Isolated Rat Pancreatic Acinar Cells Research Articles

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 (AlF4), 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.

Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K(V)LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.

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 ([Ca 2+] i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca 2+] 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 [Ca 2+] 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 [Ca 2+] i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 μM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca 2+ from acinar cells in which agonist-releasable Ca 2+ pools had been previously depleted by thapsigargin (0.5 μM) and ryanodine receptors were blocked by 50 μM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca 2+ 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.

Secretagogues cause ubiquitination and down-regulation of inositol 1,4,5-trisphosphate receptors in rat pancreatic acinar cells

Background & Aims: The action of several exocrine pancreas secretagogues depends on the second messenger inositol 1,4,5-trisphosphate (IP 3), which, via endoplasmic reticulum–located IP 3 receptors, mobilizes intracellular Ca 2+ stores. Signaling pathways like this one are regulated at multiple loci. To determine whether IP 3 receptors are one of these loci, we measured IP 3 receptor concentration, distribution, and modification in secretagogue-stimulated rat pancreatic acinar cells. Methods: Isolated rat pancreatic acinar cells were exposed to cholecystokinin and other secretagogues, or rats were injected intraperitoneally with cerulein. Then samples of cells or pancreata were probed for IP 3 receptor content and distribution as well as for ubiquitin association with IP 3 receptors. Results: Secretagogues rapidly down-regulated acinar cell IP 3 receptors both in vitro and in vivo. They also elicited receptor redistribution and caused receptors to become ubiquitinated, indicating that the ubiquitin/proteasome proteolytic pathway contributes to the down-regulation. Surprisingly, however, proteasome inhibitors did not block IP 3 receptor down-regulation, and phospholipase Cβ1 and protein kinase Cϵ also were down-regulated. Thus, secretagogues simultaneously activate an additional proteolytic pathway. Conclusions: Secretagogues rapidly down-regulate IP 3 receptors and other proteins involved in intracellular signaling by a mechanism that involves, but is not limited to, the ubiquitin/proteasome pathway. Loss of these proteins may account for the disruption of Ca 2+ mobilization that occurs in models of acute pancreatitis, and may contribute to cell adaptation under physiological conditions. GASTROENTEROLOGY 1999;116:1194-1201

Regulation of the Actin Cytoskeleton by p125 Focal Adhesion Kinase in Rat Pancreatic Acinar Cells

Background/Aims: Activation of p125 focal adhesion kinase by cholecystokinin (CCK)-8 has recently been demonstrated in pancreatic acinar cells. The purpose of this study is to examine downstream events of this kinase. Methods: Activation of p125 focal adhesion kinase in freshly isolated rat pancreatic acinar cells was determined by Western blot analysis. Actin cytoskeletal changes were visualized on TRITC-phalloidin-stained cryosections. Amylase release was measured by colorimetric assay. Results: CCK-8 caused dose-dependent activation of p125 focal adhesion kinase. Time-course analysis showed rapid activation with maximum between 5 and 10 min and stimulation still detectable after 60 min. Preincubation with 2 µM cytochalasin D specifically inhibited p125 focal adhesion kinase, but not p42 mitogen-activated protein kinase or increases in intracellular calcium concentrations. The actin cytoskeleton showed rapid reorganization after stimulation, with an initial increase in fluorescence followed by a decline after 30 min. Preincubation with cytochalasin D prevented cytoskeletal changes. Amylase release at concentrations up to 0.1 nM CCK-8 was not influenced by cytochalasin D. In contrast, supramaximal inhibition of amylase release was less pronounced after cytochalasin D incubation. Conclusion: p125 focal adhesion kinase in acinar cells appears to be part of a signalling pathway leading to changes in cellular morphology via the actin cytoskeleton. Maximal activation of this signalling pathway might participate in supramaximal inhibition of enzyme secretion.

Effect of Basic Fibroblast Growth Factor on Cholecystokinin-Induced Amylase Release and Intracellular Calcium Increase in Male Rat Pancreatic Acinar Cells

Isolated rat pancreatic acinar cells were used to investigate the effect of basic fibroblast growth factor (bFGF) on both amylase secretion and intracellular free calcium concentration ([Ca 2+] i) in response to the calcium-mobilizing secretagogue cholecystokinin-octapeptide (CCK-8). Our data show that bFGF inhibited CCK-8-induced amylase release in a concentration-dependent manner and decreased the CCK-8-induced rise in [Ca 2+] i. This inhibitory effect of bFGF on both amylase secretion and [Ca 2+] i increase in response to CCK-8 was reverted when acinar cells were pretreated with 100 μM tyrphostin A25, a tyrosine kinase inhibitor. Tyrphostin A25 also inhibited Ca 2+ influx induced by CCK-8. These results show that bFGF inhibits CCK-8-induced pancreatic response by a tyrosine kinase-dependent mechanism. A role for tyrosine phosphorylation in capacitative Ca 2+ entry is suggested.

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells.

Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840-G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 muM) and cholecystokinin-(26-33)-peptide amide (CCK8, 0.1 muM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK8-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-alpha and, to a lesser extent, PKC-epsilon. In addition, TPA-induced inhibition of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-alpha. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca2+]i. Taken together, the present findings are in agreement with the idea that PKC-alpha-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca2+]i oscillations and eventually to inhibition of high-affinity Ca2+ signalling through the native CCK receptor in pancreatic acinar cells.

Modulation of calcium signals by intracellular pH in isolated rat pancreatic acinar cells.

1. We have investigated the interactions between intracellular pH (pH1) and the intracellular free calcium concentration ([Ca2+]i) in isolated rat pancreatic acinar cells. The fluorescent dyes fura-2 and BCECF were used to measure [Ca2+]i and pHi, respectively. 2. Sodium acetate and ammonium chloride (NH4Cl) were used to acidify and alkalinize pHi, respectively. Cytosolic acidification had no effect on [Ca2+]i in resting pancreatic acinar cells, whereas cytosolic alkalinization released Ca2+ from intracellular stores. 3. Cytosolic acidification using either acetate or a CO2-HCO3(-)-buffered medium enhanced Ca2+ signals evoked by acetylcholine (ACh) and cholecystokinin (CCK). In contrast, both NH4Cl and trimethylamine (TMA) inhibited Ca2+ signals during stimulation with either ACh or CCK. This inhibitory effect was also observed in the absence of extracellular Ca2+, and was therefore not due to changes in Ca2+ entry. 4. Calcium oscillations evoked by physiological concentrations of CCK were enhanced by cytosolic acidification and inhibited by cytosolic alkalinization. 5. In order to determine the effects of pHi upon Ca2+ handling by intracellular Ca2+ stores, intraorganellar [Ca2+] was monitored using the low affinity Ca2+ indicator mag-fura-2 in permeabilized cells. Addition of NH4Cl, which is expected to alkalinize intraorganellar pH, did not alter intraorganellar [Ca2+] in permeabilized cells, suggesting that changing intraorganellar pH does not release Ca2+ from intracellular stores. Addition of NH4Cl or acetate also did not affect the rate of Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3). 6. Modification of extraorganellar ('cytosolic') pH did not affect the rate of ATP-dependent Ca2+ uptake into stores, but did modify the rate of Ca2+ release evoked by submaximal concentrations of InsP3. The rate of Ca2+ release was increased at more alkaline extraorganellar pHs. These results would suggest that manipulation of intraorganellar pH does not affect Ca2+ handling by the intracellular stores. In contrast, extraorganellar ('cytosolic') pH does affect InsP3-induced Ca2+ release from the stores. 7. In conclusion, changes in intracellular pH in pancreatic acinar cells can profoundly alter cytosolic [Ca2+]. This may shed light on earlier observations whereby cell-permeant weak acids and bases can modulate fluid secretion in epithelia.

Inhibition of cathepsin B does not affect the intracellular activation of trypsinogen by cerulein hyperstimulation in isolated rat pancreatic acinar cells.

Activation of trypsinogen is thought to trigger the autodigestive process in acute pancreatitis. The lysosomal enzyme cathepsin B was suggested to cause the activation of trypsinogen because it is known that cathepsin B is able to activate trypsinogen in special circumstances and that lysosomal and digestive enzymes are colocalized within intracellular vacuoles in the early stage of pancreatitis. As yet this hypothesis has been difficult to prove because activated trypsin is difficult to quantify in pancreatitis by conventional enzymatic measurements. We therefore employed an ELISA for trypsin activating peptide (TAP), which is a small peptide cleaved during the activation of trypsinogen and can be determined reliably. Supraphysiological concentrations of cerulein (1 nM-1 microM) resulted in a marked increase in TAP in freshly isolated pancreatic acinar cells, indicating activation of trypsinogen. This activation as determined by the TAP increase was significantly reduced by the serine protease inhibitor Fut-175 but not by the cathepsin B inhibitors E-64 and NCO-700. The concentrations of NCO-700 and E-64 abolished the cathepsin B activity of pancreatic acinar cells but did not significantly reduce the trypsin activity (after enterokinase preincubation); correspondingly the concentrations of Fut-175 used abolished the trypsin activity but did not reduce the cathepsin B activity. The results indicate that an autoactivation of trypsin rather than an activation of trypsinogen by cathepsin B triggers trypsin activation by supramaximal cerulein concentrations.

Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells.

The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.

Internalization of wheat germ agglutinin (WGA) by rat pancreatic cells in vivo and in vitro

The uptake of the lectin wheat germ agglutinin (WGA) by the rat pancreas was studied after intraperitoneal injection of FITC-labelled WGA. Strong fluorescence was seen in the interstitial space of the pancreatic parenchyma and on the basolateral surface of acinar cells after one h. Two or four h after injection there was also a fluorescence inside the acinar cells. After intraparenchymal injection of colloidal gold-labelled WGA into the pancreatic gland in situ or in vitro incubation of isolated rat pancreatic acinar cells with WGA-gold, an internalization of the lectin by receptor mediated endocytosis was observed electron microscopically. Clathrin-coated pits or vesicles could not be observed. The results suggest a direct uptake of WGA from the peritoneum into the gland by diffusion and its binding as well as internalization by the pancreatic acinar cells.

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

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

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

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

Inhibitory Effect of Thyrotropin-Releasing Hormone on Enzyme Secretion from Isolated Rat Pancreatic Acinar Cells

This study reports a direct effect of TRH on amylase secretion from isolated rat exocrine pancreatic acinar cells. TRH inhibited carbachol (10(-5) M)-stimulated amylase secretion by a maximum of 24% at a concentration of 10(-11) M (p < 0.05), but did not affect basal amylase release in concentrations from 10(-13) M to 10(-8) M. Ceruletide (3 x 10(-10) M)-stimulated amylase secretion was maximally reduced by 23% at a TRH concentration of 10(-10) M (p < 0.05). Direct stimulation of protein kinase C-mediated secretion by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was not altered by TRH. The TRH metabolite cyclo (His-Pro) did not influence basal or stimulated pancreatic secretion in vitro. These findings point to a TRH-mediated modulation of exocrine pancreatic secretion at the receptor site.

Flow cytometric measurements of intracellular Ca2+ mobilization in isolated rat pancreatic acinar cells after hormone stimulation and action of lectins

Isolated rat pancreatic acinar cells were loaded with the Ca(2+)-sensitive fluorescence dye Fluo 3 in vitro and the intracellular Ca2+ changes were analysed by flow cytometry. Morphology, viability, and loading with the dye were studied by light microscopy. Stimulation with cholecystokinin/pancreozymin (CCK) and its agonist caerulein as well as with carbamylcholine (Jestryl) led to an increase of intracellular calcium ions and a fluorescence peak. The slope and height of the Ca2+ signals were found to be influenced by preincubation of cells with some plant lectins (WGA, UEA, PHA, Con A, LCA, PNA). These effects are discussed with respect to the interaction of lectins with the carbohydrate chains of cell membrane receptors.

Oxidative and proteolytic cell injury: comparison of susceptibility of isolated rat pancreatic acinar cells versus isolated rat hepotocytes

The present study compares the susceptibility of isolated rat pancreatic acinar cells versus rat hepatocytes against xanthine oxidase-mediated oxidative injury and against proteolytic attack. Cells were incubated in solutions containing either xanthine oxidase, hypoxanthine, and chelated iron (XOD/HX solution), or various concentrations of H 2O 2 or active trypsin and chymotrypsin. Cell viability was assessed by uptake of trypan blue realease of LDH. The XOD/HX system caused a rapid generation of radicals during a few initial minutes as assessed by the deoxyribose assay, whereas the resulting damage to either type of cells developed slowly over several hours. This indication for effective cellular antioxidant defenses was supported by the finding that addition of both cell types to the XOD/HX solution significantly reduced the oxidation of deoxyribose seen in cell-free studies, There were, however, also striking differences in the behaviour of acinar cells and hepatocytes against oxidative and proteolytic attack. Pancreatic cells were more susceptible to oxidative stress than hepatocytes, whereas hepatocutes were more susceptible to proteolytic damage. In both cell types chymotrypsin caused significantly greater injury than did trypsin. This difference was small for hepatocytes and large for pancreatic cells. In both cell types catalase and glutathione peroxidase significantly reduced oxidative cell damage, suggesting that a major part of damage was mediated by H 2O 2 and hydroxyl radicals. Both cell types were also susceptible to direct damage by H 2O 2; again the susceptibility cells was greater than that of hepatocytes. In conclusion, the present results demonstrate that pancreatic cells and hepatocytes are susceptible to oxidative stress, although they have effective antioxidant capacities which are similar for both cell types. The pancreatic cell - probably due to a its large antitrypsin capacity - was markedly less susceptible to direct damage by proteases when compared with the hepatocyte. However, the high concentrations of proteases in the acinar cell might explain why these cells are more susceptible to oxidative stress than the hepatocyte, since a major part of xanthine oxidase may be generated extracellularly due to leakage of proteases from already damaged cells.

Fluorescence microscopic studies and flow cytometric measurements of lectin and hormone binding to isolated rat pancreatic acinar cells

The binding of fluorescence-labelled lectins and a fluorescence-marked hormone to the cell surface of isolated rat pancreatic acinar cells was studied by light microscopy and flow cytometric measurements. The pancreatic acinar cells were prepared by collagenase digestion. The fluorescence of cells was studied after binding of FITC-labelled WGA or UEA I as well as of FITC-marked pancreocymin/cholecystokinin (CCK-FITC) in a fluorescence microscope or FACScan. The strong binding of lectins was inhibited by preabsorption with the specific sugars. In comparison to the lectins the binding of CCK-FITC was low. There were two populations of acinar cells with different CCK-FITC binding capacity as detected by flow cytometry. The CCK-FITC cell surface fluorescence was significantly decreased by preincubation with unmarked hormone as well as with the non-labelled lectins. The inhibition of CCK-FITC binding by lectins is discussed in respect to a possible competition of the lectins and CCK for the CCK receptor.

Lectin binding studies with FITC-marked WGA and UEA I and flowcytometric measurements on isolated rat pancreatic acinar cells

Lectin binding to the glycocalyx of isolated rat pancreatic acinar cells was studied by flowcytometric measurements. The pancreatic exocrine cells were prepared after collagenase digestion and cleaned in a density gradient. The fluorescence of cells was measured in a FACScan after binding of FITC marked WGA or UEA I. The binding of lectins was inhibited by preabsorption of WGA-FITC with N-acetyl-glucosamine, sialic acid or chitinous and by preabsorption of UEA-FITC with alpha-L-fucose, respectively. Furthermore, we were able to measure a decreased WGA-FITC and UEA-FITC binding after a short preincubation of isolated cells with the peptide hormone cholecystokinin and its agonists (caerulein, pentagastrin).

Influence of colloidal bismuth subcitrate on enzyme secretion from isolated rat pancreatic acinar cells.

Bismuth salts are currently used as monotherapy or in combination with antibiotics for the treatment of Helicobacter pylori-associated peptic ulcer disease. Besides encouraging clinical results with colloidal bismuth subcitrate (CBS), there is an ongoing fear of organ toxicity with the use of bismuth salts. To study potential toxic effects of CBS under short-term exposure, we tested the influence of CBS on amylase secretion from isolated rat pancreatic acinar cells under basal conditions and following carbachol (CCh) and ceruletide (CRT) stimulation. Basal secretion was reduced by 8.9 +/- 9.6% (n = 10) (mean +/- SEM) (P < 0.05), 5.2 +/- 9.2% (P < 0.05), 9.4 +/- 6.4% (P < 0.01), and 6.2 +/- 12.2% (P < 0.05) with 0.001, 0.01, 0.1, and 1 micrograms/ml CBS, respectively. With 10 micrograms/ml and 100 micrograms/ml CBS, basal amylase secretion was increased in a dose-dependent manner, by 13.7 +/- 11.7% (P < 0.05) and 24.5 +/- 12.8% (P < 0.01). CCh (10(-5) M)- and CRT (3 x 10(-10) M)-stimulated secretory responses were not altered significantly by any of the CBS doses used. In concentrations above 1 microgram/ml, CBS increased pancreatic amylase secretion. Amylase secretion in response to secretagogues was not affected by CBS. These findings are unlikely to be associated with a toxic effect of CBS on exocrine pancreatic acinar cell function.

Secretagogue‐evoked changes in intracellular free magnesium concentrations in rat pancreatic acinar cells.

1. This study employs the fluorescent dye Mag-Fura-2 acetoxymethyl ester (AM) to measure intracellular free magnesium concentration [Mg2+]i in isolated rat pancreatic acinar cells. Initially a number of protocols were investigated to develop optimal loading conditions for the dye Mag-Fura-2 AM. The procedure yielding cells which showed minimal dye loss and no adverse compartmentalization was adopted for subsequent experiments. 2. The mean resting [Mg2+]i is 1.39 +/- 0.08 mM (n = 39). 3. Acetylcholine (ACh), cholescystokinin-octapeptide (CCK8), carbamylcholine chloride evoked marked reduction in [Mg2+]i in pancreatic acinar cells compared to resting values in the absence of secretagogues. The ACh-evoked decrease in [Mg2+]i was abolished by pre-treatment with atropine. In contrast, noradrenaline, adrenaline and histamine had no significant effect on [Mg2+]i. 4. In acinar cells loaded with the Ca(2+)-sensitive dye, Fura-2 acetoxymethyl ester (AM), ACh stimulation resulted in a marked elevation in intracellular free Ca2+ concentration [Ca2+]i. This response was blocked by pre-treatment with atropine. 5. Atomic absorption spectrophotometry was used to measure Mg2+ levels in effluent samples from pancreatic segments. Stimulation of pancreatic segments with ACh resulted in a marked elevation in Mg2+ concentrations (net efflux). On removal of ACh, Mg2+ concentration returned to resting level followed by a small net influx of Mg2+ into pancreatic tissue. 6. The results demonstrate that secretagogue-evoked alteration in [Mg2+]i may occur concurrently with Mg2+ release from pancreatic tissue.