Localization of Nucleoside Triphosphate Diphosphohydrolase-1 (NTPDase1) and NTPDase2 in Pancreas and Salivary Gland
Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary (AK,KK); Centre de Recherche en Rhumatologie et Immunology, Sainte-Foy, PQ, G1V 4G2, Canada (JL,FB,JS); Department of Visceral- and Transplantation-Surgery, Charité, Humboldt University, Berlin, Germany (OG); Biozentrum der JW Goethe-Universitaet, Zoologisches Institut AK Neurochemie, Frankfurt/M, Germany (NB); and Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts (SCR)
Correspondence to: Agnes Kittel, PhD, Dept. of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, 1450 Budapest, Hungary. E-mail: kittel{at}koki.hu
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Summary |
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Key Words: CD39 CD39L1 ecto-ATPase gastrointestinal tract
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Introduction |
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Ectonucleotidases that catalyze the hydrolysis of extracellular nucleotides could play a strategic role in modulating these nucleotide-mediated processes. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1), previously identified as ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) or CD39, is an important ectonucleotidase responsible for the sequential hydrolysis of ß-and -phosphates of tri- and diphosphonucleosides. It therefore modulates the concentrations of extracellular nucleotides in a variety of physiological systems. For example, NTPDase1 has important functions in the control of blood hemostasis and thrombosis (Kaczmarek et al. 1996
; Marcus et al. 1997
; Enjyoji et al. 1999
). NTPDase2, also named ecto-ATPase or CD39L1 has been shown in vitro to promote platelet aggregation indirectly by converting ATP to ADP, which is a specific agonist of P2Y1 and P2Y12 receptors (Sevigny et al. 2002
).
An ATPDase has been purified and characterized from the zymogen granule membrane of pig pancreas (LeBel et al. 1980; Laliberte et al. 1982
; Sevigny et al. 1995
). Immunolocalization was later performed in the porcine digestive system (Sevigny et al. 1998
) with an antiserum directed against the apyrase conserved region 4 (ACR-4) of NTPDases (Kaczmarek et al. 1996
; Sevigny et al. 1997a
). However, on the basis of these results it is difficult to define which NTPDase is in fact expressed. More recently, we have demonstrated expression of NTPDase1/CD39 in normal and transformed human pancreas by immunolocalization techniques (Kittel et al. 2002
).
Similar enzymes have been extensively studied in the salivary glands of blood-feeding arthropods, where a role in preventing blood coagulation has been ascribed to them (Ribeiro et al. 1984,1990
; Valenzuela et al. 2001
). Interestingly, several of these soluble apyrases have been cloned and shown to lack the ACR in the protein sequence (Valenzuela et al. 1998
; Valenzuela et al. 2001
) and were therefore separated into a new family of nucleotidases. The localization and role of such enzymes in mammals have not been determined.
Here we describe the distribution of NTPDase1 and NTPDase2 expression and the localization of ATP/ADPase activities in the pancreas and salivary glands of mice. The distribution and cellular localization of NTPDase1 and NTPDase2 in mouse pancreas and salivary gland provides new information regarding their roles in the regulation of nucleotide signaling pathways in the gastrointestinal tract.
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Materials and Methods |
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All chemicals were purchased from Sigma Chemical (St Louis, MO) unless otherwise indicated. Rabbit polyclonal antibody C9F was raised against mouse NTPDase1 (Braun et al. 2000; Heine et al. 2001
) and BZ34F was raised against rat NTPDase2 and has been previously described (Dranoff et al. 2002
; Sevigny et al. 2002
; Vlajkovic et al. 2002
; Braun et al. 2003
)
Male mice were used for all experiments. CD39-null (Enjyoji et al. 1999) and wild-type mice were originally from the background 129 SVJ x C57 BL/6 backcrossed seven generations onto C57 BL/6 and were compared with wild-type mice.
Brightfield Techniques
Preparation of Tissues for Cryosections
Animals were sacrificed by CO2 inhalation, organs (pancreas and submandibular salivary glands) were removed, embedded in OCT (Miles Diagnostic Division; Elkhart, IN), and immediately frozen in isopentane cooled by a mixture of ethanol and dry ice. Six-µm cryosections were cut, dried, and fixed in ice-cold acetone for 10 min.
Enzyme Histochemistry
Lead precipitation from an enzyme histochemical technique was used for demonstration of ecto-ATPase or ADPase activity (Kittel et al. 2002). After fixation and several rinses in 0.07 M Tris-maleate buffer (pH 7.4), the sections were incubated in a medium containing ATP or ADP (1 mM) as substrate, 2 mM Pb(NO3)2 (capturing agent for the liberated phosphate), 1 mM levamisole (inhibitor of alkaline phosphatases; Amersham, Poole, UK), 1 mM ouabain, (Na+,K+-ATPase inhibitor; Merck, Darmstadt, Germany), 50 µM
,ß-methylene ADP (5'-nucleotidase inhibitor), and KCl (5 mM) in Tris-maleate buffer (70 mM, pH 7.4) for 30 min at room temperature (RT). Incubation was followed by three rinses in Tris-maleate buffer. The precipitate was converted to PbS with 1% (NH4)2S (1-min incubation). After rinses in distilled water and haematoxylin staining, the sections were mounted in Aquatex (Merck) and images were obtained on an Olympus CH-30 microscope with an Olympus Camedia C-4040Z digital camera. Control experiments were performed without substrate.
Immunostaining for NTPDase1 or NTPDase2 Polyclonal Antibodies
Sectioning and fixation were carried out as described above. After rinsing with PBS, nonspecific binding sites were blocked with 7% normal goat serum in PBS for 30 min and the sections incubated overnight with C9F or BZ34F antibody at a 1:1000 dilution, at 4C. The staining was performed with Vectastain ABC elite kit (Vector Laboratories; Burlingame, CA) and 3,3'-diaminobenzidine (DAB) was applied as chromogen (SigmaAldrich; Oakville, Canada) according to the manufacturer's instructions. After washing with distilled water, sections were counterstained with Harris haematoxylin (SigmaAldrich), dehydrated, cleared in xylene, and mounted in Permount (EMS; Warrington, PA). Negative control experiments were performed using the same protocol in which the primary antibody was replaced by its preimmune serum at the same concentration (1:1000) or in the absence of primary antibody.
Investigations at Electron Microscopic Level
Enzyme Histochemistry
For electron microscopic investigation the cerium precipitation method was used. Ecto-ATPase activity was localized as described previously (Kittel et al. 1999). Animals were deeply anesthetized with sodium pentobarbital, then perfused briefly through the ascending aorta with a 0.9% NaCl solution. Perfusion fixation was performed with a cold fixative (3% paraformaldehyde (Merck), 0.5% glutaraldehyde (Taab; Aldermaston, UK), 2 mM CaCl2, and 0.25 M sucrose in 0.05 M cacodylate buffer, pH 7.4) for 30 min. Organs were removed, the samples were washed three times with cacodylatesucrose buffer (0.25 M sucrose in 0.05 M cacodylate, pH 7.4), and 70-µm vibratome sections were cut. The sections were rinsed several times in 0.07 M Tris-maleate buffer (pH 7.4), then incubated in a reaction mixture containing 3 mM CeCl3, 5 mM MnCl2, 2 mM CaCl2, 1 mM levamisol, 1 mM ouabain, 50 µM
,ß-methylene ADP, and 1 mM ATP or ADP in 0.07 M Tris-maleate buffer (pH 7.4) at 37C for 30 min. After washing with Tris-maleate buffer, the preparations were postfixed in 1% OsO4 for 30 min. After washing with distilled water, the sections were dehydrated in graded ethanol, block-stained with 2% uranyl acetate in 70% ethanol for 1 hr, and embedded in Taab 812. Ultrathin sections were examined in a Hitachi 7100 transmission electron microscope (Hitachi; Tokyo, Japan). Control reactions were performed without substrate ATP or ADP.
Immunohistochemistry
Animals were anesthetized as above. Fixation was carried out by intracardiac perfusion with PBS (137 mM NaCl, 3 mM KCl, 15 mM Na+/K-phosphate buffer, pH 7.4), 3% paraformaldehyde, and 0.5% glutaraldehyde, pH 7.4, at 4C. Vibratome sections were cut (70 µm thick), rinsed with PBS, and incubated in blocking solution (PBS containing 5% normal goat serum, 1 mg/ml bovine serum albumin) for an hour at RT. Sections were incubated with one of the polyclonal NTPDase antibodies (dilution 1:1000 in blocking solution) overnight at 4C. After several washes with PBS at RT, tissue sections were incubated with biotinylated anti-rabbit IgG antibody for 2 hr according to the ABC method and DAB was utilized as chromogen. After washing thoroughly with distilled water, sections were postfixed in 1% OsO4, dehydrated in 70% ethanol, stained with 2% uranyl acetate, and embedded in Taab 812. Negative control experiments were performed using the same protocol but with the NTPDase1 antibody omitted or preimmune serum (1:1000) used instead of the first antibody.
Enzymatic Assays
Murine tissues (see above) were collected and snap-frozen in liquid nitrogen and kept at 80C until used. Protein extracts were prepared as previously described (Sevigny et al. 1998) and tested for enzymatic activity the same day. Enzyme assays were carried out at 37C in 1 ml of 5 mM CaCl2, 80 mM Tris-HCl (pH 7.4) as described previously (Sevigny et al. 1997b
). Reactions were started by the addition of 350 µM of the substrate (ATP or ADP) and then stopped with 0.25 ml of malachite green reagent. Inorganic phosphate was estimated by the malachite green technique according to Baykov et al. (1988)
. Enzyme activity was expressed as nmoles of inorganic phosphate released per min per mg of protein. Protein concentrations were estimated by the method of Bradford using bovine serum albumin as a standard (Bradford 1976
).
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Results |
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Localization of NTPDase1 in Mouse Submandibular Salivary Gland
At light microscopic level, similar staining to that seen in pancreas was observed. Weak NTPDase1 staining was detected among mucous acini, whereas duct epithelial cells were negative. Brown deposits showed the immunoreactivity of blood vessels (Figure 2A) . Electron microscopy gave a more detailed picture of the localization of this reactivity. Many caveolae containing precipitate were visible on the basolateral aspect of the endothelial cells. Some finely dispersed precipitate was also seen on the basal membrane of the acini (Figure 2B). An acinar cell and its adjacent myoepithelial cell are shown in Figure 2C. This tight contact did not enable us to identify which membrane was stained. However, strong immunoreactivity was found in every case in the caveolae of the myoepithelial cells (Figures 2C and 2D). The plasma membrane of duct epithelial cells also showed some immunoreactivity (Figure 2D).
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As expected, cd39/ mouse salivary gland cells showed no immunoreactivity for NTPDase1 antibody (Figure 2L), but enzyme histochemical staining demonstrated some remaining ATPase activity in the myoepithelial cells and acini (Figure 2L, inset). In the absence of substrates (ATP or ADP) there was no staining (Figure 2M).
Localization of NTPDase2 in Mouse Pancreas and Submandibular Salivary Gland
In cryosections of mouse pancreas, blood vessels exhibited strong immunoreactivity for NTPDase2. Ducts were also stained, and some staining was visible in the luminal area of the acini (Figure 3A) . Electron microscopic investigation showed immunoreactivity on the basolateral aspects and in the caveolae of endothelial cells (Figurs 3B and 3E). Staining on the basal side of the acini was of variable intensity (Figures 3B, 3C, and 3E). In contrast to the light microscopic finding, electron microscopy did not show immunoreactivity for NTPDase2 in the luminal region of the acini (Figure 3D).
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Table 2 summarizes the enzyme and immunohistochemical staining obtained for selected cell types from pancreas and submandibular salivary gland. In general, the staining for NTPDase1 and NTPDase2 was similar in both tissues. However, whereas NTPDase2 antibodies gave a strong reaction in the pancreatic duct cells, no staining could be detected in the duct epithelial cells of the submandibular salivary gland. Likewise, the acini in the pancreas showed NTPDase2 staining, whereas those in the salivary gland did not.
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Discussion |
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We observed some differences in our LM and EM histochemistry. The differences in enzyme- and immunostaining at light and electron microscopic level in the mouse pancreas could potentially be explained by the high density of proteins in the zymogen granules close to the luminal membranes of the acinar cells. This feature may have masked both the epitopes and the domains responsible for the enzymatic activity. It would be expected that, in vivo, the NTPDases in the zymogen granules would not be active due to the acidic pH and the absence of water. ATPase and ADPase activity and NTPDase1 or NTPDase2 immunostaining may also have been negative because of changes in conformation during fixation. For electron microscopic studies aldehyde fixation is needed, but this treatment aggregates proteins. As a result, NTPDases could be in different conformations and not all epitopes would necessarily be detected by the antibodies.
The heterogeneity of the zymogen granule membranes described in rat pancreas almost two decades ago by Beaudoin and co-workers (1988) could provide another explanation for the occasional NTPDase1 immunostaining of the granules (Figures 1E and 1F). In another case, electron microscopy demonstrated NTPDase2 immunoreactivity at the basal membranes of the acini, while at light microscopic level some staining was also observed in the luminal region after acetone fixation of the crysections. These findings may suggest that this enzyme is expressed at the apical part of the acinar cell but that aldehyde fixation and/or high protein concentration may have prevented binding of the antibody.
Our data and data from previous reports suggest species-dependent variations in the localization of NTPDase1 in pancreas. Like our findings, an immunohistochemical study showed that apical and basolateral membranes of the acinar cells in pig pancreas were immunoreactive for an antibody directed against the common apyrase conserved region 4 (ACR 4) of NTPDase molecules (Sevigny et al. 1998). However, in human pancreatic cryosections, according to electron microscopic staining, acini were devoid of NTPDase1 immunoreactivity or ATPase activity (Kittel et al. 2002
). Only the duct cells showed immunoreactivity for NTPDase1. Electron microscopy showed staining was located in the basal foldings of the ductal epithelial cells. Sorensen and colleagues (2003)
described similar basolateral staining in the larger ducts of rat pancreas, while CD39/NTPDase1 immunofluorescence staining was localized on the luminal membranes in small intercalated/interlobular ducts. In rat, immunofluorescence showed CD39/NTPDase1 localization on the basolateral membranes of acini and also intracellularly. The differences in the localization of NTPDase1 in duct epithelial cells, depending on the size of the duct, may be of importance. Thus far we have no definitive explanation for this finding, but the localization pattern of NTPDase1 expression on the basal membrane may suggest a close interaction with P2 receptors. Interestingly, the expression level of P2Y1 receptor was found to fluctuate during the development of the pancreas, while the expression levels of some other P2 receptors (e.g., P2 x 7) were constant (Coutinho-Silva et al. 2001
).
The effects of ATP on salivary gland cells have been investigated in the past (Thyberg et al. 1982). There is evidence from pharmacological and molecular approaches for the expression of two ligand-gated ion channels, P2 x 4 and P2 x 7, and two G-protein-coupled receptors, P2Y1 and P2Y2, as detected in different salivary epithelial cell lines (Yu and Turner 1991
; Gibb et al. 1994
; Buell et al. 1996
) and in dispersed salivary gland cells (Park et al. 1997
; Gibbons et al. 2001
). The control of the concentration of the agonists of P2-receptors by NTPDases is an important factor in nucleotide signaling.
Our work is the first to show the expression pattern of NTPDases and ecto-ATPase/ADPase activity in the salivary gland of a mammal. The strongest immunoreactivity and enzyme activity belonged in every case to the membranes of myoepithelial cells and their caveolae. Caveolae are special membrane invaginations abundantly found in endothelial and smooth muscle cells. They have roles in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone, as well as in organizing signaling pathways in the cell. The presence of P2 receptors has also been detected in caveolae (Kittel, unpublished observation). In the present study, acini attached to the myoepithelial cells showed weak immunoreactivity for NTPDase1 antibodies on their basal sides, while ecto-ATPase and ecto-ADPase activity was strong at their basal membrane and infoldings.
In the cd39/ mouse salivary gland, enzyme histochemical staining demonstrated ATPase activity in the myoepithelial cells and weak activity in the acini. This finding, together with the biochemical activity data, supports the presence of NTPDase2 in this organ. NTPDase2 immunoreactivity was found in the basal membrane of endothelial cells as well as in the supporting cells of the vasculature in cd39/ salivary glands.
Finally, we must note the strong ATPase and ADPase activities of the endothelial cells of blood vessels. Staining of endothelial cells, as expected, showed the same pattern of immunoreactivity and enzyme histochemical staining as in the pancreas and other tissues (Kittel 1997,1999
; Sevigny et al. 1997b
; Braun et al. 2000
; Sevigny et al. 2002
). Interestingly, caveolae were more numerous in the endothelial cells of salivary gland than of pancreas. The caveolae of endothelial cells were labeled with NTPDase1-specific antibodies in both organs. Recent studies have reported the localization of NTPDase2 in pericytes and fibroblasts in the cardiac vasculature and liver (Dranoff et al. 2002
; Sevigny et al. 2002
). In addition to this previous finding, we have demonstrated here that NTPDase2 immunoreactivity was also observed on the basal side and in the caveolae of endothelial cells. Some of these variations could be due to the heterogeneity in the vascular endothelium, as previously reported (Ponder and Wilkinson 1983
; Pino 1986
; Steinhoff et al. 1993
). Because NTPDase1 and 2 have different expression patterns in endothelial cells, this suggests the possibility of spatial regulation of nucleotide-mediated signaling in the vasculature, as has been proposed previously (Sevigny et al. 2002
).
Taken together, the biochemical and histochemical data presented in this work demonstrate the presence both of NTPDase1 and NTPDase2 in mouse pancreas and salivary gland. Our demonstration of their different distributions in the acinar cells, ducts, and vasculature should help to clarify their respective roles in the regulation of P2 signaling pathways.
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Acknowledgments |
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We thank Mr Gyozo Goda for excellent technical assistance and the EM-TEK Kft. for their generous support.
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbracchio MP, Burnstock G (1998) Purinergic signalling: pathophysiological roles. Jpn J Pharmacol 78:113145[CrossRef][Medline]
Baykov AA, Evtushenko OA, Avaeva SM (1988) A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171:266270[Medline]
Beaudoin AR, Gilbert L, St-Jean P, Grondin G, Cabana C (1988) Heterogeneity of the zymogen granule membranes in rat pancreas. Eur J Cell Biol 47:233240[Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
Braun N, Sevigny J, Mishra SK, Robson SC, Barth SW, Gerstberger R, Hammer K, et al. (2003) Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain. Eur J Neurosci 17:13551364[CrossRef][Medline]
Braun N, Sevigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, Di Virgilio F, et al. (2000) Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur J Neurosci 12:43574366[CrossRef][Medline]
Buell G, Lewis C, Collo G, North RA, Surprenant A (1996) An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J 15:5562[Abstract]
Burnstock G (1990) Overview. Purinergic mechanisms. Ann NY Acad Sci 603:117
Chan KW, Langan MN, Sui JL, Kozak JA, Pabon A, Ladias JA, Logothetis DE (1996) A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins. J Gen Physiol 107:381397[Abstract]
Christoffersen BC, Hug MJ, Novak I (1998) Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts. Pflugers Arch 436:3339[CrossRef][Medline]
Coutinho-Silva R, Parsons M, Robson T, Burnstock G (2001) Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing. Cell Tissue Res 306:373383[CrossRef][Medline]
Dranoff JA, Kruglov EA, Robson SC, Braun N, Zimmermann H, Sevigny J (2002) The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver. Hepatology 36:11351144[CrossRef][Medline]
Enjyoji K, Sevigny J, Lin Y, Frenette PS, Christie PD, Esch JS II, Imai M, et al. (1999) Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nature Med 5:10101017[CrossRef][Medline]
Fedan JS, Frazer DG (1992) Influence of epithelium on the reactivity of guinea pig isolated, perfused trachea to bronchoactive drugs. J Pharmacol Exp Ther 262:741750[Abstract]
Fischer B, Chulkin A, Boyer JL, Harden KT, Gendron FP, Beaudoin AR, Chapal J, et al. (1999) 2-thioether 5'-O-(1-thiotriphosphate)adenosine derivatives as new insulin secretagogues acting through P2Y-receptors. J Med Chem 42:36363646[CrossRef][Medline]
Gibb CA, Singh S, Cook DI, Poronnik P, Conigrave AD (1994) A nucleotide receptor that mobilizes Ca2+ in the mouse submandibular salivary cell line ST885. Br J Pharmacol 111:11351139[Abstract]
Gibbons SJ, Washburn KB, Talamo BR (2001) P2X(7) receptors in rat parotid acinar cells: formation of large pores. J Auton Pharmacol 21:181190[CrossRef][Medline]
Hede SE, Amstrup J, Christoffersen BC, Novak I (1999) Purinoceptors evoke different electrophysiological responses in pancreatic ducts. P2Y inhibits K(+) conductance, and P2X stimulates cation conductance. J Biol Chem 274:3178431791
Heine P, Braun N, Sevigny J, Robson SC, Servos J, Zimmermann H (2001) The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2. Eur J Biochem 268:364373
Hug M, Pahl C, Novak I (1994) Effect of ATP, carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts. Pflugers Arch 426:412418[Medline]
Hug M, Pahl C, Novak I (1996) Evidence for a Na+-Ca2+ exchanger in rat pancreatic ducts. FEBS Lett 397:298302[CrossRef][Medline]
Ishiguro H, Naruse S, Kitagawa M, Hayakawa T, Case RM, Steward MC (1999) Luminal ATP stimulates fluid and HCO3-secretion in guinea-pig pancreatic duct. J Physiol (Lond) 519(pt 2):551558
Kaczmarek E, Koziak K, Sévigny J, Siegel JB, Anrather J, Beaudoin AR, Bach FH, et al. (1996) Identification and characterization of CD39 vascular ATP diphosphohydrolase. J Biol Chem 271:3311633122
Kim KC, Lee BC (1991) P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture. Br J Pharmacol 103:10531056[Abstract]
Kittel A (1997) Role of ecto-ATPases, based on histochemical investigations. Evidences and doubts. In Plesner L, Kirley T, Knowles AF, eds. Ecto-ATPases: Recent Progress on Structure and Function. New York, Plenum Publishing, 6572
Kittel A (1999) Lipopolysaccharide treatment modifies pH- and cation-dependent ecto-ATPase activity of endothelial cells. J Histochem Cytochem 47:393400
Kittel A, Kalmár B, Madarász E (1999) Effects of LPS on ecto-ATPase (NTPDase) activity and phagocytosis of cultured astrocytes. In Vanduffel L, Lemmens R, eds. Second International Workshop on Ecto-ATPases and Related Ecto-nucleotidases. Diepenbeek, Belgium, Shaker Publishing, Maastricht, The Netherlands, 158166
Kittel A, Garrido M, Varga G (2002) Localization of NTPDase1/CD39 in normal and transformed human pancreas. J Histochem Cytochem 50:549556
Laliberte JF, St-Jean P, Beaudoin AR (1982) Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase. J Biol Chem 257:38693874
LeBel D, Poirier GG, Phaneuf S, St-Jean P, Laliberte JF, Beaudoin AR (1980) Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas. J Biol Chem 255:12271233
Marcus AJ, Broekman MJ, Drosopoulos JH, Islam N, Alyonycheva TN, Safier LB, Hajjar KA, et al. (1997) The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. J Clin Invest 99:13511360
Munoz DJ, McFie C, Thorne PR (1999) Modulation of cochlear blood flow by extracellular purines. Hear Res 127:5561[CrossRef][Medline]
Park MK, Garrad RC, Weisman GA, Turner JT (1997) Changes in P2Y1 nucleotide receptor activity during the development of rat salivary glands. Am J Physiol 272:C13881393[Medline]
Pino RM (1986) The cell surface of a restrictive fenestrated endothelium. I. Distribution of lectin-receptor monosaccharides on the choriocapillaris. Cell Tissue Res 243:145155[Medline]
Ponder BA, Wilkinson MM (1983) Organ-related differences in binding of Dolichos biflorus agglutinin to vascular endothelium. Dev Biol 96:535541[Medline]
Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413492
Ribeiro JM, Sarkis JJ, Rossignol PA, Spielman A (1984) Salivary apyrase of Aedes aegypti: characterization and secretory fate. Comp Biochem Physiol B 79:8186[Medline]
Ribeiro JM, Vaughan JA, Azad AF (1990) Characterization of the salivary apyrase activity of three rodent flea species. Comp Biochem Physiol [B] 95:215219[Medline]
Rice WR, Singleton FM (1986) P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II cells. Br J Pharmacol 89:485491[Abstract]
Sevigny J, Cote YP, Beaudoin AR (1995) Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5'-p-fluorosulphonylbenzoyladenosine ATP analogue. Biochem J 312:351356[Medline]
Sevigny J, Dumas F, Beaudoin AR (1997a) Purification and identification by immunological techniques of different isoforms of mammalian ATP diphosphohydrolases. In Plesner L, Kirley TL, Knowles AF, eds. First International Workshop on Ecto-ATPases. Mar del Plata, Argentina. New York, Plenum Publishing, 143151
Sevigny J, Grondin G, Gendron FP, Roy J, Beaudoin AR (1998) Demonstration and immunolocalization of ATP diphosphohydrolase in the pig digestive system. Am J Physiol 275:G473482[Medline]
Sevigny J, Levesque FP, Grondin G, Beaudoin AR (1997b) Purification of the blood vessel ATP diphosphohydrolase, identification and localisation by immunological techniques. Biochim Biophys Acta 1334:7388[Medline]
Sevigny J, Sundberg C, Braun N, Guckelberger O, Csizmadia E, Qawi I, Imai M, et al. (2002) Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation. Blood 99:28012809
Sorensen CE, Amstrup J, Rasmussen HN, AnkorinaStark I, Novak I (2003) Rat pancreas secretes particulate ecto-nucleotidase CD39. J Physiol 551:881892
Steinhoff G, Behrend M, Schrader B, Duijvestijn AM, Wonigeit K (1993) Expression patterns of leukocyte adhesion ligand molecules on human liver endothelia. Lack of ELAM-1 and CD62 inducibility on sinusoidal endothelia and distinct distribution of VCAM-1, ICAM-1, ICAM-2, and LFA- 3. Am J Pathol 142:481488[Abstract]
Thyberg J, Sierakowska H, Edstrom JE, Burvall K, Pigon A (1982) Mitochondrial distribution and ATP levels in Chironomus salivary gland cells as related to growth, metabolic activity, and atmospheric oxygen tension. Dev Biol 90:3142[Medline]
Valenzuela JG, Belkaid Y, Rowton E, Ribeiro JM (2001) The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases. J Exp Biol 204:229237
Valenzuela JG, Charlab R, Galperin MY, Ribeiro JM (1998) Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme. J Biol Chem 273:3058330590
Vlajkovic SM, Thorne PR, Sevigny J, Robson SC, Housley GD (2002) NTPDase1 and NTPDase2 immunolocalization in mouse cochlea: implications for regulation of p2 receptor signaling. J Histochem Cytochem 50:14351442
Yu HX, Turner JT (1991) Functional studies in the human submandibular duct cell line, HSG-PA, suggest a second salivary gland receptor subtype for nucleotides. J Pharmacol Exp Ther 259:13441350[Abstract]