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The mechanisms responsible for intrapancreatic digestive enzyme activation as well as the relationship between that activation and cell injury during pancreatitis are not understood. We have employed an in vitro system in which freshly prepared pancreatic acini are exposed to a supramaximally stimulating concentration of the CCK analog caerulein to explore these issues. We find that in vitro trypsinogen activation depends on the continued presence of Ca2+ in the suspending medium and that it is half-maximal in the presence of 0.3 mM Ca2+. Caerulein-induced trypsinogen activation can be halted by removal of Ca2+ from the suspending medium or by chelation of intracellular Ca2+. Increasing intracellular Ca2+ with either ionomycin or thapsigargin does not induce trypsinogen activation. We have monitored cell injury by measuring the leakage of lactate dehydrogenase (LDH) from acini and by quantitating intercalation of propidium iodide (PI) into DNA. Leakage of LDH and intercalation of PI in response to supramaximal stimulation with caerulein can be detected only after caerulein-induced trypsinogen activation has already occurred, and these indications of cell injury can be prevented by addition of a cell-permeant protease inhibitor. Our findings indicate that caerulein-induced intra-acinar cell activation of trypsinogen depends on a rise in intracellular Ca2+, which reflects entry of Ca2+ from the suspending medium. Intra-acinar cell activation of trypsinogen is an early as well as a critical event in pancreatitis. The subsequent cell injury in this model is mediated by activated proteases.

Supramaximal stimulation of the pancreas with the CCK analog caerulein causes acute edematous pancreatitis. In this model, active trypsin can be detected in the pancreas shortly after the start of supramaximal stimulation. Incubation of pancreatic acini in vitro with a supramaximally stimulating caerulein concentration also results in rapid activation of trypsinogen. In the current study, we have used the techniques of subcellular fractionation and both light and electron microscopy immunolocalization to identify the site of trypsinogen activation and the subsequent fate of trypsin during caerulein-induced pancreatitis. We report that trypsin activity and trypsinogen-activation peptide (TAP), which is released on activation of trypsinogen, are first detectable in a heavy subcellular fraction. This fraction is enriched in digestive enzyme zymogens and lysosomal hydrolases. Subsequent to trypsinogen activation, both trypsin activity and TAP move to a soluble compartment. Immunolocalization studies indicate that trypsinogen activation occurs in cytoplasmic vacuoles that contain the lysosomal hydrolase cathepsin B. These observations suggest that, during the early stages of pancreatitis, trypsinogen is activated in subcellular organelles containing colocalized digestive enzyme zymogens and lysosomal hydrolases and that, subsequent to its activation, trypsin is released into the cytosol.

Infusion of a supramaximally stimulating dose of the pancreatic secretagogue caerulein (10 micrograms.kg-1.h-1) for 4 h induces interstitial edematous acute pancreatitis in rats. This model of acute pancreatitis is associated with evidence of acute lung injury, including sequestered neutrophils within the pulmonary microvasculature, increased microvascular permeability, and interstitial pulmonary edema. Infusion of prostaglandin E1 (PGE1; 50 ng.kg-1.min-1) along with caerulein does not alter the severity of secretagogue-induced pancreatitis, but it does reduce the severity of pancreatitis-associated acute lung injury. The rise in lung weight, lung water content, and pulmonary microvascular permeability and the sequestration of neutrophils within the pulmonary microvasculature that accompany secretagogue-induced pancreatitis are all reduced by infusion of PGE1. Infusion of PGE1 does not interfere with polymorphonuclear neutrophil sequestration in the pancreas or reduce the enhanced expression of CD11b/c receptors on circulating neutrophils. Our observations indicate that PGE1 reduces the severity of pancreatitis-associated acute lung injury by preventing neutrophil sequestration within the lung. We speculate that PGE1 interferes with neutrophil sequestration by dilating pulmonary vasculature, increasing pulmonary flow rate, and reducing neutrophil-endothelial cell interaction and attachment.

The factors that determine the severity of acute pancreatitis are unknown, but a close inverse correlation between that severity and the extent of acinar cell apoptosis that follows the triggering signal has been previously noted [A. M. Kaiser, A. K. Saluja, A. Sengupta, M. Saluja, and M. L. Steer. Am. J. Physiol. 269 (Cell Physiol. 38): C1295-C1304, 1995]. In the present studies, we have evaluated internucleosomal DNA fragmentation and apoptosis within the pancreas and the effects of inhibiting protein synthesis by cycloheximide (CHX) on these phenomena as well as on the severity of pancreatitis. We report the constitutive presence of a Ca(2+)- and Mg(2+)-dependent endonuclease activity within pancreatic nuclei that is dependent on continued protein synthesis. Furthermore, we have found that CHX administration reduces the extent of apoptosis but significantly worsens the severity of pancreatitis that follows ligation of the rat common bile-pancreatic duct. These observations are consistent with the hypothesis that apoptosis is a teleologically beneficial response to acinar cell injury during acute pancreatitis.

In an effort to elucidate factors that determine the severity of an attack of acute pancreatitis, we have quantitated the extent of necrosis and of apoptosis in five different models of experimental acute pancreatitis. Severe pancreatitis was induced by obstructing the opossum common bile-pancreatic duct, by administering to mice 12 hourly injections of a supramaximally stimulating dose of caerulein, and by feeding young female mice a choline-deficient, ethionine-supplemented diet. In each of these models of severe pancreatitis, marked necrosis but very little apoptosis was found. Mild pancreatitis was induced by obstructing the rat common bile-pancreatic duct and by infusing rats with a supramaximally stimulating dose of caerulein. In contrast to our findings in severe pancreatitis, mild pancreatitis was characterized by very little necrosis but a high degree of apoptosis. Our finding that the severity of acute pancreatitis is inversely related to the degree of apoptosis suggests that apoptosis may be a teleologically beneficial response to acinar cell injury in general and especially in acute pancreatitis.

In vivo pancreatic secretion of the lysosomal hydrolase cathepsin B was found to be increased by infusion of the secretagogue caerulein. The basal as well as caerulein-stimulated in vivo rate of cathepsin B was further increased by infusion of either chloroquine or methylamine while neither the basal nor the secretagogue-stimulated rates of amylase secretion were altered by the lysosomotropic agents. These observations indicate that neutralization of the acidic prelysosomal compartment by administration of lysosomotropic agents results in lysosomal enzyme entry, by default, into the regulated secretory pathway. In vitro stimulation of pancreatic acini with caerulein was also found to stimulate cathepsin B secretion. That in vitro rate of cathepsin B secretion stimulated by caerulein was not increased in acini prepared from animals infused with caerulein, chloroquine, or methylamine, but the in vitro rate of cathepsin B secretion stimulated by caerulein was increased in acini prepared from animals infused with caerulein plus either chloroquine or methylamine. Under these conditions, redistribution of cathepsin B from the lysosome-enriched to the zymogen granule-enriched subcellular fraction was noted, and lysosomal enzyme-containing organelles became increasingly fragile. These observations indicate that in vivo secretagogue stimulation increases the degree of diversion of lysosomal hydrolases into the regulated secretory compartment when the prelysosomal compartment has been neutralized with lysosomotropic agents.

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet for 24 h develop hemorrhagic pancreatic necrosis with a 5-day mortality rate of approximately 50%. At the end of the diet administration, the in vivo discharge of digestive enzymes is blocked, images of exocytosis and luminal membrane recycling disappear, and zymogen granules accumulate within acinar cells. The general ultrastructure, however, remains well preserved, protein synthesis is normal, and intracellular transport of secretory proteins is only slightly retarded. Thus, the CDE diet does not affect the general phenomenon of membrane fusion-fission but specifically inhibits that associated with exocytosis. Twenty-four hours after withdrawal of the CDE diet, discharge of zymogen granules into lysosomes (crinophagy) can be observed, and, 24 h latr, autophagocytosis is noted. Finally, shortly before the onset of pancreatic necrosis, cells of nearly normal appearance are noted to be scattered among cells showing varying degrees of lesion up to complete disruption. Thus, the CDE-induced pancreatic necrosis results from a sequence of events: blockade of exocytosis evolves to crinophagy and autophagy, which might lead to lysosomal activation of zymogens.

Rats infused with a dose of the secretagogue caerulein that is in excess of that which stimulates a maximal rate of pancreatic digestive enzyme secretion develop acute edematous pancreatitis. We have previously noted that infusion of this dose of caerulein (5 micrograms . kg-1 . h-1) induces the appearance of large heterogeneous vacuoles in acinar cell, blockade of exocytosis, and intracellular accumulation of digestive zymogens [O. Watanabe et al. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G457-G467, 1984 and A. Saluja et al. Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G702-G710, 1985]. The current studies were performed to further elucidate these phenomena at the electron microscopic level of resolution and employed the techniques of pulse labeling, radioautography, and immunolocalization. Rats were infused with caerulein (5 micrograms . kg-1 . h-1) for 1 h, given a pulse of [3H]phenylalanine, and killed at selected times during the subsequent 5- to 180-min postpulse period during which caerulein infusion was continued. Transport from the endoplasmic reticulum to the Golgi cisternae was not altered by supramaximal stimulation, but transport through post-Golgi elements was altered. In particular, the maturation of condensing vacuoles into zymogen granules was found to be impaired. This led to the accumulation of partially condensed vacuoles and to the development of the large vacuoles containing newly synthesized digestive zymogens as well as the lysosomal hydrolase cathepsin D. The source of the latter could be impaired sorting of lysosomal and digestive enzymes and/or fusion of vacuoles with lysosomes. At the later times after pulse labeling, mature zymogen granules were also found to fuse with these large cathepsin D-containing vacuoles by a process analogous to crinophagy. Thus these studies indicate that the large heterogeneous vacuoles that appear during supramaximal secretagogue stimulation and that contain admixed digestive zymogens and lysosomal hydrolases arise by at least two mechanisms, impaired condensing vacuole maturation and crinophagy.

The response of the intracellular pH (pHi) to stimulation of enzyme secretion in pancreatic acini was measured using the fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Acini suspended in pH 7.40 buffer demonstrated cytoplasmic alkalinization of 0.17, 0.14, and 0.15 pH units 2 min after addition of the secretagogues carbachol (10(-5) M), caerulein (10(-10) M), and bromo-A23187 (10(-6) M). Corresponding net stimulated amylase secretion over 30 min was 9.2, 10.3, and 5.6% of total content, respectively. Pretreatment of acini with atropine blocked the pHi rise induced by carbachol; addition of atropine 2 min after the carbachol did not reverse the alkalinization. Acini suspended in Ca2+ free buffer containing 0.1 or 0.2 mM ethylene glycol tetraacetic acid showed 0.21 and 0.10 pH unit alkalinization in response to caerulein (10(-10) M) and carbachol (10(-5) M) but no change in pHi after addition of bromo-A23187. Amylase release in response to increasing concentrations of caerulein was maximal at 10(-10) M, with decreasing rates of amylase release at higher drug concentrations (10(-8), 10(-7) M). Alkalinization in response to stimulation of secretion was maximal at 10(-8) M caerulein (0.30 pH units at 2 min) but was of lesser magnitude at 10(-7) M. Pancreatic acini demonstrated autoregulation of pHi over a range of external pH from 7.4 to 7.1. Net amylase release over 30 min in response to 10(-5) M carbachol was sustained at normal levels in buffers of pH varying between 7.7 and 6.5. In contrast, cytoplasmic alkalinization in response to carbachol occurred only in buffers with pH values between 7.40 and 7.10. These results indicate that amylase release occurs over a wide range of pHi and is not invariably associated with secretagogue-induced alkalinization.

Nonparallel discharge of digestive enzymes by the in vitro rabbit pancreas has been previously reported and this observation has been used to challenge the mass transport theory of enzyme synthesis and secretion by the exocrine pancreas. The current investigation reexamines the pattern of enzyme secretion from the in vitro rabbit pancreas. Secretion of alpha-amylase, trypsinogen, and chymotrypsinogen was studied from the unstimulated gland and from glands stimulated with either methacholine, cholecystokinin-pancreozymin, or the active octapeptide of cholecystokinin-pancreozymin. In all cases, only parallel discharge of alpha-amlyse, trypsinogen, and chymotrypsinogen was noted. These observations are consistent with the mass-transport theory.

The basal rate of amylase secretion from rat pancreatic lobules was found to diminish with time. This decline, which is not due to depletion of amylase stores or loss of tissue integrity, has been attributed to the build-up of amylase in the extracellular space [Ho and Rothman, Am. J. Physiol. 245 (Cell Physiol. 14): C21-C27, 1983]. This accumulation of amylase would result in a decrease in the intracellular-extracellular amylase gradient, which, according to the so-called equilibrium hypothesis, is responsible for net amylase secretion under basal conditions. Using rat pancreatic lobules and acini, we have tested the validity of this hypothesis by adding to the incubation medium at the onset of incubation either collected secretory products or purified amylase. Based on the equilibrium hypothesis, one would have predicted that these additions would also reduce the concentration gradient favoring net secretion and would result in an apparent reduction in the initial rate of basal in vitro digestive enzyme secretion. The results obtained from these studies, however, were not in accord with those predictions, since these additions did not diminish the initial rate of basal amylase secretion. Our observations therefore indicate that the decrease in the rate of basal amylase secretion from rat pancreas with time is not due to the loss of a concentration gradient favoring efflux. These results argue against the validity of the equilibrium hypothesis.

Rats infused with a supramaximally stimulating dose of the cholecystokinin-pancreozymin analogue caerulein develop acute interstitial pancreatitis (M. Lampel and H.F. Kern. Virchows Arch. A 373: 97-117, 1977). We have studied the early (30-180 min) morphological changes in pancreatic acinar cells induced by infusing caerulein (2.5 micrograms X kg-1 X h-1). The techniques of thin-section electron microscopy, freeze fracture, and enzyme and immunocytochemistry were employed. Shortly (30 min) after the onset of caerulein infusion, large vacuoles appeared in the Golgi area. After longer periods of infusion, these vacuoles further enlarged (probably by fusion with other such vacuoles as well as autophagic vacuoles) and became more widely distributed in the cytoplasm. These large vacuoles were found to be acid phosphatase positive and to be labeled by antibodies directed against digestive zymogens as well as the lysosomal enzyme cathepsin D. These observations indicate that the large vacuoles contain both digestive zymogens and lysosomal hydrolases. During caerulein infusion, morphological evidence of exocytosis at the luminal plasmalemma was reduced or absent, and evidence of basolateral exocytosis was not noted. These studies suggest that secretagogue hyperstimulation with caerulein interferes with the processes involved in condensing vacuole maturation, which normally lead to the separation of digestive zymogens and lysosomal hydrolases. As a result, both types of enzymes remain within the same compartment. This may lead to the intracellular activation of digestive enzymes by lysosomal hydrolases and be an important step in the development of acute pancreatitis.

The subcellular distribution of the lysosomal enzymes cathepsin B and D in the pancreas was evaluated in rats infused with saline (control) or a maximal (0.25 microgram . kg-1 . h-1) or a supramaximally stimulating dose (5 micrograms . kg-1 . h-1) of the secretagogue caerulein. The latter results in acute edematous pancreatitis, inhibition of digestive enzyme secretion, and the localization of digestive zymogens in organelles whose fragility has been increased by caerulein infusion [A. Saluja et al. Am. J. Physiol. 249 (gastrointest. Liver Physiol. 12): G702-G710, 1985]. Samples from control animals were found to have 29.9 +/- 1.8% of the cathepsin B activity in the pellet centrifuged at 1,300 g for 15 min (containing primarily zymogen granules) and 54.7 +/- 2.5% in the pellet centrifuged at 12,000 g for 12 min (containing primarily lysosomes and mitochondria). After supramaximal stimulation with caerulein for 3.5 h the pellet centrifuged at 1,300 g for 15 min had 55.1 +/- 2.5%, and the pellet centrifuged at 12,000 g for 12 min had 30.6 +/- 2.0% of cathepsin B activity. This redistribution was time dependent, noted within 1 h of starting caerulein infusion, and maximal after 2.5 h of infusion. Electron microscopic immunolabeling studies revealed localization of cathepsin D in discrete organelles that, in the samples from animals infused with a supramaximally stimulating dose of caerulein, were larger, more abundant, and more concentrated in the pellet centrifuged at 1,300 g for 15 min than in the controls. During infusion with supramaximal doses of caerulein, the cathepsin B-containing organelles were found to become progressively more fragile.(ABSTRACT TRUNCATED AT 250 WORDS)

A sustained increase in sympathetic nervous system (SNS) activity was induced by substituting a 10% sucrose solution for the drinking water of rats fed laboratory chow ad libitum. The effects of increased SNS activity on alpha 1-adrenergic processes in liver were examined by evaluating three alpha 1-responses, namely, phenylephrine-stimulated ouabain-sensitive 86Rb+ uptake, 45Ca2+ efflux, and glucose release. Sucrose feeding abolished phenylephrine stimulation of ouabain-sensitive 86Rb+ uptake and 45Ca2+ efflux and induced a three- to fourfold reduction in the ability of phenylephrine to stimulate glucose release from liver slices. Pretreatment with 6-hydroxydopamine markedly reduced liver norepinephrine content. When 6-hydroxydopamine was used to prevent the sucrose-induced increase in SNS activity, the changes in 86Rb+ uptake, 45Ca2+ efflux, and glucose release that otherwise followed sucrose feeding were not observed. Sucrose feeding did not alter binding of the alpha 1-antagonist [3H]prazosin to liver cell membrane alpha 1-receptors or displacement of [3H]prazosin by the alpha-agonist epinephrine. These observations suggest that sustained increases in SNS activity may have profound effects on liver alpha 1-adrenergic events that occur subsequent to hormone-receptor interaction.

Dispersed mouse pancreatic acini were loaded with the Ca2+-sensitive fluorescence probe Quin 2. Stimulation with carbamylcholine or cholecystokinin-octapeptide (CCK-OP) resulted in a rapid increase in Quin 2 fluorescence, which returned to a lower and sustained plateau level within 2 min of secretagogue stimulation. The magnitude of the initial rise in fluorescence intensity and of amylase secretion were closely related to the concentration of agonist used. Maximal fluorescence changes and amylase secretion were noted with 1 X 10(-5) M carbamylcholine and 1 X 10(-9) M CCK-OP, whereas both responses were half maximal in the presence of 5 X 10(-7) M carbamylcholine and approximately 1 X 10(-10) M CCK-OP. The resting cytosolic free Ca2+ concentration, as measured by the intensity of Quin 2 fluorescence, was calculated to be 1.03 +/- 0.12 X 10(-7) M. Cytosolic free Ca2+ rose to 1.3 +/- 0.3 X 10(-6) M after addition of 1 X 10(-5) M carbamylcholine and 1.25 +/- 0.21 X 10(-6) M following 1 X 10(-9) M CCK-OP. Amylase secretion, but not the Quin 2 fluorescence response, was attenuated at higher secretagogue concentrations. Both secretagogue-induced Quin 2 fluorescence and amylase secretion were inhibited by secretagogue antagonists. Removal of Ca2+ from the extracellular medium resulted in a 48.8 +/- 2.0% reduction of carbamylcholine-induced Quin 2 fluorescence. Following addition of carbamylcholine, CCK-OP was unable to stimulate a second increase in Quin 2 fluorescence without the intervening addition of a cholinergic antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

The earliest changes noted during the evolution of pancreatitis induced by feeding mice a choline-deficient ethionine-supplemented (CDE) diet are an increase in the number of zymogen granules in pancreatic acinar cells and an increase in digestive enzyme content of the pancreas. We have studied the processes of protein and digestive enzyme synthesis and discharge at varying times after institution of the CDE diet, a choline-deficient diet (CD), and a diet containing ethionine but not choline-deficient (E). Both the CDE and E diets increased digestive enzyme content within 12 h of their institution. Both the CDE and E diets reduced the rate of protein and amylase synthesis and caused a marked reduction in the rate of protein and amylase discharge from the pancreas. These changes were greatest and were noted earliest in the CDE diet group. A marked reduction in secretagogue-induced in vivo and in vitro amylase discharge followed ingestion of either the CDE or E diet. These studies indicate that the increased pancreatic content of digestive enzymes noted after ingestion of the CDE and E diets results from an ethionine-induced decrease in the rat of digestive enzyme discharge. This phenomenon is enhanced by simultaneous choline deficiency. Subsequent intrapancreatic activation of zymogens may couple these changes in enzyme content to the development of hemorrhagic pancreatitis.