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Experimental
INTERACTION ACTION
Cell Research 118 (1979) 171-180
OF VINBLASTINE
WITH
THE SECRETAGOGUE
OF db-CAMP IN THE RAT EXOCRINE J. F. LAUNAY,
PANCREAS
C. STOCK and J. F. GRENIER
Unite de Recherches 61 de I’INSERM de Biophysiopathologie de Plntestin, Strasbourg, France
SUMMARY It has been shown that vinblastine (VB) potentiates the secretagogue action of dibutyryl cyclic adenosine-3’,5’-monophosphate (db-CAMP) in the rat exocrine pancreas. The present work determined the step of the secretory process involved in this potentiation. To this effect, rat pancreatic fragments were pulse-labelled with L-[U-‘4C]leucine, then transferred to a chase medium. Intracellular transport of synthesized proteins from ribosomes into zymogen granules was followed by determining the specific radioactivity of proteins in subcellular fractions. Discharge of exportable proteins was studied either during the migration of proteins or after the initially synthesized proteins had reached the secretory granule population. These studies indicated that VB (5x 10m5M) diminished the rate of transport of new synthesized proteins, but stimulated the db-CAMP (2 mM)-induced enzyme release by initiating the preferential discharge of old unlabelled proteins located around the acinar lumen.
It is generally agreed that dibutyryl cyclic ticulum and are then transported, stepwise, adenosine-3’ ,5’-monophosphate (db-CAMP) from the lumen of this reticulum to the Golstimulates the discharge of exportable pro- gi complex. Finally, Golgi-derived vesicles teins in the rat exocrine pancreas [l-5]. In a that contain secretory proteins migrate to previous work [6], we have shown that this the apical cell membrane, fuse with it and effect is reversible, dose-related and spe- empty their content into the acinar lumen. The present investigation was undertaken cific. It results from the direct interaction of the nucleotide derivative with the pan- to determine the step of this secretory cycle creatic acinar cell. However, the secreta- at which VB potentiates the secretagogue gogue action of db-CAMP is low (2-4 times action of db-CAMP in the rat pancreas in the basal secretion) compared with that of vitro. The results show that VB diminishes digestive hormones and cholinergic agents the rate of intracellular transport of ex(5-7 times the basal secretion) [l, 61. It portable proteins but potentiates the dbcould be enhanced by vinblastine (VB), an CAMP-induced secretion of enzymes by agent known to disrupt microtubules, but stimulating the discharge of the most apical which does not induce any damage of the located old granules into the acinar lumen. pancreatic acinar cell [7]. Exportable proteins in the pancreas are MATERIALS AND METHODS synthesized on polysomes attached to the Male Wistar rats weighing 250-300 g were starved membranes of the rough endoplasmic re- overnight, then killed by decapitation. The pancreases E.ip
Cdl
RPS I18 11979)
were quickly removed and placed in an ice-cold buffer solution. The tissue was trimmed free of adherent fat and lymphatic nodules and cut into 8-10 pieces. The incubation was performed at 37°C in a Krebs-Ringer bicarbonate medium supplemented with o-glucose 10 mM and the amino acid mixture of Campagne & Gruber as previously described [8]. The gas phase was 0,95 %, CO, 5 %.
Pasteur pipette. filtered through I IO-mesh nylon cloth and transferred to round-bottomed 5 ml tubes which were centrifuged for 5 min at 1000 g,,,,,, to yield a tightly packed white pellet of zymogen granules. These latter were fixed for electron microscopy or resuspended in 0.2 M NaHCO,,. pH 7.4. for biochemical and radiochemical assays.
Radiochemical Incubation
procedures
In order to distinguish the three major steps of the secretory cycle, i.e. protein synthesis, intracellular transport and discharge of labelled proteins, the following experimental procedures have been elaborated. Protein synthesis. After a 10 min preincubation in standard conditions, the pancreatic fragments were incubated for 90 min in presence of L-[U-‘“Clleucine 0.5 &i/ml (320 mCi/mmole) supplemented with 2 mM db-CAMP, and/or 5 x 1OWM VB. Protein svnthesis was estimated by measuring the incorporation of labelled leucine into TCA-precipitable material of the whole tissue homogenate. Intracellular transport. After a IO min preincubation in standard conditions, the pancreatic fragments were pulse-labelled with 1 &i/ml (320 mCi/mmole) L-[UY]leucine for 4 min. They were then transferred to a chase medium containing cold leucine (0.8 mM) with or without 2 mM db-CAMP and/or 5 X IOWM VB. After a 20, 40 or 60 min ‘chase incubation’, the fragments were homogenized in 4 ml of 0.3 M sucrose and processed for isolation of subcellular fractions. Dischurge ofzymogen grunules. After a 10 min preincubation in standard conditions, the pancreatic fragments were pulse-labelled with I &i/ml (320 mCi/ mmole) L-[U-Y]leucine for 4 min. They were then transferred to a chase medium containing cold leucine (0.8 mM). In the first experimental procedure, 2 mM and/or SX 10e5M db-CAMP were added to the medium at time zero post-pulse; in the second, the fragments were incubated for 60 min in a chase medium, then transferred to their respective flasks containing db-CAMP and/or VB. After a 60 min final incubation, the pancreatic fragments were briefly washed and homogenized in 5 ml distilled water. Secretion, expressed as percent of total release from the tissue into the medium, was estimated by the determination of radioactive protein, amylase and chymotrypsinogen activities.
Isolation
of subcellular fractions
assa~.~
Samples from incubation medium, homogenates and subcellular fractions were precipitated at 4°C with 10% TCA overnight and washed twice with S% TCA. The final pellet was dissolved with 0.3 ml Soluene-350 overnight: the activitv was counted in a Packard Tricarb liquid scintillati& counter (model 3375) using the scintillation mixture Omnifluor in toluene 4 n/l (10 ml/ flask).
Biochemical
assays
Samples from incubation medium, homogenates and subcellular fractions were used for the determination of proteins, amylase and chymotrypsinogen activities by the respective methods of Lowry et al. [I I], Danielsson [12] and Reboud et al. [13]. The purity of zymogen granule and microsomal fractions was analysed by several specific markers: glucose-6-phosphatase [ 141,5’-nucleotidase [ 151,succinate dehydrogenase [l6], acid phosphatase [I71 and RNA [l8, 191.
Electron microscopy Pellets of zymogen granules and microsomes were fixed at 4°C respectively with I % osmium tetroxide in 0.3 M sucrose (unbuffered, pH -5.5) and 17~ osmium tetroxide in 0.2 M cacodvlate buffer (pH 7.4). Pancreatic fragments were fixed it room temperature in 2% glutaraldehyde buffered with 0.2 M sodium cacodylate (pH 7.4) and post-fixed with I % osmium tetroxide in the same buffer. Then pellets and pancreatic fragments were dehydrated in a graded sequence of alcohols and embedded in araldite or an Epon-Araldite mixture. Thin sections were cut with a Reichert OMU3 ultramicrotome, doubly stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope.
Statistical
anulysis
Results were expressed as the mean ? standard error of the mean. The Student’s r-tests for paired data were used to determine differences between db-CAMP- or VB-treated samples and their respective controls.
Four ml of samples of a tissue homogenate were centrifuged in I2 ml glass conical tubes for 12 min at 10°0 gmax in a Jouan centrifuge model E 95. The mitochondrial and microsomal fractions were isolated from Chemicals the suoematant according to Jamieson & Palade 191. L-[U-‘“Clleucine was obtained from CEA (Saclay), the The zymogen granule fraztion was prepared from iGe label purity was greater than 98.5%. db-CAMP was pellets ad modum Meldolesi et al. [IO] as modified by purchased from Boehringer Pharma (Mannheim). Vinus. After the removal of the supernatant, the pellets blastine was a generous gift from Eli Lilly (St Cloud). were carefully resuspended by means of a Teflon rod Omnifluor was purchased from New England Nuclear in 5 ml of 0.3 M sucrose; the suspension was centri(Frankfurt), N-Acetyl-tyrosine-ethyl ester (ATEE) fuged at 180 g,,, for 15 min to sediment all debris, and nicotinamide-adenine dinucleotide (NADH) were erythrocytes, nuclei and plasma membranes. The re- from Sigma (St Louis, MO). All other chemicals were sulting supematant (4 ml) was removed by means of a Merck products, analytical grade. E.rp Cdl
RP.\ 118 tlY7Y)
Pancreatic
Table 1. Chemistry and enzymes activities
of total homogenate,
enzymes secretion
173
zymogen granules and
microsomes prepared from rat pancreas Values given are averages. Number of experiments is shown in parentheses. Ranges are in italics. The table represents: amylase, pmoles maltose released/min at 25°C; chymotrypsin, pmoles ATEE split/min at 25°C; lipase, PEq fatty acid released/min at 25°C; succinate dehydrogenase, nmoles NADH oxidized/min at 25°C; Y-nucleotidase, pmoles Pi released/l5 min at 3PC; glucose-6-phosphatase, pmoles Pi released/l5 min at 37% acid phosphatase, pmoles paranitrophenol released/l5 min at 37°C; the values are normalized per mg protein as indicated
Protein, mg/g tissue wet cY-Amylase, pmoles/mg protein Chymotrypsinogen, pmoles/mg protein Lipase, pEq/mg protein Succinate dehydrogenase, nmoles/mg protein 5’-Nucleotidase. pmoles/mg protein Glucose-6-phosphatase, pmoles/mg protein RNA, pg/mg protein Acid phosphatase, pmoles/mg protein
Total homogenate
granules
Microsomes
176.0 (20) 162-190 71.o (20) 55-87 72.0 (20) 56-88 38.0 (20) 35-41 4.3 (5) 3.4-5.2 0.19 (5) 0.17-0.21 0.17 (12) 0.15-0.19 138.0 (8) 133-143 0.27 (5) 0.25-0.29
3.4 (20) 2.5d.3 158.0(20) 120-196 166.0 (20) 145-197 95.o (20) 78-I I2 I .4 (5) I .2-1.6 0.05 (5) 0.04-0.06 0.08 (11) 0.06-0.010 16.0 (8) 15-17 0.015 (5) 0.01-0.02
2 1.o (20) 19-23 55.o (20) 47-63 13.0 (20) I l-15 22.o (20) 20-24 1.1 (5) 0.9-1.3 0.65 (5) 0.60-0.70 0.55 (11) 0.52-0.58 221.0 (8) 215-227 0.030 (5) 0.02-0.04
Zymogen
necessary to isolate these granules as well as microsomes in relatively clean cell fracProtein synthesis tions. After a 90 min incubation of pancreatic fragPurity of subcellular fractions. After subments with L-[U-l”C]leucine, protein syncellular fractionation of pancreatic glands, thesis was estimated by the ratio of labelled the degree of purity of both zymogen granleucine incorporated into TCA-precipitable ule and microsomal fractions has been dematerial to that incorporated into the whole fined by biochemical methods and electron tissue homogenate. This ratio was 102.3? microscopy. 2.4% of control (n =7) in 2 mM db-CAMP Table 1 shows that the zymogen granule treated tissue. It was respectively 87.2_+ fraction contains digestive enzymes in 2.6% (n=7) and 89.4&3.4% (n=7) in higher concentration than the starting ho5~ 10d5 M VB-treated tissues in the presmogenate: specific activity figures calence and in the absence of 2 mM db-CAMP. culated on a protein basis show for inThus VB inhibits slightly (but significantly, stance 2.2-, 2.3-, and 25fold increases in p
Experimental
INTERACTION ACTION
Cell Research 118 (1979) 171-180
OF VINBLASTINE
WITH
THE SECRETAGOGUE
OF db-CAMP IN THE RAT EXOCRINE J. F. LAUNAY,
PANCREAS
C. STOCK and J. F. GRENIER
Unite de Recherches 61 de I’INSERM de Biophysiopathologie de Plntestin, Strasbourg, France
SUMMARY It has been shown that vinblastine (VB) potentiates the secretagogue action of dibutyryl cyclic adenosine-3’,5’-monophosphate (db-CAMP) in the rat exocrine pancreas. The present work determined the step of the secretory process involved in this potentiation. To this effect, rat pancreatic fragments were pulse-labelled with L-[U-‘4C]leucine, then transferred to a chase medium. Intracellular transport of synthesized proteins from ribosomes into zymogen granules was followed by determining the specific radioactivity of proteins in subcellular fractions. Discharge of exportable proteins was studied either during the migration of proteins or after the initially synthesized proteins had reached the secretory granule population. These studies indicated that VB (5x 10m5M) diminished the rate of transport of new synthesized proteins, but stimulated the db-CAMP (2 mM)-induced enzyme release by initiating the preferential discharge of old unlabelled proteins located around the acinar lumen.
It is generally agreed that dibutyryl cyclic ticulum and are then transported, stepwise, adenosine-3’ ,5’-monophosphate (db-CAMP) from the lumen of this reticulum to the Golstimulates the discharge of exportable pro- gi complex. Finally, Golgi-derived vesicles teins in the rat exocrine pancreas [l-5]. In a that contain secretory proteins migrate to previous work [6], we have shown that this the apical cell membrane, fuse with it and effect is reversible, dose-related and spe- empty their content into the acinar lumen. The present investigation was undertaken cific. It results from the direct interaction of the nucleotide derivative with the pan- to determine the step of this secretory cycle creatic acinar cell. However, the secreta- at which VB potentiates the secretagogue gogue action of db-CAMP is low (2-4 times action of db-CAMP in the rat pancreas in the basal secretion) compared with that of vitro. The results show that VB diminishes digestive hormones and cholinergic agents the rate of intracellular transport of ex(5-7 times the basal secretion) [l, 61. It portable proteins but potentiates the dbcould be enhanced by vinblastine (VB), an CAMP-induced secretion of enzymes by agent known to disrupt microtubules, but stimulating the discharge of the most apical which does not induce any damage of the located old granules into the acinar lumen. pancreatic acinar cell [7]. Exportable proteins in the pancreas are MATERIALS AND METHODS synthesized on polysomes attached to the Male Wistar rats weighing 250-300 g were starved membranes of the rough endoplasmic re- overnight, then killed by decapitation. The pancreases E.ip
Cdl
RPS I18 11979)
were quickly removed and placed in an ice-cold buffer solution. The tissue was trimmed free of adherent fat and lymphatic nodules and cut into 8-10 pieces. The incubation was performed at 37°C in a Krebs-Ringer bicarbonate medium supplemented with o-glucose 10 mM and the amino acid mixture of Campagne & Gruber as previously described [8]. The gas phase was 0,95 %, CO, 5 %.
Pasteur pipette. filtered through I IO-mesh nylon cloth and transferred to round-bottomed 5 ml tubes which were centrifuged for 5 min at 1000 g,,,,,, to yield a tightly packed white pellet of zymogen granules. These latter were fixed for electron microscopy or resuspended in 0.2 M NaHCO,,. pH 7.4. for biochemical and radiochemical assays.
Radiochemical Incubation
procedures
In order to distinguish the three major steps of the secretory cycle, i.e. protein synthesis, intracellular transport and discharge of labelled proteins, the following experimental procedures have been elaborated. Protein synthesis. After a 10 min preincubation in standard conditions, the pancreatic fragments were incubated for 90 min in presence of L-[U-‘“Clleucine 0.5 &i/ml (320 mCi/mmole) supplemented with 2 mM db-CAMP, and/or 5 x 1OWM VB. Protein svnthesis was estimated by measuring the incorporation of labelled leucine into TCA-precipitable material of the whole tissue homogenate. Intracellular transport. After a IO min preincubation in standard conditions, the pancreatic fragments were pulse-labelled with 1 &i/ml (320 mCi/mmole) L-[UY]leucine for 4 min. They were then transferred to a chase medium containing cold leucine (0.8 mM) with or without 2 mM db-CAMP and/or 5 X IOWM VB. After a 20, 40 or 60 min ‘chase incubation’, the fragments were homogenized in 4 ml of 0.3 M sucrose and processed for isolation of subcellular fractions. Dischurge ofzymogen grunules. After a 10 min preincubation in standard conditions, the pancreatic fragments were pulse-labelled with I &i/ml (320 mCi/ mmole) L-[U-Y]leucine for 4 min. They were then transferred to a chase medium containing cold leucine (0.8 mM). In the first experimental procedure, 2 mM and/or SX 10e5M db-CAMP were added to the medium at time zero post-pulse; in the second, the fragments were incubated for 60 min in a chase medium, then transferred to their respective flasks containing db-CAMP and/or VB. After a 60 min final incubation, the pancreatic fragments were briefly washed and homogenized in 5 ml distilled water. Secretion, expressed as percent of total release from the tissue into the medium, was estimated by the determination of radioactive protein, amylase and chymotrypsinogen activities.
Isolation
of subcellular fractions
assa~.~
Samples from incubation medium, homogenates and subcellular fractions were precipitated at 4°C with 10% TCA overnight and washed twice with S% TCA. The final pellet was dissolved with 0.3 ml Soluene-350 overnight: the activitv was counted in a Packard Tricarb liquid scintillati& counter (model 3375) using the scintillation mixture Omnifluor in toluene 4 n/l (10 ml/ flask).
Biochemical
assays
Samples from incubation medium, homogenates and subcellular fractions were used for the determination of proteins, amylase and chymotrypsinogen activities by the respective methods of Lowry et al. [I I], Danielsson [12] and Reboud et al. [13]. The purity of zymogen granule and microsomal fractions was analysed by several specific markers: glucose-6-phosphatase [ 141,5’-nucleotidase [ 151,succinate dehydrogenase [l6], acid phosphatase [I71 and RNA [l8, 191.
Electron microscopy Pellets of zymogen granules and microsomes were fixed at 4°C respectively with I % osmium tetroxide in 0.3 M sucrose (unbuffered, pH -5.5) and 17~ osmium tetroxide in 0.2 M cacodvlate buffer (pH 7.4). Pancreatic fragments were fixed it room temperature in 2% glutaraldehyde buffered with 0.2 M sodium cacodylate (pH 7.4) and post-fixed with I % osmium tetroxide in the same buffer. Then pellets and pancreatic fragments were dehydrated in a graded sequence of alcohols and embedded in araldite or an Epon-Araldite mixture. Thin sections were cut with a Reichert OMU3 ultramicrotome, doubly stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope.
Statistical
anulysis
Results were expressed as the mean ? standard error of the mean. The Student’s r-tests for paired data were used to determine differences between db-CAMP- or VB-treated samples and their respective controls.
Four ml of samples of a tissue homogenate were centrifuged in I2 ml glass conical tubes for 12 min at 10°0 gmax in a Jouan centrifuge model E 95. The mitochondrial and microsomal fractions were isolated from Chemicals the suoematant according to Jamieson & Palade 191. L-[U-‘“Clleucine was obtained from CEA (Saclay), the The zymogen granule fraztion was prepared from iGe label purity was greater than 98.5%. db-CAMP was pellets ad modum Meldolesi et al. [IO] as modified by purchased from Boehringer Pharma (Mannheim). Vinus. After the removal of the supernatant, the pellets blastine was a generous gift from Eli Lilly (St Cloud). were carefully resuspended by means of a Teflon rod Omnifluor was purchased from New England Nuclear in 5 ml of 0.3 M sucrose; the suspension was centri(Frankfurt), N-Acetyl-tyrosine-ethyl ester (ATEE) fuged at 180 g,,, for 15 min to sediment all debris, and nicotinamide-adenine dinucleotide (NADH) were erythrocytes, nuclei and plasma membranes. The re- from Sigma (St Louis, MO). All other chemicals were sulting supematant (4 ml) was removed by means of a Merck products, analytical grade. E.rp Cdl
RP.\ 118 tlY7Y)
Pancreatic
Table 1. Chemistry and enzymes activities
of total homogenate,
enzymes secretion
173
zymogen granules and
microsomes prepared from rat pancreas Values given are averages. Number of experiments is shown in parentheses. Ranges are in italics. The table represents: amylase, pmoles maltose released/min at 25°C; chymotrypsin, pmoles ATEE split/min at 25°C; lipase, PEq fatty acid released/min at 25°C; succinate dehydrogenase, nmoles NADH oxidized/min at 25°C; Y-nucleotidase, pmoles Pi released/l5 min at 3PC; glucose-6-phosphatase, pmoles Pi released/l5 min at 37% acid phosphatase, pmoles paranitrophenol released/l5 min at 37°C; the values are normalized per mg protein as indicated
Protein, mg/g tissue wet cY-Amylase, pmoles/mg protein Chymotrypsinogen, pmoles/mg protein Lipase, pEq/mg protein Succinate dehydrogenase, nmoles/mg protein 5’-Nucleotidase. pmoles/mg protein Glucose-6-phosphatase, pmoles/mg protein RNA, pg/mg protein Acid phosphatase, pmoles/mg protein
Total homogenate
granules
Microsomes
176.0 (20) 162-190 71.o (20) 55-87 72.0 (20) 56-88 38.0 (20) 35-41 4.3 (5) 3.4-5.2 0.19 (5) 0.17-0.21 0.17 (12) 0.15-0.19 138.0 (8) 133-143 0.27 (5) 0.25-0.29
3.4 (20) 2.5d.3 158.0(20) 120-196 166.0 (20) 145-197 95.o (20) 78-I I2 I .4 (5) I .2-1.6 0.05 (5) 0.04-0.06 0.08 (11) 0.06-0.010 16.0 (8) 15-17 0.015 (5) 0.01-0.02
2 1.o (20) 19-23 55.o (20) 47-63 13.0 (20) I l-15 22.o (20) 20-24 1.1 (5) 0.9-1.3 0.65 (5) 0.60-0.70 0.55 (11) 0.52-0.58 221.0 (8) 215-227 0.030 (5) 0.02-0.04
Zymogen
necessary to isolate these granules as well as microsomes in relatively clean cell fracProtein synthesis tions. After a 90 min incubation of pancreatic fragPurity of subcellular fractions. After subments with L-[U-l”C]leucine, protein syncellular fractionation of pancreatic glands, thesis was estimated by the ratio of labelled the degree of purity of both zymogen granleucine incorporated into TCA-precipitable ule and microsomal fractions has been dematerial to that incorporated into the whole fined by biochemical methods and electron tissue homogenate. This ratio was 102.3? microscopy. 2.4% of control (n =7) in 2 mM db-CAMP Table 1 shows that the zymogen granule treated tissue. It was respectively 87.2_+ fraction contains digestive enzymes in 2.6% (n=7) and 89.4&3.4% (n=7) in higher concentration than the starting ho5~ 10d5 M VB-treated tissues in the presmogenate: specific activity figures calence and in the absence of 2 mM db-CAMP. culated on a protein basis show for inThus VB inhibits slightly (but significantly, stance 2.2-, 2.3-, and 25fold increases in p
