Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules.

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5-30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.

Lazaroids protect isolated rat pancreatic acinar cells against damage induced by free radicals.

Lazaroids, 21-aminosteroids without gluco- and mineralocorticoid activity, protect against oxidative injury in nervous system cells and may therefore also have a potential for treatment of pancreatitis, where oxidative stress contributes to cell injury. The present study evaluates the protective potential of the lazaroids U-78518F, U-74500A, and U-74389F against damage to isolated pancreatic acinar cells exposed to two models of oxidative stress: (a) a XOD/HX model, consisting of xanthine oxidase, hypoxanthine, and chelated FeCl3; and (b) an ADP/Fe model, consisting of FeSO4 and the reducing agent ADP. Both models caused time-dependent cell injury as assessed by uptake of trypan blue and release of lactate dehydrogenase. Short-term peak production of free radicals in the XOD/HX model--as monitored by the deoxyribose assay--was more injurious to cells than continuous radical generation at lower levels in the ADP/Fe model. In general, lazaroids at 1-10 microM reduced oxidative damage and deoxyribose oxidation in both models. The degree of reduction of cell damage and deoxyribose oxidation depended on the type and concentration of the lazaroid and the model used. Lazaroid concentrations < 0.1 microM were ineffective, and concentrations > 50 microM even accelerated cell injury, although lazaroids still served as scavengers at high concentrations. At least part of the noxious effects of high lazaroid concentrations is due to nonspecific membrane damage because these concentrations caused cell injury also in the absence of oxidative stress. The limited range of protective concentrations has to be observed in further in vivo studies. Interestingly, acinar cells in the absence of lazaroids also reduced radical-induced deoxyribose degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypergastrinemia and blockade of gastrin-receptors on pancreatic growth in the mouse.

BACKGROUND/AIM: The present study evaluates whether endogenous gastrin regulates pancreatic growth in the mouse. MATERIALS AND METHODS: Male NMRI mice weighing 12-15 g were assigned to six groups (10 mice/group) which were treated with different combinations of 0.9% NaCl, omeprazole, a CCK-A antagonist, a CCK-B antagonist, loxiglumide, and L365, 260 for 10 days each according to different protocols. RESULTS: Omeprazole caused a marked, 10-fold increase in serum gastrin which was not affected by the gastrin antagonist, but markedly reduced by the CCK-A antagonist. The marked increase in endogenous gastrin caused by omeprazole did not promote pancreatic growth in any way. Similarly, the gastrin antagonist did not inhibit pancreatic growth. In contrast, the CCK-A antagonist significantly decreased pancreatic weight and protein content. CONCLUSIONS: The present results strongly suggest that endogenous gastrin--in contrast to CCK--does not regulate pancreatic growth in the mouse. The inhibitory effect of loxiglumide on omeprazole-induced increase in serum gastrin might be explained by recent findings which showed that CCK-A antagonists can stimulate gastric acid secretion probably due to a reduction of the inhibitory effect of basal CCK on the D-cell and its somatostatin release. Probably such a slight stimulation of gastric acid secretion caused by the CCK-A antagonist might reduce the gastrin increase caused by omeprazole's abolishment of acid secretion.

Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells.

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.

Murine systemic autoimmune disease induced by mercuric chloride (HgCl2): Hg-specific helper T-cells react to antigen stored in macrophages.

The adoptive transfer popliteal lymph node assay (PLNA) was used to demonstrate Hg-specific T-cell responses of mice that were continuously treated with HgCl2 by a regimen known to induce a systemic autoimmune disease in H-2s (murine histocompatibility complex, haplotype s) mice, but not H-2d mice. We found that spleen cells of B10.S and A.SW donors (both H-2s) responded anamnestically to HgCl2 by inducing a significant increase in cellularity in the draining PLN of the recipient: In contrast, spleen cells of HgCl2-treated DBA/2 (H-2d) donors failed to induce an increase in PLN cellularity, and spleen cells of B10.D2/n (H-2d) donors induced no changes or even diminished PLN cellularity upon re-encounter with HgCl2. Kinetic studies showed that spleen cells of B10.S donors were stimulatory from day 3 until day 14 of donor HgCl2 treatment and, when purified splenic T-cells were tested, still on day 28, the last point in time tested. The Hg-specific T-cells prepared from HgCl2-treated B10.S mice not only induced an increased cellularity, but also B-cell activation to antibody secretion in the draining PLN of the recipient. Moreover, the Hg-specific donor T-cells transferred could specifically be restimulated by killed peritoneal cells obtained from the same donors or from syngeneic donors previously treated with HgCl2. Interestingly, when killed peritoneal cells were injected as antigen the amount of Hg required for T-cell restimulation was only 1/40 of that required when free HgCl2 was used. Taken together, these results show that an HgCl2 treatment schedule designed to induce systemic autoimmune disease primes Hg-specific T-helper (Th) cells and generates immunogenic material in peritoneal cells to which the T-cells react. The possible contribution to the pathogenesis of HgCl2-induced auto-immune disease of these Hg-specific T-cells and the autoreactive T-cells reported in the literature is discussed.