Changes in zymogen granule membrane and pancreatic juice after supramaximal stimulation with caerulein in the rat

Summary Pancreatic exocrine secretion was stimulated supramaximally with an intravenous infusion of 10 μg/kg of body weight of caerulein for 10 min in four rats. Two animals were killed immediately, and two animals 120 min after the cessation of the caerulein infusion. In specimens fixed immediately after the caerulein infusion, there were irregular electron lucent structures that resembled zymogen granules and were associated with bristle-coated membranes, coated pits and coated vesicles. Two hours after the caerulein infusion there were large vacuoles that contained amorphous or membranous material and cell organelles in acinar cells. The HPLC-analysis of pancreatic juice revealed two new peaks after caerulein injection. It was concluded that supramaximal caerulein stimulation prevents normal maturation and discharge of zymogen granules leading to altered membrane recycling of secretory granules in acinar cells and to the appearance of abnormal secretory products in pancreatic juice. The novel ultrastructural findings of this study, the bristle-coated membranes and the coated pits in the membrane of dilated zymogen granules, may be related to the exceedingly high dose of caerulein administered.     

Processing of the major pancreatic zymogen granule membrane protein, GP2

INTRODUCTION: The pancreatic exocrine secretory granule, the zymogen granule, releases digestive enzymes into the intestine. GP2 is the most abundant zymogen granule membrane protein. Coincident with exocrine secretion, GP2 is released from the membrane and secreted into the pancreatic duct. AIM: To characterize changes in the structure of GP2 as it progresses through the secretory pathway. METHODOLOGY: Polarized MDCK cells that express the rat GP2 gene were used to examine the sequential processing of the polypeptide backbone. RESULTS: Within the cell, GP2 is initially proteolytically processed from a 55- to a 53-kd form at or before the trans-Golgi network. The protein is then processed to a 51-kd form, which is found on the apical plasma membrane and in secretions. Similar processing was also observed in primary rat pancreatic cultures and in MDCK cells that express human GP2. The amino-terminal sequence of human GP2 derived from pancreatic secretions was determined for two human patients and began at Gly39, revealing a potential processing site. CONCLUSIONS: In contrast to other digestive enzymes secreted by the pancreas that are activated by proteolysis in the intestine, GP2 undergoes sequential intracellular cleavage. Alterations in GP2 structure by proteolysis may regulate GP2 function at specific sites within the pancreatic cell.     

Histologic and biochemical alterations in experimental acute pancreatitis induced by supramaximal caerulein stimulation

We studied the histologic and biochemical alterations in experimental acute pancreatitis induced by supramaximal caerulein stimulation in rats. All rats received 4 subcutaneous injections of various doses of caerulein (5-50 micrograms/kg body weight) at hourly intervals over 3 h, and 9 h after the first injection all animals were killed. Subcutaneous injections of 20 micrograms/kg body weight of caerulein induced a significant increase in serum amylase activity and histologic evidence of acute interstitial pancreatitis similar to those observed with the 50 micrograms/kg body weight dosage of caerulein. Therefore, a total of 4 subcutaneous injections of 20 micrograms/kg body weight of caerulein was chosen to study the time-course of structural and biochemical alterations in caerulein-induced acute pancreatitis. Serum amylase activity reached a maximal value of 10-fold increase over the basal values at 6 h, and then decreased gradually to normal values at 18 h after the first injection. Remarkable interstitial edema and cytoplasmic vacuoles in acinar cells were the earliest histologic alterations. Cellular infiltration was prominent at 9-12 h after the first injection. Although these histologic changes almost completely disappeared after 24 h, the reduction in the number of zymogen granules was still detectable by electron microscopic examination even after 7 days. DNA content in the pancreas showed no significant changes following the induction of acute pancreatitis, whereas a moderate to marked reduction in enzyme content persisted after 7 days. Within 14 days after the initiation of the injections, both structural and biochemical changes had completely disappeared.     

Histologic and biochemical alterations in experimental acute pancreatitis induced by supramaximal caerulein stimulation

Summary We studied the histologic and biochemical alterations in experimental acute pancreatitis induced by supramaximal caerulein stimulation in rats. All rats received 4 subcutaneous injections of various doses of caerulein (5–50 μg/kg body weight) at hourly intervals over 3 h, and 9 h after the first injection all animals were killed. Subcutaneous injections of 20 μg/kg body weight of caerulein induced a significant increase in serum amylase activity and histologic evidence of acute interstitial pancreatitis similar to those observed with the 50 μg/kg body weight dosage of caerulein. Therefore, a total of 4 subcutaneous injections of 20 μg/kg body weight of caerulein was chosen to study the time-course of structural and biochemical alterations in caerulein-induced acute pancreatitis. Serum amylase activity reached a maximal value of 10-fold increase over the basal values at 6 h, and then decreased gradually to normal values at 18 h after the first injection. Remarkable interstitial edema and cytoplasmic vacuoles in acinar cells were the earliest histologic alterations. Cellular infiltration was prominent at 9–12 h after the first injection. Although these histologic changes almost completely disappeared after 24 h, the reduction in the number of zymogen granules was still detectable by electron microscopic examination even after 7 days. DNA content in the pancreas showed no significant changes following the induction of acute pancreatitis, whereas a moderate to marked reduction in enzyme content persisted after 7 days. Within 14 days after the initiation of the injections, both structural and biochemical changes had completely disappeared. *** DIRECT SUPPORT *** A00DX035 00005     

Exocytosis occurs at the lateral plasma membrane of the pancreatic acinar cell during supramaximal secretagogue stimulation

In vitro and in vivo studies indicate that the secretory response to both caerulein and carbamylcholine stimulation is biphasic. Over the range of submaximal to maximal concentrations of secretagogues, discharge of exocrine proteins in vitro into the incubation medium and in vivo into the pancreatic duct increased and morphologic analysis indicated that exocytosis of zymogen granules occurred exclusively at the luminal membrane. Under in vivo conditions, supramaximal stimulation with caerulein or carbamylcholine resulted in a dose-dependent decrease in amylase release into the pancreatic duct and increase in the appearance of amylase in the blood circulation. Under in vitro or in vivo conditions, supramaximal secretagogue stimulation resulted in marked inhibition of exocytotic activity at the luminal plasma membrane, the appearance of intergranule contacts and fusions within the cytoplasm, and the appearance of exocytotic activity at the lateral plasma membrane. Lateral exocytotic images were observed with individual and fused zymogen granules and autophagic vacuoles. This redirection in the final step of the secretory pathway provides in part the biological basis for the increased appearance of pancreatic (pro)enzymes in the interstitial fluid and serum during supramaximal secretagogue stimulation.     

Patch clamped single pancreatic zymogen granules: direct measurements of ion channel activities at the granule membrane.

BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.     

The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

Background  

GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria.     

Acetylcholine-induced zymogen granule exocytosis: comparison between acini and single pancreatic acinar cells

INTRODUCTION: Numerous studies have been carried out on the agonist-evoked calcium responses of single pancreatic acinar cells; however, several reports have shown that dissociation of the exocrine pancreas into predominantly single cells has an adverse effect on agonist-evoked amylase secretion. AIMS AND METHODOLOGY: To determine whether single acinar cells behave in an anomalous manner compared with cells within an intact acinus, we measured exocytosis in both single acinar cells and acini (2-5 cells) present in the same preparation. Exocytosis of individual zymogen granules was quantified in real-time by using the technique of continuous time-differential analysis of brightfield digital images. RESULTS: Basal rates of exocytosis were low in both single cells and intact acini. Application of 10 microM acetylcholine for 6 minutes stimulated a biphasic secretory response in acinar cells. Additionally, we found that exocytotic events occur repetitively in specific locations within the apical domain; i.e., there are exocytotic "hot spots." There were no statistically significant differences between the exocytotic rates, nor were there any differences in the characteristics of the exocytotic hot spots of single cells compared with those of acini. CONCLUSION: We conclude that time-differential analysis of brightfield images appears to be a useful tool for the investigation of the role of gap junctions in zymogen granule exocytosis and that single acinar cells provide a reasonable model for studies of acinar cell signaling and secretion.     

Changes in serum pancreatic enzymes after hormonal stimulation in chronic pancreatitis

Eighteen patients with chronic pancreatitis and 12 healthy controls were subjected to hormonal stimulation by continuous secretin plus cerulein intravenous infusion or a rapid injection of secretin. In both tests total serum amylase, lipase, and TLI (trypsin-like immunoreactive substances) levels were measured. Continuous intravenous infusion does not bring about changes in the serum levels of the enzymes studied; rapid injection of secretin, however, induces changes in the serum levels of TLI and lipase (but not amylase) which makes it possible to distinguish patients with chronic pancreatitis in its early stages from advanced chronic pancreatitis but is of doubtful value in distinguishing healthy subjects from those suffering with chronic pancreatitis.     

Role of endogenous platelet-activating factor in the regulation of pancreatic blood flow during caerulein stimulation

The role of endogenous platelet-activating factor (PAF) in the control of pancreatic blood flow during caerulein stimulation was investigated. Pancreatic blood flow in anesthetized rats was measured continuously by laser Doppler flowmetry for 2h during the intravenous infusion of caerulein (0.25 μg/kg per h). Pancreatic blood flow showed a gradual, consistent, and significant increase, reaching 114.2±2.3% of the basal value after 120 min. Changes in pancreatic blood flow induced by caerulein were completely inhibited by a cholecystokinin (CCK) antagonist (loxiglumide, 5 mg/kg per h, i.v.) and by a specific PAF antagonist (CV-6209, 1 mg/kg, i.v.-bolus). Systemic blood pressure remained stable in all groups. These results suggest an important role of endogenously yielded PAF in regulating pancreatic blood flow during caerulein stimulation to the pancreas.     

Chronic ethanol consumption increases the fragility of rat pancreatic zymogen granules.

Intracellular activation of pancreatic digestive enzymes by lysosomal hydrolases is thought to be an early event in the pathogenesis of pancreatic injury. As ethanol excess is an important association of pancreatitis, experimental work has been directed towards exploring possible mechanisms whereby ethanol may facilitate contact between inactive digestive enzyme precursors and lysosomal enzymes. The aim of this study was to find out if chronic ethanol administration increases the fragility of rat pancreatic zymogen granules. Sixteen male Sprague-Dawley rats were pair fed ethanol and control liquid diets for four weeks. Zymogen granule fragility was then assessed in pancreatic homogenate by determination of (a) latency and (b) per cent supernatant enzyme after sedimentation of zymogen granules. Amylase was used as a zymogen granule marker enzyme. Latency was significantly reduced in pancreatic homogenates of ethanol fed animals suggesting increased zymogen granule fragility. In support of this finding, there was a trend towards increased supernatant enzyme after ethanol feeding. In conclusion, administration of ethanol increases the fragility of pancreatic zymogen granules in the absence of morphological evidence of pancreatic injury. It is proposed that zymogen granule fragility may play an early part in the pathogenesis of alcoholic pancreatitis by permitting contact between digestive and lysosomal enzymes.     

Haemodynamic and exocrine effects of caerulein at submaximal and supramaximal levels on the rat pancreas: role of cholecystokinin receptor subtypes.

BACKGROUND: We have investigated the involvement of cholecystokinin (CCK) receptor subtypes in haemodynamic changes in the pancreas of anaesthetized rats during submaximal and supramaximal stimulation with the CCK analogue, caerulein. METHODS: For submaximal stimulation, caerulein (0.4 nmol/kg/h) was infused intravenously, while acute pancreatitis was induced by intraperitoneal injections of high doses of caerulein (3 x 25 nmol/kg). Pancreatic blood flow was measured by hydrogen clearance. RESULTS: Low caerulein doses increased pancreatic blood flow by 26 +/- 8% and vascular conductance by 24 +/- 4%. This effect was mimicked by the CCK2 agonist gastrin-17. All effects were abolished by a CCK2 antagonist while a CCK1 antagonist remained inactive. Conversely, amylase output by caerulein was abolished by CCK1 receptor blockade, but not by inhibition of CCK2 receptors. During caerulein-induced pancreatitis, vascular conductance increased by 109 +/- 26% and remained elevated throughout the experiment; vascular flow initially increased by 62 +/- 27% and then returned to baseline. The vascular effects were prevented by a CCK2 receptor antagonist, while the induction of pancreatitis was due to CCK1 receptor stimulation. CONCLUSIONS: Caerulein increases pancreatic vascular flow via activation of CCK2 receptors. This effect occurs both at submaximal and at supramaximal levels of exocrine stimulation.     

Protein profiles in rabbit pancreatic juice analyzed by HPLC after stimulation of secretion by secretin and cerulein

This study analyzed the secretory pattern of pancreatic proteins released from the rabbit pancreas after acute stimulation of secretion by the cholecystokinin analog cerulein. To facilitate this, a new analytical approach utilizing high performance liquid chromatography (HPLC) was considered. Secretin (0.1 CU/kg x h) was intravenously infused in anaesthetized rabbits in combination with cerulein (0.05, 0.2 or 0.05 followed by 0.2 ug/kg x h) over 3 hours. Pancreatic juice was collected from the main pancreatic duct. The release of protein, amylase, trypsin and chymotrypsin was measured by conventional photometric methods, and the protein profiles were analyzed by reversed phase HPLC. Separation of pancreatic juice proteins by HPLC (Nucleosil 300-7 RP column; injection of 50 ul aliquots of samples normalized to 10 mg/ml protein concentration) resulted in a resolution of up to 16 peaks. Peaks representing amylase, prolipase, prophospholipase A2, procarboxypeptidases, chymotrypsinogen, trypsinogen, and glycoproteins were identified with some certainty by SDS-gel electrophoresis. Secretin infusion produced a small and short lasting rise in total protein secretion but lead to a persistent increase of fluid flow. The release of enzymes followed a mainly parallel pattern according to the photometric measurements. The resolution of the whole profile of pancreatic juice proteins by HPLC demonstrated only minor variations without a consistent or increasing tendency towards a preferential release of individual enzymes. Since even microheterogenities in the samples became apparent after HPLC, this approach would be sensitive enough to mirror effects like nonparallel release of enzymes.     

Alcohol and zymogen activation in the pancreatic acinar cell

SUMMARY: Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiologic concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and pancreatitis. Supraphysiologic concentrations of CCK also cause zymogen activation in isolated pancreatic acini. This activation first occurs in a nonzymogen granule compartment that contains lysosomal markers. A low pH environment may also be needed for activation. To examine the ability of alcohols to sensitize the acinar cell to CCK, the conversion of zymogens to active enzymes in isolated acini was assayed. Alcohols, including 35 mmol/L ethanol, sensitized acini to CCK induced activation. The sensitization increased with chain length and was less in branched compared with unbranched alcohols. The relationship of alcohol's structure to sensitization may be related to the mechanism of sensitization.