Heat shock protein 70 prevents secretagogue-induced cell injury in the pancreas by preventing intracellular trypsinogen activation

Abstract

Rodents given a supramaximally stimulating dose of cholecystokinin or its analogue cerulein develop acute pancreatitis with acinar cell injury, pancreatic inflammation, and intrapancreatic digestive enzyme (i.e., trypsinogen) activation. Prior thermal stress is associated with heat shock protein 70 (HSP70) expression and protection against cerulein-induced pancreatitis. However, thermal stress can also induce expression of other HSPs. The current studies were performed using an in vitro system to determine whether HSP70 can actually mediate protection against pancreatitis and, if so, to define the mechanism underlying that protection. We show that in vitro exposure of freshly prepared rat pancreas fragments to a supramaximally stimulating dose of cerulein results in changes similar to those noted in cerulein-induced pancreatitis, i.e., intra-acinar cell trypsinogen activation and acinar cell injury. Short-term culture of the fragments results in HSP70 expression and loss of the pancreatitis-like changes noted after addition of cerulein. The culture-induced enhanced HSP70 expression can be prevented by addition of either the flavonoid antioxidant quercetin or an antisense oligonucleotide to HSP70. Under these latter conditions, addition of a supramaximally stimulating concentration of cerulein results in trypsinogen activation and acinar cell injury. These findings indicate that the protection against cerulein-induced pancreatitis that follows culture-induced (and possibly thermal) stress is mediated by HSP70. They suggest that the HSP acts by preventing trypsinogen activation within acinar cells.

Figure 1

Effect of duration of culture and incubation with oligonucleotides on the viability of pancreas fragments as assessed by MTT assay and amylase secretion. (a) MTT assay. S I and S II represent sense oligonucleotides, AS I and AS II represent antisense nucleotides, and Q represents quercetin as described in the text. Values are mean ± SEM obtained from at least three separate experiments and expressed as percent of control (0 hours) values. ^AP < 0.05 compared with freshly prepared (0 hours) samples. (b) Amylase release into the medium in response to 0.1 nM cerulein was quantitated as described in the text. Values are mean ± SEM for at least three separate experiments, each consisting of three or more wells in each group. ^AP < 0.05 when cerulein-stimulated amylase secretion is compared with basal values at each time point indicated.

Figure 2

Effect of duration of culture and incubation with oligonucleotides on dose response for cerulein-stimulated amylase secretion from pancreas fragments. Amylase release into the medium was quantitated as described in the text. Values are mean ± SEM for at least three separate experiments at each cerulein concentration in each group. Cer, cerulein.

Figure 3

Effect of duration of culture on the ability of supramaximal cerulein to cause trypsinogen activation (as assessed by measuring trypsin activity and TAP levels) and cell injury (LDH leakage) in pancreas fragments. Values are mean ± SEM obtained from at least three independent experiments and expressed as percent of maximal response obtained when freshly prepared pancreas fragments are stimulated with supramaximal dose of cerulein. ^AP < 0.05 when values obtained from cerulein-stimulated freshly prepared fragments are compared with the basal values.

Figure 4

Effect of duration of culture on HSP70 expression in pancreas fragments, as assessed by Western blotting. Relative optical densities were quantitated as described in the text and are expressed as mean ± SEM for at least three independent experiments in each group. ^AP < 0.05 compared with control (0 hour) value.

Figure 5

Effect of incubation of pancreas fragments with quercetin or antisense/sense HSP70 oligonucleotides on HSP expression. (a) C₀, freshly prepared pancreas fragments; C₁₂, pancreas fragments after 12 hours of culture; Q, pancreas fragments after 12 hours of culture with quercetin (50 μM). HSP70 protein expression was evaluated by Western blotting, and relative optical densities are expressed as mean ± SEM for at least three separate experiments in each group. ^AP < 0.01 when quercetin-incubated fragments are compared with untreated 12-hour control fragments. (b) C₀, freshly prepared pancreas fragments; C₁₂, pancreas fragments after 12 hours of culture; S I, pancreas fragments incubated with sense oligonucleotide S I (1 μM) for 12 hours; AS I, pancreas fragments incubated with 14-mer antisense oligonucleotide AS I (1 μM) for 12 hours; S II, pancreas fragments incubated with sense oligonucleotide S II (1 μM) for 12 hours; AS II, pancreas fragments incubated with 18-mer antisense oligonucleotide AS II (1 μM) for 12 hours. Expression of indicated HSPs was assessed by Western blotting. Relative optical densities of HSP70 bands are expressed as mean ± SEM for at least three separate experiments in each group. ^AP < 0.01 when antisense oligonucleotide-incubated fragments are compared with untreated 12-hour control fragments.

Figure 6

Effect of incubation of pancreas fragments with quercetin (50 μM) on trypsinogen activation. Trypsin activity in the homogenates was measured according to the method described in the text. Values are mean ± SEM obtained from at least three separate experiments. ^AP < 0.05 when cerulein-treated freshly prepared fragment values are compared with basal values. ^BP < 0.05 when quercetin-incubated cerulein-stimulated fragments are compared with cerulein-stimulated 12-hour control fragments.

Figure 7

Effect of incubation of pancreas fragments with antisense/sense HSP70 oligonucleotides (1 μM) on trypsinogen activation and cell injury as assessed by LDH release into the medium. Trypsin activity (a), TAP levels (b), and LDH leakage (c) were measured as described in text. Values are mean ± SEM obtained from at least three independent experiments and are expressed as percent of maximal response to cerulein stimulation in freshly prepared pancreas fragments. ^AP < 0.05 when cerulein-treated freshly prepared fragment values are compared with the basal values. ^BP < 0.05 when antisense oligonucleotide-incubated cerulein-stimulated fragments are compared with cerulein-stimulated 12-hour control fragments.

Figure 8

Light microscopic colocalization of TAP and cathepsin B in rat pancreas fragments, 30 minutes after stimulation with 0.1 μM cerulein. (a) Freshly prepared fragments. (b) Fragments after 12 hours of culture. (c) Fragments incubated with AS I nucleotide for 12 hours. Double-labeling fluorescence microscopy of paraffin-embedded sections was carried out with TAP antibody/FITC-conjugated anti-rabbit IgG and anti-cathepsin B/rhodamine-conjugated anti-goat IgG. The areas positive for both TAP and cathepsin B appear as yellow dots. Arrowheads in a and c indicate the areas positive for both cathepsin B and TAP. The images shown are representative of several examined in each group in three independent experiments.