Acinar inflammatory response to lipid derivatives generated in necrotic fat during acute pancreatitis.

Lipids play a role in acute pancreatitis (AP) progression. We investigate the ability of pancreatic acinar cells to trigger inflammatory response in the presence of lipid compounds generated in necrotic areas of peripancreatic adipose tissue (AT) during AP induced in rats by 5% sodium taurocholate. Lipid composition of AT was analyzed by HPLC-mass spectrometry. Acinar inflammatory response to total lipids as well as to either the free fatty acid (FFA) fraction or their chlorinated products (Cl-FFAs) was evaluated. For this, mRNA expression of chemokine (C-C motif) ligand 2 (CCL2) and P-selectin as well as the activation of MAPKs, NF-κB and STAT-3 were analyzed in pancreatic acini. Myeloperoxidase (MPO) activity, as an inducer of Cl-FFA generation, was also analyzed in AT. MPO activity significantly increased in necrotic (AT-N) induced changes in lipid composition of necrotic fat, such as increase in FFA and phospholipid (PL) content, generation of Cl-FFAs and increases in saturated FFAs and in the poly-:mono-unsaturated FFA ratio. Total lipids from AT-N induced overexpression of CCL2 and P-selectin in pancreatic acini as well as MAPKs phosphorylation and activation of NF-κB and STAT3. FFAs, but not Cl-FFAs, up-regulated CCL2 and P-selectin in acinar cells. We conclude that FFAs are capable of up-regulating inflammatory mediators in pancreatic acini and given that they are highly produced during AP, mainly may contribute to the inflammatory response triggered in acinar cells by fat necrosis. No role is played by Cl-FFAs generated as a result of neutrophil infiltration.

Mechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pancreatitis.

This study aimed to investigate the role of therapeutic dexamethasone (Dex) treatment on the mechanisms underlying chemokine expression during mild and severe acute pancreatitis (AP) experimentally induced in rats. Regardless of the AP severity, Dex (1 mg/kg), administered 1 h after AP, reduced the acinar cell activation of extracellular signal-regulated kinase (ERK) and c-Jun-NH(2)-terminal kinase (JNK) but failed to reduce p38-mitogen-activated protein kinase (MAPK) in severe AP. In both AP models, Dex inhibited the activation of nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT) factors. All of this resulted in pancreatic down-regulation of the chemokines monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC). Lower plasma chemokine levels as well as decreased amylasemia, hematocrit and plasma interleukin-1beta (Il-1beta) levels were found either in mild or severe AP treated with Dex. Pancreatic neutrophil infiltration was attenuated by Dex in mild but not in severe AP. In conclusion, by targeting MAPKs, NF-kappaB and STAT3 pathways, Dex treatment down-regulated the chemokine expression in different cell sources during mild and severe AP, resulting in decreased severity of the disease.

The role of redox status on chemokine expression in acute pancreatitis.

This study focused on the involvement of oxidative stress in the mechanisms mediating chemokine production in different cell sources during mild and severe acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) and 3.5% NaTc, respectively. N-Acetylcysteine (NAC) was used as antioxidant treatment. Pancreatic glutathione depletion, acinar overexpression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC), and activation of p38MAPK, NF-kappaB and STAT3 were found in both AP models. NAC reduced the depletion of glutathione in BPDO- but not in NaTc-induced AP, in which oxidative stress overwhelmed the antioxidant capability of NAC. As a result, inhibition of the acinar chemokine expression and signalling pathways occurs in mild, but not in severe AP. However, MCP-1 and CINC expressions in whole pancreas and plasma chemokine levels were not reduced by NAC, even in BPDO-induced AP, suggesting that in addition to acini, other pancreatic cells produced chemokines by antioxidant resistant mechanisms. The high Il-6 plasma levels found during AP, both in NAC-treated and non-treated rats, pointed out cytokines as activating factors of chemokine expression in non-acinar cells. In conclusion, from early AP oxidant-mediated MAPK, NF-kappaB and STAT3 activation triggers the chemokine expression in acini but not in non-acinar cells.

Application of capillary zone electrophoresis to the characterisation of the human milk protein profile and its evolution throughout lactation.

This work describes the use of capillary zone electrophoresis for the characterisation of human milk proteins. The major proteins were identified following different strategies, such as the treatment with enzymes for selective protein modification. Using this method we studied the proteins in human milk from different donors throughout lactation. Qualitative and quantitative differences in the composition of the individual proteins were observed. The different beta-casein phosphoforms were separated and quantified. The average proportion of the 0P:1P:2P:3P:4P:5P was, approximately, 3:6:9:4:10:2. The evolution of the ratio of the different beta-casein phosphoforms during lactation is reported.

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.

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells.

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.

Cholecystokinin regulates glycoprotein membrane composition of rat pancreatic zymogen granules.

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the alterations in the carbohydrate membrane composition under both chronic CCK stimulation and long-term CCK blockade for 3, 7 and 15 days. By flow cytometry using FITC-WGA--which specifically binds to N-acetylglucosamine and sialic acid--we measured the amount of WGA molecules bound to each individual granule. Parallel studies on pancreatic secretion were also carried out. CCK treatment displayed a differential effect on two zymogen granule subpopulations (Z1 and Z2) identified by flow cytometry on the basis of their light scatter properties: no effects on Z2 zymogen granules were observed in CCK-treated rats, while Z1 granules showed a significant increase in WGA binding at day + 7 which coincides with an increase in protein secretion in response to the hormone. On the contrary, a significant decrease in the amount of WGA receptors was observed in zymogen granule membrane of both the Z1 and Z2 subsets of rats subjected to a long-term CCK blockade. Again, these changes parallel to the reduction observed in protein secretion. Our results suggest that glycoconjugates of zymogen granule membrane involved in CCK-regulated exocytosis contain N-acetylglucosamine and sialic acid residues whose quantities are regulated by CCK.

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.

Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome.

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.

Cross-talk between TLR4 and PPARγ pathways in the arachidonic acid-induced inflammatory response in pancreatic acini.

Arachidonic acid (AA) is generally associated with inflammation in different settings. We assess the molecular mechanisms involved in the inflammatory response exerted by AA on pancreatic acini as an approach to acute pancreatitis (AP). Celecoxib (COX-2 inhibitor), TAK-242 (TLR4 inhibitor) and 15d-PGJ2 (PPARγ agonist) were used to ascertain the signaling pathways. In addition, we examine the effects of TAK-242 and 15d-PGJ2 on AP induced in rats by bile-pancreatic duct obstruction (BPDO). To carry out in vitro studies, acini were isolated from pancreas of control rats. Generation of PGE2 and TXB2, activation of pro-inflammatory pathways (MAPKs, NF-κB, and JAK/STAT3) and overexpression of CCL2 and P-selectin was found in AA-treated acini. In addition, AA up-regulated TLR4 and down-regulated PPARγ expression. Celecoxib prevented the up-regulation of CCL2 and P-selectin but did not show any effect on the AA-mediated changes in TLR4 and PPARγ expression. TAK-242, reduced the generation of AA metabolites and repressed both the cascade of pro-inflammatory events which led to CCL2 and P-selectin overexpression as well as the AA-induced PPARγ down-regulation. Thus, TLR4 acts as upstream activating pro-inflammatory and inhibiting anti-inflammatory pathways. 15d-PGJ2 down-regulated TLR4 expression and hence prevented the synthesis of AA metabolites and the inflammatory response mediated by them. Reciprocal negative cross-talk between TLR4 and PPARγ pathways is evidenced. In vivo experiments showed that TAK-242 and 15d-PGJ2 treatments reduced the inflammatory response in BPDO-induced AP. We conclude that through TLR4-dependent mechanisms, AA up-regulated CCL2 and P-selectin in pancreatic acini, partly mediated by the generation of PGE2 and TXB2, which activated pro-inflammatory pathways, but also directly by down-regulating PPARγ expression with anti-inflammatory activity. In vitro and in vivo studies support the role of TLR4 in AP and the use of TLR4 inhibitors and PPARγ agonists in AP treatment.

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.

Adrenalectomy induces a decrease in the light scatter properties and amylase content of isolated zymogen granules from rat pancreas as analyzed by flow cytometry.

The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90 degrees light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one-R1 (95.45 +/- 1.21%)-containing zymogen granules with a smaller mean size and complexity, and a minor population-R2 (4.45 +/- 0.24%)-the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P < 0.05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P < 0.001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11.25 +/- 0.75% and 15.25 +/- 1.15% (adrenalectomized rats at days +3 and +7 respectively) versus 4.45 +/- 0.24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size.

Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats.

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.

Glucocorticoids regulate L-fucose glycoconjugates in rat pancreatic zymogen granules.

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the influence of glucocorticoid levels on saccharide membrane composition. The following animal groups were used: (1) control rats; (2) rats treated with hydrocortisone (1, 10 and 25 mg/kg/day) for 1, 3 and 8 days; (3) postadrenalectomized rats at days +1, +3 and +8; and (4) adrenalectomized rats receiving hydrocortisone therapy (10 mg/kg/day) for 8 days. By flow cytometry, fluoresceinated (FITC) lectins were used to measure the amount of Concanavalin A (Con A) (specific for D-mannose), wheat germ agglutinin (WGA) (specific for N-acetyl-D-glucosamine) and sialic acids and Tetragonolobus purpureus (TP) (specific for L-fucose) bound to individual zymogen granules from two subpopulations, Z1 and Z2, identified on the basis of their forward and side scatter properties. The molar ratio of the different FITC-lectins revealed significant differences in the glycoconjugate composition of Z1 and Z2 granules, the Z1 granules showing higher ratios of N-acetyl-D-glucosamine:L-fucose and N-acetyl-D-glucosamine:D-mannose, both in control, adrenalectomized and hydrocortisone-treated rats. It was also observed that N-acetyl-D-glucosamine and/or sialic acids were more abundant than L-fucose and D-mannose in the zymogen granule membrane. Z1 and Z2 granules had different glycosylation patterns. Neither adrenalectomy nor hydrocortisone treatments varied the Con A binding to zymogen granules. An increase in WGA binding was only induced by administration of very high doses of hydrocortisone (25 mg/kg/day) for 8 days, an effect not directly related to glucocorticoids. In contrast, a correlation between the FITC-TP labelling and glucocorticoid levels can be established, so that, in a time-dose dependent way, an increase was observed in zymogen granules of rats treated with hydrocortisone while a decreased TP binding was found in adrenalectomized rats-an effect which was reversed with hydrocortisone therapy. Therefore, glucocorticoids exert a direct influence on the saccharide composition of rat pancreatic zymogen granules, regulating the amount of L-fucose glycoconjugates, with Z2 granules more sensitive than Z1 ones.

Hydrocortisone induces an increase of amylase content in individual zymogen granules from rat pancreas.

This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses.

Response of exocrine pancreatic secretion to CCK in adrenalectomized rats.

A study was made of the effect of adrenalectomy over different periods of time (6, 15 and 21 days) on exocrine pancreatic secretion in the rat in basal conditions and under stimulation with CCK. It was observed that adrenalectomy does not alter the rate of pancreatic flow but the response capacity to CCK is depressed. The secretion of total protein and amylase decreases significantly after sixth day, reaching the lowest levels after 21 days. Despite this, after 6 days the adrenalectomized rats showed the same capacity of response to CCK as the non-adrenalectomized animals, while after longer periods of time (15 and 21 days) the response to CCK was inhibited. The fact that the lack of glucocorticoids prevents the maturation of zymogen granules seems to be the main reason why the acinar cells do not increase protein secretion in response to CCK at 15 and 21 days after adrenalectomy. It is concluded that the sensitivity of exocrine pancreas to CCK and the amount of zymogen granules in the acinar cells decrease as a function of the time over which the animals are deprived of glucocorticoids.

Exocrine pancreatic response to CCK in rats after long-term hydrocortisone administration.

The effects of prolonged administration of hydrocortisone (10 mg/kg/day) on exocrine pancreatic secretion were examined, analyzing the quantitative and qualitative variations in secretion at 7, 18 and 30 days of treatment. The weight of the pancreata was found to increase during the period of hydrocortisone treatment. After the 7th day of treatment the hormone significantly decreases basal exocrine pancreatic secretion, maintaining similar values up to the 30th day of treatment. Hydrocortisone enhances the pancreatic response to CCK since the percent increase in acinar secretion under stimulation compared to basal secretion surpassed control levels in all cases. However, the inhibitory effect on secretion shown by hydrocortisone opposes and surpasses the stimulatory effect of the secretagogue, secretion being reduced in the treated animals. Hydrocortisone especially affected the amylase fraction of the juice, to a larger extent inhibiting the isoenzyme A1, with an IEP of 8.5 when the animals were treated over 30 days; thus, a considerable reduction was observed in the amylase secreted both in resting conditions and under stimulation with CCK. There is a possibility that chronic treatment with glucocorticoids may sensitize the acinar cells, alter the composition of the pancreatic juice and inhibit secretion, effects that may involve pancreatic dysfunction.

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.