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)
Infusion of a supramaximal dose of caerulein results in acute interstitial pancreatitis in rats. We report studies of in vivo pancreatic acinar cell function during the initial 3.5 h of supramaximal stimulation with caerulein (5 micrograms X kg-1 X h-1). Amino acid [( 3H]phenylalanine) uptake was not altered, and there was no change in the rate or extent of protein synthesis or in intracellular transport of in vivo pulse-labeled proteins from microsome to zymogen granule-enriched fractions. However, the discharge of labeled protein was markedly inhibited. Radioautographic studies indicated that the pulse-labeled proteins retained in the gland were not located extracellularly but had accumulated within acinar cells, with a preferential distribution at the cell apex (presumably in zymogen granules) and in large vacuoles that form within the cell during hyperstimulation. Supramaximal stimulation with caerulein also caused increasing amounts of amylase and labeled proteins to be recovered in the postmicrosomal fraction. These findings suggest that supramaximal stimulation causes digestive enzymes to become localized in organelles that are fragile and subject to disruption during tissue homogenization. These organelles may be the vacuoles noted in morphological studies and believed to represent immature condensing vacuoles and/or crinophagic vacuoles.
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.
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