Cell membrane resorption in the rat exocrine pancreas cell after in vivo stimulation of the secretion, as studied by in vitro incubation with extracellular space markers.

Pancreatic secretion in the rat was stimulated in vivo by pilocarpine injection causing 90% of the storage granules to be discharged within 2 h. Incubation in vitro with [(14)C]sorbitol indicated that maximal ingestion of this extracellular space marker occurred 3 h after secretogogue injection. Morphological cell membrane measurements on cells with stimulated secretion revealed a simultaneous decrease in amount of membrane bordering the microvilli at the cell apex, lamellar processes, and infoldings present at the latero-basal face of these cells. In 3-h stimulated cells, having the average zymogen granule content characteristic for that phase of secretion, ferritin treatment in vitro showed that the infoldings and related fragmentation vesicles had ingested ferritin and could consequently be considered as being transport vehicles for redundant cell membrane. During stimulated secretion numerous vesicles and vacuoles appeared in the apical cytoplasm. Part of these structures were postulated to be related to the Golgi complex and were discussed in relation to secretory protein transport. Another part of these structures was assumed to have an endocytotic nature, although they never contained ferritin.

Unstimulated secretion of protein from rat exocrine pancreas cells.

Our earlier work demonstrated that the rate of protein synthesis in the exocrine cells of the rat pancreas is constant in different physiological states, including prolonged fasting. In this study we have followed the fate of the protein in the pancreatic cells of the fasting animal in vivo as well as in vitro. The data were obtained by quantitative radioautography and by biochemical determinations. In nonanesthesized, fasting rats, without cannulated pancreatic duct, some 80% of the proteins synthesized at a given time leaves the cell within 12 hr by way of secretion, intracellular breakdown not being important. Two mechanisms of fasting secretion exist. The first, starting at a slow rate after 20 min, is inferred to result from fortuitous contacts of young secretory granules with the apical cell membrane. The rate of secretion is the same in vivo as in vitro, at least during the first 4 hr after pulse labeling. Within 7 hr about 20% of the total amount of newly synthesized protein has left the cell. The second mechanism consists of an orderly movement of the mass of secretory granules towards the apical cell membrane as caused by the continuous assembly of new granules. The granules that come into contact with the cell membrane are discharged. It takes about 7-12 hr for secretory protein transported in this way to reach the cell membrane. The addition of new secretory granules to those present is essential for the second mechanism, for the blockade of protein synthesis by cycloheximide decreases the rate of this phase of secretion without interfering with the secretory process proper. Atropin does not inhibit the fasting secretion in vitro, nor does extensive washing of the tissue slices, excluding possible secretagogues as important factors in fasting secretion.

Non-parallel secretion of newly synthesized rat pancreatic proteins after feeding a diet containing raw soybean flour.

The relative rate of secretion of rat pancreatic proteins was studied in vivo using a double label method. Rats were injected with [3H]leucine and after different time intervals with [14C]leucine. At a fixed time after administration of the second precursor the animals were killed, and the pancreatic proteins were separated by polyacrylamide gel electrophoresis. The dpm of tritium to dpm of 14C ratio of several identified enzymes was assessed. The percentage secretion of a newly synthesized secretory protein was derived from the difference between the actual 3H/14C ratio and the 3H/14C ratio that was found for non-secretory proteins. In pancreata of rats fed with a standard diet several identified proteins, viz. three trypsinogens, chymotrypsinogen and three amylases were secreted in "parallel". When a diet containing raw soybean flour was fed, the secretory pattern for the amylases differed from that of the other proteins.

Effect of diet composition on the protein synthetic pattern of the rat pancreas after a feeding period of five days.

Rats were fed for five days on a protein-rich and on a carbohydrate-rich diet, respectively. One half of the pancreas of these rats was incubated with [3H]leucine and the other half with [14C]leucine and extracts from these pancreas halves were prepared. Mixtures of the differently labeled extracts were subjected to electrophoresis towards the anode as well as towards the cathode on a polyacrylamide gel containing urea at pH 8.5. Several secretory enzymes could be identified on the gels. Along the gels the 3H : 14C ratio was determined in 1 mm slices. The results show that after five days of feeding a diet there is some adaptation to diet composition. Generally rather small changes in synthetic rate occur. Only one component, the cathodic chymotrypsinogen shows an important difference in synthetic rate under the two circumstances.

Fate of the major zymogen granule membrane-associated glycoproteins from rat pancreas. A biochemical and immunocytochemical study.

A glycoprotein resembling the major zymogen granules membrane-associated glycoprotein (GP-2) from the exocrine rat pancreas was detected in a 200 000 g sedimentable subfraction from pancreatic secretion. This glycoprotein had a slightly smaller molecular weight than GP-2 (70 000 D versus 75 000 D), gave a line of identity with GP-2 in a double immunodiffusion test against anti GP-2, and tryptic peptide charts of both glycoproteins were largely similar. Immunofluorescence cytochemistry showed that GP-2 antigens were exclusively located in the exocrine pancreas, where they were preferentially found in the perimeter of secretory granules and in the acinar lumina. These results suggest the possibility of loss of GP-2 from exocrine cells during secretion and raise doubts as to the status of GP-2 as a true membrane glycoprotein.

In situ localization of galactosyltransferase in surface mucous cells of the rat stomach.

Cryostate sections of the rat stomach fundus were incubated in the presence of uridine 5'-diphosphate-[3H]-galactose. The radioactivity in the surface mucous epithelial cells was shown by autoradiography to be specifically incorporated into a supranuclear area, the area where in these cells the Glogi system is situated. The incorporation lasted only 5-6 min and was Mn++ dependent. Galactose was probably incorporated into a beta-glycosidic bond.

Changes in rat pancreatic protein synthesis after a single feeding with diets containing raw or heated soybeans.

The patterns of protein synthesis following the administration of a single meal containing defatted ground soybeans (RSD) or heated defatted ground soybeans (HSD) were compared. A double label method was used so that the determination of the relative rate of synthesis of an enzyme could not be obscured by a possible alteration of the activity or quantity of the enzyme. Rats were given one meal of RSD or HSD and were subsequently killed at different times after the meal. Eight hours after the meal, the relative rate of synthesis of one of the three trypsinogens was substantially increased with RSD feeding, whereas that of the amylases and one chymotrypsinogen were somewhat lower. The synthetic rate of lipase, ribonclease, proelastase, another chymotrypsinogen and of two trypsinogens was unaffected when feeding RSD is compared to feeding HSD. The relative rate of synthesis of one of the trypsinogens was unaffected 8 hours after RSD feeding, but was increased 16 hours after RSD feeding. Actinomycin D could suppress the effects of RSD feeding on the protein synthetic rate of some, but not of all, secretory proteins.

Inactivation of rat pancreas amylase during electrophoresis in the presence or absence of urea.

Rat pancreas amylase activity was irreversibly destroyed when subjected to electrophoresis. The measure of enzyme inactivation was dependent on the duration of electrophoresis. In the presence of urea the amylase denaturating effect was enhanced.

Circadian rhythm of protein synthesis activity in the exocrine pancreas of fed and starved rats.

Rats, either fed a solid standard chow ad libitum or starved for 24 h prior to the beginning of the experiments, were kept in a normal light-dark cycle of 12 h. At certain times over a total period of 24 h, three animals of each group were killed, their pancreata were excised and the protein synthetic capacity was determined, measuring both the amount of cellular RNA and the percentage of ribosomes active in protein synthesis. A clear-cut circadian rhythm in the synthetic activity was found, well expressed in the fed animals but less evident and shifted in time in the starved ones. The RNA/DNA ratios in the two sets of animals remained fairly constant throughout the day, but were somewhat lower in the fasted rats.

Secretion of newly synthesized proteins by isolated liver cells.

Suspensions of isolated liver cells were incubated with [3H]leucine as a precursor. The appearance of radioactive albumin and alpha 1-acid glycoprotein in the incubation medium was determined. After about 20 min both albumin and alpha 1-acid glycoprotein were secreted in a linear way. When fucose and galactose were used as radioactive precursors, time lags of about 10 min could be observed.

Effect of feeding diets of different composition on the protein synthetic pattern of the rat pancreas.

Rats were fed a protein-rich or carbohydrate-rich diet for 3 weeks. By means of a double-label technique, we compared the protein-synthesizing patterns under these conditions. After 3 weeks of feeding, the relative rates of all the identified secretory enzymes and of some unidentified components had changed. The synthetic rates of the proteolytic proenzymes and of ribonuclease were increased after feeding a protein-rich diet, whereas those of the amylases and of lipase were lowered. The protein synthetic patterns of the rat pancreas under two extreme feeding conditions were compared to that of the pancreas of rats constantly fed a balanced diet of an intermediate composition. The results indicated that changes in relative rate of synthesis are mainly provoked by the protein-rich diet. Only the synthetic rate of the anodic chymotrypsinogen was strongly influenced by the carbohydrate-rich diet.

The proportion of ribosomes engaged in rat pancreatic polysomal structures as influenced by pilocarpine injection.

Rats intravenously injected with pilocarpine at a concentration of 4 mg per 100 g body weight were killed after various time intervals and the relative numbers of ribosomes per cell active in protein synthesis were determined. It was found that until 1 h after pilocarpine injection there is a significant decrease in the number of ribosomes engaged in protein synthesis, whereas 2--3 h after pilocarpine injection, the number of active ribosomes equals that of control animals. After a short incubation in vitro, the same relative number of active ribosomes is always found, independent of the value determined in the pancreas directly after the death of the animal.

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.

Intracellular transport of the major glycoprotein of zymogen granule membranes in the rat pancreas. Demonstration of high turnover at the plasma membrane.

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP-2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse-chase experiments were performed. The incorporation of the first newly synthesized GP-2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP-2 reaches the cell surface within the same time span. After 6-8 h chase considerable more newly synthesized GP-2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP-2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane. GP-2 is the only protein that appears in discernable quantity in the plasma membrane during 1-4 h after a pulse label. Nevertheless GP-2 comprises only a small percentage of externally 125I-iodinated plasma membrane proteins. We conclude that GP-2 has a high turnover rate at the plasma membrane level. Treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.

Biosynthesis of the major glycoprotein associated with zymogen-granule membranes in the pancreas.

The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.