JEJUNAL ADENYLATE CYCLASE ACTIVITY IN HUMAN SUBJECTS DURING VIRAL GASTROENTERITIS

Transcription

1 GASTROENTEROL()(;Y 70: Copyright 1976 by The Williams & Wilkins Co. Vol. 70. No.3 Printed in U.S.A. JEJUNAL ADENYLATE CYCLASE ACTIVITY IN HUMAN SUBJECTS DURING VIRAL GASTROENTERITIS ARNOLD G. LEVY, M.D., LAWRENCE WIDERLITE, M.D., CHARLES J. SCHWARTZ, M.D., RAPHAEL DOLIN, M.D., NEIL R. BLACKLOW, M.D., JERRY D. GARDNER, M.D., DANIEL V. KIMBERG, M.D., AND JERRY S. TRIER, M.D. Section on Gastroenterology. Digest ive.. Diseases Branch. National Institute o f Arthritis. M etabolism. and Digestive Diseases. and the Laboratory of Clinical Investigation. National Institute of Allergy and Infectious Diseases. National Institutes of Health. Bethesda. Maryland; and Departments of Medicine. Boston Univ ersity Medical Center. Peter Bent Brigham Hospital. Beth Israel Hospital. and Harvard Medical School. Bo ston. Massachusetts The histopathological changes thatoccur in the jejunal mucosa of humans infected with the Norwalk or Hawaii agent of acute infectious nonbacterial gastroenteritis ("viral" gastroenteritis) have been well characterized. The pathogenesis of diarrhea in this syndrome remains unknown; however, recent reports have suggested a possible role for the adenyl ate cyclase system. In this combined paper, two groups of investigators working independently and employing slightly different techniques report that: (1) there is marked interindividual variation in the apparent specific activity of adenyl ate cyclase in human jejunal biopsy tissue; (2,) such variation can be minimized by expressing enzyme activity as a fraction of maximal that can be stimulated by 10-2 M sodium fluoride; and (3) adenyl ate cyclase activity in jejunal mucosa is not increased during diarrhea or illness in human viral gastroenteritis, therefore suggesting no role for the adenylate cyclase system in the pathogenesis of diarrhea in this common clinical entity. Increased adenylate cyclase activity in small intestinal mucosa has beeh observed during diarrhea produced by Vibrio cholerae 1 a - or Escherichia c o l 4-6 ~. This increased activity and the consequent rise in cellular cyclic AMP appears to produce net intraluminal accumulation of fluid and electrolytes Recent studies 9 10 have raised the possibility of a similar mechanism for the pathogenesis of diarrhea in piglets caused by the transmissible gastroenteritis virus. Severe diarrhea may also be part of the symptom complex of the acute infectious nonbacterial gastroenteritis produced by two putative viruses, the Norwalk and the Hawaii agents Whereas the histopathology caused by these agents has been well described, the pathogenesis of diarrhea and the possible role of the adenylate cyclase system remain unknown. In this report two groups of investigators from the National Institutes of Health (group 1) and from Boston University, Beth Israel, and Peter Bent Brigham Hospitals (group 2) working independ- Received June Accepted September Address requests for reprints to: Dr. J erry D. Gardner. Building 10. Room 9D-15. National Institutes of Healt h. Bethesda. Maryland This work was supported in part by Grants AM AM AM RR and by Contract DADA C-2071 from the United States Army Medical Research and Development Command. The authors thank Thomas Conlon and Patricia Ware for their technical assistance with the adenylate cyclase assays. and Blanche Fors for her excellent secretarial assistance in preparing the manuscript. 321 entlyand employing slightly different assay techniques havecomhined their results from studies in which adenylate cyclase activity was d ~ t ~ r in m jejunal i n e d mucosa 6f 23 adult human volunteers before, during, and after acute illness induced by Norwalk or Hawaii agent. Methods Volunteers Human volunteers for group 1 were 14 males and females, ages 19 to 21 years, and for group 2 were 9 males, ages 25 to 50 years. All volunteers had normal physical examinations, and none had known gastrointestinal or liver disease. For each subject complete blood count, urinalysis, liver chemistries, blood urea nitrogen, creatinine, chest X-ray, and electrocardiogram were within normal limits. Written informed consent was obtained before all procedures. Studies by group 1 were performed at the Clinical Center of the National Institutes of Health; under close hospital supervision, as described previously;,. volunteers for group 2 were maintained in strict isolation in the Boston University Clinical Research Center at Boston City HospitaL These studies were approved by committees on human experimentation at the National Institutes of Health or at Boston University School of Medicine, Harvard Medical School, and the Depart ment of the Army. Administration of Inoculum The inoculum containing Norwalk or Hawaii agent was a stool filtrate obtained from a previous volunteer study and was shown to be free of bacterial. fungal, parasitic, or other viral agents by conventional safety tests. '1 Subjects were fasted overnight and then given 2 g of NaHCO s by mouth, followed by

2 322 LEVY ETAL. Vol. 70, No to 5.0 ml of either Norwalk or Hawaii filtrate which had been diluted in 30 ml of water. Food was then withheld for 3 hr. Definition of Clinical Illness Any volunteer who developed documented vomiting and/or diarrhea (unformed stool) along with one or more typical symptoms of illness, e.g., abdominal discomfort, anorexia, malaise, or fever (>38 C), was defined as having definite illness. Intestinal Biopsies Each group obtained peroral intestinal biopsies with a multipurpose biopsy tube ls positioned fluoroscopically in the proximal jejunum. Group 1 obtained biopsies before, 48 to 60 hr after, and 2 to 3 weeks after administration of inoculum. (In those volunteers who became ill, the first biopsy performed after administration of inoculum corresponded to 24 to 36 hr after onset of illness.) Group 2 obtained biopsies at the same intervals, except that the third specimen was obtained 5 days after onset of illness. In all biopsies, 3 or 4 pieces of tissue were obtained. One piece was processed for histological assessment, and the remaining pieces were assayed for adenyl ate cyclase activity. Adenylate Cyclase Assay Group 1. To assay for adenylate cyclase activity, biopsy specimens were immediately placed in 50 mm Tris-HCI, ph 7.4, at 4 C, and homogenized by 20 strokes of a pestle in a Ten Broeck glass, hand homogenizer. Homogenization of tissue was kept constant (4 min), as was the interval between homogenization and initiation of the adenylate cyclase assay (1 min). Protein concentration of the homogenate was determined using the method of Lowry et al. l with bovine serum albumin as a standard. Adenylate cyclase activity was determined by measuring the formation of 32P-Iabeled cyclic AMP from [a- 32 P]ATP (New England Nuclear Corp., Boston, Mass.). The final reaction mixture (70 /-ll) contained: 50 mm Tris-HCI, ph 7.4; 5.0 mm MgCI 2 ; 9 mm theophylline; 10 mm KCI; 1.25 mm ATP; [a- 32 P]ATP (approximately 10 6 cpm); ATP-regenerating system (14 /-lg of creatine phosphokinase; 10 mm creatine phosphate); fresh tissue homogenate (30 to 110 /-lg of protein); and 10-2 M sodium fluoride or equivalent volume of buffer "control." Incubation was carried out for 10 min in a Dubnoff metabolic shaker at 37 C. The reaction was stopped by adding 100 /-ll of a solution containing 2.5% sodium dodecyl sulfate, 40 mm ATP, and 12.5 mm [3H] cyclic AMP (New England Nuclear Corp., Boston, Mass.) (approximately 20,000 counts per min). Assay "blanks" were prepared by omitting tissue homogenate or by adding tissue homogenate after the reaction was stopped; both methods gave equivalent blank values (4 to 7 counts per min above machine background). Isolation and detection of cyclic [ 32 P]AMP was performed using the procedure described by Salomon, et al. 20 Liquid scintillation counting was performed using a Packard model 3320 liquid scintillation counter. Results were corrected for recovery of [3H]cyclic AMP, which was 70 to 80%. Adenylate cyclase activity was constant for at least 10 min at 37 C and was a linear function of protein concentration. Group 2. Biopsy specimens were immediately immersed in ice-cold Krebs-Ringer bicarbonate solution (ph 7.4; containing (mm] Na, 144; K, 10; Ca, 1.25; Mg, 1.1; CI, 127; HC03. 25; H 2PO., 0.3; and HPO. 1.65). The tissue was transferred to a chilled ground glass I-ml microtissue homogenizer (Kontes Glass Co., Vineland, N. J.) containing 0.3 ml of 20 mm glycylglycine and 1 mm MgSO. (ph 7.8) and homogenized with four strokes of a tightly fitting glass pestle. The interval between performance of biopsy and homogenization of tissue' was less than 1 hr; the interval between time of homogenization and initiation of the assay for adenylate cyclase was kept constant at 45 min. Protein concentration of the homogenate was determined using the method of Lowry et al. 10 with bovine serum albumin as a standard. Adenylate cyclase activity was determined by measuring the formation of "C-Iabeled cyclic AMP from [8- H C]ATP. The final reaction mixture (50 /-ll) contained: 50 mm Tris-HCI, ph 7.4; 5.0 mm MgCI 2 ; 10 mm caffeine; 0.2 mm ATP; 1 /-lci of [8-UC]adenosine triphosphate (New England Nuclear Corp., Boston, Mass.); ATP regenerating Eystem (10 mm phosphoenolpyruvate; 12.5 /-lg of pyruvate kinase); fresh tissue homogenate (150 to 250 /-lg of protein); and 10-2 M sodium fluoride or equivalent volume of buffer "control." Incubations were for 5 min at 37 C. The reaction was terminated by adding 0.2 ml of a solution containing 60 /-lg of cyclic AMP and 0.25 /-lci of cyclic [3H]AMP (Schwarz/Mann, Van Nuys, Calif.) as a recovery marker, and boiling for 3 min. Isolation and detection of cyclic ["C]AMP was performed using a method21 slightly different from that employed by group 1. Liquid scintillation counting was performed using an Intertechnique SL40 liquid scintillation spectrometer. Results were corrected for recovery of [3H]cyclic AMP, which was 40 to 50%. Blank values for' [HC] were usually 4 to 6 cpm above machine background. Adenylate cyclase activity was constant for at least 5 min at 37 C and was a linear function of protein concentration. Results Jejunal adenylate cyclase activity (pmoles per min per mg of protein) determined by group 1 was considerably greater than that determined by group 2 (tables 1 and 2). This difference can be completely accounted for by group 1 having used a higher substrate concentration (ATP = 1.25 mm, versus 0.2 mm by group 2) and a shorter time interval between homogenization of tissue and initiation of cyclase assay (1 min versus 45 min by group 2). After homogenization, jejunal adenyl ate cyclase activity decreases at a rate of 3.2% per min. 22 The comparability of results from the two groups is further demonstrated by the lack of a difference between results when preinoculation adenyl ate cyclase activity is expressed as basal to fluoride ratio (mean by group 1,0.38; mean by group 2, DAD). Each group found substantial interindividual variation in basal as well as fluoride-stimulated adenylate cyclase activity (tables 1 and 2). This variation was minimized by expressing basal enzyme activity as a fraction of maximal activity stimulated by 10-2 M sodium fluoride (compare ratios of standard deviation to mean in tables 1 and 2). Elevation of such a ratio has been demonstrated previously in jejunal biopsies from patients during illness with Asiatic cholera. 3 In the present studies, statistical analyses (paired t-tests) were performed using the basal to fluoride ratios. Group 1. There was no significant difference between basal to fluoride ratios (table 1) from preinoculation biopsies and from biopsies obtained 2 to 3 weeks after inoculation in volunteers in any of the four clinical categories: those who had received Hawaii agent and became ill (P > 0.9), or remained well (P > 0.05); and

3 March 1976 JEJUNAL ADENYLATE CYCLASE ACTIVITY IN HUMANS 323 TABLE 1. Jejunal adenylate cyclase activity with Hawaii or Norwalk agent infection (group 1)a Pre-inoculation hr after inoculation 2-3 wk after inoculation Clinical features Volunteer no. Adenylate Basal Adenylate Basal Adenylate Basal cyclase cyclase cyclase Diarrhea Vomiting Fever activityb Fluoride activityb Fluoride activity' Fluoride Hawaii agent (Standard deviation/ (1.51) (0.33) (0.53) Norwalk agent (Standard deviation/ (0.69) (0.37) (0.74) (0.31) (0.81) (0.49) (0.47) (0.63) (0.30) a Assay ATP concentration ~ 1.25 mm. b pmoles of cyclic AMP formed per min per mg of protein. e Nausea. d Abdominal cramps. TABLE 2. Jejunal adenylate cyclase activity with Norwalk agent infection (group 2)a Volunteer no. Pre-inoculation 24 to 36 hr. after onset 4 to 5 days after onset of illness of illness Clinical features Adenylate Basal Adenylate Basal Adenylate Basal cyclase cyclase cyclase Diarrhea Vomiting Fever activity' Fluoride activity' Fluoride activity' Fluoride Norwalk agent (Standard deviation/ (0.76) (0.26) (0.82) (0.40) (0.89) (0.:32) a Assay ATP concentration ~ 0.2 mm. b pmoles of cyclic AMP formed per min per mg of protein. those who had received Norwalk agent and became ill (P > 0.9), or remained well (P > 0.5). Furthermore, there are no significant differences between means of the above ratios (preinoculation and 2 to 3 weeks after inoculation) and ratios obtained 48 to 60 hours after inoculation in three of the four clinical categories: those who received Hawaii agent and became ill (P > 0.9), or remained well (P > 0.4); and those who received Norwalk agent and became ill (P > 0.2). There was a significant decrease (P < 0.01), however, in the basal to fluoride ratio in volunteers who received Norwalk agent and did not become ill. Histology was assessed in group 1 patients challenged with Hawaii agent. The typical mucosal lesion was observed 48 to 60 hr after inoculation in patients 1, 2, and 3 with overt clinical illness (table 1). Mucosal

4 324 LEVY ETAL. Vol. 70, No.3 morphology remained normal in the 3 patients who remained asymptomatic and in patient 4 who developed vomiting. Histology was not obtained in group 1 patients challenged with Norwalk agent. Group 2. There was no significant difference (P > 0.7) between basal to f1uoride ratios obtained from preinoculation biopsies and from biopsies obtained 4 to 5 days after illness. Furthermore, there was no significant difference (P > 0.5) between the means of these values and values obtained 24 to 36 hr after onset of illness. The morphology of base line intestinal biopsies obtained from 9 volunteers who subsequently received Norwalk agent was normal. Biopsies obtained from all 9 individuals after Norwalk agent challenge showed the characteristic lesion of acute infectious non bacterial gastroenteritis,12-15 confirming the clinical evidence that these individuals had gastroenteritis (table 2). Discussion In this study, despite strict attention to experimental conditions, two independent groups of investigators employing slightly different techniques confirmed a previous report22 of marked interindividual variation in adenylate cyclase activity (expressed on a per milligram of protein basis) in human jejunal mucosal biopsy specimens. These results are in contrast to those of Chen et al. 3 who found minimal interindividual variation in basal as well as f1uoride-stimulated adenyl ate cyclase activity in biopsies from patients with Asiatic cholera, both during and after recovery from illness. The time from biopsy to homogenization of tissue and from homogenization to initiation of assay was kept constant by both groups in the present study; therefore, variations in these intervals could not account for the interindividual variation in cyclase activity observed in the present study. Whereas Chen et al. 3 found decreased specific activity of adenylate cyclase with protein concentrations less than 20 or greater than 50 J.Lg per J.LI, we failed to observe such variation at protein concentrations of 21 to 79 J.Lg per 50 J.LI (group 1) or 150 to 250 J.Lg per 50 J.LI (group 2). Thus, varying protein concentrations do not account for the interindividual variations in cyclase activity observed in the present studies. The reason or reasons for the discrepancy between our results and those of Chen et al. remains unclear. Peroral inoculation of normal volunteers with Norwalk or Hawaii agent of acute infectious non bacterial gastroenteritis produced clinically indistinguishable syndromes which were typical of naturally occurring illness attributed to "viral gastroneteritis, ".22 "winter vomiting disease, "24 or acute infectious nonbacterial gastroenteritis. 25 Jejunal biopsies from volunteers receiving Norwalk agent13, 14 or Hawaii agent reveal similar, reversible histopathological lesions during diarrheal illness. In some cases, mucosal lesions may be associated with illness not characterized by diarrhea (e.g., vomiting, malaise, anorexia, and fever),15 or may occur in the absence of illness. 12, 14 The precise pathophysiology of diarrhea induced by viral agents is not known. Altered transport of water and electrolytes, similar to that produced through the adenylate cyclase system by Vibrio cholerae and Esche-. richia coli,4. 7, 8 has recently been demonstrated in the proximal jejunum of piglets experimentally infected with transmissible gastroenteritis virus; 9 however, adenyl ate cyclase activity was not altered in either small or large intestine of acutely infected piglets. Of interest is the capability of this virus to elevate concentrations of cyclic AMP in cultured porcine kidney cells. 10 In the present study, we found no elevation of intestinal mucosal adenylate cyclase activity in either Norwalk- or Hawaii-induced illness. The cause of the decrease in cyclase activity in volunteers (studied by group 1) who did not become ill 48 hr after administration of Norwalk agent remains unknown; however, changes in enzyme activity did not correlate with the volunteers' clinical status. It is conceivable that adenylate cyclase activity could have been elevated during disease with either Norwalk or Hawaii agent, but that activation was reversed during homogenization and dilution required for the cyclase assay. Measurement of cyclic AMP content in biopsy specimens would be of interest in this regard. REFERENCES 1. Pierce NF, Greenough WB III, Carpenter CCJ Jr: Vibrio cholerae enterotoxin and its mode of action. Bact Reviews 85:1-13, Kimberg DV, Field M, Johnson J, et al: Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J Clin Invest 50: , Chen LC. Rohde JE, Sharp GWG: Properties of adenyl cyclase from human jejunal mucosa during naturally acquired cholera and convalescence. J Clin Invest 51: , Guerrant RL, Ganguly U, Casper AGT, et al: Effect of Escherichia coli on fluid transport across canine small bowel. J Clin Invest 52: , Kantor HS, Tao P, Wisdom C: Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture. Infect Immun 9: , Kantor HS, Tao P, Gorbach SL: Stimulation of intestinal adenyl cyclase by Escherichia coli enterotoxin: comparison of strains from an infant and an adult with diarrhea. J Infect Dis 129: 1-9, Moon HW, Whipp SC, Baetz AL: Comparative effects of enterotoxins from Escherichia coli and Vibrio cholerae on rabbit and swine small intestine. Lab Invest 25: , Banwell JG, Sherr H: Effect of bacterial enterotoxins on the gastrointestinal tract. Gastroenterology 65: , Butler DG, Gall DG, Kelly MH, et al: Transmissible gastroenteritis-mechanisms responsible for diarrhea in an acute viral enteritis in piglets. J Clin Invest 53: , Mishra NK, Ryan WL, Chaudhuri SN: Elevation of the intracellular levels of cyclic adenosine monophosphate during in vitro infection of Transmissible Gastroenteritis Virus. Arch Ges Virusforsch 41: , Blacklow NR, Dolin R, Fedson DS, et al: Acute infectious non-bacterial gastroenteritis: etiology and pathogenesis. Ann Intern Med 76: , Schreiber DS, Blacklow NR, Trier JS: The small intestinal lesion induced by Hawaii agent acute infectious non bacterial gastroenteritis. J Infect Dis 129: , Agus SG, Dolin R, Wyatt RG, et al: Acute infectious non bacterial gastroenteritis: intestinal histopathology. Ann Intern Med 79: 18-25, 1973

5 March 1976 JEJUNAL ADENYLATE CYCLASE ACTIVITY IN HUMANS Schreiber DS. Blacklow NR, Trier JS: The mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis. N Engl.J Med 288: , Dolin R, Levy AG, Wyatt RG, et al: Viral gastroenteritis induced by the Hawaii agent: jejunal histopathology and serologic response. Am J Med 59: , Dolin R, Blacklow NR, DuPont H, et al: Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. J Infect Dis 123: , Knight V: The use of volunteers in medical virology. Prog Med Virol 6:1-26, Brandborg LL, Rubin GE, Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37:1-16, Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent..jbiol Chern 193: , Salomon Y, Londos C, Rodbell M: A highly sensitive adenyl ate cyclase assay. Anal Biochem 58: , Schwartz CJ, Kimberg DV, Ware P: Adenylate cyclase in intestinal crypt and villus cells: stimulation by cholera enterotoxin and prostaglandin El. Gastroenterology 68:94-104, Klaeveman HL, Conlon TP, Levy AG, et al: Effects of gastrointestinal hormones on adenyl ate cyclase activity in human jejunal mucosa. Gastroenterology 68: , Cheever FS: Viral agents in gastrointestinal disease. Med Clin North Am 51: , Zahorsky J: Hyperemesis hiemis or the winter vomiting disease. Arch Pediatr 46: , Dingle JH, McCorkle LP, Badger GD, et al: A study of illness in a group of Cleveland families. XIII. Clinical description of acute non bacterial gastroenteritis. Am J Hyg 64::