Enzyme load in pancreatic acinar cells is increased in the early stages of acute pancreatitis induced by duct obstruction in rats

Abstract

Trypsinogen and amylase content has been analysed by flow cytometry in individual pancreatic cells from rats with acute pancreatitis induced by pancreatic duct obstruction, from the earliest stages to 48 h after obstruction. Parallel morphological studies of the pancreas by electron microscopy and analysis of various parameters for the diagnosis of pancreatitis will allow research into the possible relationship between intracellular enzyme load and the severity of pancreatitis. Progressive increases in amylase activity in ascites and plasma, the volume of ascites, haematocrit, vacuolization, oedema and macrophage infiltration were observed between 1.5 h and 12 h after duct obstruction. A progressive increase in enzyme content was also observed in individual acinar cells at this stage. Interestingly, the larger increase was for trypsinogen, so that the trypsinogen/amylase ratio was significantly increased in all acinar cells by 12 h after duct obstruction. This represents a risk factor for the development of pancreatitis. Sections of pancreas taken from rats that had duct obstruction for 48 h showed massive dilatation and disorganization of the endoplasmic reticulum, focal apoptosis and necrosis. These severe alterations would affect enzyme synthesis, as reflected by the significant decrease in the intracellular enzyme load observed at this stage. However, not all acinar cells were affected equally by the damage induced by pancreatitis: R(1) cells appeared to be more sensitive than R(2) cells. In conclusion, intracellular accumulation of digestive enzymes occurs at early stages of pancreatitis, and this effect is proportionally greater for trypsinogen, a finding that could explain the degree of severity achieved in the course of pancreatitis.

Cholecystokinin blockade triggers earlier and enhanced intra-acinar oxygen free radical generation in acute pancreatitis induced by pancreatic duct obstruction in rats

“…Since we studied the time course of OFR generation in acinar cells at different stages of AP, we were able to observe differences in the oxidative response developed by the two cell populations that could be differentiated by flow cytometry on the basis of light scatter properties [26,44]. Whereas no differences between R1 and R2 cells were found in untreated rats with PDO at any stage of AP, asynchronous generation of OFR could be observed when L364,718 was administered.…”

“…After this isolation procedure, mostly single cells [(25-30) × 10 6 /pancreas] were obtained, as well as some small clusters consisting of up to five cells. The labelling of isolated cells with specific antisera against digestive enzymes assessed the purity of acinar cells as 99 % [26]. Cell viability monitored with Trypan Blue was always greater than 95 %, and this was not significantly decreased by H 2 O 2 , at least during the duration of the experiments for verifying stability in DHR dye.…”

“…Whereas no differences between R1 and R2 cells were found in untreated rats with PDO at any stage of AP, asynchronous generation of OFR could be observed when L364,718 was administered. With regard to enzyme overload [26] and disturbances in Ca 2+ homoeostasis [35,44], previous studies have concluded that R1 cells appear to be the most sensitive to damage produced by PDO. This notion is supported by the present results, indicating that the detrimental effects of CCK blockade on the course of AP are apparent earlier in R1 than in R2 cells.…”

Asynchronous impairment of calcium homoeostasis in different acinar cells after pancreatic duct obstruction in rat

“…Leucocytes infiltrating the pancreas were excluded on the basis of their unique high-light scatter properties [forward or low light scatter (FSC)\side or 90m light scatter (SSC)]. The labelling of cells with specific antisera against digestive enzymes [17] was used to assess the purity of cells in the samples. Gated cells (99 % ; cells selected by their FSC and SSC properties, as well as their fluorescence intensity) were stained positively for pancreatic enzymes and were considered acinar cells.…”

“…Two different subsets of acinar cells have been reported by us in previous studies [17][18][19] that were termed R1 and R2. They can be clearly differentiated on the basis of light scatter properties ; R1 cells display lower forward and side scatter than R2 cells.…”