Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform).

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.

The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression.

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes.

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

The mixed lineage kinase DLK is oligomerized by tissue transglutaminase during apoptosis.

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

Purification, characterization, and localization of an ATP diphosphohydrolase in porcine kidney.

Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.

Identification, characterization, and immunolocalization of a nucleoside triphosphate diphosphohydrolase in pig liver.

Different isoforms of nucleoside triphosphate diphosphohydrolases (NTPDases; EC 3.6.1.5), also identified as ATP diphosphohydrolases, have been previously described in mammalian tissues. We report here the biochemical characterization of NTPDases in the pig liver. Optimum pH of catalysis is more acidic for this enzyme than for NTPDases (neutral or alkaline pH) found in other mammalian tissues. It is less sensitive to bile salts than the bovine spleen NTPDase. Calculated Km values for ATP and ADP (31 and 21 microM, respectively) are slightly higher than those reported for the latter enzyme. Electrophoretograms of these enzymes also show different migration patterns. Western blots with Ringo, an antibody that recognizes the different isoforms of mammalian NTPDases, show a small but reproducible difference in estimated molecular masses (75 kDa for liver vs 78 kDa for spleen NTPDase). A second antibody, generated against a different sequence of NTPDase I, does not recognize the liver enzyme, thereby indicating some differences in primary structure. Immunolocalization produced a strong signal on hepatocytes, epithelial cells of the bile duct system, and vascular cells. Immunoreactivity was variable among hepatocytes of different lobules and among hepatocytes within a given lobule. In general, those located in the perilobular zone were more reactive than those located in the central zone and in the periphery of the centrolobular vein.

Identification and immunolocalization of two isoforms of ATP-diphosphohydrolase (ATPDase) in the pig immune system.

The occurrence of a variety of purine receptors in the immune system indicates that extracellular purines play important functional roles. Extracellular purine concentrations are, in great part, determined by ectonucleotidases, namely, the ATP diphosphohydrolase, also identified as CD39, a lymphocyte cell surface marker. The latter enzyme converts triphospho- and diphosphonucleosides to nucleoside monophosphates. In this study, high levels of ATPase and ADPase activities have been found in homogenates of the different pig lymphoid organs. Specific activities decreased in the following order: spleen > bone marrow > thymus > lymph glands. The parallel decrease in ATPase and ADPase activities, in the presence of sodium azide, indicated that an ATP diphosphohydrolase (ATPDase) was responsible for these activities. Particulate fractions, prepared from the different lymphoid organs by ultracentrifugation on a sucrose cushion, showed about a 10-fold enrichment of ATPDase activity. Identity of ATPDase was confirmed by electrophoretograms of the particulate fractions and Western immunoblots, with an antibody that recognizes ATPDases from different sources. Two isoforms of ATPDase were found (I and II), corresponding to molecular masses of 78,000 and 54,000, respectively, as estimated by SDS-PAGE. Immunohistochemical localization was carried out on these different organs: In spleen, reaction was found in both white and red pulps. A particularly intense reaction was put in evidence in nervous fibers of this organ. Immunolocalization also showed positive reactions with tonsilar lymphoid structures, diffuse lymphoid tissues, and nodules associated with stomach, duodenum, jejunum, and ileum. In addition, our observations establish the presence of ATPDase in lymphocytes and macrophages of the pig immune system.

Localization of the mixed-lineage kinase DLK/MUK/ZPK to the Golgi apparatus in NIH 3T3 cells.

DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus. (J Histochem Cytochem 47:1287-1296, 1999)

Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)

Zonal induction of mixed lineage kinase ZPK/DLK/MUK gene expression in regenerating mouse liver.

ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

Demonstration and immunolocalization of ATP diphosphohydrolase in the pig digestive system.

Two isoforms of ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) have been previously characterized, purified, and identified. This enzyme is an ectonucleotidase that catalyzes the sequential release of gamma- and beta-phosphate groups of triphospho- and diphosphonucleosides. One of its putative roles is to modulate the extracellular concentrations of purines in different physiological systems. The purpose of this study was to define, identify, and localize these two isoforms of ATPDase in the pig digestive system. ATPDase activity was measured in pig stomach, duodenum, pancreas, and parotid gland. Enzyme assays, electrophoretograms, and Western blots with a polyclonal antibody that recognizes both isoforms demonstrate the presence of ATPDase in these organs. Immunolocalization showed intense reactions with gastric glands (parietal and chief cells), intestine (columnar epithelial cells), parotid gland, and pancreas. Smooth muscle cells all along the digestive tract were also highly reactive. Considering the variety of purinoceptors associated with the digestive system, the ATPDase is strategically positioned to modulate purine-mediated actions such as electrolyte secretion, glandular secretion, smooth muscle contraction, and blood flow.

Membrane dipeptidase in the pig exocrine pancreas. Ultrastructural localization and secretion.

The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse.

This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2Kb-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products alpha-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.

Purification, characterization, and localization of two ATP diphosphohydrolase isoforms in bovine heart.

Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the following steps: differential centrifugation, sucrose cushion centrifugation, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromatographies. The purified enzyme has an optimum pH of catalysis of 7.5 and requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme, with ADP as the substrate, is 29 microM, and the apparent maximal velocity is 1.6 mumol.min-1.mg protein-1. Substrate specificity, heat-inactivation curves, and copurification of adenosinetriphosphatase (ATPase) and adenosinediphosphatase (ADPase) activities confirmed the identity of the purified enzyme as an ATPDase. In addition, polyacrylamide gel electrophoresis, under nondenaturing conditions, showed identical migration patterns for the protein involved in ATPase and ADPase activities. Western blot analysis, with an antibody that specifically recognizes the NH2-terminal sequence of pig pancreas ATPDase and specifically reacts with bovine and human ATPDases, showed cross-reactivity with the purified ATPDase isoforms from the bovine heart. Immunocytochemical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardial cells. Immunoreactivity was variable, producing a mosaic-like aspect. As expected, smooth muscle cells and endothelial cells of coronary vessels were highly reactive. This ectoenzyme could play a protective role against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vasodilator, especially in hypoxic or ischemic conditions that favor the release of ATP.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box.

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Purification and immunohistochemical localization of the ATP diphosphohydrolase in bovine lungs.

We have recently described different isoforms of mammalian ATP diphosphohydrolase (ATPDase; EC 3.6.1.5). In the present study, we purified the lung ATPDase by column chromatographies followed by polyacrylamide gel electrophoresis under nondenaturing conditions. The active polypeptide that has a molecular mass of 78 kDa was identified by affinity labeling to the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA), followed by detection on Western blot with an antibody specific for FSBA. N-glycosidase F treatment shifted the molecular mass of the 78-kDa polypeptide down to 54 kDa, indicating that the enzyme bears approximately 6-12 NH2-linked oligosaccharide chains. A polyclonal antibody raised against the pancreas ATPDase, which specifically recognized the 78-kDa glycoprotein on Western blot, was used to carry out an immunological survey of the enzyme distribution in bovine lungs. Immunoreactivity was detected on airway epithelia from the trachea down to alveolar cells, airway and vascular smooth muscle cells, submucous glands, chondrocytes, leucocytes, as well as endothelial and mesothelial cells. Such a wide distribution suggests that the ATPDase may affect a variety of physiological effects mediated by extracellular nucleotides, such as airway smooth muscle tone, surfactant secretion, platelet aggregation, and inflammation.

Purification of the blood vessel ATP diphosphohydrolase, identification and localisation by immunological techniques.

ATP diphosphohydrolase (ATPDase) or apyrase (EC 3.6.1.5), an enzyme that hydrolyses the gamma and beta phosphate residues of triphospho- and diphosphonucleosides, has been purified from the bovine aorta media. A particulate fraction was isolated by differential, and sucrose cushion centrifugations, producing a 33-fold enrichment in ADPase activity. Solubilization of the enzyme from the particulate fraction with Triton X-100 caused a partial loss of activity. The solubilized enzyme was purified by DEAE-agarose, Affi-Gel blue and Concanavalin A column chromatographies yielding an additional 138-fold enrichment of the enzyme. The enzyme preparation was further purified by PAGE under non-denaturing conditions, followed by its detection on the gel. The active band was cut out and separated by SDS/PAGE. Overstaining with silver nitrate revealed a single band corresponding to a molecular mass of 78000. Presence of an ATP binding site on the latter protein was demonstrated by labelling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an analogue of ATP, followed by its detection by a Western blot technique. Labelling specificity was demonstrated by competition experiments with Ca-ATP and Ca-ADP. An antiserum directed against the N-terminal sequence of the pig pancreas ATPDase (54 kDa) cross-reacted with the bovine aorta ATPDase at 78 kDa. Digestion of the ATPDase with N-glycosidase F caused a marked shift of the molecular mass, thereby showing multiple N-oligosaccharide chains. Immunohistochemical localisation confirmed the presence of ATPDase on both endothelial and smooth muscle cells.

Localization of rat pancreatitis-associated protein during bile salt-induced pancreatitis.

BACKGROUND & AIMS: Pancreatitis-associated protein (PAP), which is overexpressed in pancreatic acinar cells, appears in pancreatic juice and serum after acute pancreatitis. The aim of this study was to examine intracellular localization of PAP and amylase in healthy rat pancreas and pancreatitis pancreas to ascertain PAP transport from the rough endoplasmic reticulum to the zymogen granules into the acinar lumen. METHODS: Control rats and rats with taurocholate-induced pancreatitis were killed after 24 hours. Pancreata prepared for light and electron microscopy were used for amylase and PAP detection with specific antibodies. RESULTS: Induced acute pancreatitis disturbed the gross histology and ultrastructure of the acinar cells with the formation of new intracellular fibrous material into the cytoplasm, which was also found into the acinar lumen. PAP is almost absent from normal acinar cells; after acute pancreatitis, it appears in rough endoplasmic reticulum, it is strongly present in normal and abnormal zymogen granules, and it remains an important component of the fibrous material. Except for its exclusive presence in fibrous material, PAP is always colocalized with amylase in the other cell compartments. CONCLUSIONS: These observations show that the accumulated PAP into the acinar cells in response to acute pancreatitis behaves like all the other secretory proteins with the exception that it also accumulates in a new fibrillous cellular structure also found in the acinar lumen.

Models of exocrine pancreatic pathologies: a microscopical point of view.

The objective of this review is to emphasize the contribution of optical and electronic microscopy to the study of the normal and pathological rat pancreas. These basic techniques used to explore the pancreatic morphology are capital to precisely localize specific enzymes or proteins in cellular subcompartments, to establish the normal or the pathological state of the gland, and to detect and describe unusual structures that appear under certain experimental conditions.