Original Paper
Digestion 1992;51:193-197
Division of Experimental Surgery, Clinic of Surgery, Medical Academy of Magdeburg, FRG
Effect of Phospholipase A 2and Ethanol on the Survival of Acinar Cells Isolated from the Rat Pancreas
Keyw ords
Abstract
Acute pancreatitis Acinar cells Phospholipase A2 Ethanol Pathogenesis
The study was undertaken to investigate the effect of phospho lipase A2 (PLA2) on the viability of isolated pancreatic acinar cells (PACs) and its possible synergy with ethanol. The sur vival of PACs may be reduced by PLA2 in a dose-dependent manner. In the presence of 0.25 gg PLA2-( 106 cells)-1, all cells died within less than 4 h of incubation at 37 °C. A quantity of 180 mM ethanol, which alone was not lethal for PACs, increased the rate of cell death induced by PLA2 at a certain concentration of this enzyme. The present results imply that (1) PLA2 is able to damage PACs in the absence of additional substrates, and (2) at an appropriate activity of PLA2, ethanol may amplify the toxic effects of this lytic enzyme and force autodigestion of the pancreas.
It is widely accepted that phospholipase A2 (PLA2) contributes to the pathogenesis of acute pancreatitis [1-3]. It has deleterious effects when introduced into tissue. Imme diate acinar and duct cell necrosis follows an intraductal injection of this enzyme into the pancreatic duct, and the pathomorphological findings have some similarity to those ob served in human acute pancreatitis [4], In former studies, we introduced a model of isolated pancreatic acinar cells (PACs) to
Received: March 11,1991 Received in revised form: February 10, 1992
simulate initial steps of the pathogenesis of acute pancreatitis [5, 6], Therefore, PACs were incubated in the presence of various pancreatitis-relevant noxae. In contrast to the whole pancreatic tissue and other complex in vitro preparations, this model has the advan tage of all cells being in uniform contact with the noxious agent. Consequently, a kinetic analysis of cell death (in vitro simulation of acinar cell necrosis) is possible.
Dr. Gerald Letko Division of Experimental Surgery Clinic of Surgery. Medical Academy Leipziger Str. 44 D-O-3090 Magdeburg (FRG)
© 1992 S. Kargcr AG, Basel 0012-2823/92 0514-0193S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
G. Letko M. Siech
Incubation, min
Fig. 1 . Time course of acinar cell damage. Isolated PACs ( I mg dry weight = l .5 • 106 cells) were incubated at 37 °C in a shaking bath in Eagle's medium (o) with 0.05 (A). 0.25 (v) or 0.5 |ig PLAr (106 cells)-' (□). An aliquot o f this suspension was added (■) to exclude toxic effects of ammonium sulfate present as a preser vative in the PLAi suspension as a further control after preceding denaturation.
Fig. 2. Influence of ethanol and PLAi on survival of isolated PACs incubated as described in the legend to Figure I . o = Control; • = ethanol ( 180 m.\7); A = PLA; [0.05 pg-(106 cells)-1]; ▲ = PLA: plus ethanol; □ = PLAi [0.5 pig- (106 cells)- '];■ = PLA: plus ethanol.
To continue this approach, we studied the influence of PLA? on the survival of PACs and its possible synergy with ethanol, an im portant etiological factor in acute pancreatitis [7], in the induction of acinar cell damage.
with PLA: and/or ethanol at the beginning of the incu bation. At specified times, samples of incubates were withdrawn, and the number of intact cells was deter mined by trypan blue exclusion. Well-prepared suspen sions had an initial viability of 90-95% and lost less than 10% of this during the 4-hour incubation in the presence of oxygen. Statistical analysis was performed by testing the slope of the regression line of cell viability against time in a modified t test [8].
Materials and Methods
194
Letko/Siech
Results
The typical time course of cell decline of PACs incubated in Eagle’s medium at 37 °C is shown in figure 1. Survival of PACs may be reduced by PLA2 in a dose-dependent man ner. In the presence of 0.25 pg PLAi’OO6 cells)-1, all cells died within less than 4 h of incubation (fig. 1). If isolated PACs were sup plemented with 180 mM ethanol, there was
Survival o f Acinar Cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
Collagenase for isolation of PACs and PLAi (from porcine pancreas; 700 U-(mg protein)-1 were obtained from Bochringer Mannheim. FRG. All other chemi cals were of analytical grade. Female albino laboratory rats (140—180 g) were used in all experiments. The animals were fasted 24 h before the operation and were anesthetized by an intraperitoneal injection of hexobarbital sodium (125 mg/kg body weight). PACs were isolated by a procedure based on collagenase digestion as recently described [6]. The isolated cells were incubated in Eagle’s medium (MEM) and gassed with oxygen at 37°C in a shaking water bath. In the study groups, the cells were supplemented
no measurable effect on cell survival (fig. 2). To study the influence of this ethanol concen tration in conjunction with the action of PLA2 on the viability of PACs. ethanol was com bined with various PLA2 activities. Figure 2 shows the time course of cell decline in a rep resentative experiment. It becomes evident from this plot that the presence of ethanol (180 mM) increased the cell damage induced by PLA2. For statistical analysis, a transfor mation of such plots was performed. The time course observed in the presence of PLA2alone was set to 100% (fig. 3). The differences re sulting from additional applications of etha nol were plotted versus time. The significance of the differences was evaluated by linear regression and statistical analysis. The response of PACs incubated with identical amounts of ethanol differed depend ing on the PLA2 activities applied. While 0.25 pig PLA2-(106 cells)-1 had no significant effect on cell survival, 0.5 ptg PLA2-(106 cells)-1 amplified cell death significantly (p < 0.02).
There is no doubt that PLA2 is involved in the pathogenesis of acute pancreatitis [1,2.9] although to date, there is no satisfactory mechanism to explain the activation of the proenzyme within the gland without the in volvement of pancreatic trypsin [10], PLA2 is a ubiquitous enzyme, and in addition to its role as a pancreatic digestive enzyme, it is a constituent of granulocytes, macrophages, tis sue membranes and organelles [9]. The mem brane-bound enzyme does not need trypsin for activation, however, it is calcium-dependent and involved in the metabolic turnover of membrane phospholipids [11], For exam ple, an increase in intracellular free Ca2+, by enhancement of the membrane permeability to calcium by lipid peroxidation products
Fig. 3. Influence of ethanol and PLA2 on survival o f isolated PACs incubated as described in the legend to figure 1. Independent experiments (n = 5-7) are compiled after transformation of individual time courses. To this aim, the data o f the PLA2 curves were set to 100%, and the data o f the corresponding curves (PLA? plus ethanol) were plotted as deviations from these ‘standardized’ curves. A = Transformation of PLA2 [0.25 pig-(106 cells)-1] curves to 100%: ▲= rela tive deviation ofPLA2 plus ethanol curves (nonsignifi cant); □= transformation ofPLA 2 [0.5 pig-( 10° cells)-1] curves to 100%: ■ = relative deviation of PLA2 plus ethanol curves (p < 0.02).
[ 12], may activate this membrane PLA2 and lead to phospholipid depletion with resulting cell death [11], To study the effect of active PLA2on PACs we incubated these cells (20-106 cells-ml-1 = 12.5 mg dry weight-ml-1) with increasing
195
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
Discussion
196
Letko/Sicch
are in accordance with in vivo findings of aci nar cell damage after intraductal PLA2 instil lation reported by Aho and Nevalainen [4], The amount of porcine pancreas PLA2 [~ 5 pg-(mgdry weight)-1] is comparable with that enzyme activity which led to total cell death within the 4-hour incubation period (fig. 1). Ethanol is increasingly cited as a cause of acute pancreatitis [7], In our experimental studies, it was necessary, however, to com bine ethanol uptake with other factors to induce this disease [6, 15]. Recently, it has been shown that with a combination of tem porary ductal obstruction and stimulation of secretion (induces pancreatic juice edema [16] and a single ethanol administration, the majority of rats will develop signs of acute pancreatitis [15]. In our present study, we used a relatively high concentration of ethanol. Nevertheless, it became evident that ethanol in these con centrations, which alone are not lethal for PACs (fig. 2), may act together under certain circumstances with PLA2 in increasing the rate of cell death (fig. 2, 3). As demonstrated in isolated acini with a spin label, ethanol may potentiate alterations in membrane compo nents (phospholipids) induced by other fac tors [17], Using this method, the increased PLA2 activity has a better access to its sub strates and membrane phospholipids [ 18]. It cannot be excluded that membrane frag ments of damaged cells may serve as donors of lecithin, a substrate of PLA2. But control experiments with low PLA2 activities (not shown) or with ethanol alone (fig. 3) hint at being of minimum contribution to further cell damage. In conclusion, our results demon strate that (i) PLA2 is able to damage PACs without simultaneous presence of additional substrates and (ii) at the appropriate activity of PLA2, ethanol may amplify the dangerous potential of this lytic enzyme and force auto digestion of the pancreatic gland.
Survival o f Acinar Cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
amounts of PLAi (obtained from porcine pancreas). According to Nagai et al. [13], 1 mg of rat pancreas contains 55 pg PLAi, which corresponds to 0.209 pg PLAj-Cmg dry weight)-1. While 0.08 pg PLA2 -(mg dry weight)-1 slightly reduced the viability of PACs, only in the presence of 0.4 pg PLA2 -(mg dry weight)-1 did all cells die within 4 h of incuba tion at 37 °C (fig. 1). This observation shows that PLA2 may damage acinar cells and con firms the original work of Creutzfeldt and Schmidt [3], Together with the older litera ture, our findings contradict the publication of Nagai et al. [13]. In their study with the iso lated rat pancreatic acini, bee venom PLA2 [0.88-88 pg-(mg dry weight)-1] did not seri ously alter the cellular integrity during a 30min incubation, as indicated by the missing release of [35S]methionine-labelcd proteins [ 13], The same was true if the incubates were supplemented with lecithin, a favored sub strate of this enzyme. Hietaranta et al. [14] have performed experiments similar to those of Nagai et al. [13], however, they used hu man PLA2 in their study and aspartylamino transferase as an indicator of cell damage. Their findings were comparable to those of Nagai et al. [13]. The different results obtained in both types of experiments may have resulted from differences in the experimental design. Some of these divergences are: (i) the time of incu bation; (ii) the sources of the enzyme; (iii) the cells with plasma membranes permeable to the dye (molecular weight = 961) in the trypan blue exclusion test were regarded as dead; with respect to the release of macromolecules, as radioactivity-labeled proteins, these cells could still appear to be intact, and (iv) while isolated cells were in uniform contact with PLA2 in acini, the areas of cell-to-cell contacts are excluded from enzymic attack. On the other hand, the results presented in this paper
References 8 Sachs L: Angewandte Statistik. Ber lin. Springer. 1974. 9 Nevalainen TJ: The role of phos pholipase Aj in human acute pan creatitis. Klin Wochenschr 1989;67: 180-182. 10 Lankisch PG: Pathogenesis of pan creatic inflammation: in Galzer G, Ranson JHC (eds): Acute Pancreati tis. Experimental and Clinical As pects o f Pathogenesis and Manage ment. London, Baillière Tindall. 1988, pp 182-193. 11 Färber JL: Membrane injury and calcium homeostasis in pathogene sis o f coagulative necrosis. Lab In vest 1982:47:114-123. 12 Kagan VE. Archipenko YV. Bitov VB. Kozlov YP: Modification o f the enzymatic systems for Ca2t trans port in sarcoplasmic reticulum upon lipid peroxidation. Biochem USSR 1983:48:320-330. 13 Nagai H, Heinrich H. Wünsch PH. Fischbach W, Mössner J: Role of pancreatic enzymes and their sub strates in autodigestion o f the pan creas. In vitro studies with isolated rat pancreatic acini. Gastroenterol ogy 1989:96:838-847.
14 Hietaranta AJ. Laszik ZG, Aho HJ. Kortesuo PJ, Nevalainen TJ: The role o f phospholipase A i in pan creatic acinar cell injury. Int J Pan creatol 1991;8:187-201. 15 Letko G. Nosofsky T, Lessel W. Siech M: Transition o f rat pan creatic juice edema into acute pan creatitis by single ethanol adminis tration. Pathol Res Pract 1991:187: 247-250. 16 Becker V: Speichclddcm und Pankreatitis. Aktuel Chir 1988:23:183— 189. 17 Ponnapa BC. Hoek JB. Waring AJ, Rubin E: Effect o f ethanol on amy lase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol fed rats. Biochem Pharmacol 1987:36:69— 79. 18 Van Kuijk FJGM. Sebanian A, Handelmann FJ, Dratz EA: A new role for phospholipase Ai: Protec tion o f membranes from lipid perox idation damage. Trends Biochem Sci 1987:12:31-33.
197
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1 Gram D: Acute necrotising pancre atitis - A role for enterokinasc. Int J Pancreatol 1986; 1:16 7 - 183. 2 Nevalainen TJ: Phospholipase A 2 in acute pancreatitis. Scand J Gas troenterol 1988;23:897-904. 3 Creutzfcldt W. Schmidt H: Aetiol ogy and pathogenesis o f pancreatitis (current concepts). Scand J Gas troenterol I970:5(suppl 6):47-62. 4 Aho HJ, Nevalainen TJ: Experi mental pancreatitis in the rat. Light and electron microscopical observa tions on early pancreatic lesions in duced by intraductal injection of trypsin, phospholipase A 2 , lysolecithin and non-ionic detergent. Vir chows Arch [B] 1982:40:347-356. 5 Letko G, Falkenberg B. Wilhelm W: Effect o f trypsin, chymotrypsin. and uncoupling on survival o f isolated acinar cells from rat pancreas. Int J Pancreatol 1989;4:431-441. 6 Letko G, Spormann H. Sokolowski A, Schulz HU: Pancreatic acinar cells: Isolation, characterization and application in physiologic and pathophysiologic studies, with spe cial reference to acute pancreatitis. Exp Pathol 1988:34:10-22. 7 Singh M. Simsek H: Ethanol and the pancreas. Current status. Gastroen terology 1990:98:1051-1062.
Digestion 1992;51:193-197
Division of Experimental Surgery, Clinic of Surgery, Medical Academy of Magdeburg, FRG
Effect of Phospholipase A 2and Ethanol on the Survival of Acinar Cells Isolated from the Rat Pancreas
Keyw ords
Abstract
Acute pancreatitis Acinar cells Phospholipase A2 Ethanol Pathogenesis
The study was undertaken to investigate the effect of phospho lipase A2 (PLA2) on the viability of isolated pancreatic acinar cells (PACs) and its possible synergy with ethanol. The sur vival of PACs may be reduced by PLA2 in a dose-dependent manner. In the presence of 0.25 gg PLA2-( 106 cells)-1, all cells died within less than 4 h of incubation at 37 °C. A quantity of 180 mM ethanol, which alone was not lethal for PACs, increased the rate of cell death induced by PLA2 at a certain concentration of this enzyme. The present results imply that (1) PLA2 is able to damage PACs in the absence of additional substrates, and (2) at an appropriate activity of PLA2, ethanol may amplify the toxic effects of this lytic enzyme and force autodigestion of the pancreas.
It is widely accepted that phospholipase A2 (PLA2) contributes to the pathogenesis of acute pancreatitis [1-3]. It has deleterious effects when introduced into tissue. Imme diate acinar and duct cell necrosis follows an intraductal injection of this enzyme into the pancreatic duct, and the pathomorphological findings have some similarity to those ob served in human acute pancreatitis [4], In former studies, we introduced a model of isolated pancreatic acinar cells (PACs) to
Received: March 11,1991 Received in revised form: February 10, 1992
simulate initial steps of the pathogenesis of acute pancreatitis [5, 6], Therefore, PACs were incubated in the presence of various pancreatitis-relevant noxae. In contrast to the whole pancreatic tissue and other complex in vitro preparations, this model has the advan tage of all cells being in uniform contact with the noxious agent. Consequently, a kinetic analysis of cell death (in vitro simulation of acinar cell necrosis) is possible.
Dr. Gerald Letko Division of Experimental Surgery Clinic of Surgery. Medical Academy Leipziger Str. 44 D-O-3090 Magdeburg (FRG)
© 1992 S. Kargcr AG, Basel 0012-2823/92 0514-0193S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
G. Letko M. Siech
Incubation, min
Fig. 1 . Time course of acinar cell damage. Isolated PACs ( I mg dry weight = l .5 • 106 cells) were incubated at 37 °C in a shaking bath in Eagle's medium (o) with 0.05 (A). 0.25 (v) or 0.5 |ig PLAr (106 cells)-' (□). An aliquot o f this suspension was added (■) to exclude toxic effects of ammonium sulfate present as a preser vative in the PLAi suspension as a further control after preceding denaturation.
Fig. 2. Influence of ethanol and PLAi on survival of isolated PACs incubated as described in the legend to Figure I . o = Control; • = ethanol ( 180 m.\7); A = PLA; [0.05 pg-(106 cells)-1]; ▲ = PLA: plus ethanol; □ = PLAi [0.5 pig- (106 cells)- '];■ = PLA: plus ethanol.
To continue this approach, we studied the influence of PLA? on the survival of PACs and its possible synergy with ethanol, an im portant etiological factor in acute pancreatitis [7], in the induction of acinar cell damage.
with PLA: and/or ethanol at the beginning of the incu bation. At specified times, samples of incubates were withdrawn, and the number of intact cells was deter mined by trypan blue exclusion. Well-prepared suspen sions had an initial viability of 90-95% and lost less than 10% of this during the 4-hour incubation in the presence of oxygen. Statistical analysis was performed by testing the slope of the regression line of cell viability against time in a modified t test [8].
Materials and Methods
194
Letko/Siech
Results
The typical time course of cell decline of PACs incubated in Eagle’s medium at 37 °C is shown in figure 1. Survival of PACs may be reduced by PLA2 in a dose-dependent man ner. In the presence of 0.25 pg PLAi’OO6 cells)-1, all cells died within less than 4 h of incubation (fig. 1). If isolated PACs were sup plemented with 180 mM ethanol, there was
Survival o f Acinar Cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
Collagenase for isolation of PACs and PLAi (from porcine pancreas; 700 U-(mg protein)-1 were obtained from Bochringer Mannheim. FRG. All other chemi cals were of analytical grade. Female albino laboratory rats (140—180 g) were used in all experiments. The animals were fasted 24 h before the operation and were anesthetized by an intraperitoneal injection of hexobarbital sodium (125 mg/kg body weight). PACs were isolated by a procedure based on collagenase digestion as recently described [6]. The isolated cells were incubated in Eagle’s medium (MEM) and gassed with oxygen at 37°C in a shaking water bath. In the study groups, the cells were supplemented
no measurable effect on cell survival (fig. 2). To study the influence of this ethanol concen tration in conjunction with the action of PLA2 on the viability of PACs. ethanol was com bined with various PLA2 activities. Figure 2 shows the time course of cell decline in a rep resentative experiment. It becomes evident from this plot that the presence of ethanol (180 mM) increased the cell damage induced by PLA2. For statistical analysis, a transfor mation of such plots was performed. The time course observed in the presence of PLA2alone was set to 100% (fig. 3). The differences re sulting from additional applications of etha nol were plotted versus time. The significance of the differences was evaluated by linear regression and statistical analysis. The response of PACs incubated with identical amounts of ethanol differed depend ing on the PLA2 activities applied. While 0.25 pig PLA2-(106 cells)-1 had no significant effect on cell survival, 0.5 ptg PLA2-(106 cells)-1 amplified cell death significantly (p < 0.02).
There is no doubt that PLA2 is involved in the pathogenesis of acute pancreatitis [1,2.9] although to date, there is no satisfactory mechanism to explain the activation of the proenzyme within the gland without the in volvement of pancreatic trypsin [10], PLA2 is a ubiquitous enzyme, and in addition to its role as a pancreatic digestive enzyme, it is a constituent of granulocytes, macrophages, tis sue membranes and organelles [9]. The mem brane-bound enzyme does not need trypsin for activation, however, it is calcium-dependent and involved in the metabolic turnover of membrane phospholipids [11], For exam ple, an increase in intracellular free Ca2+, by enhancement of the membrane permeability to calcium by lipid peroxidation products
Fig. 3. Influence of ethanol and PLA2 on survival o f isolated PACs incubated as described in the legend to figure 1. Independent experiments (n = 5-7) are compiled after transformation of individual time courses. To this aim, the data o f the PLA2 curves were set to 100%, and the data o f the corresponding curves (PLA? plus ethanol) were plotted as deviations from these ‘standardized’ curves. A = Transformation of PLA2 [0.25 pig-(106 cells)-1] curves to 100%: ▲= rela tive deviation ofPLA2 plus ethanol curves (nonsignifi cant); □= transformation ofPLA 2 [0.5 pig-( 10° cells)-1] curves to 100%: ■ = relative deviation of PLA2 plus ethanol curves (p < 0.02).
[ 12], may activate this membrane PLA2 and lead to phospholipid depletion with resulting cell death [11], To study the effect of active PLA2on PACs we incubated these cells (20-106 cells-ml-1 = 12.5 mg dry weight-ml-1) with increasing
195
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
Discussion
196
Letko/Sicch
are in accordance with in vivo findings of aci nar cell damage after intraductal PLA2 instil lation reported by Aho and Nevalainen [4], The amount of porcine pancreas PLA2 [~ 5 pg-(mgdry weight)-1] is comparable with that enzyme activity which led to total cell death within the 4-hour incubation period (fig. 1). Ethanol is increasingly cited as a cause of acute pancreatitis [7], In our experimental studies, it was necessary, however, to com bine ethanol uptake with other factors to induce this disease [6, 15]. Recently, it has been shown that with a combination of tem porary ductal obstruction and stimulation of secretion (induces pancreatic juice edema [16] and a single ethanol administration, the majority of rats will develop signs of acute pancreatitis [15]. In our present study, we used a relatively high concentration of ethanol. Nevertheless, it became evident that ethanol in these con centrations, which alone are not lethal for PACs (fig. 2), may act together under certain circumstances with PLA2 in increasing the rate of cell death (fig. 2, 3). As demonstrated in isolated acini with a spin label, ethanol may potentiate alterations in membrane compo nents (phospholipids) induced by other fac tors [17], Using this method, the increased PLA2 activity has a better access to its sub strates and membrane phospholipids [ 18]. It cannot be excluded that membrane frag ments of damaged cells may serve as donors of lecithin, a substrate of PLA2. But control experiments with low PLA2 activities (not shown) or with ethanol alone (fig. 3) hint at being of minimum contribution to further cell damage. In conclusion, our results demon strate that (i) PLA2 is able to damage PACs without simultaneous presence of additional substrates and (ii) at the appropriate activity of PLA2, ethanol may amplify the dangerous potential of this lytic enzyme and force auto digestion of the pancreatic gland.
Survival o f Acinar Cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
amounts of PLAi (obtained from porcine pancreas). According to Nagai et al. [13], 1 mg of rat pancreas contains 55 pg PLAi, which corresponds to 0.209 pg PLAj-Cmg dry weight)-1. While 0.08 pg PLA2 -(mg dry weight)-1 slightly reduced the viability of PACs, only in the presence of 0.4 pg PLA2 -(mg dry weight)-1 did all cells die within 4 h of incuba tion at 37 °C (fig. 1). This observation shows that PLA2 may damage acinar cells and con firms the original work of Creutzfeldt and Schmidt [3], Together with the older litera ture, our findings contradict the publication of Nagai et al. [13]. In their study with the iso lated rat pancreatic acini, bee venom PLA2 [0.88-88 pg-(mg dry weight)-1] did not seri ously alter the cellular integrity during a 30min incubation, as indicated by the missing release of [35S]methionine-labelcd proteins [ 13], The same was true if the incubates were supplemented with lecithin, a favored sub strate of this enzyme. Hietaranta et al. [14] have performed experiments similar to those of Nagai et al. [13], however, they used hu man PLA2 in their study and aspartylamino transferase as an indicator of cell damage. Their findings were comparable to those of Nagai et al. [13]. The different results obtained in both types of experiments may have resulted from differences in the experimental design. Some of these divergences are: (i) the time of incu bation; (ii) the sources of the enzyme; (iii) the cells with plasma membranes permeable to the dye (molecular weight = 961) in the trypan blue exclusion test were regarded as dead; with respect to the release of macromolecules, as radioactivity-labeled proteins, these cells could still appear to be intact, and (iv) while isolated cells were in uniform contact with PLA2 in acini, the areas of cell-to-cell contacts are excluded from enzymic attack. On the other hand, the results presented in this paper
References 8 Sachs L: Angewandte Statistik. Ber lin. Springer. 1974. 9 Nevalainen TJ: The role of phos pholipase Aj in human acute pan creatitis. Klin Wochenschr 1989;67: 180-182. 10 Lankisch PG: Pathogenesis of pan creatic inflammation: in Galzer G, Ranson JHC (eds): Acute Pancreati tis. Experimental and Clinical As pects o f Pathogenesis and Manage ment. London, Baillière Tindall. 1988, pp 182-193. 11 Färber JL: Membrane injury and calcium homeostasis in pathogene sis o f coagulative necrosis. Lab In vest 1982:47:114-123. 12 Kagan VE. Archipenko YV. Bitov VB. Kozlov YP: Modification o f the enzymatic systems for Ca2t trans port in sarcoplasmic reticulum upon lipid peroxidation. Biochem USSR 1983:48:320-330. 13 Nagai H, Heinrich H. Wünsch PH. Fischbach W, Mössner J: Role of pancreatic enzymes and their sub strates in autodigestion o f the pan creas. In vitro studies with isolated rat pancreatic acini. Gastroenterol ogy 1989:96:838-847.
14 Hietaranta AJ. Laszik ZG, Aho HJ. Kortesuo PJ, Nevalainen TJ: The role o f phospholipase A i in pan creatic acinar cell injury. Int J Pan creatol 1991;8:187-201. 15 Letko G. Nosofsky T, Lessel W. Siech M: Transition o f rat pan creatic juice edema into acute pan creatitis by single ethanol adminis tration. Pathol Res Pract 1991:187: 247-250. 16 Becker V: Speichclddcm und Pankreatitis. Aktuel Chir 1988:23:183— 189. 17 Ponnapa BC. Hoek JB. Waring AJ, Rubin E: Effect o f ethanol on amy lase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol fed rats. Biochem Pharmacol 1987:36:69— 79. 18 Van Kuijk FJGM. Sebanian A, Handelmann FJ, Dratz EA: A new role for phospholipase Ai: Protec tion o f membranes from lipid perox idation damage. Trends Biochem Sci 1987:12:31-33.
197
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:35:47 PM
1 Gram D: Acute necrotising pancre atitis - A role for enterokinasc. Int J Pancreatol 1986; 1:16 7 - 183. 2 Nevalainen TJ: Phospholipase A 2 in acute pancreatitis. Scand J Gas troenterol 1988;23:897-904. 3 Creutzfcldt W. Schmidt H: Aetiol ogy and pathogenesis o f pancreatitis (current concepts). Scand J Gas troenterol I970:5(suppl 6):47-62. 4 Aho HJ, Nevalainen TJ: Experi mental pancreatitis in the rat. Light and electron microscopical observa tions on early pancreatic lesions in duced by intraductal injection of trypsin, phospholipase A 2 , lysolecithin and non-ionic detergent. Vir chows Arch [B] 1982:40:347-356. 5 Letko G, Falkenberg B. Wilhelm W: Effect o f trypsin, chymotrypsin. and uncoupling on survival o f isolated acinar cells from rat pancreas. Int J Pancreatol 1989;4:431-441. 6 Letko G, Spormann H. Sokolowski A, Schulz HU: Pancreatic acinar cells: Isolation, characterization and application in physiologic and pathophysiologic studies, with spe cial reference to acute pancreatitis. Exp Pathol 1988:34:10-22. 7 Singh M. Simsek H: Ethanol and the pancreas. Current status. Gastroen terology 1990:98:1051-1062.
