Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B

GASTROENTEROLOGY 1997;113:304–310

Cerulein-Induced In Vitro Activation of Trypsinogen in Rat Pancreatic Acini Is Mediated by Cathepsin B ASHOK K. SALUJA, ERIN A. DONOVAN, KENJI YAMANAKA, YOSHIKAZU YAMAGUCHI, BERND HOFBAUER, and MICHAEL L. STEER Department of Surgery, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts

Background & Aims: One of the central, unresolved issues in the pathogenesis of acute pancreatitis is the uncertainty regarding the mechanisms responsible for the premature intrapancreatic activation of digestive enzyme zymogens. The aim of the current study was to develop and characterize an in vitro system that might mimic the events leading to trypsinogen activation within the pancreas during pancreatitis. Methods: Activation of trypsinogen in response to stimulation with cerulein was quantitated in isolated rat pancreatic acini. Results: Activation of trypsinogen was detected within 10 minutes of exposing isolated rat pancreatic acini, in Ca2/-containing buffer, to a supramaximally stimulating concentration of cerulein in vitro. Complete inhibition of pancreatic cathepsin B activity with E-64d, a specific, potent and irreversible cathepsin B inhibitor, prevents cerulein-induced in vitro trypsinogen activation. Conclusions: In vitro activation of trypsinogen can be detected when pancreatic acini are exposed to a supramaximally stimulating dose of cerulein. The results using this in vitro system support the hypothesis that the appearance of active trypsin within the pancreas during the early stages of cerulein-induced pancreatitis reflects activation of trypsinogen by the lysosomal hydrolase cathepsin B.

T

he exocrine pancreas synthesizes and secretes a variety of digestive enzyme zymogens that, under physiological conditions, become activated only after they reach the duodenum. Acute pancreatitis is believed to result from the premature activation of some or all of these zymogens within the pancreas itself and to reflect autodigestive injury to the gland.1 Although activated digestive enzymes have been detected within the pancreas during acute pancreatitis,2 – 4 one of the central unresolved issues concerning the pathogenesis of acute pancreatitis has been the continuing uncertainty regarding the mechanisms by which intrapancreatic activation of digestive enzyme zymogens might occur. We have suggested previously that zymogen activation may be the result of colocalization within cytoplasmic / 5e1e$$0011

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organelles of digestive enzyme zymogens with the lysosomal hydrolase cathepsin B.5 Such colocalization has been observed,6 – 11 and cathepsin B can activate trypsinogen.12,13 However, the observation made by us as well as others14,15 that administration of the cathepsin B inhibitor E-64 to animals does not protect against several forms of experimental pancreatitis has raised concerns regarding the role of cathepsin B in the evolution of acute pancreatitis. The present study was designed to further explore this issue. The experiments were based on our recently reported observation16 that trypsinogen activation within the pancreas occurs within 10 minutes of infusing rats with a supramaximally stimulating dose of the cholecystokinin (CCK) analogue cerulein. Infusion of a supramaximally stimulating concentration of cerulein for 1–3 hours results in interstitial edematous pancreatitis with evidence of inflammation and patchy necrosis, but trypsinogen activation can be detected within the pancreas before evidence of either biochemical or morphological injury to acinar cells is noted. The rapidity with which trypsinogen becomes activated in vivo after supramaximal stimulation with cerulein suggested to us that in vitro studies might be designed to explore the mechanism of trypsinogen activation. We reasoned that it may be possible to induce trypsinogen activation in vitro by supramaximal stimulation with cerulein and that such an in vitro reductionist approach may prove useful in studies designed to evaluate the effect of inhibiting cathepsin B on the activation of trypsinogen. We report that in vitro activation of trypsinogen occurs when isolated pancreatic acini are incubated in Ca2/-containing buffer with a supramaximally stimulating concentration of cerulein. This activation is both time and cerulein concentration dependent. Cerulein-induced in vitro activation of trypsinogen is prevented when cathepsin B activity is totally inhibited by the cell-permeant cathepsin B inhibitor E-64d. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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Materials and Methods Male Wistar rats weighing about 100–125 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). Cerulein and CCK-JMV-180 [Boc-tyr(SO3)-NleGly-Trp-Nle-Asp-(2-phenylester)] were purchased from Research Plus (Bayonne, NJ), and collagenase was obtained from Worthington Biochemical (Freehold, NJ). E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and E-64d [(2S,3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] were purchased from Sigma Chemical Co. (St. Louis, MO). The trypsin substrate Boc-Gln-Ala-Arg-MCA was purchased from Peptides International (Louisville, KY). All other reagents were of the highest purity commercially available.

Preparation of Pancreatic Acini Dispersed rat pancreatic acini were prepared by collagenase digestion and gentle shearing as described previously.17,18 Acini were resuspended in HEPES-Ringer buffer (pH 7.4), which contained 115 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4 , 1 mmol/L CaCl2 , 10 mmol/L HEPES, 15 mmol/L glucose, and 0.1% bovine serum albumin. The buffer was saturated with O2 . Viability of acini was ú95% as assessed by trypan blue exclusion. In some experiments, extracellular [Ca2/] was altered by washing acini and suspending them in the same buffer but with or without 1 mmol/L CaCl2 . After incubation of suspended acini with various agents, the cells were washed twice with HEPES-Ringer buffer and homogenized in cold buffer with a motorized glass-Teflon homogenizer (Polytron; Brinkman Inst., Westbury, NY). The homogenate was centrifuged (50g for 2 minutes), and the supernatant was used for the assay of trypsin and lactate dehydrogenase activities.

Assays Amylase activity was measured as described previously.19 Cathepsin B activity was determined according to McDonald and Ellis20 with minor modifications9 using CBZarginyl-arginine-B-napthylamide, a highly specific substrate of cathepsin B activity. Trypsin activity was measured fluorimetrically using Boc-Gln-Ala-Arg-MCA as the substrate according to the method of Kawabata et al.21 For that measurement, 200 mL of the sample was added to a cuvette containing assay buffer (50 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, 1 mmol/L CaCl2 , and 0.1% bovine serum albumin). The reaction was initiated by the addition of substrate, and the fluorescence emitted at 440 nm after excitation at 380 nm was monitored. Trypsin activity in the samples was calculated using a standard curve generated by assaying purified trypsin obtained from Worthington Biochemical. Maximal values for trypsin activity after cerulein-induced in vitro trypsinogen activation were in the range of 0.8–1.5 mg trypsin/mg pancreatic DNA. To allow for pooling of data from experiments in which there was considerable variability of this value, the data are expressed as the percent of the maximal value obtained for that experiment.

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Unless otherwise noted, the maximal value obtained for each experiment was defined as the value obtained when acini were exposed to 0.1 mmol/L cerulein for 30 minutes. To calculate fractional trypsinogen activation, an aliquot of acinar cell homogenate was incubated with enteropeptidase for 1 hour, and trypsin activity was quantitated. DNA was quantified fluorimetrically using Hoechst dye 33258 (Sigma) and calf thymus DNA as described previously.22

Data Presentation The results reported represent means { SEM for multiple determinations from at least three separate acini preparations. In the figures, vertical bars denote SEM, and the absence of such bars indicate that the SEM was too small to show. The significance of the changes was evaluated using Student’s t test when the data consisted of two groups only or by analysis of variance (ANOVA) when comparing three or more groups. If ANOVA indicated significant differences, the data were analyzed by using Tukey’s method as a post hoc test for the difference between the groups. Values of P õ 0.05 were considered significant.

Results and Discussion We have recently reported the observation that trypsinogen becomes activated within the pancreas during the very early stages of cerulein-induced pancreatitis and long before evidence of either morphological or biochemical injury to the gland can be detected.16 In the current studies, we extended these investigations in an attempt to develop an in vitro system that might mimic the events leading to trypsinogen activation within the pancreas during pancreatitis. We hoped that such an in vitro system might serve as a powerful tool with which to probe the mechanisms responsible for intrapancreatic activation of digestive enzymes in pancreatitis. At the outset, we concluded that an in vitro system mimicking in vivo trypsinogen activation during cerulein-induced pancreatitis should meet certain predefined criteria. First, the dose-response relationship between cerulein-induced pancreatitis, as well as cerulein-induced trypsinogen activation in vivo, should be similar to that for cerulein-induced in vitro activation of trypsinogen. Second, because cerulein-induced pancreatitis and in vivo trypsinogen activation require interaction of an agonist with the low-affinity state of the acinar cell CCK-A receptors,16,23 a low-affinity receptor antagonist such as CCK-JMV-180 should cause little if any in vitro activation of trypsinogen. Third, agents such as CCK-JMV180 and L-364,718, which are antagonists at the lowaffinity state of the CCK-A receptor, should prevent in vitro trypsinogen activation by cerulein just as they prevent both cerulein-induced pancreatitis and cerulein-induced in vivo activation of trypsinogen. Fourth, in vitro WBS-Gastro

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Figure 1. Effect of cerulein on amylase secretion and trypsinogen activation. Rat pancreatic acini were incubated for 30 minutes in the presence of different concentrations of cerulein. The fraction of amylase content secreted into the medium (s) and trypsin activity in the acinar cells (●) were quantitated as described in the text. The results are expressed as the percent of maximal response. The maximal rate of amylase secretion (23% { 2% of total content) was noted in the presence of 0.1 nmol/L cerulein. The maximal extent of trypsinogen activation (1.2 { 0.5 mg trypsin/mg DNA) was noted in the presence of 0.1 mmol/L cerulein. Maximal activation of trypsinogen was 0.07% { 0.03% of the total trypsinogen present in the acinar cell. Results shown are mean values obtained from at least four different experiments. *P õ 0.05 compared with zero time.

activation of trypsinogen by cerulein, like its in vivo counterpart, should be rapid (i.e., occur within 10 minutes) and should precede evidence of cell injury. The results of studies evaluating these four criteria are shown in Figures 1–4. Previous studies reported by our group23 and others24 have clearly shown that ceruleininduced pancreatitis occurs when doses of cerulein are infused that are in excess of those that elicit a maximal rate of digestive enzyme secretion. We recently reported the observation that in vivo intrapancreatic activation of trypsinogen requires infusion of supramaximally stimulating doses of cerulein and that it does not occur when submaximal doses of the secretagogue are administered.16 As shown in Figure 1, in vitro activation of trypsinogen is also noted when acini are exposed to maximally or supramaximally stimulating concentrations of cerulein but not when lower concentrations are used. The specific activity of trypsin after activation brought about by a 30-minute exposure of acini to 0.1 mmol/L cerulein was 1.2 { 0.5 mg trypsin/mg DNA, a value similar to the value that we16 and others have reported for in vivo intrapancreatic trypsin activity during cerulein-induced pancreatitis in rats. However, contrary to the in vivo situation in which the pancreatic homogenates obtained / 5e1e$$0011

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Figure 2. Effect of CCK-JMV-180 and L-364,718 on cerulein-induced activation of trypsinogen. Rat pancreatic acini were incubated for 30 minutes in the presence of cerulein (CER; 0.1 mmol/L), CCK-JMV-180 (JMV; 0.1 mmol/L) alone or in combination with cerulein (CER / JMV) and L-364,718 (L-18; 1 mmol/L) in combination with cerulein (CER / L-18). Trypsin activity in the homogenized acini was quantitated as described in the text. The results are expressed as a percent of the response observed with 0.1 mmol/L cerulein alone. Results shown are the means { SEM obtained from at least four different experiments. *P õ 0.01 compared with cerulein alone.

Figure 3. Effect of extracellular calcium on cerulein-induced activation of trypsinogen. Rat pancreatic acini were incubated with either buffer alone or buffer containing 0.1 mmol/L cerulein (CER) in the presence or absence of 1 mmol/L CaCl2 for 30 minutes, and the trypsin activity in the homogenized acini was measured as described in the text. Results are expressed as the percent of maximal activity obtained in each experiment. Values shown are expressed as means { SEM obtained from at least four different experiments. *P õ 0.05 compared with basal.

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Figure 4. Time course of cerulein-induced activation of trypsinogen. Rat pancreatic acini were incubated with either buffer alone (●) or buffer containing 0.1 mmol/L cerulein (j) for varying times, and the trypsin activity in the homogenized acini was measured as described in the text. Results are expressed as the percent of maximal activity obtained in each experiment. Values shown are expressed as means { SEM obtained from at least four different experiments. *P õ 0.05 compared with zero time.

from control rats did not have any detectable trypsin activity,16 in vitro incubation of acini under basal conditions resulted in a small but significant activation of trypsinogen. This basal activation, most likely, represents trypsinogen activation that occurred in the process of preparing the acini. Pancreatic acinar cells are known to possess CCK-A receptors that exist in at least two affinity states: a highaffinity state that mediates digestive enzyme secretion and a lower affinity state that mediates the inhibition of secretion.25,26 We have previously reported the observation that cerulein-induced pancreatitis,23 as well as cerulein-induced in vivo trypsinogen activation,16 require interaction of an agonist with the lower affinity state of the acinar cell CCK-A receptor. Cerulein is such an agonist, and as shown in Figure 1, concentrations of cerulein that occupy the lower affinity state of acinar cell CCKA receptors can induce in vitro trypsinogen activation. On the other hand, CCK-JMV-180 is a low-affinity receptor antagonist. It neither induces pancreatitis in rats when administered in supramaximally stimulating doses23 nor causes in vivo trypsinogen activation in rat pancreatic acini.16 As required by criterion 2 above, supramaximally stimulating concentrations of CCK-JMV180 cause only minimal in vitro activation of trypsinogen (Figure 2). Cerulein-induced pancreatitis as well as in vivo tryp/ 5e1e$$0011

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sinogen activation can be prevented by infusion of either supramaximally stimulating doses of CCK-JMV-180 or of pharmacologically effective doses of the CCK-A receptor-antagonist L-364,718 along with the cerulein. Criteria 3 above would require that in vitro cerulein-induced trypsinogen activation be prevented by inclusion of either CCK-JMV-180 or of L-364,718 in the incubation mixture. As shown in figure 2, when a supramaximally stimulating concentration of CCK-JMV-180 (0.1 mmol/L) is combined with a supramaximally stimulating concentration of cerulein, in vitro trypsinogen activation is prevented. Inclusion of L-364,718 along with cerulein also prevents in vitro trypsinogen activation (Figure 2). The response of acinar cells to CCK stimulation is known to require Ca2/ in the extracellular medium and to involve an increase in intracellular free Ca2/ levels.25 As shown in Figure 3, in vitro cerulein-induced trypsinogen activation does not occur when acini suspended in nominally Ca2/-free buffer are studied. However, when acini are incubated with 1 mmol/L Ca2/, significant activation is noted. These observations indicate that ceruleininduced in vitro trypsinogen activation, like other CCK-induced acinar cell responses,25 is dependent on the presence of Ca2/ in the extracellular medium. Criteria 4 would require that in vitro cerulein-induced activation of trypsinogen should occur within 10 minutes of the onset of supramaximal cerulein stimulation and should precede evidence of cell injury. As shown in Figure 4, trypsinogen activation can actually be detected within 5 minutes of exposure to cerulein, is clearly observed within 10 minutes of supramaximal stimulation, and is maximal within 20 minutes of supramaximal stimulation with cerulein. Incubation of acini for less than 30 minutes with 0.1 mmol/L cerulein does not appear to affect cell viability, i.e., lactate dehydrogenase leakage from acini is not increased within that time (not shown). Thus, these observations indicate that, indeed, the requirements of criterion 4 have been met, i.e., in vitro cerulein-induced trypsinogen activation is rapid, and it precedes evidence of cell injury. Taken together, these observations indicate that each of the four criteria that we established for an appropriate in vitro system that mimicked intrapancreatic activation of trypsinogen during cerulein-induced pancreatitis have now been fulfilled. To our knowledge, this is the first report of an in vitro system in which supramaximally stimulating concentrations of cerulein result in the appearance of active trypsin in pancreatic acini. The only previously reported attempt to develop an in vitro model system for studies of digestive enzyme activation during pancreatitis was that study recently described by Leach WBS-Gastro

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et al.27 They noted that conversion of the zymogen procarboxypeptidase A1 to a lower-molecular-mass form could be detected when pancreatic acini were exposed in vitro to supramaximally stimulating concentrations of cerulein.27 That conversion presumably reflects conversion of the zymogen into the activated enzyme carboxypeptidase A, and in that sense, the in vitro events and the system described by Leach et al. may be similar to those reported in the current article. However, there are important differences between the in vitro activation system described by Leach et al.27 and that reported in the current article. Leach et al. did not note an increase in carboxypeptidase A1 activity after in vitro exposure of acini to supramaximally stimulating concentrations of cerulein. Rather, they found that the electrophoretic mobility of procarboxypeptidase A1 was changed and that the protein appeared to migrate as would be expected for the activated zymogen. Their observations, therefore, do not unambiguously indicate that zymogen activation had actually occurred because there may be other explanations for the altered electrophoretic mobility that they observed. The second and perhaps more important difference between the phenomenon observed by Leach et al. and that noted in our studies has to do with the relationship between the in vitro events

Figure 5. Effect of E-64d on cathepsin B activity and cerulein-induced activation of trypsinogen. After a 30-minute preincubation with 1 mmol/L cathepsin B inhibitor E-64d, rat pancreatic acini were further incubated with 0.1 mmol/L cerulein (CER) along with E-64d for 30 minutes, and cathepsin B ( ) and trypsin (j) activities in the homogenized acini were measured as described in the text. Results are expressed as the percent of activity obtained after incubation with cerulein alone. Values shown are expressed as means { SEM obtained from at least three different experiments.

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and the changes that occur in vivo during cerulein-induced pancreatitis. In our studies, in vivo activation of trypsinogen was observed when samples taken from animals infused with a supramaximally stimulating dose of cerulein were evaluated,16 and the characteristics of that in vivo trypsinogen activation closely parallel those noted for in vitro trypsinogen activation. In contrast, conversion of procarboxypeptidase A1 to a lower-molecularmass protein consistent with the activated zymogen is not detected when pancreas samples taken from animals with cerulein-induced pancreatitis are evaluated.28 Thus, the relevance of the changes noted by Leach et al. to the events that occur during the early stages of pancreatitis is not clear. Although there is general agreement that intrapancreatic digestive enzyme activation is an early and important event in the evolution of acute pancreatitis, the mechanisms responsible for that activation are not known. We have suggested previously that the premature intrapancreatic activation of digestive enzymes in pancreatitis might involve the lysosomal hydrolase cathepsin B.5 The suggestion was based on the observation that, in many models of experimental pancreatitis, digestive enzyme zymogens are colocalized with lysosomal hy-

Figure 6. Effect of E-64 on cathepsin B activity and cerulein-induced activation of trypsinogen. After a 30-minute preincubation with the cathepsin B inhibitor E-64 (0.1 or 1.0 mmol/L), rat pancreatic acini were further incubated with 0.1 mmol/L cerulein (CER) along with E64 for 30 minutes. Cathepsin B ( ) and trypsin (j) activities were measured in the homogenized acini as described in the text. Results are expressed as the percent of activity obtained after incubation with cerulein alone. Values shown are expressed as means { SEM obtained from at least three different experiments with each concentration of E-64.

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Figure 7. Effects of E-64d on amylase secretion and trypsin activity. (A ) Rat pancreatic acini were incubated for 30 minutes with different concentrations of cerulein in the presence (m) or absence (●) of 1 mmol/L E-64d. The fraction of amylase content secreted into the medium was quantitated as described in the text. (B ) Different amounts of commercial trypsin were assayed for trypsin activity in the presence (m) or absence (●) of 1 mmol/L E-64d. Trypsin activity is shown in relative fluorescence units. Data shown are typical of three separate experiments.

drolases within cytoplasmic vacuoles.6 – 11 The fact that the lysosomal hydrolase cathepsin B can activate trypsinogen12,13 and that trypsin can activate the remaining zymogens clearly supported that suggestion. On the other hand, the finding that the cathepsin B inhibitor E64 did not protect against several forms of experimental pancreatitis14,15 raised concerns regarding the validity of the so-called ‘‘colocalization’’ hypothesis. Administration of E-64 to animals, however, results in only incomplete inhibition of pancreatic cathepsin B activity,14,15 and for that reason, it has not been possible to define the effects of total cathepsin B inhibition in the pancreas using intact animals. In the current studies, we further explored this issue by taking advantage of the in vitro system of ceruleininduced trypsinogen activation. We hoped that the use of that reductionist system might allow us to achieve total inhibition of acinar cell cathepsin B activity and that, under those conditions, we could evaluate the effects on trypsinogen activation of inhibiting cathepsin B. In our initial studies, the cathepsin B inhibitor E-64 was used, but we found that, even in vitro, E-64 did not completely inhibit acinar cell cathepsin B activity. We therefore turned to the agent E-64d. E-64d, like E-64, is a specific, potent, and irreversible inhibitor of cathepsin B, but it has been specifically designed to be more permeant to intracellular organelles than E-64. 29 As shown in Figure 5, incubation of pancreatic acini with 1 mmol/L E-64d results in complete inhibition of cathepsin B activity. Furthermore, cerulein-induced in vitro activation of trypsinogen is completely prevented when concentrations of E-64d that completely inhibit cathepsin B are used. On the other hand, when cathepsin B activity / 5e1e$$0011

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is only incompletely inhibited by either lower concentrations of E-64d (Figure 5) or by E-64 (Figure 6), cerulein-induced trypsinogen activation persists. Although these results support the role of cathepsin B in cerulein-induced activation of trypsinogen, it is possible that these inhibitors block the trypsinogen activation by inhibiting the activity of some other thiol protease. However, our recent results, which indicate that CA-074, a much more specific inhibitor of cathepsin B, also prevented cerulein-induced activation of trypsinogen (Saluja and Steer, unpublished data, October 1997), lend further support to the conclusion that cathepsin B mediates cerulein-induced activation of trypsinogen. To rule out the possibility that inhibition of cerulein-induced activation of trypsinogen by E-64d could be caused by its nonspecific effects on cerulein-stimulated digestive enzyme secretion or on trypsin activity itself, we studied the effects of E-64d on cerulein-stimulated amylase secretion from acini and on in vitro trypsin activity. As shown in Figure 7, E-64d by itself had no effect on either trypsin activity or cerulein-induced amylase secretion from acinar cells. Taken together, the observations reported in this article indicate that cerulein-induced in vitro activation of trypsinogen is dependent on cathepsin B activity. Relatively low levels of cathepsin B activity appear to be sufficient for cerulein-induced trypsinogen activation to proceed, and this may explain the failure of agents such as E-64 to protect against pancreatitis induced by supramaximal stimulation with cerulein and against pancreatitis induced by other means. The observation that intrapancreatic activation of trypsinogen is mediated by cathepsin B during cerulein-inWBS-Gastro

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duced pancreatitis lends further support to the colocalization hypothesis, which proposes that colocalization of digestive enzyme zymogens and lysosomal hydrolases is necessary but by itself is not sufficient to induce pancreatitis. 30 Furthermore, these results suggest that interventions that completely inhibit pancreatic cathepsin B may be of benefit in either the treatment or prevention of pancreatitis.

15. 16.

17.

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Received June 7, 1996. Accepted March 7, 1997. Address requests for reprints to: Michael L. Steer, M.D., Department of Surgery, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. Fax: (617) 667-2978. Supported by grant DK 31396 from the National Institutes of Health.

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