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Effect of misoprostol in preventing stress-induced intestinal fluid secretion in rats
Prostaglandins Lcukotrienes and Essential Fatty Acids (1989) 38.43-48 0 Longman Group UK Ltd 1989
Effect of Misoprostbl in Preventing Intestinal Fluid Secretion in Rats
Stress-Induced
L. R. EMPEY and R. N. FEDORAK University of Alberta, Department of Medicine, Division of Gastroenterology, 579 Robert Research Building, Edmonton, Alberta, Canada, T6G 2C2 (Correspondence to RNFI
Newton
function by central nervous Abstract - Psychological stress may alter gastrointestinal system controlled alteration of local intestinal mediators. Prostaglandins have been shown to prevent epithelial damage to various noxious stimuli. The purpose of this study was to determine (a) if wrap restraint stress altered in vivo intestinal fluid absorption in rats, and (b) if the prostaglandin E, analogue, misoprostol, could correct observed fluid malabsorption. In vivo loop studies demonstrated net fluid secretion in the ileum and colon of cold wrap restraint stressed rats. In cold wrap restraint stressed rats, misoprostol reversed net secretion to absorption, but it had no effect on fluid absorption in controls. Mild wrap restraint stress did not alter in vivo fluid absorption. We conclude that cold wrap restraint stress is accompanied by net intestinal fluid secretion that can be effectively reversed with misoprostol. Abbreviations: LT, Leukotriene; PG, Prostaglandin.
Introduction
time, the direct effect of stress on gastrointestinal function (7). The effect of psychological stress on the gastrointestinal tract has recently become an area of interest for physicians, physiologists, and psychologists. Stress has been shown to alter gastric acid secretion (8), participate in the development of gastritis (9), alter intestinal motility in patients with irritable bowel syndrome (10, ll), and change fluid and electrolyte absorption in the non-diseased intestine as well as in Crohn’s disease, ulcerative colitis, and celiac sprue (1, 12-14). The examination of the effect of psychological stress on gastrointestinal func-
Emotional disturbances, such as stress and anxiety, have been associated with gastrointestinal dysfunction which is characterized by abdominal pain, bloating, and diarrhea (l-6). The correlation between psychological stress and gastrointestinal physiological alterations was initially reported by William Beaumont in 1833 in his observation of gastric secretion in a gastric fistula which had occurred as a result of a gunshot wound to Alexis Saint Martin. Beaumont observed that the stomach produced a significantly greater amount of gastric acid under duress, and his study correlated, for the first 43
.
44
PROSTAGLANDINS
tion has made use of a number of wellcharacterized animal models (1, 15-17). Neverthe mechanisms of stress-induced theless, alteration of intestinal function remain poorly understood. Advances in the understanding of the role of eicosanoids in gastrointestinal health and disease have led to the proposal that the products of cycle-oxygenase metabolism, prostaglandins (PG), are protective to the intestine while the products of 5-lipoxygenase metabolism, Leukotrienes (LT), are injurious (18-22). In the stomach, prostaglandins have been demonstrated to provide a mucosal cytoprotective effect through stimulation of mucus secretion, enhancement of mucosal blood flow, stimulation of alkaline secretion, maintenance of mucosal sulphahydryl groups, and stimulation of surface-active phospholipids (23-29). A similar cytoprotective role of prostaglandins in the colon during acetic acidinduction of colitis has recently been demonstrated (18). However, the role of eicosanoids in stress-induced gastrointestinal physiological alterations remains to be determined. Recently, cold restraint stress in rats has been shown to cause gastrointestinal mucosal injury associated with a decrease in gastric mucosal prostaglandin EZ synthesis (17). In this study, we examined the effect of mild wrap restraint stress and the more severe cold wrap restraint stress on in vivo basal fluid absorption in the rat jejunum, ileum and colon. We then determined the effect of the long-acting prostaglandin Ei analogue, Misoprostol, on stress-induced alterations of intestinal fluid absorption. Materials and Methods Materials
Misoprostol [(+) methyl (llalpha,l3E)-11,16dihydroxy-16-methyl-9-oxoprost-13-en-1-oate] is a synthetic prostaglandin Ei methyl ester analogue and is insoluble in water (29). Misoprostol for oral instillation was prepared by finely crushing a 200 pg Misoprostol (Cytotec@) tablet and suspending the powder in 10 ml 1% methyl cellulose; final concentration was (20 pg/ml). The misoprostol suspension was stored in 1 ml aliquotes at -70°C until the day of the experiment. The misoprostol used in this study was a gift from G. D. Searle Co. Of Canada Limited. Methyl cellulose, 15 centipoises, was purchased from Sigma Chemical Co., St. Louis, MO.
LEUKOTRIENES
AND ESSENTIAL
FATTY
ACIDS
Xylazine (RompumR), 20 mg/ml, was purchased from Bayvet Division of Chemagro Ltd., Etobicoke, ON. Ketamine hydrochloride (Ro arseticR), 100 mg/ml, was purchased from Rogar PSTB Inc., London, ON. Sodium Pentobarbitol (Somnotol’), 65 mg’ml, was purchased from M.T.C. Pharmaceuticals, Mississauga, ON. Atropine Sulfate, 0.6 mg/ml, was purchased from GLAXO Laboratories, Montreal, QUE. The remainder of the chemicals were reagent grade and purchased from Sigma Chemical Co. Mild wrap restraint stress
Non-fasting male Sprague-Dawley rats (250-300 g, Biotron, University of Alberta, Edmonton AB) were lightly anesthetized with ether and wrap restrained in a manner similar to the method used by Senay and Levine (15). Briefly, the forelimbs and shoulders were taped to the body with a harness of paper masking tape secured to and wrapped around the thoracic trunk. Animals recovered from the ether in 3-5 minutes. During the restraint period, movement of the forelimbs was restricted, preventing grooming but allowing for some movement in the cage (16). Care was taken during the restraint procedure to prevent injury and to allow the limbs to rest in a neutral position. Animals were restrained in this manner for 1 h, after which they were anaesthetized and their in vivo basal fluid absorption examined as described below. Cold wrap restraint stress
Non-fasting male Sprague-Dawley rats, 250-300 g, were lightly anesthetized with ether in preparation for cold wrap restraint stress in the manner described by Williams et al (16). Briefly, the forelimbs and shoulders were attached to the thoracic trunk with a harness of paper masking tape and the hind limbs were then taped to the body in a similar fashion. The animals were placed back in the cage in the prone position and kept at 4°C for 2.5 h, after which they were anaesthetized and their in vivo intestinal fluid absorption examined. During the restraint period, the animals were completely immobilized, remaining in one position without struggling. Once again, care was taken during the restraint procedure to prevent injury and to allow the limbs to rest in a neutral position. Stress-free studies
None of these animals were exposed to mild wrap restraint or cold wrap restraint stress. Rats were allowed ad libitum access to standard rat
MISOPROSTOL
PREVENTS
STRESS-INDUCED
INTESTINAL
FLUID SECRETION
chow, (Wayne Rodent BloxR, Continental Grain Co., Chicago IL) and water and housed in a 12 h light-dark cycled animal care facility. Animals were allowed 7 d to acclimate to the environment before experiments were performed. Stress-free control animals were anaesthetized with ether but were not wrapped. Misoprostol administration The animal group requiring misoprostol pretreatment was prepared in the following manner: Rats were anesthetized with a Xylazine (5 mg/kg), Ketamine Hydrochloride (70 mgtkg) mixture, which was administered intraperitoneally to achieve a short-acting, manageable anesthetic plane. Oral-gastric incubation was achieved using a length of polyethylene tubing with an outer diameter of 1.09 mm, (PE-20, Becton, Dickinson and Co., Parsippany, NJ). The oral-gastric tube was carefully placed into the stomach with the animal in an upright position, thus preventing aspiration or regurgitation of fluid. One ml of the prepared misoprostol suspension (12.5 pg/‘kg) was instilled, via the tube, into the stomach daily for 5 consecutive days. Preliminary studies with methylene blue-labelled suspension indicated the entire volume remained in the stomach following instillation and did not reflux into the lungs (data not shown). Age-matched control animals were treated with identical daily instillations of 1 ml of 1% methyl cellulose suspension without misoprostol. Animals from both the misoprostol pre-treated and the age-matched control groups were then randomly divided into groups which received cold wrap restraint stress or served as non-stressed controls. In vivo fluid absorption studies Study animals were anesthetized with pentobarbitol (55 mg/kg) and atropine (0.5 mg/kg) and kept warm with a thermostatic heat lamp. In vivo intestinal fluid absorption studies were performed as previously described (30). The intestinal tract was exposed through a midline incision. An occluding ligature was placed at the caecal ascending junction and the colonic luminal contents were flushed out the rectum with a warm 154 mM sodium chloride solution instilled via a cannula inserted through an incision just distal to the occluding ligature; residual saline was emptied by gentle manual expression. A colonic loop of approximately 15 cm in length, beginning 2 cm distal to the caecal-colonic junc-
45
tion and extending to the peritoneal reflection, was created with ligatures. The small intestine was handled in a similar manner: occluding ligatures were placed at the Ligament of Treitz and the ileal-caecal valve, and incisions through the gut wall were made distal and proximal to these ligatures, respectively. A cannula was inserted through the proximal incision, and the luminal contents were flushed through the distal incision with warm 154 mM sodium chloride solution. Loops of approximately 15 cm length were created with ligatures in the jejunum (beginning 2 cm distal to the Ligament of and in the ileum (beginning 2 cm Treitz), proximal to the ileal-caecal valve). In isolating the loops, care was taken not to compromise mesenteric, vascular or neural continuity. A 27 gauge needle was inserted obliquely through the outer muscle layer along the antimesenteric border and 2 ml of 154 mM NaCl, prewarmed to 37°C was instilled into each empty loop. No fluid leakage was detected and the loops were only mildly distended. The viscera were returned to the abdominal cavity and the incision was closed. The rats were allowed to recover from the anesthesia. Sixty minutes after abdominal closure, the animals were given a pentobarbitol overdose (250 mg/kg iv) and the loops removed. The length of each loop was measured and the loop was weighed, both full and empty, to determine the residual intraluminal volume. The loops were then dried in a 50” oven for 24 h. Results were expressed as the difference between initial and residual luminal loop volume per gram dry weight or per centimeter of bowel. Statistical Analysis Statistical analysis of multiple comparisons was performed by analysis of variance. This was done by multi-varied profile analysis of repeated measures employing a microcomputer’s statistical software package (EPISTAT 2, Round Rock, TX). When overall analysis revealed a significant interaction, the student’s t-test was used to examine the significance of differences. Results Animals groups Mild wrap restrained rats could move around the cage by using their hind limbs, but were prevented from grooming and feeding. Throughout the period of restraint, the rats attempted to remove the harness restraining their forelimbs.
46
PROSTAGLANDINS
Cold wrap restrained rats were completely immobilized and remained in the prone position at the bottom of the cage without moving throughout the restraint period. Body temperature remained constant at 37°C. In neither restraint group did macroscopic evidence of small or large intestinal ulceration occur. Intestinal j?uid absorption in vivo As shown in Figure 1, non-stressed age-matched control animals demonstrated a similar degree of in vivo net basal fluid absorption in the jejunum, ileum and colon. Mild wrap restraint stressed animals did not have altered intestinal fluid absorption, as compared to non-stressed, agematched control animals. Results were the same, whether they were expressed as volume per centimeter of bowel or per gram dry weight (data not shown). As shown in Figure 2, pretreatment with
LEUKOTRIENES
AND ESSENTIAL
FATIY
ACIDS
misoprostol alone for 5 days did not alter net in vivo fluid absorption in the jejunum, ileum or colon as compared to non-treated controls. Cold wrap restraint stress alone reversed net intestinal fluid absorption to net secretion in the ileum and colon but not in the jejunum. Pre-treatment with misoprostol for 5 consecutive days prior to cold wrap restraint stress reversed net in vivo fluid secretion in the ileum and colon to control values. Discussion Physical or psychological stress may alter gastrointestinal physiological function in humans and in animals (l-6, 31, 32). Some of these stress conditions have been associated with altered gastric acid secretion, gastritis, peptic ulcer, and altered intestinal motility in irritable 1
A. Jo/unum
STRESS-FREE
JEJUNUU
COLON
IWJY
MILD RESTRAINT
STRESS
+ + JEJUNUM
ILEUM
COLON
Fig. 1 Effect of mild wrap restraint stress on net intestinal basal fluid absorption in viva. Results in jejunum (open bar), ileum (hatched bar) and colon (solid bar) are compared to non-stressed age-matched control rats and expressed as &q/h. No significant alteration in net intestinal basal fluid absorption is apparent with mild wrap restraint stress. Values are mean + for nr4 rats.
+ +
Fig. 2 Effect of misoprostai treatment on cold wrap restraint stress-induced alterations in net intestinal basal fluid absorption in vivo. Results in A. jejunum (open bars), B. ileum (hatched bars) and C. colon (solid bars) were compared in the presence of absence of misoprostai, 12.5 &kg/d. Misoprostoi pretreatment reserved cold wrap restraint stress-induced net intestinal fluid in ileum and colon. Values are mean f SEM for n=6 rats. * p < 0.001 compared to ail other groups.
MISOPROSTOL PREVENTS STRESS-INDUCED
INTESTINAL FLUID SECRETION
bowel syndrome (8-11, 33, 34). Psychological stress during dichotomous listening significantly reduced human jejunal fluid absorption through a cholinergic parasympathetic nervous mechanism (1). In animal studies, restraint stressinduced changes in intestinal transit and transport were partially mediated by endogenous central nervous system corticotropin-releasing factor (31, 33) but not by other pituitary or adrenally derived factors (16). Cerebroventricular administration of a specific corticotropinreleasing factor receptor antagonist, prevented gastrointestinal secretory and motor responses elicited by partial body restraint (1, 33). Although these recent studies implied that corticotropin-releasing factor exerted its central nervous system action by a receptor-mediated pathway, they did not elucidate the specific site of action within the brain or the local mediators of the intestinal injury. Recently, prostaglandins have been identified to confer enhancement of gastrointestinal enterocyte resistance against a number of specific damaging events. This cytoprotective effect has been shown to occur through stimulation of mucus secretion, enhancement of mucosal blood flow, stimulation of alkaline secretion, stimulation of deoxyribonucleic acid synthesis, maintenance of mucosal sulphahydryl groups, stabilization of tissue lysosomes and stimulation of surface-active phopholipids (23-29). Recently, it has been suggested that the cytoprotective effect of prostaglandins is mediated not by surface epithelial protection, but by prevention of deeper mucosal injury (34-36, 18). Indeed, psychological stress has been shown to reduce intestinal blood flow (32) and lower endogenous prostaglandin levels (17)) both elements known to play an important role in the transport of fluid and electrolytes across the intestine. Using wrap restraint as the stress-initiating stimulus, we, thus, examined the effect of long-acting prostaglandin El analogue, misoprostol, on stress-induced intestinal fluid secretion in rats. In this study, mild wrap restraint stress did not alter basal in uivo net intestinal fluid absorption in any of the tested segments. Williams et al, however, demonstrated that a similar stress model stimulated large intestinal transit and increased fecal excretion (16). The method of in vivo isolated intestinal loops used in this study effectively eliminates motility, affording a direct assessment of intestinal transport and may
PROST
c
47
explain the differences identified. During the more severe cold wrap-restraint stress, basal in vivo intestinal fluid absorption changed to net secretion in the ileum and colon. In contrast, the jejunal absorption remained unaltered, and may reflect the high permeability of the jejunal epithelium. Thzse changes in ileal and colonic fluid transport were seen in the absence of intestinal ulceration. The development of diminished intestinal fluid absorption may explain the increased fecal excretion seen by Williams et al during cold wrap restraint stress compared to mild wrap restraint stress (16). In the present study, orally administered longacting prostaglandin El analogue, misoprostol, reversed the net intestinal secretory response in ileum and colon of cold wrap restraint stressed rats to near control levels. This effect of misoprostol was seen following pretreatment once daily for 5 d with 12.5 pg/kg/d, a total daily dose similar to that used in humans. It is unlikely that direct stimulation of intestinal sodium or chloride transport plays a role, since the presence of misoprostol alone did not alter intestinal fluid absorption in the non-stressed control state. The mechanism of action of misoprostol in preventing stress-induced alterations in intestinal fluid absorption remains to be determined. It is unlikely that intraluminally administered prostaglandin El analogues alter the release of endogenous central nervous system mediators that may initiate and co-ordinate gastrointestinal secretory and motor response during stress (31, 33). It is more likely that misoprostol has its effect at a local level, perhaps by enhancement of mucosal blood flow (32), repletion of diminished endogenous prostaglandin levels (17), or by stabilization of stress induced mast cells (37). Acknowledgements Dr Fedorak is the recipient of a Clinical Investigatorship from the Alberta Heritage Foundation for Medical Research. This work is supported by a grant from the Alberta Heritage Foundation for Medical Research. The authors express appreciation to G. D. Searle Co. of Canada Limited for their generous support and provision of Misoprostol for purposes of this study, and to Mrs M Pollard for her expert secretarial support.
References 1. Barclay G R, Turnberg L A, The effect of physiological stress on salt and water transport in the human jejunum. Gastroenterology 93: 91-98, -1987. 2. McRae S, Younger K. Thompson D G, Wingate D L, Sustained mental stress alters human jejunal motor
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DROSTAGLANDINS
activity. Gut 23: 404-409. 1982. Furnell .l R. Dutton P V. Alleviation of toddlers diarrhoea by environmental management. Journal of Psychosomatic Research 30(3): 283-288. 1986. Bommelaer G. Rouch M. Dapoigny M, Pais P, Toutnut R et al. Epidemiology of intestinal functional disorders in an apparently healthy population. Gastroenterology and Clinical Biology IO: 7-12, 1986. Almy T P, Kern F, Tulin M, Alterations in colonic function in man under stress. Gastroenterology 12: 425-436. 1949. Lucas C E, Sugawa C. Riddle J, Rector F. Rosenburg B. Walt A J, Natural history and surgical dilemma of “stress” gastric bleeding. Archives of Surgery 102: 266, 1970. Beaumont W, Experiments and observation on the gastric juices and the physiology of digestion. Dover Publications, New York. 1833. Alp M H, Court J H. Grant A K. Personality pattern and emotional stress in the genesis of gastric ulcer. Gut 11: 733, 1970. Skillman J J, Bushnell L S, Goldman H. Silen W, Respiratory failure, hypotension, sepsis and jaundice: A clinical syndrome associated with lethal hemorrhage from acute stress ulceration of the stomach. American Journal of Surgery 123: 25, 1971. Latimer P, Sarna S. Campbell D, Latimer M. Daniel E E, Colonic motor and myoelectric activity a comparative study of normal subjects, psychoneurotic patients and patients with IBS. Gastroenterology 88: 893-901, 1981. Krag E et al. Irritable bowel syndrome: current concepts and future trends. Scandinavian Journal of Gastroenterology 109: 107-115, 1985. Mekhjian H S. Switz D M. Melnyk C S. Rankin G B. Brooks R K, Clinical Features and natural history of Crohn’s disease. Gastroenterology 77: 898, 1979. Binder H J, Absorption and secretion of water and electrolytes by small and large intestine p. 1040 in Gastrointestinal Disease: Pathophysiology Diagnosis Management. 4th ed. (M H Sleisenger. J S Fordtran, eds) W B Saunders Company, 1989. Fordtran J S. Rector F C, Locklear T W, Ewton M F, Water and solute movement in the small intestine of patients with sprue. Journal of Clinical Investigation 46: 287, 1967. Senay S E, Levine B T. Synergism between cold and restraint for rapid production of stress ulcers in rats. Proceedings of the Society of Experimental Bioiogica! Medicine 124: 1221-1223. 1967. Williams C L, Villar R G, Peterson J M, Burks T F. Stress induced changes in intestinal transit in the rat: A model for irritable syndrome. Gastroenterology 94: 611-621, 1988. Avunduk C, Palokowski G F, Eastwood G L. Effects of stress on gastric mucosal prostaglandin synthesis in intact. adrenalectomized. and sham operated rats. Gastroenterology 94: A-15, 1988. Empey L R, Macarthur C, Jewel1 L D, Fedorak R N. Cytoprotective effect of prostaglandin E, analogue in acetic acid colitis. Gastroenterology 94: A-616. 1988. Wallace J L. Whittle B J, Boughton-Smith N K, Prostaglandin protection of rat colonic mucosa from damage induced by ethanol. Digestive Diseases and Sciences 30: 866-876. 1985. Robert A. Lancaster C, Davis J P. Field S 0. Wikrema
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AND ESSENTIAL
FATTY
ACIDS
Sinha A J. Thornburgh B A. Cytoprotection by prostaglandin occurs in spite of penetration of absolute ethanol into the gastric mucosa. Gastroenterology 88: 328-333, 1985. Psaila J V, Myers B, Jones I R, Rhodes J. Effect of prostaglandin PGE, on alcohol-induced ulceration in the rat colon. Digestion 35: 224-228, 1Y86. MacNaughton W K. Morris G P and Wallace J L, Inhibition of leukotriene synthesis markedly enhances healing in a rat model of inflammatory bowel disease. Gastroenterology 94: A618, 1988. Ferguson W W, Edmonds A W, Straling V R. Wagensteen S L, Protective effect of PGE, on lysosomal enzyme release in serotonin-induced gastric ulceration. Annals of Surgery 177: 648-654, 1973. Schiessel R, Matthews J, Barzilai A, Merhar A, Silen W. PGE: stimulates gastric chloride transport: possible key to cytoprotection. Nature 283: 671-673. 1980. Gilberyson T J. Ruwart M J. Stryd R P et al. l6,16dimethyl PGE- increases dry organ weights of the stomach, small-intestine and colon in rats. Gastroenterology 82: 1066, 1982. Reinhart W H. Muller 0. Halter F. influence of long term 16.16-dimethyl prostaglandin E2 treatment on the rat gastrointestinal mucosa. Gastroenterology 85: 1003-1010, 1983. Lewis G P. lmmunoregulatory activity of metabolites of arachidonic acid and their role in inflammation. British Medical Bulletin 39: 243-248. 1983. Miller T A, Jacobson E D, Gastrointestinal cytoprotection by prostaglandins. Gut 20: 75-87, 1979. Bauer R F. Misoprostol preclinical pharmacology. Digestive Diseases and Sciences 30(11): 118-125, 1985. Chang E B, Fedorak RN, Field M, Experimental diabetic diarrhea in rats: intestinal mucosal denervation hypersensitivity and treatment with clonidine. Gastroenterology 91: 564-567. 1986. Lenz H J, Raedler A. Greten H. Vale W W. Rivier J E, Stress-induced gastrointestinal secretory and motor responses in rats are mediated by endogenous corticotropin-releasing factor. Gastroenterology 95: 1510-1517. 1988. Guth P H, Leung F W, Physiology of the gastric circulation. p. 1031-1053 in Physiology of the gastrointestinal tract. (Johnson L R. ed) Raven, New York 1987. Williams C L. Peterson J M, Villar R G. Burks T F. Corticotropin-releasing factor directly mediates colonic responses to stress. American Journal of Physiology 253: G582-586, 1987. Lacy E R, Ito S. Microscopic analysis of ethanol damage to rat gastric mucosa after treatment with a prostaglandin. Gastroenterology 83: 619-625, 1982. Wallace J L, Morris G P, Krausse E J, Greaves S E, Reduction by cytoprotective agents of ethanol-induced damage to the rat gastric mucosa: a correlated morphological and physiological study. Canadian Journal of Physiology and Pharmacoloav 60: 1686-1699. 1982. Tarnawski A. Hollander D: Stachura J, Krause W J. Gergely H, Prostaglandin protection of the gastric mucosa against alcohol injury - a dvnamic time-related process. Role of the mucosaj proliferative zone. Gastroenterologv 88: 334-352. 1958. ReimannH J, Lewin J, Schmidt U. Wendt P, Blueml G. Dajani E Z, Misoprostal prevents damage to the gastric mucosa by stabilizing the mast cells. Prostaglandins 33: 105-106, 1987.
Effect of Misoprostbl in Preventing Intestinal Fluid Secretion in Rats
Stress-Induced
L. R. EMPEY and R. N. FEDORAK University of Alberta, Department of Medicine, Division of Gastroenterology, 579 Robert Research Building, Edmonton, Alberta, Canada, T6G 2C2 (Correspondence to RNFI
Newton
function by central nervous Abstract - Psychological stress may alter gastrointestinal system controlled alteration of local intestinal mediators. Prostaglandins have been shown to prevent epithelial damage to various noxious stimuli. The purpose of this study was to determine (a) if wrap restraint stress altered in vivo intestinal fluid absorption in rats, and (b) if the prostaglandin E, analogue, misoprostol, could correct observed fluid malabsorption. In vivo loop studies demonstrated net fluid secretion in the ileum and colon of cold wrap restraint stressed rats. In cold wrap restraint stressed rats, misoprostol reversed net secretion to absorption, but it had no effect on fluid absorption in controls. Mild wrap restraint stress did not alter in vivo fluid absorption. We conclude that cold wrap restraint stress is accompanied by net intestinal fluid secretion that can be effectively reversed with misoprostol. Abbreviations: LT, Leukotriene; PG, Prostaglandin.
Introduction
time, the direct effect of stress on gastrointestinal function (7). The effect of psychological stress on the gastrointestinal tract has recently become an area of interest for physicians, physiologists, and psychologists. Stress has been shown to alter gastric acid secretion (8), participate in the development of gastritis (9), alter intestinal motility in patients with irritable bowel syndrome (10, ll), and change fluid and electrolyte absorption in the non-diseased intestine as well as in Crohn’s disease, ulcerative colitis, and celiac sprue (1, 12-14). The examination of the effect of psychological stress on gastrointestinal func-
Emotional disturbances, such as stress and anxiety, have been associated with gastrointestinal dysfunction which is characterized by abdominal pain, bloating, and diarrhea (l-6). The correlation between psychological stress and gastrointestinal physiological alterations was initially reported by William Beaumont in 1833 in his observation of gastric secretion in a gastric fistula which had occurred as a result of a gunshot wound to Alexis Saint Martin. Beaumont observed that the stomach produced a significantly greater amount of gastric acid under duress, and his study correlated, for the first 43
.
44
PROSTAGLANDINS
tion has made use of a number of wellcharacterized animal models (1, 15-17). Neverthe mechanisms of stress-induced theless, alteration of intestinal function remain poorly understood. Advances in the understanding of the role of eicosanoids in gastrointestinal health and disease have led to the proposal that the products of cycle-oxygenase metabolism, prostaglandins (PG), are protective to the intestine while the products of 5-lipoxygenase metabolism, Leukotrienes (LT), are injurious (18-22). In the stomach, prostaglandins have been demonstrated to provide a mucosal cytoprotective effect through stimulation of mucus secretion, enhancement of mucosal blood flow, stimulation of alkaline secretion, maintenance of mucosal sulphahydryl groups, and stimulation of surface-active phospholipids (23-29). A similar cytoprotective role of prostaglandins in the colon during acetic acidinduction of colitis has recently been demonstrated (18). However, the role of eicosanoids in stress-induced gastrointestinal physiological alterations remains to be determined. Recently, cold restraint stress in rats has been shown to cause gastrointestinal mucosal injury associated with a decrease in gastric mucosal prostaglandin EZ synthesis (17). In this study, we examined the effect of mild wrap restraint stress and the more severe cold wrap restraint stress on in vivo basal fluid absorption in the rat jejunum, ileum and colon. We then determined the effect of the long-acting prostaglandin Ei analogue, Misoprostol, on stress-induced alterations of intestinal fluid absorption. Materials and Methods Materials
Misoprostol [(+) methyl (llalpha,l3E)-11,16dihydroxy-16-methyl-9-oxoprost-13-en-1-oate] is a synthetic prostaglandin Ei methyl ester analogue and is insoluble in water (29). Misoprostol for oral instillation was prepared by finely crushing a 200 pg Misoprostol (Cytotec@) tablet and suspending the powder in 10 ml 1% methyl cellulose; final concentration was (20 pg/ml). The misoprostol suspension was stored in 1 ml aliquotes at -70°C until the day of the experiment. The misoprostol used in this study was a gift from G. D. Searle Co. Of Canada Limited. Methyl cellulose, 15 centipoises, was purchased from Sigma Chemical Co., St. Louis, MO.
LEUKOTRIENES
AND ESSENTIAL
FATTY
ACIDS
Xylazine (RompumR), 20 mg/ml, was purchased from Bayvet Division of Chemagro Ltd., Etobicoke, ON. Ketamine hydrochloride (Ro arseticR), 100 mg/ml, was purchased from Rogar PSTB Inc., London, ON. Sodium Pentobarbitol (Somnotol’), 65 mg’ml, was purchased from M.T.C. Pharmaceuticals, Mississauga, ON. Atropine Sulfate, 0.6 mg/ml, was purchased from GLAXO Laboratories, Montreal, QUE. The remainder of the chemicals were reagent grade and purchased from Sigma Chemical Co. Mild wrap restraint stress
Non-fasting male Sprague-Dawley rats (250-300 g, Biotron, University of Alberta, Edmonton AB) were lightly anesthetized with ether and wrap restrained in a manner similar to the method used by Senay and Levine (15). Briefly, the forelimbs and shoulders were taped to the body with a harness of paper masking tape secured to and wrapped around the thoracic trunk. Animals recovered from the ether in 3-5 minutes. During the restraint period, movement of the forelimbs was restricted, preventing grooming but allowing for some movement in the cage (16). Care was taken during the restraint procedure to prevent injury and to allow the limbs to rest in a neutral position. Animals were restrained in this manner for 1 h, after which they were anaesthetized and their in vivo basal fluid absorption examined as described below. Cold wrap restraint stress
Non-fasting male Sprague-Dawley rats, 250-300 g, were lightly anesthetized with ether in preparation for cold wrap restraint stress in the manner described by Williams et al (16). Briefly, the forelimbs and shoulders were attached to the thoracic trunk with a harness of paper masking tape and the hind limbs were then taped to the body in a similar fashion. The animals were placed back in the cage in the prone position and kept at 4°C for 2.5 h, after which they were anaesthetized and their in vivo intestinal fluid absorption examined. During the restraint period, the animals were completely immobilized, remaining in one position without struggling. Once again, care was taken during the restraint procedure to prevent injury and to allow the limbs to rest in a neutral position. Stress-free studies
None of these animals were exposed to mild wrap restraint or cold wrap restraint stress. Rats were allowed ad libitum access to standard rat
MISOPROSTOL
PREVENTS
STRESS-INDUCED
INTESTINAL
FLUID SECRETION
chow, (Wayne Rodent BloxR, Continental Grain Co., Chicago IL) and water and housed in a 12 h light-dark cycled animal care facility. Animals were allowed 7 d to acclimate to the environment before experiments were performed. Stress-free control animals were anaesthetized with ether but were not wrapped. Misoprostol administration The animal group requiring misoprostol pretreatment was prepared in the following manner: Rats were anesthetized with a Xylazine (5 mg/kg), Ketamine Hydrochloride (70 mgtkg) mixture, which was administered intraperitoneally to achieve a short-acting, manageable anesthetic plane. Oral-gastric incubation was achieved using a length of polyethylene tubing with an outer diameter of 1.09 mm, (PE-20, Becton, Dickinson and Co., Parsippany, NJ). The oral-gastric tube was carefully placed into the stomach with the animal in an upright position, thus preventing aspiration or regurgitation of fluid. One ml of the prepared misoprostol suspension (12.5 pg/‘kg) was instilled, via the tube, into the stomach daily for 5 consecutive days. Preliminary studies with methylene blue-labelled suspension indicated the entire volume remained in the stomach following instillation and did not reflux into the lungs (data not shown). Age-matched control animals were treated with identical daily instillations of 1 ml of 1% methyl cellulose suspension without misoprostol. Animals from both the misoprostol pre-treated and the age-matched control groups were then randomly divided into groups which received cold wrap restraint stress or served as non-stressed controls. In vivo fluid absorption studies Study animals were anesthetized with pentobarbitol (55 mg/kg) and atropine (0.5 mg/kg) and kept warm with a thermostatic heat lamp. In vivo intestinal fluid absorption studies were performed as previously described (30). The intestinal tract was exposed through a midline incision. An occluding ligature was placed at the caecal ascending junction and the colonic luminal contents were flushed out the rectum with a warm 154 mM sodium chloride solution instilled via a cannula inserted through an incision just distal to the occluding ligature; residual saline was emptied by gentle manual expression. A colonic loop of approximately 15 cm in length, beginning 2 cm distal to the caecal-colonic junc-
45
tion and extending to the peritoneal reflection, was created with ligatures. The small intestine was handled in a similar manner: occluding ligatures were placed at the Ligament of Treitz and the ileal-caecal valve, and incisions through the gut wall were made distal and proximal to these ligatures, respectively. A cannula was inserted through the proximal incision, and the luminal contents were flushed through the distal incision with warm 154 mM sodium chloride solution. Loops of approximately 15 cm length were created with ligatures in the jejunum (beginning 2 cm distal to the Ligament of and in the ileum (beginning 2 cm Treitz), proximal to the ileal-caecal valve). In isolating the loops, care was taken not to compromise mesenteric, vascular or neural continuity. A 27 gauge needle was inserted obliquely through the outer muscle layer along the antimesenteric border and 2 ml of 154 mM NaCl, prewarmed to 37°C was instilled into each empty loop. No fluid leakage was detected and the loops were only mildly distended. The viscera were returned to the abdominal cavity and the incision was closed. The rats were allowed to recover from the anesthesia. Sixty minutes after abdominal closure, the animals were given a pentobarbitol overdose (250 mg/kg iv) and the loops removed. The length of each loop was measured and the loop was weighed, both full and empty, to determine the residual intraluminal volume. The loops were then dried in a 50” oven for 24 h. Results were expressed as the difference between initial and residual luminal loop volume per gram dry weight or per centimeter of bowel. Statistical Analysis Statistical analysis of multiple comparisons was performed by analysis of variance. This was done by multi-varied profile analysis of repeated measures employing a microcomputer’s statistical software package (EPISTAT 2, Round Rock, TX). When overall analysis revealed a significant interaction, the student’s t-test was used to examine the significance of differences. Results Animals groups Mild wrap restrained rats could move around the cage by using their hind limbs, but were prevented from grooming and feeding. Throughout the period of restraint, the rats attempted to remove the harness restraining their forelimbs.
46
PROSTAGLANDINS
Cold wrap restrained rats were completely immobilized and remained in the prone position at the bottom of the cage without moving throughout the restraint period. Body temperature remained constant at 37°C. In neither restraint group did macroscopic evidence of small or large intestinal ulceration occur. Intestinal j?uid absorption in vivo As shown in Figure 1, non-stressed age-matched control animals demonstrated a similar degree of in vivo net basal fluid absorption in the jejunum, ileum and colon. Mild wrap restraint stressed animals did not have altered intestinal fluid absorption, as compared to non-stressed, agematched control animals. Results were the same, whether they were expressed as volume per centimeter of bowel or per gram dry weight (data not shown). As shown in Figure 2, pretreatment with
LEUKOTRIENES
AND ESSENTIAL
FATIY
ACIDS
misoprostol alone for 5 days did not alter net in vivo fluid absorption in the jejunum, ileum or colon as compared to non-treated controls. Cold wrap restraint stress alone reversed net intestinal fluid absorption to net secretion in the ileum and colon but not in the jejunum. Pre-treatment with misoprostol for 5 consecutive days prior to cold wrap restraint stress reversed net in vivo fluid secretion in the ileum and colon to control values. Discussion Physical or psychological stress may alter gastrointestinal physiological function in humans and in animals (l-6, 31, 32). Some of these stress conditions have been associated with altered gastric acid secretion, gastritis, peptic ulcer, and altered intestinal motility in irritable 1
A. Jo/unum
STRESS-FREE
JEJUNUU
COLON
IWJY
MILD RESTRAINT
STRESS
+ + JEJUNUM
ILEUM
COLON
Fig. 1 Effect of mild wrap restraint stress on net intestinal basal fluid absorption in viva. Results in jejunum (open bar), ileum (hatched bar) and colon (solid bar) are compared to non-stressed age-matched control rats and expressed as &q/h. No significant alteration in net intestinal basal fluid absorption is apparent with mild wrap restraint stress. Values are mean + for nr4 rats.
+ +
Fig. 2 Effect of misoprostai treatment on cold wrap restraint stress-induced alterations in net intestinal basal fluid absorption in vivo. Results in A. jejunum (open bars), B. ileum (hatched bars) and C. colon (solid bars) were compared in the presence of absence of misoprostai, 12.5 &kg/d. Misoprostoi pretreatment reserved cold wrap restraint stress-induced net intestinal fluid in ileum and colon. Values are mean f SEM for n=6 rats. * p < 0.001 compared to ail other groups.
MISOPROSTOL PREVENTS STRESS-INDUCED
INTESTINAL FLUID SECRETION
bowel syndrome (8-11, 33, 34). Psychological stress during dichotomous listening significantly reduced human jejunal fluid absorption through a cholinergic parasympathetic nervous mechanism (1). In animal studies, restraint stressinduced changes in intestinal transit and transport were partially mediated by endogenous central nervous system corticotropin-releasing factor (31, 33) but not by other pituitary or adrenally derived factors (16). Cerebroventricular administration of a specific corticotropinreleasing factor receptor antagonist, prevented gastrointestinal secretory and motor responses elicited by partial body restraint (1, 33). Although these recent studies implied that corticotropin-releasing factor exerted its central nervous system action by a receptor-mediated pathway, they did not elucidate the specific site of action within the brain or the local mediators of the intestinal injury. Recently, prostaglandins have been identified to confer enhancement of gastrointestinal enterocyte resistance against a number of specific damaging events. This cytoprotective effect has been shown to occur through stimulation of mucus secretion, enhancement of mucosal blood flow, stimulation of alkaline secretion, stimulation of deoxyribonucleic acid synthesis, maintenance of mucosal sulphahydryl groups, stabilization of tissue lysosomes and stimulation of surface-active phopholipids (23-29). Recently, it has been suggested that the cytoprotective effect of prostaglandins is mediated not by surface epithelial protection, but by prevention of deeper mucosal injury (34-36, 18). Indeed, psychological stress has been shown to reduce intestinal blood flow (32) and lower endogenous prostaglandin levels (17)) both elements known to play an important role in the transport of fluid and electrolytes across the intestine. Using wrap restraint as the stress-initiating stimulus, we, thus, examined the effect of long-acting prostaglandin El analogue, misoprostol, on stress-induced intestinal fluid secretion in rats. In this study, mild wrap restraint stress did not alter basal in uivo net intestinal fluid absorption in any of the tested segments. Williams et al, however, demonstrated that a similar stress model stimulated large intestinal transit and increased fecal excretion (16). The method of in vivo isolated intestinal loops used in this study effectively eliminates motility, affording a direct assessment of intestinal transport and may
PROST
c
47
explain the differences identified. During the more severe cold wrap-restraint stress, basal in vivo intestinal fluid absorption changed to net secretion in the ileum and colon. In contrast, the jejunal absorption remained unaltered, and may reflect the high permeability of the jejunal epithelium. Thzse changes in ileal and colonic fluid transport were seen in the absence of intestinal ulceration. The development of diminished intestinal fluid absorption may explain the increased fecal excretion seen by Williams et al during cold wrap restraint stress compared to mild wrap restraint stress (16). In the present study, orally administered longacting prostaglandin El analogue, misoprostol, reversed the net intestinal secretory response in ileum and colon of cold wrap restraint stressed rats to near control levels. This effect of misoprostol was seen following pretreatment once daily for 5 d with 12.5 pg/kg/d, a total daily dose similar to that used in humans. It is unlikely that direct stimulation of intestinal sodium or chloride transport plays a role, since the presence of misoprostol alone did not alter intestinal fluid absorption in the non-stressed control state. The mechanism of action of misoprostol in preventing stress-induced alterations in intestinal fluid absorption remains to be determined. It is unlikely that intraluminally administered prostaglandin El analogues alter the release of endogenous central nervous system mediators that may initiate and co-ordinate gastrointestinal secretory and motor response during stress (31, 33). It is more likely that misoprostol has its effect at a local level, perhaps by enhancement of mucosal blood flow (32), repletion of diminished endogenous prostaglandin levels (17), or by stabilization of stress induced mast cells (37). Acknowledgements Dr Fedorak is the recipient of a Clinical Investigatorship from the Alberta Heritage Foundation for Medical Research. This work is supported by a grant from the Alberta Heritage Foundation for Medical Research. The authors express appreciation to G. D. Searle Co. of Canada Limited for their generous support and provision of Misoprostol for purposes of this study, and to Mrs M Pollard for her expert secretarial support.
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