ATP sensitive K+ conductance in pancreatic zymogen granules: Block by glyburide and activation by diazoxide

Summary The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb⁺ > K⁺ > Na⁺ > Cs⁺ > Li⁺ > N-methyl glucamine⁺. The conductance could be inhibited by Ca²⁺, Mg²⁺, Ba²⁺, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 mm tetra-ethyl ammonium or 5mm 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K⁺ conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K⁺ channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K⁺ conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K⁺ channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells.This research was supported by grants from the Cystic Fibrosis Foundation (ZO298) and NIH (DK-39658). F.T. is recipient of a Fellowship from the American Cystic Fibrosis Foundation. K.C.V. is a participant of a summer research program for undergraduate students from Knox College, Galesburg, IL.     

Effects of epidermal growth factor and calcium omission on cholecystokinin-stimulated Cl- conductance in rat pancreatic zymogen granules.

Evidence suggests that cholecystokinin-octapeptide (CCK-8)-induced activation of a Cl- conductance in the membrane of zymogen granules (ZG) is closely related to pancreatic enzyme secretion. Following stimulation of isolated pancreatic acinar cells with increasing concentrations of CCK-8, the Cl- conductance in the ZG from these acini increased, reached a maximum of 40 +/- 7% above basal Cl- conductance at 10(-12) M CCK-8, and then decreased at CCK-8 concentrations higher than 10(-9) M to a level comparable to the basal Cl- conductance. We had interpreted the inhibitory action of high CCK-8 concentrations to be due to the generation of high concentrations of diacylglycerol and/or its metabolites by an "overstimulation" of phospholipase C at supramaximal CCK-8 concentrations. We now show that EGF abolishes the downstroke of the dose response curve for CCK-8-induced ZG Cl- conductance and shifts the stimulatory response to higher CCK-8 concentrations. Similarly in a nominally "Ca(2+)-free buffer" (free [Ca2+] approximately 0.2 nM), stimulated Cl- conductance at 10(-12) M CCK-8 is nearly abolished and the decreased Cl- conductance at 10(-8) M CCK-8 is increased to the level of maximal stimulation at 10(-12) M CCK-8. We conclude that both EGF and low [Ca2+] affect CCK-8-induced ZG Cl- conductance by decreasing phospholipase C activity.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Lysophospholipids in pig pancreatic zymogen granules in relation to exocytosis.

A hypothesis to explain the stimulatory role of cyclic AMP (adenosine 3':5'-monophosphate) in pancreatic enzyme secretion. has been tested. In this hypothesis cyclic AMP would activate a phospholipase activity, which would lead to a locally increased lysophospholipid formation, resulting in a fusion between the zymogen granule membrane and the apical plasma membrane. Cyclic AMP added to isolated pig pancreatic zymogen granules leads to an increased lysis of these granules, but the slowness of this effect makes its physiological significance dubious. In pancreatic homogenates or zymogen granules no stimulating effect of cyclic AMP on lipase of phospholipase activity could be demonstrated. Isolated zymogen granules have a high lysophospholipid content (27% of total phospholipids), consisting of the 1-acyl and 2-acyl forms of lysophosphatidylcholine and lysophosphatidylethanolamine. Experiments with radioactive phosphatidylcholine indicate that the lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. It is concluded that these experiments do not lend support to the above hypothesis for the mechanism of action of cyclic AMP in pancreatic enzyme secretion     

Evidence for two conductance/exchange pathways for chloride in rat parotid secretory granules

Electron probe x-ray microanalysis was used to determine that bromide is localized to rat parotid secretory granules at early stages of an in situ Cl/Br washout experiment. Chloride efflux and bromide influx across the secretory granule membrane occurred with a time order of minutes. Since the Cl washout data indicated minimal Cl binding within the granule, and therefore minimal Br binding, the Br localization results suggest the presence of two or more anion conductance/exchange pathways in the granule membrane for the Cl (Br) ion.     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between a HCO3- -permeable conductance and a CLCA protein from rat pancreatic zymogen granules

Ca(2+)-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca(2+)-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca(2+)-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca(2+) (2.5-50 microM) increased the conductance for I(-), NO(3)(-), Br(-), or HCO(3)(-), but not for Cl(-), as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K(+)-salts. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate (100 microM) or 25 microM dithiothreitol strongly inhibited Ca(2+)-dependent lysis. The permeability sequence, Ca(2+) dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca(2+)-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca(2+)-activated HCO(3)(-) conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion.     

Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Ultrastructural immunocytochemical localization of l-glutamate decarboxylase and GABA in rat pancreatic zymogen granules

Summary The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, l-glutamate decarboxylase (GAD) and gamma aminobutyrate--ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7±0.5 gold particles/m² over the cytoplasm, 36.6±14.1 gold particles/ m² over the zymogen granules, and 2.9±2.1 gold particles /m² over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4±2.5 gold particles/m² over the zymogen granules, 4.7±1.1 gold particles/m² over the mitochondria and 6.3±0.5 gold particles/m² over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p< 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate --ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.     

Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc

The dynamicaspects of exocytosis, especially in the normal acinar tissue en bloc,have remained unclear. We visualized exocytosis directly in the tissueof the exocrine pancreas of rodents by video-enhancedcontrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol orcholecystokinin caused many of the zymogen granules in the apical poleto disappear abruptly. The exocytotic transients of individual granuleswere completed in 0.48-0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of ~0.06 µm/sor less during stimulation. In the tissue preparation, granules locatedfar from the apical pole frequently moved back and forth for 1-7µm without showing exocytosis. Colchicine suppressed this movementand the late phase of the secretory response. Real-time (VEC-DIC)observation of granule dynamics revealed that the initial step ofexocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.

     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis

To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptide mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis.     

In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Purified preparations of pancreatic zymogen granules have the peculiar property of lysing instantaneously at neutral pH, a property clearly irreconcilable with the cytoplasmic pH of the acinar cell. Two important factors known for regulating the stability of secretory granules are calcium and pH. Fluorescence microscopy of acinar cells in the presence of weak bases showed that zymogen granules have an acidic pH. In vivo, abolition of the delta pH by NH4Cl did not induce any lysis of the granules. In vitro, with purified granules, an acidic intragranular pH was measured. This delta pH was produced by a Donnan potential. The importance for granule stability of keeping the intragranular pH acidic has been confirmed in vitro by addition of K+ and nigericin to the suspension medium. These conditions produced alkalinization of the granule matrix and caused instantaneous solubilization of the granules. Concentrations of 15 mM total, and 10 mM free calcium were measured in purified granules. The importance of intragranular Ca2+ was evaluated by means of the ionophore A23187 which induced calcium efflux and granule lysis. The lysis induced by the calcium ionophore was in direct relation with the calcium efflux, since addition of Ca2+ to the medium, at concentrations corresponding to that measured in the granule, relieved the effect. The role of calcium-binding sites on the cytoplasmic surface of the granules was investigated with Ca2+, EGTA, and La3+. Calcium did not have any damaging effects; EGTA induced a slight lysis, while lanthanum yielded a strong and spontaneous lysis at micromolar concentrations. In addition to calcium-binding sites, La3+ would bind to specific sites on the granule that would be directly coupled to maintenance of its stability. These findings suggest that the intragranular acidic pH and calcium are both important for the in vitro stability of the zymogen granule and that purified granules have lost, in the course of purification, some cytoplasmic factors that in vivo, control the permeability of the membrane to protons, and chloride more particularly. Calcium-binding sites and other specific sites probed with La3+, presumably on proteins at the surface of the granule, are also believed to have key roles in preserving the integrity of the membrane and the resulting stability of the granule.     

Histochemical staining of zymogen granules of pancreatic acinar cells using a permanganate-HID-technique

Summary Using a permanganate-HID technique specific staining of cystine-containing zymogen granules of exocrine pancreas is possible. This method is based upon breaking and oxidation of disulphide bonds into sulphonic acids which subsequently are demonstrable with the HID-reaction.N,N-dimethyl-p-phenylendiamine-dihydrochloride was obtained from Fluka AG/ Basel; N,N-dimethyl-m-phenylendiamine-dihydrochloride from Schuchardt/Munich.     

Proteomic analysis of pancreatic zymogen granules: identification of new granule proteins

The composition of zymogen granules from rat pancreas was determined by LC-MS/MS. Enriched intragranular content, peripheral membrane, and integral membrane protein fractions were analyzed after one-dimensional SDS-PAGE and tryptic digestion of gel slices. A total of 371 proteins was identified with high confidence, including 84 previously identified granule proteins. The 287 remaining proteins included 37 GTP-binding proteins and effectors, 8 tetraspan membrane proteins, and 22 channels and transporters. Seven proteins, pantophysin, cyclic nucleotide phosphodiesterase, carboxypeptidase D, ecto-nucleotide phosphodiesterase 3, aminopeptidase N, ral, and the potassium channel TWIK-2, were confirmed by immunofluorescence microscopy or by immunoblotting to be new zymogen granule membrane proteins.     

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules.

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.