Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas

Abstract

Background and aim: Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues.

Methods: In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, beta adrenergic stimulation) stress.

Results: Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis.

Conclusions: These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.

Figure 1

Effect of thermal stress and β adrenergic stimulation on pancreatic heat shock protein 70 (HSP70) expression. Rats were exposed to either thermal stress (A) or β adrenergic stimulation (B), as described in the text. At the indicated times, the animals were sacrificed, the pancreata were homogenised, and HSP70 expression was evaluated by western blotting, as described in the methods section. Relative optical densities are expressed as mean (SEM) values of at least three animals at each time point.

Figure 2

Effect of prior thermal stress on the severity of caerulein induced pancreatitis: 12 hours after exposure to thermal stress, pancreatitis was induced by caerulein administration and its severity was quantitated, as described in the text, in control animals, in control animals exposed to hyperthermia, in rats given caerulein, and in rats given caerulein after prior exposure to hyperthermia. Results are expressed as a per cent of the increase noted in non-stressed animals given caerulein (that is, maximal increase). Results represent mean (SEM) values for at least three animals at each time point. *Significantly different (p<0.05) from non-stressed caerulein treated rats. MPO, myeloperoxidase.

Figure 3

Effect of β adrenergic stimulation on the severity of caerulein induced pancreatitis: 24 hours after administration of isoproterenol, pancreatitis was induced by caerulein administration and its severity was quantitated, as described in the text, in control animals, in control animals injected with isoproterenol, in rats given caerulein, and in rats given caerulein after administration of isoproterenol. Results are expressed as a per cent of the increase noted in non-stressed animals given caerulein (that is, maximal increase). Results represent mean (SEM) values for at least three animals at each time point. *Significantly different (p<0.05) from non-stressed caerulein treated rats. MPO, myeloperoxidase.

Figure 4

Effect of hyperthermia and β adrenergic stimulation on caerulein induced intrapancreatic trypsinogen activation: pancreatitis was induced in rats after prior stress induced by hyperthermia (A) or administration of isoproterenol (B), as described in the text. Trypsinogen activation was monitored either by quantitation of trypsin activity or by measurement of trypsinogen activation peptide (TAP), as described in the text. Results are expressed as a per cent of the increase noted in non-stressed animals given caerulein (that is, maximal increase). Results represent mean (SEM) values for at least three animals at each time point. *Significantly different (p<0.05) from non-stressed caerulein treated rats.

Figure 5

Immunolocalisation of HSP70 in the pancreas. Pancreatic tissues obtained from control rats (A), from rats 12 hours after exposure to hyperthermia (B), from rats 24 hours after β adrenergic stimulation (C), from rats 36 hours after prior thermal stress (D), or from rats given caerulein alone (E) or caerulein after prior thermal or non-thermal stress (F, G) were processed for heat shock protein 70 (HSP70) localisation, as described in the text. While there was very little HSP70 expression in control pancreas (A), exposure of rats to hyperthermia (B) or β adrenergic stimulation (C) resulted in significant upregulation of HSP70 expression. HSP70 expression was similar to that seen in control samples 36 hours after prior thermal stress (D). Administration of caerulein to control animals (E) resulted in modest expression of HSP70 with profound vacuolisation and oedema. Prior thermal stress (F) or isoproterenol treatment (G) significantly decreased caerulein induced vacuolisation and oedema. No immunostaining was seen in sections where the primary antibody was omitted (H). Results shown are representative of three different experiments for each experimental condition.

Figure 6

Caerulein stimulated amylase secretion from rat pancreatic acini. Dispersed pancreatic acini were prepared from control rats, rats pre-exposed to hyperthermia, or rats stimulated with isoproterenol and were incubated with various concentrations of caerulein for 30 minutes. Amylase release into the medium was determined as described in the text. Results are expressed as per cent of total amylase content in acini. Results represent mean (SEM) values obtained from three separate experiments.