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

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

Internalization of cationized ferritin by isolated pancreatic acinar cells

The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis. Dedicated to Professor P. Heinrich on the occasion of his 60th birthday     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis.     

Calcium-magnesium interactions in pancreatic acinar cells.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and digital imaging of Ca2+- or Mg2+-sensitive fluorescent dyes as well as Mg2+-sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) -induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal independent of the electrochemical gradients for Mg2+, Ca2+, Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone-induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK-induced [Mg2+]i decrease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentrations profoundly affects acinar cell Ca2+ signaling.     

Rat pancreas adenylate cyclase V. Its presence in isolated rat pancreatic acinar cells.

(1) In order to determine the cellular localization of the secretin- and pancreozymin-sensitive adenylate cyclase in rat pancreas, the occurence of this enzyme system has been investigated in isolated pancreatic cells. (2) Digestion of rat pancreatic lobules with collagenase yields a preparation of isolated cells which upon differential morphological analysis appears to consist for 97% of acinar cells and to contain for fewer centro-acinar and ductal cells than undissociated lobules. (3) Expressed per mg protein, the isolated cells contain the same amount of DNA, chymotrypsin and lactic dehydrogenase as the undissociated tissue. The stimulated adenylate cyclase activity is nearly entirely recovered in the isolated acinar cells, as is also the case for the low Km adenosine 3',5-cyclic monophosphate phosphodiesterase activity and the adenosine 3',5'-cyclic monophosphate (cyclic AMP) content. Marked losses are noted for the basal adenylate cyclase and the high Km cyclic AMP phosphodiesterase activities. (4) Washing the isolated acinar cells in Krebs-Ringer bicarbonate medium containing 10 mM 1-methyl-3-isobutylxanthine causes a cyclic AMP level 2.6 times that in cells washed in Krebs-Ringer bicarbonate alone. The cyclic AMP level is further increased by subsequently incubating the cells for 10 min in the presence of 3-10(-7) M pancreozymin-C-octapeptide or secretin to values 1.7 or 4.7 times the control level in cells incubated for 10 min with 1-methyl-3-isobutylxanthine alone. (5) It is suggested that the adenylate cyclase of the acinar cells may be involved, with another factor, in the stimulation of enzyme secretion, whereas a ductular cyclase would function in the regulation of the bicarbonate-dependent fluid secretion.     

Chloroquine alters the processing of secretin in isolated rat pancreatic acinar cells

In this study we considered the effect of chloroquine on the processing and intracellular distribution of internalized secretin radioligand in acinar cells. Chloroquine (100 microM) had no effect on the total amount of 125I-secretin bound but had marked effects on the processing of this radioligand in acinar cells. After an initial 60 min of radioligand binding in the presence and absence of chloroquine, cells were washed free of unbound radioligand, resuspended and then processed for different times at 37 degrees C. During 60, 120 and 180 min of processing, the amount of internalized radioligand in the presence of 100 microM chloroquine was increased by 116, 194 and 273%, respectively, compared to untreated control samples. Chloroquine also increased the amount of intact 125I-secretin radioligand within the cell as measured by rebinding to pancreatic plasma membranes. After 120 and 180 min of processing, intact peptide within the acinar cell was 25 and 66% greater in the presence of this agent than in control samples (P less than or equal to 0.01). To determine if chloroquine affected intracellular localization of the secretin radioligand, we measured the amount of radioactivity in soluble and particulate fractions of cell homogenates. Chloroquine decreased radioactivity entering particulate fractions of the cell by greater than 35% after 120 and 180 min of processing (P less than or equal to 0.01). This study demonstrates that (1) chloroquine inhibits the intracellular degradation of secretin in acinar cells and (2) chloroquine alters intracellular localization of this peptide during processing.     

Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function

OBJECTIVES: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen-activated protein kinase (MAPK) signalling and exocrine secretion. MATERIALS AND METHODS: A nicotine dose- and time-response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. RESULTS: Nicotine treatment caused dose-dependent activation of extracellular signal-regulated kinases (ERK1/2), the maxima occurring at 100 micro m and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine-induced cell proliferation occurred at 24 h, and UO126-treatment significantly reduced this response. Exposure of cells to 100 microm nicotine for 6 min significantly enhanced both baseline and cholecystokinin-stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. CONCLUSIONS: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.     

Comparative effects of epidermal growth factor and carbachol on phosphoinositide synthesis and breakdown in pancreatic acinar cells.

Carbachol (CCh) and epidermal growth factor (EGF) elicited a concentration-dependent increase in [32P]phosphatidyl-inositol-4-phosphate (PtdIns-4P) formation in homogenates derived from agonist-stimulated rat pancreatic acini. The combination of CCh and EGF produced a response which was not synergistic or additive. EGF, unlike CCh, failed to cause [32P]PtdIns-4,5P2 breakdown, suggesting different mechanisms involved in the stimulation of [32P]PtdIns-4P formation induced by EGF and CCh. We conclude that PtdIns kinase represents a key component of the signaling pathways utilized by EGF and CCh in exocrine pancreas.     

Photoaffinity labeling with GTP-gamma-azidoanilide of a cholera toxin-sensitive 40 kDa protein from pancreatic acinar cells

In isolated pancreatic acinar plasma membranes a 40 kDa protein was labeled with the photoreactive GTP-analogue [alpha 32P] GTP-gamma-azidoanilide. Increased incorporation of the photolabel into the 40 kDa protein was obtained in the presence of increasing concentrations of cholecystokinin-octapeptide (10(-8) - 10(-5) M) but not with carbachol. Adenylyl cyclase activating hormones such as vasoactive intestinal polypeptide and secretin had no effect. Pretreatment of plasma membranes with cholera toxin reduced incorporation of GTP-gamma-azidoanilide into the 40 kDa protein by about 30%. This reduction was reversed if ADP-ribosylation by cholera toxin was performed in the presence of cholecystokinin, whereas carbachol had no effect. The data indicate that a cholera toxin-sensitive 40 kDa GTP-binding protein is involved in functionally coupling cholecystokinin- but not muscarinic acetylcholine-receptors to phospholipase C.     

Somatostatin inhibition of phosphoinositides turnover in isolated rat acinar pancreatic cells: interaction with bombesin.

The effects of somatostatin-14 and bombesin on [3H]inositol phosphate accumulation were studied in 24 h myo-[3H]inositol-prelabeled cultured rat acinar cells. Bombesin, 10 nM, stimulated basal formation of phosphatidyl monophosphate (InsP1), phosphatidyl 4,5-biphosphate (InsP2) and inositol 1,4,5-triphosphate (InsP3) by 128 +/- 5.2%, 147 +/- 10% and 155 +/- 5%, respectively. At 5 s, the ED50 value for InsP3 stimulation was 0.70 +/- 0.2 nM. This stimulation was partly blocked (64 +/- 0.04% inhibition) by 10 ng/ml Bordetella pertussis toxin. In contrast to bombesin, somatostatin, 10 nM, inhibited basal InsP1, InsP2 and InsP3 formation. At 5 s, the inhibition degree for InsP3 was 18 +/- 2.5% and the IC50s values 1 +/- 0.09 nM, 1 +/- 0.12 nM and 0.07 +/- 0.005 nM for InsP1, InsP2 and InsP3, respectively. Bombesin-stimulated InsP3 formation was also inhibited by somatostatin. At 5 s, the inhibition degree was 85 +/- 3.5% at 10 nM and the IC50 value, 0.10 +/- 0.05 nM. Furthermore, somatostatin inhibition of bombesin stimulation was partly blocked (66 +/- 4% inhibition) by Bordetella pertussis toxin. These data therefore suggest that the acinar pancreatic cells contain a somatostatin receptor exerting a negative control on basal and bombesin receptor-stimulated phosphatidyl inositol turnover.     

Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells

In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively. Our results show that stimulation of cells with cholecystokinin led to release of Ca2+ from intracellular stores that then accumulated into mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered; in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases in mitochondrial Ca2+ and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca2+ that could in turn activate the mitochondrial machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca2+ can act as a regulator of mitochondrial activity.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Ultrastructural and functional changes in pancreatic acinar cells during autolysis.

Effects of anoxemic cell injury on rat pancreatic acinar cells were studied in a preparation where tissue samples were incubated at temperature between 18-20 degrees C in a moist atmosphere for 0, 0.5, 1, 3, 6, 12, and 24 h in vitro. Electron microscopy revealed that disintegration of acinar cells began by swelling of various cell compartments and gradual breakdown of cell membranes. Zymogen granules remained morphologically intact for at least 3 h. There were no signs of increased autophagic activity during the period of observation. Myelin figures and other membranous remnants of disintegrated cells, together with individual cells and cell organelles whose morphology was relatively well preserved were seen even after w4 h incubation. The secretory response of acinar cells to pancreozymin stimulation, as measured by amylase release into the incubation medium in vitro, decreased progressively closer to zero during 12 h autolysis. No active trypsin could be detected in the tissue samples during the 24 h observation time. It was concluded that during hypoxic autolysis at room temperature between 18-20 degrees C in vitro: 1. Acinar cell disintegration results from breakdown of cellular membranes, 2. autophagocytosis is not involved, 3. most of zymogen granules remain morphologically intact even at the time when cell membranes show evidence of damage, 4. there is no trypsin activation taking place in the tissue, and 5. the acinar cells are capable of responding to secretory stimulation for 3 to 6 h after removal of the tissue from the experimental animal.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.