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Anatomy↗
Biomedical subjects
A R Beaudoin
Publications and source records attributed to A R Beaudoin.
Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.
The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.
Animals↗
Shedding of vesicular material from the cell surface of eukaryotic cells: different cellular phenomena.
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Animals↗
Identification and localization of ATP-diphosphohydrolase (apyrase) in bovine aorta: relevance to vascular tone and platelet aggregation.
In this work, we confirm the existence of an ATP-diphosphohydrolase (apyrase) in bovine aorta and we show that its properties are different from the previously described pancreas ATP-diphosphohydrolase. Hence the aorta enzyme should be considered as a novel type of apyrase. The demonstration is based on pH dependency profiles, heat denaturation curves, 60Co irradiation-inactivation curves and enzyme localization after polyacrylamide gel electrophoresis under non-denaturing conditions. In addition, the irradiation-inactivation curves clearly showed that for both pancreas and aorta enzymes preparations, the same catalytic site is responsible for the hydrolysis of ATP and ADP. The molecular masses of enzymes calculated with this method are 132 +/- 19 kDa (mean +/- S.D.) and 189 +/- 30 kDa (mean +/- S.D.) for the pancreas and aorta enzymes, respectively. Preliminary observations on isolated bovine brain capillaries revealed a high level of enzyme activity strongly suggesting that an ATP-diphosphohydrolase is associated with endothelial cells. The presence of the enzyme on this type of cells was confirmed with pulmonary endothelial cells in culture. Considering the high proportions of smooth muscle cells relative to endothelial cells and the high level of enzyme activity in the aorta preparation, an ATP-diphosphohydrolase activity is definitely present in smooth muscle cells. The ATP-diphosphohydrolase activities described above could regulate the relative concentrations of purine nucleotides both in the plasma and within the vascular wall and hence could play a role both in platelet aggregation and in the control of vascular tone.
Animals↗
Endothelin-1 induces prostacyclin release from bovine aortic endothelial cells.
The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.
6-Ketoprostaglandin F1 alpha↗
Secretory pathways in animal cells: with emphasis on pancreatic acinar cells.
Studies over the past three decades have clearly established the existence of at least two distinct pathways for the intracellular transport and release of secretory proteins by animal cells. These have been identified as the regulated and constitutive pathways. Many observations have indicated that in certain cells, such as those of the exocrine pancreas and parotid glands at least, these pathways coexist in the same cells. Although the general scheme of protein transport within these pathways is well established, many fundamental aspects of intracellular transport remain to be unraveled. How are proteins transported through the endoplasmic reticulum? How are the transitional vesicles formed and what are the underlying mechanisms involved in their fusion with the cis-Golgi cisterna? Even the general mode of transfer through the Golgi stack is debated: Is there a diffusion through the stack by flow through intercisternal tubules and openings or is there a vesicle transfer system where membrane quanta hop from one cisterna to the other? What is the fate of secretory proteins in the trans-Golgi area and by what mechanisms is a fraction of newly synthesized molecules of a given secretory protein released spontaneously while the majority of such nascent molecules are diverted into a secretory granule compartment? In this review, we have examined these and other aspects of intracellular transport of secretory proteins using pancreatic acinar cells as our reference model and we present some evidence to support the existence of a paragranular pathway of secretion associated with secretory granule maturation.
Animals↗
Alterations of gamma-glutamyltranspeptidase associated with human pancreatic cancer.
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Humans↗
Ultrastructural localization of GP2 in acinar cells of pancreas: presence of GP2 in endocytic and exocytic compartments.
GP2 is a glycoprotein found in pancreatic acinar cells. Its subcellular distribution suggests that it may be involved both in exocytosis and endocytosis. Immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of zymogen granules, on Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the lumina of acini. In addition, this protein was observed in small vacuoles and tubular structures previously identified as "basal lysosomes," "snake-like tubules," and in lysosomes. Because the latter group of structures are involved in endocytosis, it is possible that GP2 may be involved in this phenomenon. GP2 was readily detectable in pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated zymogen granules also caused release of GP2 in a sedimentable form which, by electron microscopy, appeared as a fibrillar structure. Immunocytochemical localization of amylase was studied in parallel with GP2 and was found in the secretory product to be associated with thread-like structures, presumably the pancreatic thread protein. The physiological significance of these observations is discussed.
Animals↗
Marked differences in immunocytological localization of [3H]estradiol-binding protein in rat pancreatic acinar tumor cells compared to normal acinar cells.
[3H]Estradiol can bind to a specific protein in normal rat pancreatic acinar cells. Electron microscopic immunocytochemical analysis has shown this protein to be localized primarily in the rough endoplasmic reticulum and mitochondria. Rat exocrine pancreatic tumor cell lines, whether grown in tissue culture (AR42J) or as a tumor mass after sc injection into rats (DSL-2), lacked detectable amounts of this [3H]estradiol-binding protein (EBP), as determined by the dextran-coated charcoal assay. Furthermore, primary exocrine pancreatic neoplasms induced with the carcinogen azaserine contained little or no detectable [3H]estradiol-binding activity. However, electron immunocytochemical studies of transformed cells indicated the presence of material that cross-reacted with antibodies prepared against the [3H]EBP. The immunopositive reaction in transformed cells was localized almost exclusively in lipid granules. Such lipid organelles in normal acinar cells, although present less frequently than in transformed cells, have never been observed to contain EBP-like immunopositive material. Presumably, the aberrant localization of EBP in these acinar tumor cells results in loss of function of this protein, which in normal pancreatic acinar cells appears to exert a modulating influence on zymogen granule formation and the process of secretion.
Animals↗
Fatty acid and enzymatic compositional changes in the pancreas of rats fed dietary n-3 and n-6 polyunsaturated fatty acids.
The influence of n-3 and n-6 PUFA on the fatty acid composition and the enzyme content of zymogen granules of the normal exocrine pancreas was tested on rats. The animals were fed on different diets comprising 5% fish oil (FO), safflower oil (SFO), and evening primrose oil (EPO) used singly or in combination as dietary fats. The results were compared with those from animals fed 5% hydrogenated beef tallow (HBT). The fatty acid composition and digestive enzyme content were analyzed after a 6-wk feeding period. Differences in the pancreatic fatty acid profiles were related to the fatty acid composition of the ingested fats. Equivalent levels of n-3 fatty acids and 20:3n-6 were obtained with either EPO or FO fed singly or in combination. Similar results were observed with SFO/FO. Higher C20:3n-6/C20:4n-6 ratios were obtained with the oil mixtures. An increase in amylase levels, but a decrease in serine protease (Band 21 kdalton) levels, was associated with EPO. An elevation in procarboxypeptidase levels paralleled an increase in 18:0 levels, whereas the proportion of lipase (Band 49 kdalton) varied inversely with the proportion of C20:3n-6. The SFO/FO mixture elevated the proportions of protease II and proelastase. These results suggest that specific fatty acids influence the proportion of specific digestive enzymes in the zymogen granules.
Analysis of Variance↗
Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis.
Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.
Animals↗
Cytochemical localization of beta-NADPase and TMPase in B cells of pancreas.
Cytochemical staining methods for beta-nicotinamide adenine dinucleotide phosphatase (beta-NADPase) and trimetaphosphatase (TMPase) were applied to B cells of rat pancreas. beta-NADPase was localized in one or two intermediary Golgi saccules, lysosomes, multigranular bodies, and in a variable percentage of secretory granules. It was also found in elongated tubules comparable to those previously reported in the acinar cell of pancreas. TMPase reaction product was not detected in Golgi saccules, but was present in lysosomes, multigranular bodies, some secretory granules, and elongated tubules. These observations have shown that beta-NADPase and TMPase activities are present in B cells of pancreas; that these enzymes are present in "multigranular bodies" and lysosomes supporting previous evidence that these structures may be involved in the degradation of insulin; and that elongated tubules similar to those described in the exocrine cell and muscle cells are also present in an endocrine cell.
Acid Anhydride Hydrolases↗
Influence of steroids on the exocrine pancreas: presence of laminated bodies in the acinar lumen following castration and adrenalectomy.
The influences of combined adrenalectomy and castration and of a replacement therapy with estradiol and glucocorticoids were examined in the rat pancreas. At the light microscope level, the surgical treatment changed the overall shape of the acini and caused widening of intralobular and interlobular spaces as well as partial depletion of zymogen granules. A significant reduction of the acinar lumen size was observed in castrated-adrenalectomized (CasX-AdreX) animals. This could be reversed by a replacement therapy with glucocorticoids. Moreover, triamcinolone even caused a significant lumen enlargement as compared to sham-operated animals. Such an enlargement was not observed with dexamethasone. Both these glucocorticoids caused an increase in zymogen granule content of the acinar cell. At the ultrastructural level, hitherto undescribed type of precipitates was observed in the acinar lumen of CasX-AdreX rats. Precipitates that exhibited a laminated aspect were identified as luminal laminated bodies. The latter could have some interest from the etiological point of view of certain pancreatic diseases, such as cystic fibrosis and pancreatitis. Replacement therapy with estradiol did not affect the frequency of these precipitates, but glucocorticoid had a significantly inhibitory effect. From our observation, it is clear that some steroid exert a profound influence on the acinar cell and that a replacement therapy with glucocorticoids or estradiol do not totally restore the normal aspect of the pancreas acinar tissue. It suggests that some unknown factors from adrenals or testis are still required in addition to glucocorticoids and estrogens for the maintenance of the structural integrity of the gland.
Adrenalectomy↗
Type of dietary lipids exerts a major influence on the secretory activity of the exocrine pancreas: medium-term studies.
A possible influence of the type of dietary lipids on the pancreas exocrine function was tested on rats. For this purpose, four groups of rats were fed on different diets comprising 5% of different types of lipids: fish oil, evening primose oil, hydrogenated beef tallow, and a mixture of fish oil and evening primose oil. After a 6 week feeding period, the secretory activity of the pancreas was measured. Under resting conditions, rats fed hydrogenated beef tallow release different proportions of amylase, lipase, and serine proteases as compared to rats fed unsaturated lipids. In stimulated conditions, there was no significant difference in the relative proportions of enzymes secreted by the pancreas among the different groups of rats but the secretory response to cerulein stimulation from rats fed saturated lipids was increased by more than 40%. These results demonstrate that the type of dietary lipids exerts a major influence on the secretory activity of the pancreas.
Animals↗
Pancreatic juice composition: new views about the cellular mechanisms that control the concentration of digestive and nondigestive proteins.
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Animals↗
Immunocytochemical localization of the [3H]estradiol-binding protein in rat pancreatic acinar cells.
Significant amounts of an estradiol-binding protein (EBP) are present in pancreatic acinar cells. This protein differs from the one found in female reproductive tissues and secondary sex organs (which is commonly referred to as estrogen receptor). EBP has now been purified from rat pancreas and was used as an antigen to induce polyclonal antibodies in rabbits. The antiserum obtained was purified initially by ammonium sulfate fractionation and then still further by interaction with a protein fraction from pancreas that was devoid of estradiol-binding activity. The latter procedure was used to precipitate nonspecific immunoglobulin Gs. Western blot analysis demonstrated that the anti-EBP antibody reacted specifically with a doublet of protein bands having mol wt of 64K and 66K. When this purified antibody was used as an immunocytochemical probe in conjunction with protein-A-gold, acinar cells were labeled on the surface of the endoplasmic reticulum, on the plasma membrane, and in mitochondria. This specific labeling pattern was not observed when preimmune serum was used. No labeling was observed over the nucleus, Golgi apparatus, or zymogen granules with purified anti-EBP antibodies. The unexpected distribution of EBP in both the endoplasmic reticulum and mitochondria is discussed.
Animals↗
The asynchronous transport of secretory proteins in the exocrine pancreas. Compatibility with the hypothesis of a paragranular pathway?
An asynchronous transport of individual secretory proteins has been recently described in the pancreas. This asynchrony was observed in both unstimulated and stimulated conditions. It has also been proposed that unstimulated and stimulated secretions correspond to distinct secretory processes. Indeed according to that hypothesis, under resting conditions, a small fraction of the newly synthesized secretory proteins are channeled into a paragranular (vesicle) pathway while the residual proteins are packaged in the zymogen granules. These zymogen granules eventually move to the cell surface where their content is extruded by exocytosis. Under stimulated conditions the latter process is accelerated. Since the same type of asynchrony is observed under resting and stimulated conditions in the pancreatic juice, one can wonder if the hypothesis of a paragranular pathway is compatible with the observed asynchrony. In this review, an explanation is presented to account for the facts that following pulse and chase labelling, two waves of labelled proteins are released under resting secretions and secondly that asynchrony is maintained in both resting and stimulated conditions.
Animals↗
A developmental toxicity evaluation of gossypol.
In utero development was analyzed in pregnancies that resulted from matings between gossypol-treated male rats and untreated female rats, and in pregnancies in which gossypol was administered to the pregnant rat only. Gossypol treatment of males had no effect on the outcome of pregnancy. There was no significant effect on resorption, fetal growth, or malformation rate. Similarly, gossypol administered to pregnant dams at stages during organogenesis had no observable effect on pregnancy. Under the conditions of this experiment, gossypol administered to either the breeding male rat or the pregnant female rat had no demonstrable adverse effect on development in utero.
Animals↗