Co-Variation of Serum Osteoprotegerin and Pigment-Epithelial Derived Factor as Biomarker of Pancreatic Cancer.

Pancreatic cancer is one the most lethal cancers. Currently, there are reliable predictive markers to assess cancer development. Widely used CA19-9 molecular marker has been less effective in the diagnosis of early stages of cancer. Objective: To study if the soluble Osteoprotegerin (OPG) and pigment-epithelial derived factor (PEDF) levels in serum will be an indicator of cancer progression. Methods: Soluble OPG and PEDF were measured from human pancreatic cancer patients by ELISA. Results: We show that while OPG has been less predictive features, PEDF is more sensitive than CA19-9 in cancer detection. More importantly, PEDF and CA19-9 as combined markers showed higher sensitivity in stratifying early stages of pancreatic cancer. Conclusion: Results from the pilot studies suggest that PEDF is useful biomarker for pancreatic cancer.

Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca(2+) responses in pancreatic acinar cells.

Bile acids are known to induce Ca(2+) signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca(2+)](i) responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca(2+)](i) responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K gamma-isoform also decreased [Ca(2+)](i) responses to bile acids. Depletion of CCK-sensitive intracellular Ca(2+) pools or application of caffeine inhibited bile acid-induced [Ca(2+)](i) signals, indicating that bile acids release Ca(2+) from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca(2+) in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca(2+) reloading into the ER. Bile acids inhibited Ca(2+) reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP(3), facilitate bile acid-induced [Ca(2+)](i) responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca(2+) reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca(2+)](i) increases and trypsinogen activation mediate key pathological processes in this disorder.

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase.

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.

Localized pancreatic NF-kappaB activation and inflammatory response in taurocholate-induced pancreatitis.

Transcription factor nuclear factor-kappaB (NF-kappaB) is activated in cerulein pancreatitis and mediates cytokine expression. The role of transcription factor activation in other models of pancreatitis has not been established. Here we report upregulation of NF-kappaB and inflammatory molecules, and their correlation with local pancreatic injury, in a model of severe pancreatitis. Rats received intraductal infusion of taurocholate or saline, and the pancreatic head and tail were analyzed separately. NF-kappaB and activator protein-1 (AP-1) activation were assessed by gel shift assay, and mRNA expression of interleukin-6, tumor necrosis factor-alpha, KC, monocyte chemoattractant protein-1, and inducible nitric oxide synthase was assessed by semiquantitative RT-PCR. Morphological damage and trypsin activation were much greater in the pancreatic head than tail, in parallel with a stronger activation of NF-kappaB and cytokine mRNA. Saline infusion mildly affected these parameters. AP-1 was strongly activated in both pancreatic segments after either taurocholate or saline infusion. NF-kappaB inhibition with N-acetylcysteine ameliorated the local inflammatory response. Correlation between localized NF-kappaB activation, cytokine upregulation, and tissue damage suggests a key role for NF-kappaB in the development of the inflammatory response of acute pancreatitis.

Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase.

Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-kappaB inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-kappaB in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.

Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells.

Neutrophil infiltration into the pancreas is a key event in pancreatitis. Here we show that intercellular adhesion molecule-1 (ICAM-1), which regulates neutrophil adhesion, is present on rat pancreatic acinar cells, is upregulated by a hormone (cerulein) and mediates direct binding of neutrophils to acinar cells. ICAM-1 was upregulated in pancreas of rats with experimental pancreatitis induced by supramaximal doses of cerulein. Furthermore, cerulein time and dose dependently stimulated expression of ICAM-1 mRNA and protein in isolated pancreatic acinar cells. Inhibitory analysis showed that activation of transcription factor nuclear factor-kappaB (NF-kappaB) was involved in ICAM-1 upregulation by cerulein, but NF-kappaB did not mediate basal expression of ICAM-1 mRNA in acinar cells. With an adhesion assay, we found that neutrophils bind to isolated pancreatic acinar cells and that cerulein upregulates the extent of adhesion. Neutralizing ICAM-1 antibody blocked neutrophil binding to both control and cerulein-stimulated acinar cells, suggesting ICAM-1 involvement in this adhesion. Thus the acinar cell is capable of targeting neutrophils to its surface, a process that may be important for inflammatory and cell death responses in pancreatitis and other pancreatic disorders.

Multiple isoforms of the ryanodine receptor are expressed in rat pancreatic acinar cells.

Regulation of cytosolic Ca(2+) is important for a variety of cell functions. The ryanodine receptor (RyR) is a Ca(2+) channel that conducts Ca(2+) from internal pools to the cytoplasm. To demonstrate the presence of the RyR in the pancreatic acinar cell, we performed reverse transcriptase (RT)-PCR, Western blot, immunocytochemistry and microscopic Ca(2+)-release measurements on these cells. RT-PCR showed the presence of mRNA for RyR isoforms 1, 2 and 3 in both rat pancreas and dispersed pancreatic acini. Furthermore, mRNA expression for RyR isoforms 1 and 2 was demonstrated by RT-PCR in individual pancreatic acinar cells selected under the microscope. Western-blot analysis of acinar cell immunoprecipitates, using antibodies against RyR1 and RyR2, showed a high-molecular-mass (>250 kDa) protein band that was much less intense when immunoprecipitated in the presence of RyR peptide. Functionally, permeablized acinar cells stimulated with the RyR activator, palmitoyl-CoA, released Ca(2+) from both basolateral and apical regions. These data show that pancreatic acinar cells express multiple isoforms of the RyR and that there are functional receptors throughout the cell.

Endoplasmic reticulum Ca(2+)-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells.

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca(2+)-ATPase thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca(2+)-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca(2+)-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca(2+)-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca(2+)-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide.

BACKGROUND & AIMS: Although alcoholism is a major cause of pancreatitis, the pathogenesis of this disorder remains obscure. Failure to produce experimental alcoholic pancreatitis suggests that ethanol may only increase predisposition to pancreatitis. This study sought to develop a model of ethanol pancreatitis by determining if an ethanol diet sensitizes rats to pancreatitis caused by cholecystokinin octapeptide (CCK-8). METHODS: Rats were fed intragastrically either control or ethanol diet for 2 or 6 weeks. The animals were then infused for 6 hours with either saline or CCK-8 at a dose of 3000 pmol. kg(-1). h(-1), which by itself did not induce pancreatitis. The following parameters were measured: serum amylase and lipase levels, pancreatic weight, inflammatory infiltration, number of apoptotic acinar cells, pancreatic messenger RNA (mRNA) expression of cytokines and chemokines, and nuclear factor (NF)-kappaB activity. RESULTS: All measures of pancreatitis, as well as NF-kappaB activity and mRNA expression for tumor necrosis factor alpha, interleukin 6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and inducible nitric oxide synthase, were significantly increased only in rats treated with ethanol plus CCK-8. CONCLUSIONS: An ethanol diet sensitizes rats to pancreatitis caused by CCK-8. The combined action of ethanol and CCK-8 results in NF-kappaB activation and up-regulation of proinflammatory cytokines and chemokines in the pancreas. These mechanisms may contribute to the development of alcoholic pancreatitis.

Early NF-kappaB activation is associated with hormone-induced pancreatitis.

Inflammation and cell death are critical to pathogenesis of acute pancreatitis. Here we show that transcription factor nuclear factor-kappaB (NF-kappaB), which regulates these processes, is activated and plays a role in rat cerulein pancreatitis. NF-kappaB was strongly activated in the pancreas within 30 min of cerulein infusion; a second phase of NF-kappaB activation was prominent at 3-6 h. This biphasic kinetics could result from observed transient degradation of the inhibitory protein IkappaBalpha and slower but sustained degradation of IkappaBbeta. The hormone also caused NF-kappaB translocation and IkappaB degradation in vitro in dispersed pancreatic acini. Both p65/p50 and p50/p50, but not c-Rel, NF-kappaB complexes were manifest in pancreatitis and in isolated acini. Coinfusion of CCK JMV-180, which abolishes pancreatitis, prevented cerulein-induced NF-kappaB activation. The second but not early phase of NF-kappaB activation was inhibited by a neutralizing tumor necrosis factor-alpha antibody. Antioxidant N-acetylcysteine (NAC) blocked NF-kappaB activation and significantly improved parameters of pancreatitis. In particular, NAC inhibited intrapancreatic trypsin activation and mRNA expression of cytokines interleukin-6 and KC, which were dramatically induced by cerulein. The results suggest that NF-kappaB activation is an important early event that may contribute to inflammatory and cell death responses in acute pancreatitis.

nNOS and Ca2+ influx in rat pancreatic acinar and submandibular salivary gland cells.

Regulation of agonist-activated Ca2+ influx by the NOS pathway through generation of cGMP is being found in an increasing number of cell types. In the present work, we examined the role of the NOS pathway in agonist-evoked [Ca2+]i oscillations and attempted to identify the NOS isoform most likely to regulate Ca2+ influx. For this, we first show that two Ca(2+)-mobilizing agonists acting on pancreatic acinar cells, bombesin (BS) and the cholecystokinin analog CCK-JMV-180 (CCKJ), evokes different type of [Ca2+]i oscillations. The BS-evoked [Ca2+]i oscillations rapidly became acutely dependent on the presence of extracellular Ca2+, whereas the CCKJ-evoked oscillations continue for long periods of time in the absence of Ca2+ influx. This differential behavior allowed us to isolate Ca2+ influx and study its regulation while controlling for non specific effects on all other Ca2+ transporting events involved in generating [Ca2+]i oscillations. Inhibitors of selective steps in the NOS pathway inhibited agonist-induced cGMP production. The inhibitors were then used to show that scavenging NO with reduced hemoglobin, inhibition of guanylyl cyclase with 1H-[1,2,4] oxadiazolo[4,3-a] quinoxaline-1-one (ODQ) and inhibition of protein kinase G with Rp-8-pCPT-cGMPS inhibited [Ca2+]i oscillations evoked by BS but not those evoked by CCKJ. These findings were extended to duct and acinar cells of the SMG. In these cells, Ca(2+)-mobilizing agonists stimulate large Ca2+ influx, which was inhibited by all inhibitors of the NOS pathway. Western blot analysis and immunolocalization revealed that the cells did not express iNOS, eNOS was expressed only in blood vessels and capillaries whereas nNOS was expressed at high levels next to the plasma membrane of all cells. Accordingly, the nNOS inhibitor 7-nitroindazole (7-NI) inhibited BS- but not CCKJ-evoked [Ca2+]i oscillations and Ca2+ influx into SMG acinar and duct cells. Thus, together, our findings favor nNOS as the isoform activated by the Ca2+ released from internal stores to generate cGMP and regulate Ca2+ influx.

Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis.

The aim of this study was to determine whether tumor necrosis factor-alpha (TNFalpha) and receptors for TNFalpha are expressed in the exocrine pancreas, and whether pancreatic acinar cells release and respond to TNFalpha. Reverse transcription PCR, immunoprecipitation, and Western blot analysis demonstrated the presence of TNFalpha and 55- and 75-kD TNFalpha receptors in pancreas from control rats, rats with experimental pancreatitis induced by supramaximal doses of cerulein, and in isolated pancreatic acini. Immunohistochemistry showed TNFalpha presence in pancreatic acinar cells. ELISA and bioassay measurements of TNFalpha indicated its release from pancreatic acinar cells during incubation in primary culture. Acinar cells responded to TNFalpha. TNFalpha potentiated NF-kappaB translocation into the nucleus and stimulated apoptosis in isolated acini while not affecting LDH release. In vivo studies demonstrated that neutralization of TNFalpha with an antibody produced a mild improvement in the parameters of cerulein-induced pancreatitis. However, TNFalpha neutralization greatly inhibited apoptosis in a modification of the cerulein model of pancreatitis which is associated with a high percentage of apoptotic cell death. The results indicate that pancreatic acinar cells produce, release, and respond to TNFalpha. This cytokine regulates apoptosis in both isolated pancreatic acini and experimental pancreatitis.

Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell.

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined. METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed. RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules. CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.

The role of calcium in the regulation of protein synthesis in the exocrine pancreas.

The present study was designed to examine the role of Ca2+ in the regulation of digestive enzyme synthesis, to determine whether changes in intracellular Ca2+ stores or cytosolic Ca2+ caused the observed effects, and to establish the steps in the pathway of protein synthesis where the regulation occurs. Protein synthesis, polysome size, and the ratio of completed to nascent polypeptides were measured as a function of Ca2+ in the intracellular stores and the cytoplasm of pancreatic acinar cells. Rat acini and rabbit pancreatic lobules were incubated in media containing 1 mM CaCl2 with the following additives: cholecystokinin (CCK) octapeptide; the inhibitors of microsomal Ca2+ ATPase, thapsigargin (THP) and 2,5-di(tertbutyl)-hydroquinone (BHQ); the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA); an inhibitor of translational initiation, 7-methylguanosine 5'-triphosphate; and an inhibitor of translation elongation, cyclohexamide. THP and BHQ depleted intracellular pools of Ca2+ and caused a sustained elevation in cytosolic [Ca2+]. Under these conditions, the polysome size diminished, and the ratio of completed proteins increased twofold relative to nascent polypeptides despite an overall decrease in net protein synthesis (55.3 +/- 2.7% of control). These effects paralleled those caused by incubation with 1 nM CCK. Incubation of pancreatic acini with BAPTA plus THP or BHQ depleted the pool [Ca2+] without changing the cytosolic [Ca2+]. In addition, these agents decreased the net protein synthesis (30.1 +/- 3.6% compared to control) and polysome size and increased the ratio of completed to nascent polypeptides to 2:1. These results suggest that depletion of intracellular stores of Ca2+ without changes in cytosolic [Ca2+] decreases protein synthesis at translational initiation.

Acyl-coenzyme A causes Ca2+ release in pancreatic acinar cells.

The regulation of cytosolic Ca2+ is important for a variety of cell functions. One non-inositol 1,4,5-trisphosphate (IP3) compound that may regulate Ca2+ is palmitoyl-coenzyme A (CoA), a fatty acid-CoA that is reported to cause Ca2+ release from intracellular stores of oocytes, myocytes, and hepatocytes. To study the role of palmitoyl-CoA in the pancreatic acinar cell, rat pancreatic acini were isolated by collagenase digestion, permeablized with streptolysin O, and the release of Ca2+ from internal stores was measured with fura-2. Palmitoyl-CoA released Ca2+ from internal stores (EC50 = 14 microM). The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP3-sensitive Ca2+ pool. The effects of submaximal doses of IP3 or cyclic ADP-ribose plus palmitoyl-CoA were additive. Fatty acid-CoA derivatives with carbon chain lengths of 16-18 were the most potent and efficacious. Ryanodine and caffeine or elevated resting [Ca2+] sensitized the Ca2+ pool to the actions of palmitoyl-CoA. Fatty acid-CoA levels in pancreatic acini were measured by extraction with 2-propanol/acetonitrile, followed by separation and quantification using reverse phase high performance liquid chromatography, and were found to be 10.17 +/- 0.93 nmol/mg protein. These data suggest the presence of an IP3-insensitive palmitoyl-CoA-sensitive Ca2+ store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca2+-induced Ca2+ release.

The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis.

BACKGROUND & AIMS: Pancreatitis is characterized by inflammation and death of acinar cells. Death can occur by either necrosis or apoptosis. The initial injury may cause expression of cytokines that mediate activation and infiltration of neutrophils. The aim of this study was to assess the effect of neutrophils and platelet-activating factor (PAF) in cell death responses. METHODS: The effects of neutrophil depletion with antineutrophil serum (ANS) and a PAF antagonist (BN52021) were measured in the cerulein model of pancreatitis. Rats received a 6-hour intravenous infusion of cerulein either alone or after treatment with ANS, BN52021, or both. RESULTS: Cerulein-induced pancreatitis was characterized by neutrophilic infiltration, vacuolization of acinar cells, and foci of necrosis. Treatment with ANS and BN52021 prevented the inflammatory response caused by cerulein and decreased the cell damage. Treatment with ANS increased apoptosis in cerulein-infused animals. When BN52021 was added, apoptosis was abolished. The measurement of PAF in pancreatic tissue showed a ninefold increase with cerulein treatment alone and a 14-fold increase in cerulein-infused, neutrophil-depleted animals. CONCLUSIONS: The results indicate that cerulein stimulates pancreatic production of PAF. PAF mediates both apoptosis and neutrophil chemotaxis in the pancreas. Neutrophils in turn may convert acinar cells undergoing apoptosis into necrotic cells.

Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat.

BACKGROUND & AIMS: Mechanisms of cell death in human and experimental pancreatitis remain poorly understood. The aim of this study was to determine the mechanisms of cell death, apoptosis vs. necrosis, in models of pancreatitis induced by pancreatic duct ligation in the opossum and rat. METHODS: Morphological changes were shown by light and electron microscopy, chromatin condensation and DNA breaks were assessed using Hoechst 33258 staining and DNA nickend labeling, and DNA fragmentation was characterized b gel electrophoresis. RESULTS: Cells with morphology of both necrosis and apoptosis were found in pancreata from duct-ligated animals; however, the apoptosis-necrosis ratio was different for the opossum and the rat. Both apoptotic and necrotic cells stained for breaks in DNA, suggesting that both apoptosis and necrosis are associated with DNA fragmentation and that DNA end labeling cannot specifically detect apoptotic cells. No significant oligonucleosomal DNA fragmentation was found in pancreas from duct-ligated animals, although the endonuclease responsible for oligonucleosomal DNA fragmentation was present in pancreatic nuclei. CONCLUSIONS: Duct ligation resulted predominantly in apoptosis in the rat and necrosis in the opossum. The differences in types of cell death in the two models may result from differences in inflammatory cell infiltration.

Isolation of subcellular agonist-sensitive calcium stores from the pancreatic acinar cell.

The purpose of the present study was to develop a technique to identify, isolate and partially purify these membrane bound compartments for further characterizations of their Ca2+ transport and storage mechanisms. We 45Ca(2+)-loaded the agonist-sensitive Ca2+ stores in rat pancreatic acini. The loading was accomplished by first depleting the stores with carbachol stimulation followed by the addition of 45Ca2+ and atropine to the extracellular media. After homogenization of the 45Ca(2+)-loaded acini, subcellular fractions were resolved on sucrose and Nycodenz gradients. 45Ca2+ fluxes were minimized during these procedures by inclusion in the media of LaCl3. Five subcellular fractions were identified that specifically accumulated 45Ca2+ after carbachol stimulation. Electron microscopic observations of the fractions demonstrated that three of the fractions consisted of rough membrane vesicles; that one consisted of a mixture of rough and smooth membrane vesicles; and that one consisted of smooth membrane vesicles. All fractions were enriched in glucose-6-phosphatase. All 5 fractions demonstrated ATP dependent 45Ca2+ uptake. By Western blot analysis, all fractions contained calnexin, p58, sarcoplasmic reticulum type Ca(2+)-ATPase, and IP3 receptor. These results demonstrated that the 45Ca(2+)-loading technique can be used to isolate and characterize distinct compartments of the agonist-sensitive Ca2+ store in the pancreatic acinar cell.