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Effects of Prostaglandins and Cholera Enterotoxin

on Intestinal Mucosal Cyclic AMPAccumulation

EVIDENCEAGAINST AN ESSENTIAL ROLEFOR

PROSTAGLANDINSIN THE ACTION OF TOXIN

DANIEL V. KIMBERG, MICHAEL FIELD, ELAINE GERSHON,andANroN HENDERSON

From the Department of Medicine, Harvard Medical School and TheGastrointestinal Unit of the Department of Medicine, Beth Israel Hospital,Boston, Massachusetts 02215

A B S T R A C T Both cholera enterotoxin and certainprostaglandins have been shown to stimulate intestinalfluid secretion in vivo, to cause ion flux changes in vitrosimilar to those caused by addition of cyclic 3',5'-adeno-sine monophosphate (cyclic AMP), and to activate in-testinal mucosal adenyl cyclase. It has been suggestedthat the effects of the enterotoxin on intestinal cyclicAMPmetabolism may be indirect, and that locally syn-thesized prostaglandins may serve as required intermedi-ates for the effects of the enterotoxin in activating in-testinal mucosal adenyl cyclase. In order to clarify cer-tain aspects of the mechanisms by which these two agentsalter intestinal mucosal cyclic AMPmetabolism and iontransport, their effects on cyclic AMPaccumulation inrabbit ileal mucosa were examined in vitro. Addition of5 Ag per ml (75 Ag per 150 mg mucosa) of purifiedcholera enterotoxin produced a peak increase in cyclicAMPlevel in 3 h but there was a time delay of at least30 min before any effect was observed. Inhibition ofcyclic nucleotide 'phosphodiesterase with theophyllinefailed to reduce this time delay. In contrast, addition ofprostaglandin E1 (PGE1) increased the cyclic AMPlevel rapidly, a peak effect being observed in 2 min. Thetime of the peak prostaglandin-induced changes in cyclicAMP level and short-circuit current correlated closely.A maximal increment in cyclic AMPlevel was achievedwith 5 X 10' M PGE1. When 10' M PGE1 was addedto mucosa already maximally stimulated with choleratoxin, the resulting cyclic AMP. level was equal to the

Dr. Kimberg is the recipient of a Research Career De-velopment Award, AM-19377.

Received for publication 29 March 1973 and in revisedform 24 August 1973.

sum of the levels reached when each agent was addedalone. Furthermore, the effects of the enterotoxin onmucosal cyclic AMP levels were not influenced by in-domethacin under conditions where mucosal prosta-glandins synthesis was inhibited. The results suggestthat endogenous prostaglandins do not provide an es-sential link in the activation of intestinal mucosal adenylcyclase by cholera enterotoxin. The present study alsoindicates that the effect of cholera enterotoxin on in-testinal mucosal cyclic AMPmetabolism involves a defi-nite time delay which is not due to cyclic nucleotidephosphodiesterase activity.

INTRODUCTION

There has been a great deal of recent interest in eluci-dating the mechanism of action of cholera enterotoxinin the intestine (see references 1-3 for reviews). It isnow known that the massive outpouring of fluid andelectrolyte is of small intestinal origin (4, 5), that itoccurs in the absence of significant alterations in smallbowel morphology (6, 7), and that an active secretoryprocess provides the driving force for these intestinallosses (8-10). Several recent studies strongly suggestthat cholera enterotoxin produces its effects by stimu-lating intestinal mucosal adenyl cyclase, thereby alter-ing the concentration of cyclic 3',5'-adenosine monophos-phate (cyclic AMP)1 (11-13). Both cyclic AMPandcholera enterotoxin have similar effects on ion trans-port when added in vitro to isolated ileal mucosa (10-

1Abbreviations used in this paper: cyclic AMP, cyclic3',5'-adenosine monophosphate; PG, prostaglandin; SCC,short-circuit current.

The Journal of Clinical Investigation Volume 53 March 1974-941-949 941

14). Evidence has also been presented that the twoagents act upon the same secretory mechanism (10).Furthernmore, clholera enterotoxin has been shown toincrease adenyl cyclase activity and cyclic AMP levelsin the intestinal mucosa with a time pattern similar tothat of its effect on fluid transport (13, 15).

The mechanism by which cholera enterotoxin acti-vates intestinal mucosal a(lenyl cyclase is largely un-known. After addition of toxin, adenyl cyclase activityincreases slowly over 2-4 h (11, 15). There may also bean initial time lag of 15-45 min before any change inadenyl cyclase activity, cyclic AMIP level, or ion trans-port occurs (1-4, 10-13, 15). Because of the inevitabledifficuilty in mieasuring iniitial simall clhaniges, however,both the existence of suclh a time lag and its durationare uncertain. If an irreducible interval between contactof toxin with the mucosa and initiation of a cyclicAMP-related event does indeed exist, this fact hasimportant implications with respect to the mechanism bywhiclh the toxin acts. One possible explanation for atime lag is the presence of sufficient cyclic nucleotidephosphodiesterase activity to prevent the cyclic ANIPlevel from increasing in response to a small incrementin adenyl cyclase activity. If that is the case, it shouldbe possible to shorten the time lag by inhibiting phos-phodiesterase activity with theophylline. Another pos-sible explanation for a time lag is that cholera entero-toxin may affect the synthesis or degradation of anothercompound which interacts more directly with adenylcyclase. The cellular level of this compound would firsteither have to increase or decrease sufficiently before achange in adenyl cy clase activity could occur. Asidefrom bacterial toxins, the prostaglandins are the onlyother group of compounds which has been shown to en-hance intestinal mucosal adenyl cyclase (11, 12) and tostimulate small bowel wvater and electrolyte secretion.The latter has been demonstrated both in vivo (16, 17)and in vitro (18).

Recently, it was suggested that the effects of the en-terotoxin on intestinal cyclic AMPmetabolism may beindirect, with an increase in locally synthesized prosta-glandins serving as the required intermediates responsi-ble for activating mucosal cyclase (19, 20). The previ-ously reported additive effect of prostaglandin El(PGE,) and cholera enterotoxin on intestinal mucosaladenyl cyclase (11, 12) argues against this hypothesis,but, taken alone, this evidence is less than conclusive.Effects on the adenyl cyclase activity of broken-cellpreparations may not be representative of events in theintact cell. Furthermore, neither of the two prior studiesconcerned with adenyl cyclase activity demonstrated thata maximally stimulating amount of toxin had been em-ployed (11, 12).

Tlhe lpresent study was uin(lertaken in or(ler to clarify

some of these issues. More specifically, experimentswere designed to (a) determine the time and dose-re-sponse patterns of mucosal cyclic AMP levels after ilvitro additions of cholera enterotoxin and PGE1; (b)determine whether the time interval between toxin addi-tion and the first increase in cyclic AMP level can beshortened by adding a larger amount of toxin or byinlhibiting cyclic nucleotide phosphodiesterase activitywvith theophylline; (c) determine the relationship be-tween the effects of PGEi on mucosal cyclic AMPlevelsand on short-circuit current (SCC), the latter being tisedas a measure of cyclic AMP-dependent secretory re-sl)olse; and (d) explore further the relationship betweenprostaglandin and toxin-indtuced inicreases in mucosalcyclic AMP levels, by establishing wvhether their effectin combination is competitive, additive, or synergistic,and by determining whether inhibition of prostaglan(libiosynthesis will interfere with the action of toxin.

METHODSIncubation of tissutes. New Zealand white male rabbits

that weighed between 2 and 3 kg were killed by a blow tothe head, and the distal ileum was removed, opened longi-tudinally, and rinsed in a Krebs-Ringer solution (pH 7.4)which was gassed with 95% 02-5% C02. The Krebs-Ringersolution contained the following ions in millimoles perliter: Na, 141; K, 10; Ca, 1.25; Mg, 1.1; Cl, 127; HCO:,25; H2PO4, 0.3; and HPO4, 1.65. The ileum was strippe(dof muscularis, and three to six segments of mucosa, eachapproximately 50 mg in wet weight, were added to 50-mlErlenmeyer flasks containing between 7.2 and 7.5 ml of theKrebs-Ringer solution and additions of purified choleraenterotoxin,2 PGE,,8 indomethacin, acetylsalicylic acid (as-pirin), or the appropriate solvents as indicated below.Flasks were incubated at 37C in a metabolic water bathoscillating at a rate of 80 rpm while being continluouslygassed with 95% 02-5% C02 which was first bubbledthrough water to minimize evaporative water loss.

Cyclic AMP measurements. At the termination of in-cubation, individual segments of mucosa were rapidly re-moved from the flasks, and each was placed in a conicalcentrifuge tube containing 5 ml of ice-cold 5% trichloro-acetic acid with 0.8 nCi of [3H]cyclic AMP (24 Ci permmol) as a recovery marker. The tissues were homogenizedwith a Teflon pestle in a glass homogenizer, and the tri-chloroacetic acid precipitates were then centrifuged at 4,000g for 20 min at 4C. The supernates were decanted into40-ml conical centrifuge tubes, and 0.5 ml of 1 N HCI wasthen added. The trichloroacetic acid was extracted fivetimes with 10 ml of diethyl ether, after which the samplesw-ere evaporated to dryness at 50'C using an Evapo-Mixevaporator (Buchler Instruments Div., Nuclear-Chicago

2 Purified cholera enterotoxin, prepared according to theprocedure described in J. Infect. Dis., 121 (Suppl.) : 563,1970 and under contract for the National Institute of Al-lergy and Infectious Diseases by R. A. Finkelstein, Ph.D.,The University of Texas Southwestern Medical School,Dallas, Texas.

'We thank Dr. John Pike of the Upjohn Company,Kalamazoo, Michigan for a generouis suipply of prosta-glandin El,.

942 D. V. Kimberg, M. Field, E. Ger