Effect of long-term CCK blockade on the pancreatic acinar cell renewal in rats with acute pancreatitis

This study determines the effect of 7-day pretreatment with L364,718 (a potent cholecystokinin (CCK) receptor antagonist) on pancreatic cell turnover during the course of acute pancreatitis (AP) induced in the rat by bile-pancreatic duct obstruction (BPDO). Cell cycle distribution and apoptosis were analyzed by flow cytometry using propidium iodide (PI) and Annexin V staining. Besides altering the pancreatic redox status, long-term CCK blockade inhibited the normal proliferation of acinar cells as indicated by the significant increase in G(0)/G(1)-phase cells and the decrease in G(2)/M-cells found in control rats treated with L364,718 for 7 days. A progressive depletion in pancreatic GSH was found from 3 to 24h after BPDO with similar values in L364,718-pretreated and non-treated rats, which led to a maximum peak in malondialdehyde (MDA) levels 6h after BPDO. However, plasma amylase activity and ascites volume indicated higher severity of AP in L364,718-pretreated rats. CCK blockade enhanced the alterations that appear in cell cycle distribution of acinar cells during AP demonstrated by the significantly higher increase in G(0)/G(1)-cells and decrease in S-cells found in L364,718-treated rats 48h after BPDO. Our results indicate that the renewal of acinar cells deleted by apoptosis 48h after BPDO worsens if CCK is blocked before inducing AP.     

Alterations in the glycoconjugates of pancreatic cell membrane induced by acute pancreatitis

The alterations that progressively appear in plasma membrane glycoconjugates of rat pancreatic cells at different stages of acute pancreatitis induced by duct obstruction have been analyzed on individual cells by flow cytometry using the fluoresceinated lectins, wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and Concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine, L-fucose and D-mannose, respectively. Two populations of pancreatic cells were differentiated according to the forward scatter (size), which showed different density of saccharidic terminals located at external positions in the glycoconjugates of the plasma membrane. A significant increase in WGA and TP binding was found 1.5 h after pancreatic obstruction, which could be due to the fusion of zymogen granules with the plasma membrane as suggested by the basolateral exocytosis observed by electron microscopy at this stage. The most external sugar residues of membrane glycoconjugates are removed 12 h after pancreatic duct obstruction as a consequence of an advanced state of pancreatitis. The hydrolytic process reaches greater depths in the membrane 48 h after obstruction. At this stage a significant decrease in WGA, TP and ConA binding was found in all pancreatic cells, indicating the loss of N-acetyl D-glucosamine and/or sialic acid, L-fucose and even D-mannose which is located in the core of the glycan. The results provide information about the progressive degradation induced by acute pancreatitis in pancreatic cell membrane glycoconjugates.     

N-acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis

Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N-acetyl-l-cysteine (NAC) to prevent the generation of OFR and the changes in the cell-cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow-cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF-alpha plasma levels were also measured. The distribution of acinar cells throughout the different cell-cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO-induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper-amylasaemia and maintenance of plasma TNF-alpha levels at values not significantly different from controls were found. NAC administration inhibited progression of cell-cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S-phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO-induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell-cycle phases. Prevention of OFR generation of acinar cells in rats with PDO-induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.     

Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction

The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.     

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.     

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.     

Response of rat exocrine pancreas to high-fat and high-carbohydrate diets

Intake of diets with high fat content is a risk factor for acute pancreatitis and pancreatic cancer. The underlying mechanisms leading to the development of these diseases due to high fat intake are currently unknown. The current study was designed in rats to determine the physiologic and pathological consequences of a highfat diet that contained excess amounts of cottonseed oil or a high-carbohydrate diet that contained high amounts of sucrose on the exocrine pancreas. Rats were maintained on the diets for 4 weeks, and a cannula was inserted into the right jugular vein and one into the pancreatic duct for collection of pancreatic juice. Volume of the pancreatic juice and concentrations of amylase, lipase, and trypsinogen in the pancreatic juice were measured before and after infusions of CCK-8. Results showed that basal and CCK-stimulated pancreatic outputs of volume, amylase and lipase but not trypsinogen, were significantly elevated in intact rats given a high-fat diet when compared with rats given a high-carbohydrate diet. Forty-eight hours later, rats were sacrificed, and parts of the pancreas were removed for isolation of pancreatic acinar cells and for histopathologic studies. Pancreatic acini isolated from rats on a high-fat diet showed significantly lower basal and CCK-stimulated amylase release when compared with those on a high-carbohydrate diet. Histology of the pancreas of rats on a high-carbohydrate diet appeared normal; however, the pancreas of rats on high-fat diet showed significant alterations in exocrine pancreas. These results showed abnormalities in the exocrine pancreas of rats on a high-fat diet, that were not found in rats on a high-carbohydrate diet; further, they support the contention that a high-fat diet has a deleterious effect on the pancreas.     

Pro- and anti-inflammatory response of acinar cells during acute pancreatitis. Effect of N-acetyl cysteine

We investigate the ability of acinar cells to produce tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) at different stages of acute pancreatitis (AP). Since oxidative stress is involved in the inflammatory response, the effect of N-acetyl cysteine (NAC) has also been evaluated. AP was induced in rats by bile-pancreatic duct obstruction (BPDO). NAC (50 mg/kg) was administered 1h before and 1h after BPDO. Acinar cells were incubated for 4 h at 37 degrees C in 5% CO2 atmosphere in absence and presence of 24-h BPDO-PAAF (20%, v/v) as stimulant agent. Acinar production of TNF-alpha and IL-10 was analysed by flow cytometry. Plasma amylase activity and histological studies of the pancreas indicated the severity of AP. PAAF significantly stimulated the acinar production of TNF-alpha and IL-10 in control rats. TNF-alpha production was also significantly stimulated in acinar cells of rats with AP, although a decrease in the pro-inflammatory response was found from 6 h after BPDO onwards. However, acinar cells failed to produce IL-10 from 3 h after BPDO. The protective effect of NAC treatment against oxidative cell damage reduced the pancreatic injury and maintained and enhanced the ability of acinar cells to produce IL-10 at early AP stages. As long as acinar cells were not severely damaged in the course of AP, greater ability to produce cytokines in response to PAAF was found in those with higher forward scatter (R2 cells). We suggest that the capability of acinar cells to maintain an appropriate balance between the production of pro- and anti-inflammatory mediators could contribute to determine the degree of severity of AP.     

Recovery of exocrine pancreas six months following pancreatitis induction with L-arginine in streptozotocin-diabetic rats.

The aim of the present work was to investigate the laboratory and morphologic alterations in the pancreas 6 months after pancreatitis induction with L-arginine (Arg) in normal and streptozotocin (STZ)-diabetic rats. The amylase content of the pancreas was significantly decreased in the Arg-treated groups vs. the control group. No significant changes were observed in the DNA, soluble protein and lipase contents of the pancreas. In the STZ-treated groups, the serum glucose level was significantly elevated, whereas the serum immunoreactive insulin (IRI) level was significantly decreased vs. the control group. In these treated groups, the amylase content of the pancreas was also significantly decreased, but that of trypsinogen was significantly elevated vs. the control group. Histologic sections revealed periductal fibroses, adipose tissue and tubular complexes in the Arg-treated rats, but centroacinar hyperplasia was not observed in these groups. No alterations were observed on histological examination in the diabetic rats vs. normal rats 6 months following pancreatitis induction. In conclusion, a major restitution of the pancreatic enzyme content, but moderate histologic alterations were detected 6 months following pancreatitis induction with Arg. The diabetic state appeared to shift the normal pancreatic enzyme content (decreased amylase and increased trypsinogen) in this long-term study, but not to modify the recovery of the exocrine pancreas 6 months following Arg-induced pancreatitis.     

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Effect of secretin on pancreatic juice proteins in caerulein-induced acute pancreatitis in the rat

Nine hours after the start of treatment with caerulein in rats, an increase in the weight of the pancreas and an increase in serum amylase levels were observed. Likewise, a significant increase in endogenous secretin occurred in rats with acute pancreatitis. A dramatic reduction in the secretion of total protein and amylase was also observed. A partial recovery of this latter effect was achieved after an infusion of high doses of secretin. Under our experimental conditions, the volume of secretion did not vary in caerulein-treated rats wtih respect to controls, either in resting conditions or under secretin stimulation, which indicates that the ductular cells were not significantly affected. Isoelectrofocusing (IEF) and crossed-immunoelectrophoresis (CIE) studies revealed important alterations in the proteins of the pancreatic juice of rats with caerulein-induced acute pancreatitis. Trypsinogen appeared to be particularly affected, showing an increase in the T2 acidic form with an IEP of 4.4 and a decrease in the basic form T3 with an IEP of 8.0, which splits in other forms with a clear antigenic community. A hydrolase was also observed with an IEP of 6.2. In this sense, secretin administration may also be said to induce a significant improvement in established acute pancreatitis, since it tended to normalize the structure and proportion of the proteins secreted.     

Occurrence of pancreatic ductal cell dysplasia in rats fed with a high fat diet and ethanol

The effects of alcohol and diet on acute pancreatitis were studied in 192 male Wistar rats. The animals were fed with standard laboratory food up to three months of age and, after that, were divided into four groups of 48 animals, each group receiving a different diet: standard, fat-rich, protein-rich or carbohydrate-rich. In each diet group, 24 animals obtained 15% (v/v) ethanol in their drinking solution while the other 24 rats had water ad libitum. The diet period lasted for 12 weeks, after which acute experimental pancreatitis was induced under diethyl ether anesthesia by ductal injection of rat bile into the pancreatic ducts. Moderate or severe ductal cell dysplasia developed in three of the 15 survivors in the group fed with a high-fat diet and 15% ethanol in their drinking solution. Mild acute pancreatitis was histologically found in 13 rats and moderate pancreatitis in one rat in this group. One rat did not show any pancreatic parenchymal changes. Two of the rats with ductal cell dysplasia had mild pancreatitis and the pancreas of the third rat was normal in this respect. Dysplastic changes were not found in any other experimental group used in the study. The observation is statistically significant at p less than 0.025 level. The results indicate that alcohol and a high fat diet together might have a carcinogenic effect on pancreatic ductal epithelium in rats.     

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.     

The Novel Cytokine Interleukin-33 Activates Acinar Cell Proinflammatory Pathways and Induces Acute Pancreatic Inflammation in Mice

Background

Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown.

Methods/Results

In duct ligation-induced acute pancreatitis in mice and rats, we found that (a) IL-33 concentration was increased in the pancreas; (b) mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c) plasma histamine and pancreatic substance P concentrations were increased; and (d) pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK) and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α). Also, IL-33 activated ERK in human pancreatic tissue.

Significance

As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation.

Conclusion

IL-33 is induced in acute pancreatitis, activates acinar cell proinflammatory pathways and exacerbates acute pancreatic inflammation.     

通过G418处理离体纯化小鼠胰腺上皮细胞

目的：探索一种能有效去除成纤维细胞,筛选有较强增殖能力上皮细胞的方法。方法：本研究利用成纤维细胞对G418敏感的特性,在成体小鼠胰腺细胞分离后,采用悬浮细胞直接接种到含30μg/mL G418的培养基中,或细胞贴壁生长汇合至50-70%后用30至100μg/mL之间不同浓度G418处理24h、48h或72h两种方案进行上皮细胞的纯化。结果：在直接接种法培养处理中,存活的大部分细胞为成纤维细胞,上皮细胞的生长受到抑制,无法得到纯化的胰腺上皮集落;而在细胞贴壁生长汇合至50-70%后经G418处理,成纤维细胞随着处理浓度的增加死亡率也在逐渐上升,其中50μg/mL G418处理72小时对去除成纤维细胞效果最佳。结论：G418处理能够有效去除成纤维细胞,分离纯化出一群在离体条件下具有强增殖能力、形成大上皮细胞集落的细胞。该分离纯化方法为今后进一步研究成体胰腺干/祖细胞增殖与分化调控机制等问题奠定基础。     

Mouse embryonic stem cells that survive γ-rays exposure maintain pluripotent differentiation potential and genome stability

This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.     

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.     

Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342

Proper synchronization of donor nuclei has been shown to have a major influence on the developmental potential of nuclear transfer embryos. In the present study, a protocol was established to synchronize porcine fetal fibroblasts in the G2 stage of the cell cycle. Cell cycle analyses were performed by flow cytometry. Cells were pre-synchronized by serum deprivation or aphidicoline-treatment; then incubated in medium containing 0.1 microg/ml Hoechst 33342 (H342). The fluorochrome H342 has been shown to be a topoisomerase-inhibitor that can inhibit progression through the cell cycle. There was no significant difference in the percentage of fibroblasts in G2/M whether cells were pre-synchronized in medium supplemented with 0.1% serum for 48h or 0.5% serum for 6 days. Compared with controls, pre-synchronization in early S-phase before incubation in H342 increased the proportion of G2/M fibroblasts; also an increase from 0 and 6 versus 12h culture in complete medium before incubation in H342 resulted in an increased percentage of cells in G2/M at the end of the synchronization period (9.3 and 13.1% versus 33.7%; P<0.001). Neither an increase in the concentration of H342 (0.1-1.0 microg/ml) nor a longer exposure time (12h versus 18h versus 24h) increased the proportion of G2/M fibroblasts. The protocol established in this study arrested porcine fibroblasts reversibly in the G2/M-stage and is therefore suitable to provide synchronized cells for nuclear transfer experiments.     

Minor role of oxidative stress during intermediate phase of acute pancreatitis in rats

Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies have focused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats; taurocholate (3% intraductal infusion) and cerulein (10 microg/kg/h), were used to study markers of oxidative stress: Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde, and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-2'-deoxyguanosine in pancreas. In both models, pancreatic glutathione depleted by 36-46% and pancreatic ascorbic acid depleted by 36-40% (p <.05). In the taurocholate model, plasma glutathione was depleted by 34% (p <.05), but there were no significant changes in plasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and no significant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, or pancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-2'-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitis models.