lines refer to untreated control tissues. From Reference 27.
6.
7.
8. >)
.
.
.
‘‘a
9.
00.
.
10.
Co
0.1
FIG.
7. Effect
mM
Mn
1.0 mM
Mn
11.
of heat-stable
enterotoxin on guanylate cyclase activity in a crude membrane fraction derived from isolated ileal epitheial cells. Assay conditions are those described by Field et al. (27). Results are shown for two Mn2 concentrations (5 mM Mg2’ also present). GTP, guanosine triphosphate.
12. 13.
cGMP concentration are indeed responsible for the associated changes in active ion transport has not been proven, but seems likely. E. coil ST may ultimately prove to be one of a class of bacterial enterotoxins that exert their diarrheagenic effect by stimulating the enzyme guanybate cyclase.
14.
The observations the author has described concerning the actions of heat-stable enterotoxin of E. coil were the work of several investigators: W. J. Laird, who not only provided us with toxin but also participated in transport studies; L. H. Graf, P. L. Smith, M. Rao, and S. Guandalini. Their contributions were much greater than the author’s own.
16.
References 1.
C. L. Heat-labile and heat-stable forms the enterotoxin from E. coil strains enteropathogenic GYLES,
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15.
17.
18. of
ENTEROTOXINS
Ann. N.Y. Acad. Sci. 176: 3 14, 1971. B., B. JACOBS AND R. MITRA. Antitoxin responses to infections with enterotoxigenic Escherichia coil. J. Infect. Diseases 129: 330, 1974. SACK, D. A., M. H. MERSON, J. G. WELLS, R. B. SACK AND G. K. MORRIS. Diarrhea associated with heat-stable enterotoxin-producing strains of E. coil. Lancet 2: 239, 1975. RYDER, R. W., I. K. WACHSMUTH, A. E. BUXTON, D. G. EVANS, H. C. DuPoNT, E. MASON AND F. F. Biutam. Infantile diarrhea produced by heat-stable enterotoxigenic E. co/i. New Engl. J. Med. 295: 849, 1976. GYLES, C. L. Relationships among heat-labile enterotoxins of Escherichia coil and Vibrio cho/erae. J. Infect. Diseases 129: 277, 1974. EVANS, D. J., L. C. CHEN, G. T. CIJRLIN AND D. G. EVANS. Stimulation of adenyl cyclase by Escherichia coil enterotoxin. Nature New Biol. 236: 137, 1977. KuPsi-zIN, F. A., AND R. F. ENGERT. Immunological interrelationships between cholera toxin and the heat-labile and heat-stable enterotoxins of coliform bacteria. Infect. Immun. 18: 110, 1977. FIELD, M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3’, 5’-AMP. Am. J. Physiol. 221: 992, 1971. USSING, H. H., AND K. ZERAHN. Active transport of sodium as the source of electric current in the shortcircuited isolated frog skin. Acta Phys. Scand. 23: 110, 1951. FIELD, M. Cholera toxin, adenylate cyclase, and the process of active secretion in the small intestine: the pathogenesis of diarrhea in cholera. In: Physiology of Membrane Disorders, part 5, Clinical Disorders of Membrane Transport Processes, edited by T. E. Andreoli, J. F. Hoffman, and D. D. Fanestil. New York: Plenum Press, 1978, p. 877. SCHULTZ, S. G. Sodium-coupled solute transport by small intestine: a status report. Am. J. Physiol. 233: E249, 1977. FIELD, M. Intestinal secretion. Gastroenterology 66: 1063, 1974. SHEERIN, H. E., AND M. FIELD. Ileal mucosal cyclic AMP and CI secretion: Serosal vs mucosal addition of cholera toxin. Am. J. Physiol. 232: E2 10, 1976. NELLANS, H. N., R. A. FRIZZELL AND S. G. SCHULTZ. Coupled sodium-chloride influxes across the brush border of rabbit ileum. Am. J. Physiol. 225: 467, 1973. HIRSCHHORN, N., J. L. KINZIE, D. B. SACHAR, R. S. NORTHRUP, J. 0. TAYLOR, S. Z. ARMAD AND R. A. PHILLIPS. Decrease in net stool output in cholera during intestinal perfusion with glucose-containing solutions. New EngI. J. Med. 279: 176, 1968. NELLANS, H. N., R. A. FRIZZELL AND S. G. SCHULTZ. Effect of acetazolumide on sodium and chloride transport by in vitro rabbit ileum. Am. J. Physiol. 228: 1808, 1975. CASE, R. M., M. JOHNSON, T. SCRATCHARD AND H. S. A. SHERRATr. Cyclic adenosine 3’, 5’-monophosphate concentration in the pancreas following stimulation by secretin, cholecystokinin-pancreozymin and acetylcholine. J. Physiol. 223: 669, 1972. SCHWARTZ, D. J., D. V. KJMBERG, H. E. SHEERIN, M. FIELD AND S. I. SAID. Vasoactive intestinal peptide stimulation of adenylate cyclase and active dccSACK,
pigs.
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R.
196
FIELD trolyte secretion 54: 536, 1974.
19.
20.
21.
22.
23.
in intestinal
mucosa.
J. Clin.
Invest.
N. R., R. D0LIN, D. S. FEDSON, H. Dupor, R. S. NORTHRUP, R. B. HORNICK AND R. M. CHANOCK. Acute infectious nonbacterial gastroenteritis: Etiology and pathogenesis. Ann. Internal Med. 76: 993, 1972. KERZNER, B., J. MCCLUNG, J. KELLY, M. BUTLER, D. GALL AND R. HAMILTON. Intestinal secretion in acute viral enteritis. A function of crypt-type enterocytes? Gastroenterology 68: 909, 1975. GILL, D. M. Mechanism of action of cholera toxin. In: Advances in Cyclic Nucleotide Research, edited by P. Greengard and G. A. Robison. New York: Raven Press, 1977, vol 8, p. 85. CASSEL, D., AND Z. SELINGER. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. USA 74: 3307, 1977. PIERCE, N. F. Protection against challenge with Escherichia co/i. Heat-labile enterotoxin by immunization of rats with cholera toxin/toxoid. Infect. Immun. 18:
338, 24.
BLACKLOW,
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25.
26.
27.
28.
1977.
R. S., J. F. SAGIN, W. A. PIERZCHALA, G. BONDE, B. A. RUBIN AND H. TIr. Activation of heat-labile Escherichia coil enterotoxin by trypsin. J. Infect. Diseases 133: 541, 1976. GYLES, C. L., S. PALCHANDHURI AND W. K. Ms. Naturally occurring plasmid carrying genes for enterotoxin production and drug resistance. Science 198: 198, 1977. ALDERETE, J. F., AND D. C. ROBERTSON. Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coil. Infect. Immun. 19: 1021, 1978. FIELD, M., L. H. GRAF, W. J. LAIRD AND P. L. SMITH. Heat-stable enterotoxin of Escherichia co/i: in vitro effects on guanylate cyclase activity, cyclic GMP concentration and ion transport in small intestine. Proc. Natl. Acad. Sci. USA 75: 2800, 1978. HUGHES, J. M., F. MURAD, B. CHANG AND R. L. Gumuw.i’r. Role of cyclic GMP in the action of heat-stable enterotoxin of Escherichia co/i. Nature 271: 755, 1978. RAPPAPORT,
Field,3
of action co/i
of cholera 2
M.D. ABSTRACT
Current
including
how
properties
of
separate probably
in
transfer
triphosphatase, basis
for
both
heat-labile
processes is inhibited
cells.
Cholera
mucosal
and
this
stimulates
its enterotoxicity.
other
with
diar-
How the small intestine secretes: the relation between diarrhea in a patient with cholera and active secretion in an Ussing chamber.
The
American
cholera can 1 liter/hr.
JournalofC/inica/
Nutrition
have The
massive excreted
32: JANUARY
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/1/189/4666372 by University of Glasgow user on 26 April 2018
by E.
co/i
toxins
from
NAD
guanylate
32:
189-196,
secretion their
results represses cyclase,
and
enterotoxins.
Two
secretory from
the
effect A,
by
subunitguanosine
adenylate which
1)
is stimulated,
to a membrane-bound
normally
mucosal
exert
intestine,
structures
monophosphate:
anion
stimulation
the
coli
3’,5’-adenosine
2) active
which
in small on
Escherichia
cyclic
This
Nuir.
and
and
ribose
enzyme,
J. C/in.
rheal disease-one heat labile (1, 2) and the other heat stable (1, 3, 4). The former is immunologically cross-reactive with cholera toxin (5) and, like cholera toxin, stimulates adenylate cyclase (6). The latter is not neutralized by cholera antitoxin (7) and heretofore there has been little insight into its mode of action. The purpose of this report is 2-fold: first, summarize current information on intestinal water and electrolyte transport in order to provide a conceptual framework for understanding how enterotoxins, in particubar, and enteric infection, in general, can stimulate intestinal secretion. Second, summarize current information on cholera toxin and the E. coli enterotoxins, including some of our own recent studies with the heat-stable enterotoxin of E. coli.
A patient with diarrhea exceeding
cyclase.
ofsecretion
cells
heat-labile
intestinal
Am.
mammals
altered
diphosphate
mechanism
monophosphate heat-stable
in villus
Certain entenc bacteria excrete substances that interact with the intestinal epitheium and cause the transport of water and electrobytes to reverse from absorption to secretion. Cholera is a classic example of this process and has been extensively studied. Two enterotoxins have been identified among the extracellular products of Escherichia coli isolated humans
are
and
inhibiting
enterotoxin
and
adenylate
of adenosine
on the
3’, 5’-adenosine
transport
thereby
E. co/i
is reviewed
cyclic
of NaCl
ion crypt
by
and
intestinal
catalyzed stable
cholera
absorption
stimulating
information
it is altered
active
coupled
from
and
cyclase. appears
Heatto be the
1979.
fluid is nearly protein-free and has an dcctrobyte composition similar to that of serum except that the chloride concentration is lower and the bicarbonate concentration higher. Dehydration, metabolic acidosis and their consequences may develop but, if water and electrolyte losses are adequately replaced, no fever or other evidence of tissue necrosis or systemic involvement appears and recovery is universal. The diarrhea originates in the small intestine, all of which is usually involved. There is neither epitheial invasion by bacteria nor evidence of damage to epitheial cells, even at the ultrastructural bevel. There is also no impairment of absorptive processes for nutrients such as sugars and amino acids. This diarrheal process can be reproduced in animals by innoculating the small intestine with either cholera organisms, a bacteria-free culture filtrate or the purified toxin (see below). At about the time that these cardinal features of clinical cholera gained general acceptance, the effects of cyclic 3’, 5’-adenosine monophosphate (cAMP) on an in vitro preparation of small intestine (8) were demonstrated, thereby setting the stage for much of the subsequent research into the cellular and molecular changes through which Vibrio I
From
the
Departments
of Medicine
logical cago,
and Physiological Sciences, Chicago, Illinois 60637. 2 Supported by National Institute AI-09029 and AM-2l345. 3
1979,
and
Pharmaco-
University of
Health
of
Chi-
Grants
Professor. pp.
189-196.
Printed
in U.S.A.
189
FIELD
190
brane couples the transmembrane movement of glucose to that of Na; the Na that enters with glucose is then pumped out of the cell into the lateral space; in the process adenosine triphosphate is hydrolyzed by Na, K-activated adenosine triphosphatase located in the basolateral membrane. The driving force for gl ucose absorption, therefore is the electrochemical potential difference for Na across the brush-border membrane. This difference, which consists of both a difference in Na’ concentration (intracellular Na” is low) and in electric potential (the cell interior is dcctronegative), favors the influx and restricts the efflux of Na and also glucose, whose movement is coupled to that of Na. Once glucose accumulates in the absorptive cell, it diffuses across the basolateral border by a process that may be carrier-mediated but is not coupled to Na’. A corollary of the Na’dependence ofglucose absorption is the stimulation of Na’ (and secondarily of C1 and water) absorption by luminal glucose. Absorption of neutral amino acids is also coupled to that of Na (1 1). As shown in Figure 1, the addition of theophylline, which inhibits enzymatic degradation of cAMP, to the serosal side of the ileal mucosa, causes a rapid and sustained increase in PD and 5CC. The same result can be obtained by adding cAMP itself. Figure 1 also illustrates the changes in net fluxes that develop under short-circuit conditions: the
cho/erae enterotoxin exerts its secretory effect. I will briefly describe the experimental preparation and some of its ion transport properties so that subsequent comments on underlying mechanisms will make sense: rabbit ileum can be stripped of muscle and mounted in an “Ussing” chamber (9) as a flat sheet separating two identical, oxygenated, bicarbonate-buffered Ringer solutions. Under basal conditions, 1 to 4 mV electric potential difference (PD) across the ileab mucosa (serosa-positive) can be measured (10). For ion flux measurements, this PD is usually nuffifled (short-circuited) by passing current from an external source through the tissue. When the epitheium is short-circuited, all net ion fluxes can be attributed to active transport. A net flux is determined as the difference between opposing unidirectional fluxes measured with radioisotopes. The unidirectional fluxes are often larger than the net fluxes because thejunctions between cells are highly permeable to monovalent ions. Normally the ileum generates net absorptive fluxes of Na’ and Cl and a larger net secretory flux of HCO (10). The PD and short-circuit current (5CC) are due mainly to the latter. If glucose is added to the buminal side of the tissue, the PD and SCC increase very quickly due to stimulation of active Na’ absorption (I 1). The relation between glucose and Na transports can be explained as follows (1 1): a carrier in the brush border mem-
6
C”
E
4
C.) #{188}.
-4--
C..)
2
+Theo
(I)
30
0
FIG. (J,),
and
theophylline.
I. Effects residual From
60
90
minutes of theophylline ion flux (Jet) Reference
on SSC corresponds
and
net ion fluxes. to the
12.
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/1/189/4666372 by University of Glasgow user on 26 April 2018
actual
time
The
inset
period
showing over
which
net Na flux fluxes
were
(J,) , net measured.
Cl flux Theo,
CHOLERA
AND
ESCHERICHIA
-5 -5
2
HOURS
FIG. vitro.
2. Effects
From
AFTER
ofcholera Reference 13.
TOXIN
toxin
AOOED
on transmural
PD
in
direction of net C1 transport reverses and, in the absence of luminal glucose, net absorption of Na” disappears. Under some circumstances, net Na secretion develops. The residual ion flux (that portion of the 5CC not accounted for by Na” and Cb), which is a measure of HCO secretion (10), either remains the same or increases. In general, the increase in 5CC represents the difference between the change in net anion flux (Cl and HCO) and the change in net Na flux. Since these changes have all been measured under short-circuit conditions, there appears to be a change in the active transport of each of these ions. The sum of these effects constitutes a large driving force for the secretion not only of electrolytes but also of water, which follows the ion movements passively. The action of cAMP and theophylline is closely mimicked by that of cholera toxin ( 10). The time course is different (Fig. 2), since the activation of adenylate cyclase by cholera toxin is slow, but the end result is the same. Indeed, theophylline, added 4 hr after cholera toxin, evokes little further change in PD. A detailed consideration of the cellular mechanisms responsible for these alterations of ion transport is beyond the scope of this presentation and, indeed, these mechanisms are as yet only very imperfectly understood. I would nonetheless like to briefly and, of necessity, superficially state my current understanding of these mechanisms since the way they are viewed affects our thinking not only about enterotoxic diarrheas but also about diarrheas associated with invasive and destructive enteric organisms. Normally, a
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COLI
ENTEROTOXINS
191
major part of salt and water absorption from the small intestine appears to be a consequence of the presence in the brush-border membrane ofa channel or carrier that accepts and transbocates the ion pair, NaC1 (14). In the absence of Na’, Cl uptake is diminished and, in the absence of Cl, Na uptake is diminished by the same amount. The Na that enters the cell along with C1, is then actively extruded across the basolateral membrane by the ouabain-inhibitable Na’s pump. The efflux of C1 from cell to lumen over the coupled pathway is thus restricted by the bow intracellular Na concentration and therefore C1 accumulates in the intestinal epithelial cell above ebectrochemical equilibrium and diffuses out of the cell across the basolateral membrane (the latter is presumably far more permeable to free C1 than is the brush border). This mechanism for Cl absorption is directly analogous to that for glucose absorption (see above) and is another example of how the intestinal epitheial cell uses the potential energy in the Na gradient for the transport of a variety of other solutes. Coupled uptake of NaCl across the brush border is inhibited by cAMP, which thereby inhibits “active” Cl absorption and also a large portion of active Na absorption (14). Indeed, in the absence of nutrients which specifically stimulate Na absorption, the majority of salt and water absorption in the small intestine probably occurs by this cAMP-inhibitable mechanism. It is important to bear in mind that this action of cAMP appears to be specific for coupled NaCl transbocation across the brush border and does not constitute a general inhibition of all absorptive processes. The capacity of the Na pump to respond to an increase in Na influx is not impaired and the Na-coupled absorptive processes for sugars and neutral amino acids are also not impaired. Fluid losses of cholera patients can be replaced orally if glucose is added to the replacement solution (15). Although cAMP plays shift
from
the antiabsorptive a significant role absorption
to
secretion,
effect of in the overall it cannot
by itself explain net secretion. Either an underlying secretory process is unmasked through the inhibition of coupled NaC1 absorption or cAMP must have a second effect, directly stimulating active secretion. That the latter alternative is the correct one has been
FIELD
192
demonstrated with pharmacologic agents (acetazobomide, furosemide) which, when placed on the luminab side of the intestine, inhibit coupled NaCl absorption without stimulating secretion ((16); M. Field, unpublished data). Figure 3 summarizes these separate actions of cAMP. The antiabsorptive action is exerted on villus cells, (these are the only cells accessible to measurements of mitial rates of Na’ and Cl uptake, which were made over 45 sec (14)). There is considerable indirect, but nonetheless compelling evidence that the secretory action of cAMP is exerted mainly or exclusively on cells in the crypts of Lieberkuhn (10). The crypt portion of the epitheium, in addition to being the site for cell renewal, also appears to function as an exocrine gland. cAMP, which is an intracelbular mediator for the stimulation by secretin of pancreatic NaHCO3 and water secretion (17), appears also to be an intracellular mediator of secretion in the small intestine, responding to both intraluminal stimuli (i.e., cholera toxin) and hormonal stimuli (i.e., vasoactive intestinal peptide ( 1 8)). Just as the “resting” pancreas still maintains a low rate of secretion, the intestinal crypts, in the absence of extraordinary stimuli such as cholera toxin, may do the same. This “basal” secretion is normally more than offset by the absorptive activities of the villus cells and is therefore difficult to quantitate. If villus cells are damaged or destroyed, however, some LUMEN
VILLUS
BLOOD
CELL
CRYPT
CRYPT
FIG. transport adenosine
CELL
LUMEN
3. Postulated separate actions of cAMP on ion by small intestine. See text for details. ATP, triphoSphate; ADP, adenosine diphoSphate.
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degree of underlying secretion may be unmasked. In viral enteritis, for example, viruses seem to preferentially invade villus cells and crypt cells are often spared (19, 20). Indeed, partial villus atrophy and associated malabsorption of nutrients has been described (19). The watery diarrhea sometimes observed in viral enteritis could therefore be due to the unmasking of crypt cell secretion (20). The mechanism for active anion secretion appears to be fundamentally the same in intestinal crypts as in a variety of other secretory epitheia and is not well understood. A model for active Cl secretion that is closely rebated to the model for coupled NaC1 absorption has recently been proposed (10). Molecular mechanism and heat-labile E. coil
of action enterotoxins
of cholera
Cholera toxin exerts its secretory effect by stimulating the activity of intestinal mucosal adenylate cyclase, thereby increasing the concentration of cAMP in intestinal epitheiab cells (10). The time course of these changes is the same as that of the resulting alterations of ion transport (10). The structure, cell membrane receptor, and intracellular action of cholera toxin have recently been reviewed ( 10, 2 1). It is a protein of 84,000 MW consisting of three peptide chains, A1, A2, and B which weigh about 24,000, 5,000 and 1,000 daltons, respectively. Each toxin molecule contains one each of the A chains and five B chains. A1 and A2 are connected by a single disulfide bond. The B chains can be isolated as a single subunit, which is known as “choberagenoid”. Choleragenoid does not stimulate adenybate cyclase but binds to cell membrane receptors in a manner indistinguishable from that ofthe full toxin molecule. The cell receptor for cholera toxin and choleragenoid is the monosiaboganglioside, Gmi. The bond between Gmi and B subunits is very tight, dissociation constants in the range of bO having been reported. The A subunit (A1 + A2) is nontoxic when added to intact cells but highly active when added to lysates. This toxicity resides in the A1 peptide: by itself A2 is nontoxic, and it does not enhance the toxicity of A1 in lysates. Adenylate cyclase stimulation by cholera toxin therefore involves attachment of the B subunits to cell surface receptors, followed by
CHOLERA
insertion through
AND
ESCHERICHIA
and possibly where it interacts with adenylate cyclase. This reorientation of the A1 peptide in the cell membrane is temperature-dependent and slow. At least 15 miii transpires before detectable stimulation ofthe intestinal cyclase develops whereas addition of A1 to a lysate produces measurable enzyme activation within seconds. As shown by Gill (21), adenylate cyclase activation by the A1 peptide of cholera toxin requires NAD and is greatly facilitated by one or more cytosol macromolecules. Like the active fragment of diptheria toxin, the A1 peptide of cholera toxin splits NAD into adenosine diphosphoribose and nicotinamide, the former being covalently bonded to a receptor protein in the plasma membrane. As shown by Cassel and Selinger (22), the receptor protein is a guanosine triphosphatase, or part of a guanosine triphosphatase, which regulates the activity of adenylate cyclase by converting this enzyme from its active, guanosine triphosphate-bound form to an inactive form. A1 peptide inactivates this guanosine triphosphatase activity. These reactions are summarized in Figure 4. Some E. co/i produce a heat-labile enterotoxin (LT) which is similar to and possibly identical with cholera toxin. This toxin can be neutralized with cholera antitoxin and also with anticholeragenoid (23). It contains a 24,000 dalton subunit which is an ADPR transferase (D. M. Gill, personal communication). It is possible, as suggested by Rappaport et al. (24), that E. coli secrete LT in the form of a larger protein that is then activated by bacterial and possibly mammaian proteases. The genetic information for LT production is contained in a plasmid (25) and therefore toxicity can be transmitted from one strain of E. coli to another. Purification heat-stable
COLI
of the A1 peptide into the plasma membrane,
and biological actions of the enterotoxin (ST) of E. coil
In addition to LT, ST has been isolated from diarrheagenic strains of E. coli (1, 3, 4). Toxigenic E. co/i can produce both LT and ST, LT alone, or ST alone. As is the case with LT, the genetic information for ST production has been found in a pbasmid and a single plasmid can carry genes for both LT and ST production (25).
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193
ENTEROTOXINS
GTP
(AlP
cAMP
GDP NA
NAD
FIG. 4. Postulated A1 subunit of cholera guanosine
GDP,
triphosphate;
adenosine
molecular mode of action toxin. See text for details. ATP,
adenosine
of the GTP,
triphosphate;
diphosphate.
Purification and composition of ST has recently been described by Alderete and Robertson (26). ST appears to be a peptide of 4400 to 5 100 dabtons. Amino acid analysis reveals a high proportion of cystine and three tyrosines. A single N-terminal residue, glycine, was observed. The purified toxin is stable to heating (100 C, 30 mm) and does not lose biological activity after treatment with pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, or phospholipase C. Organic
solvents
(acetone,
phenol,
chloroform,
methanol) have no effect on its biological activity. The purified toxin is stable to acid (pH 1.0) but loses biological activity in alkali (pH 9.0). Neither lipopolysaccharide nor lipid could be detected in the purified material, but a positive carbohydrate reaction was obtained. Periodate oxidation did not, however, diminish the biological activity of ST. ST is a poor antigen but neutralization of ST activity was observed with serum from rabbits immunized with ST. The purification technique of Alderete and Robertson (26) used the following sequence: ultrafiltration through PM-bO and UM-2 Diaflo membranes, acetone fractionation, chloroform-methanol extraction, preparative gel electrophoresis, ion-exchange chromatography on DEAE agarose, and gel filtration on Bio-GEL P-l0. An alternative procedure for partial purification developed by W. J. Laird and D. M. Gill (unpublished data) was used to prepare ST for our own in vitro studies: chromatography on Amberlite XAD-2, elution with 99% methanol and 1% acetic acid,
FIELD
194
gel filtration on Sephadex LH-20, and elution with water. One ng of the purified ST of Alderete and Robertson and 10 ng of the Laird and Gill preparation elicit a half-maximal secretory response in a suckling mouse (one mouse unit). We have examined the in vitro effects of ST on ion transport and cyclic nucleotide metabolism (27). Addition of ST to the luminal side of rabbit ileal mucosa mounted in an Ussing Chamber produced a rapid increase in PD and 5CC that was slowly reversed by removing ST from the buminal bathing solution (Fig. 5, upper panel). Electrical responses could be obtained with as little as 0. 1 mouse unit/ml, which, according to Alderete and Robertson (26), is equivalent to 0. 1 ng/mb. Increasing concentrations of ST caused corresponding increases in PD (Lower panel). Addition of theophylli.ne after 60 mouse units of ST per ml produced a substantial further increase in PD that was, however, smaller than that produced by theophylline in the absence ofST. These electrical changes are associated with changes in unidirectional and net Na” and Cl fluxes under short-circuit conditions that are qualitatively identical with, although 25 to 50% smaller in magnitude than, those produced by theo-
U) 0 >
Minutes
FIG.
5. Effect of ST on transepithelial PD. The upand lower panels give results for separate tissues from the same animal. ST was added on the mucosal side. The specified increments in concentration are in mouse units per milliliter. Theophylline (THEO) was added to the serosal side. Reversibility was examined by replacing the solution in the mucosal-side reservoir several times, each change representing a 1: 10 dilution of the prior concentration. From Reference 27.
per
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phylline or cAMP ((27) and S. Guandalini and M. Field, unpublished data). ST did not alter cAMP concentration but produced a rapid and persistent elevation of cyclic guanosine triphosphate (cGMP) concentration (Fig. 6). cGMP concentration increased linearly with the square root of ST concentration over the entire range of concentrations tested (up to 266 mouse units/mb, which produced a 46-fold elevation). Furthermore, at the low end of their dose-response curves, electrical and cGMP responses showed similar sensitivities to ST. For cxample, 0.3 mouse unit/mi, which produced about one-third of the maximal electrical response, caused about a 4-fold elevation of cGMP concentration. Theophylline produced about a 3-fold elevation of cGMP concentration in both ST-treated and control intestine (Fig. 6), suggesting that ST does not affect cGMP phosphodiesterase activity. In order to ascertain that ST does indeed stimulate guanybate cyclase, we determined the activity of this enzyme is a crude membrane fraction from epitheiai cells isolated from rabbit ileum. As shown in Figure 7, addition of ST to the in vitro assay stimulated enzyme activity, the relative stimulation being more marked at low (0. 1 mM) than at high (1 mM) Mn2 concentration. The sensitivity of this membrane assay to ST was considerably lower than that of the intact cell assays, although, because of the small assay volume required, the total amounts of toxin added were similar. ST-containing E. coli culture filtrates also stimulate cGMP accumulation in mouse and rabbit small intestine in vivo, as recently reported by Hughes et al. (28). These investigations also showed that intraluminal 8-bromo-cGMP stimulated fluid secretion without increasing cAMP concentration. Preliminary reports from several laboratories suggest that, unlike cholera toxin and LT, ST stimulation of a nucleotide cyclase may be specific
for
intestine
(including
colon).
Guan-
ybate cyclase stimulation by ST could not be demonstrated in a variety of other tissues. Thus the intestine may have a unique receptor, a unique guanylate cyclase, or an enzyme that is needed to activate the toxin. These studies demonstrate another target enzyme for bacterial enterotoxins. That the ST-induced increments in intestinal mucosal
CHOLERA
AND
ESCHERICHIA
COLI for
cAMP
2.
C
4)
4,
0
0
3.
0.
E
0.
I
4.
6 35
5
FIG. and
cGMP
6. Effect
Minutes of heat-stable
concentrations
in
5.
enterotoxin rabbit
ileal
on cAMP mucosa.
ST
added at time zero; theophylline (Theo) added at 30 min. ST concentrations in mouse units per milliliter are shown. Dashed lines refer to untreated control tissues. From Reference 27.
6.
7.
8. >)
.
.
.
‘‘a
9.
00.
.
10.
Co
0.1
FIG.
7. Effect
mM
Mn
1.0 mM
Mn
11.
of heat-stable
enterotoxin on guanylate cyclase activity in a crude membrane fraction derived from isolated ileal epitheial cells. Assay conditions are those described by Field et al. (27). Results are shown for two Mn2 concentrations (5 mM Mg2’ also present). GTP, guanosine triphosphate.
12. 13.
cGMP concentration are indeed responsible for the associated changes in active ion transport has not been proven, but seems likely. E. coil ST may ultimately prove to be one of a class of bacterial enterotoxins that exert their diarrheagenic effect by stimulating the enzyme guanybate cyclase.
14.
The observations the author has described concerning the actions of heat-stable enterotoxin of E. coil were the work of several investigators: W. J. Laird, who not only provided us with toxin but also participated in transport studies; L. H. Graf, P. L. Smith, M. Rao, and S. Guandalini. Their contributions were much greater than the author’s own.
16.
References 1.
C. L. Heat-labile and heat-stable forms the enterotoxin from E. coil strains enteropathogenic GYLES,
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15.
17.
18. of
ENTEROTOXINS
Ann. N.Y. Acad. Sci. 176: 3 14, 1971. B., B. JACOBS AND R. MITRA. Antitoxin responses to infections with enterotoxigenic Escherichia coil. J. Infect. Diseases 129: 330, 1974. SACK, D. A., M. H. MERSON, J. G. WELLS, R. B. SACK AND G. K. MORRIS. Diarrhea associated with heat-stable enterotoxin-producing strains of E. coil. Lancet 2: 239, 1975. RYDER, R. W., I. K. WACHSMUTH, A. E. BUXTON, D. G. EVANS, H. C. DuPoNT, E. MASON AND F. F. Biutam. Infantile diarrhea produced by heat-stable enterotoxigenic E. co/i. New Engl. J. Med. 295: 849, 1976. GYLES, C. L. Relationships among heat-labile enterotoxins of Escherichia coil and Vibrio cho/erae. J. Infect. Diseases 129: 277, 1974. EVANS, D. J., L. C. CHEN, G. T. CIJRLIN AND D. G. EVANS. Stimulation of adenyl cyclase by Escherichia coil enterotoxin. Nature New Biol. 236: 137, 1977. KuPsi-zIN, F. A., AND R. F. ENGERT. Immunological interrelationships between cholera toxin and the heat-labile and heat-stable enterotoxins of coliform bacteria. Infect. Immun. 18: 110, 1977. FIELD, M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3’, 5’-AMP. Am. J. Physiol. 221: 992, 1971. USSING, H. H., AND K. ZERAHN. Active transport of sodium as the source of electric current in the shortcircuited isolated frog skin. Acta Phys. Scand. 23: 110, 1951. FIELD, M. Cholera toxin, adenylate cyclase, and the process of active secretion in the small intestine: the pathogenesis of diarrhea in cholera. In: Physiology of Membrane Disorders, part 5, Clinical Disorders of Membrane Transport Processes, edited by T. E. Andreoli, J. F. Hoffman, and D. D. Fanestil. New York: Plenum Press, 1978, p. 877. SCHULTZ, S. G. Sodium-coupled solute transport by small intestine: a status report. Am. J. Physiol. 233: E249, 1977. FIELD, M. Intestinal secretion. Gastroenterology 66: 1063, 1974. SHEERIN, H. E., AND M. FIELD. Ileal mucosal cyclic AMP and CI secretion: Serosal vs mucosal addition of cholera toxin. Am. J. Physiol. 232: E2 10, 1976. NELLANS, H. N., R. A. FRIZZELL AND S. G. SCHULTZ. Coupled sodium-chloride influxes across the brush border of rabbit ileum. Am. J. Physiol. 225: 467, 1973. HIRSCHHORN, N., J. L. KINZIE, D. B. SACHAR, R. S. NORTHRUP, J. 0. TAYLOR, S. Z. ARMAD AND R. A. PHILLIPS. Decrease in net stool output in cholera during intestinal perfusion with glucose-containing solutions. New EngI. J. Med. 279: 176, 1968. NELLANS, H. N., R. A. FRIZZELL AND S. G. SCHULTZ. Effect of acetazolumide on sodium and chloride transport by in vitro rabbit ileum. Am. J. Physiol. 228: 1808, 1975. CASE, R. M., M. JOHNSON, T. SCRATCHARD AND H. S. A. SHERRATr. Cyclic adenosine 3’, 5’-monophosphate concentration in the pancreas following stimulation by secretin, cholecystokinin-pancreozymin and acetylcholine. J. Physiol. 223: 669, 1972. SCHWARTZ, D. J., D. V. KJMBERG, H. E. SHEERIN, M. FIELD AND S. I. SAID. Vasoactive intestinal peptide stimulation of adenylate cyclase and active dccSACK,
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FIELD trolyte secretion 54: 536, 1974.
19.
20.
21.
22.
23.
in intestinal
mucosa.
J. Clin.
Invest.
N. R., R. D0LIN, D. S. FEDSON, H. Dupor, R. S. NORTHRUP, R. B. HORNICK AND R. M. CHANOCK. Acute infectious nonbacterial gastroenteritis: Etiology and pathogenesis. Ann. Internal Med. 76: 993, 1972. KERZNER, B., J. MCCLUNG, J. KELLY, M. BUTLER, D. GALL AND R. HAMILTON. Intestinal secretion in acute viral enteritis. A function of crypt-type enterocytes? Gastroenterology 68: 909, 1975. GILL, D. M. Mechanism of action of cholera toxin. In: Advances in Cyclic Nucleotide Research, edited by P. Greengard and G. A. Robison. New York: Raven Press, 1977, vol 8, p. 85. CASSEL, D., AND Z. SELINGER. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. USA 74: 3307, 1977. PIERCE, N. F. Protection against challenge with Escherichia co/i. Heat-labile enterotoxin by immunization of rats with cholera toxin/toxoid. Infect. Immun. 18:
338, 24.
BLACKLOW,
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25.
26.
27.
28.
1977.
R. S., J. F. SAGIN, W. A. PIERZCHALA, G. BONDE, B. A. RUBIN AND H. TIr. Activation of heat-labile Escherichia coil enterotoxin by trypsin. J. Infect. Diseases 133: 541, 1976. GYLES, C. L., S. PALCHANDHURI AND W. K. Ms. Naturally occurring plasmid carrying genes for enterotoxin production and drug resistance. Science 198: 198, 1977. ALDERETE, J. F., AND D. C. ROBERTSON. Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coil. Infect. Immun. 19: 1021, 1978. FIELD, M., L. H. GRAF, W. J. LAIRD AND P. L. SMITH. Heat-stable enterotoxin of Escherichia co/i: in vitro effects on guanylate cyclase activity, cyclic GMP concentration and ion transport in small intestine. Proc. Natl. Acad. Sci. USA 75: 2800, 1978. HUGHES, J. M., F. MURAD, B. CHANG AND R. L. Gumuw.i’r. Role of cyclic GMP in the action of heat-stable enterotoxin of Escherichia co/i. Nature 271: 755, 1978. RAPPAPORT,
