Rat Exocrine Pancreatic Cells Research Articles

Effect of GTP and Ca 2+ on inositol 1,4,5-trisphosphate induced Ca 2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca 2+ and GTP on the release of Ca 2+ from the inositol 1,4,5-trisphosphate (IP 3) sensitive Ca 2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca 2+ released due to IP 3 directly correlated with the amount of stored Ca 2+ and was found to be inversely proportional to the medium free Ca 2+ concentration. Ca 2+ release induced by 0.18 μM IP 3 was half maximally inhibited at 0.5 μM free Ca 2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca 2+ release on its own, but a single addition of GTP (20 μM) abolished the apparent desensitization of the Ca 2+ release which was observed during repeated IP 3 applications. This effect of GTP was reversible. GTPγS could not replace GTP. Desensitization still occurred when GTPγS was added prior to GTP. The reported data indicate that GTP, stored Ca 2+ and cytosolic free Ca 2+ modulate the IP 3 induced Ca 2+ release.

Protein disulfide-isomerase in rat exocrine pancreatic cells is exported from the endoplasmic reticulum despite possessing the retention signal.

Recently we found by immunogold electron microscopy that protein disulfide-isomerase (PDI), a major resident protein in the lumen of the endoplasmic reticulum (ER) of many cells, is exceptionally localized in rat exocrine pancreatic cells not only in the ER but also in plasma membranes and other organelles along secretory pathway (Akagi, S., Yamamoto, A., Yoshimori, T., Masaki, R., Ogawa, R., and Tashiro, Y. (1988) J. Histochem. Cytochem. 36, 1069-1074). These observations suggest that another type of PDI, e.g. one with a defective ER retention signal, might exist and be transported in the exocrine pancreatic cells. We therefore compared biochemical and immunochemical properties of the transported PDI with the authentic ER resident PDI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, urea-polyacrylamide gel electrophoresis, and isoelectric focusing showed that the former was indistinguishable from the latter. We prepared a polyclonal antibody against the synthetic hexapeptide, which corresponds to the carboxyl terminus of PDI containing the putative ER retention signal "KDEL." The epitopes of this antibody (anti-KDEL antibody) were located within the KDEL sequence. Anti-KDEL antibody reacted with PDI in both the plasma membranes and the ER of rat pancreatic cells in immunoblot analysis as well as in immunogold electron microscopy. These results suggest that PDI exported from the ER to the plasma membranes in rat exocrine pancreatic cells possesses the KDEL sequence.

Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas

The non-steroidal anti-inflammatory drugs, flufenamic acid, mefenamic acid and niflumic acid, block Ca 2+-activated non-selective cation channels in inside-out patches from the basolateral membrane of rat exocrine pancreatic cells. Half-maximal inhibition was about 10 μM for flufenamic acid and mefenamic acid, whereas niflumic acid was less potent (IC 50 about 50 μM). Indomethacin, aspirin, diltiazem and ibuprofen (100μM) had not effect. It is concluded that the inhibitory effect of flufenamate, mefenamate and niflumate is dependent on the specific structure, consisting of two phenyl rings linked by an amino bridge.

Developmental change of Ca2+-ATPase activity on plasma membrane of rat pancreas during perinatal period.

Cat+-ATPase activity in rat pancreatic exocrine cells and endocrine cells during perinatal development was cytochemically examined at the electron microscopic level. The intense enzyme activity on the plasma membrane of endocrine cells was observed from the beginning of differentiation of endocrine cells, as well as in adult rats. However, no activity was seen on the plasma membrane of exocrine cells in prenatal rats. Ca2+-ATPase activity on the basolateral plasma membrane of exocrine cells appeared after birth, whereas pancreatic acini and zymogen granules were fully developed during the late embryonic stage. It seemed that the appearance of Ca2+-ATPase activity on the plasma membrane corresponded to the beginning of exocrine secretory function. These results suggest that Ca2+-ATPase activity on plasma membrane of exocrine cells may be deeply involved in the secretory function and/or its regulation.

Changes in the intercellular junction of rat exocrine pancreatic cells induced by long term ethanol ingestion.

The findings of electron microscopic observation of the intercellular junctions of pancreatic exocrine cells from male Wistar rats that had been given ethanol ad libitum in place of water are described. In control animals, numerous tight junctions having a distinct pentalamellar structure were observed in the well developed interdigitations of the baso-lateral domain of both the ductal and centroacinar cells. This type of junction differed structurally as well as topographically from the occluding junctions situated in the apical part of the lateral domain and exhibited a tri-lamellar rather than a penta-lamellar structure. Further, this type of junction was not observed in the interdigitations between the acinar and centroacinar cells or in those between acinar cells. These specialized membrane structures became hypertrophic after 1 and 2 months of ethanol ingestion. That is, the interdigitated cell processes became elongated but the distribution density of the tight junctions was similar to that in control animals, so that the total number of tight junctions increased. After 3 months of ethanol ingestion, the interdigitated cell processes atrophied, the intercellular spaces widened, and the number of tight junctions markedly decreased. Thereafter, these changes were observed continuously in ethanol-treated animals. However, the occluding and adhering junctions were not markedly affected by ethanol intake.

Three-dimensional distribution of acid phosphatase-positive thread-like structures "nematolysosomes" in rat pancreatic exocrine cells.

Three-dimensional distribution of acid phosphatase-positive thread-like structures "nematolysosomes" in rat pancreatic exocrine cells.

Agonist‐induced changes in cell membrane capacitance and conductance in dialysed pancreatic acinar cells of rats.

1. Single acinar cells enzymatically isolated from the rat pancreas were subjected to tight-seal whole-cell recordings. Changes in cell membrane capacitance and conductance were simultaneously recorded using a phase-sensitive detection method. 2. Acetylcholine (ACh, 0.05-0.5 microM) and cholecystokinin octapeptide (CCK-8, 10-50 pM) concomitantly induced transient increases in cell membrane current, capacitance and conductance only when cytosolic Ca2+ was weakly chelated by EGTA (70 microM). These responses were prolonged when the cells were dialysed with a solution containing GTP gamma S (a stable analogue of GTP, 50-100 microM), whereas they were inhibited by dialysing with that containing GDP beta S (a stable analogue of GDP). These results suggest that a type of guanine-nucleotide-binding protein (G-protein) could be involved in ACh- or CCK-receptor signalling. 3. The ACh- or CCK-induced responses (with or without GTP gamma S in the cytosol) were all abolished when a high dose of EGTA (1-2 mM) was injected into the acinar cells. In addition, A23187, a calcium ionophore, induced sustained responses when the cytosolic Ca2+ was weakly buffered by 70 microM-EGTA. These results suggest that the secretagogues regulate the changes in cell membrane capacitance and conductance via an increase and decrease of cytosolic Ca2+ concentration. 4. Oscillatory changes in cell membrane conductance and capacitance were consistently observed even without applying secretagogues when the cells were dialysed with a solution containing GTP gamma S (50-100 microM) and cytosolic free Ca2+ ions weakly buffered at about 10(-6) M with a low dose of EGTA and CaCl2. 5. The peak amplitude of changes in cell membrane capacitance induced by ACh or CCK-8, with or without GTP gamma S in the cytosol, varied between 200 and 1000 fF, thereby suggesting that 20-100 zymogen granules can fuse with the luminal cell membrane in response to these agonists in rat exocrine pancreatic acinar cells.

Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue.

Second messengers have been implicated in the control of communication between cells of various tissues and of a number of cell lines. To assess whether protein kinase C (PKC) is involved in the regulation of gap junctions between primary differentiated cells, we studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on PKC translocation and junctional conductance of rat pancreatic exocrine cells. Our results show that although TPA induced the translocation of PKC from a "cytosolic" to a "microsomal" fraction within minutes, it failed to block the junctional conductance of acinar cell pairs up to 30 min after application. By contrast, analogous experiments on a liver-derived cell line (WB cells) showed that TPA-induced PKC translocation was paralleled by a marked and irreversible inhibition of intercellular coupling. These results indicate that, in contrast to the effects on transformed or dedifferentiated permanent cell lines, PKC is not involved in gating gap junctional channels between primary differentiated secretory cells of the pancreas.

Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein.

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells.

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.

A histochemical study of variations in the localization of 5'-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period.

The circadian variation of 5'-nucleotidase (AMPase) activity was studied in rat pancreatic exocrine cells. The localization of this enzyme, often associated with the plasmalemma, was studied by ultracytochemical methods at six time points over the 24-h period. The localization of AMPase activity exhibited a clear-cut circadian variation. During the light span strong activity was observed on the luminal plasmalemma, negative or weak activity on the baso-lateral plasmalemma and clearly visible activity on intracellular structures such as cytoplasmic vacuoles (fragmentation-like vesicles), dilated rims of the Golgi cisternae (or cisternal ends of the Golgi stacks), condensing vacuoles and lysosomal bodies. During the dark span the activity was detectable only on the baso-lateral plasmalemma. The fact that AMPase activity could not be found on the luminal plasmalemma during the dark span suggests that the luminal membranes may be replaced by the membranes of secretory granules, which do not display AMPase activity. The intracellular localization of AMPase activity during the light span, especially at 08.00 h, includes all cytoplasmic compartments which have hitherto been associated with the intracellular pathway for membrane retrieval from the plasmalemma. Moreover, the appearance of the activity in the dilated rims of the Golgi stack and condensing vacuoles indicates that these compartments may constitute a functional unit.

Attempts to quantitate immunocytochemistry at the electron microscope level.

The ability to localize intracellular macromolecules in situ by high resolution techniques has been made possible by the development of antibody labelling of thin sections obtained either from tissues embedded in an hydrophilic matrix, or by ultracryotomy or from conventional plastic embedded tissue. When particle-tagged immunological reagents are used to visualize intracellular antigens, quantitative information can be obtained by combining particle counts with morphometric estimations of compartment volume. Various detection systems have been used successfully for quantitation, which include ferritin-conjugated antibodies, biotin-avidin-ferritin complexes and, more recently, gold-protein A conjugates. Examples of the use of these techniques the localization of secretory proteins in pancreatic exocrine cells, opsin and a large membrane protein in photoreceptor cells of frog retina, and contractile proteins in skeletal muscle are given. Quantitative data obtained by morphometric analysis, both in bovine and rat pancreatic exocrine cells, are compared with values assessed by biochemical methods.

Immunoferritin localization of amylase and chymotrypsinogen in stimulated and unstimulated rat exocrine pancreatic cells

The precise localization of secretory proteins in exocrine pancreatic cells has not yet been elucidated. Especially the role of the Golgi complex in protein processing remains a matter of controversy. Based on morphological and autoradiographical work with the frog pancreas, we described proteins moving from the peripheral elements at the cis (immature) side of the Golgi complex across the stack of Golgi cisternae towards the condensing vacuoles at the trans (mature) side of the Golgi complex. Jamieson and Palade described a similar pathway in the stimulated Guinae pig pancreas, but suggested that in unstimulated cells the Golgi cisternae are bypassed and that vesicles carry proteins directly from the RER to the condensing vacuoles. Novikoff et al. postulate from their study on pancreatic cells of 4 mammalian species including Guinea pig and rat that condensing vacuoles can originate directly from specialized regions of the ER (so-called GERL) at the trans side of the Golgi complex. This makes the role of the peripheral elements and the Golgi cisternae in protein processing even more doubtful.

Ultrastructural alterations of the cytomembranes of rat pancreatic exocrine cells induced by treatment with 2-acetylaminofluorene

Male rats of the Leeds albino strain were fed a diet containing 0·05% of 2-acetylaminofluorene ( 2-AAF). The rats were killed after 3–4 weeks and 8–10 months of treatment, and also up to 6 months after the end of a 10 month period of 2-AAF-feeding, and their pancreatic acinar cells studied by electron microscopy. 2-AAF appeared to produce a relatively specific lesion within these cells, being largely confined to the granular endoplasmic reticulum and perinuclear cisternae. This consisted mainly of cisternal dilatation and the appearance of intracisternal granules and appeared to be permanent in character, since it persisted up to 6 months after treatment had been withdrawn. The functional activity of the pancreatic acinar cells appeared to be unaffected by 2-AAF. The possible nature of the 2-AAF lesion is discussed.