ATP-consuming and ATP-generating Enzymes Secreted by Pancreas

Abstract

Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either [ 3 H]AMP to adenosine. Along with highly active hydrolytic enzymes, there were also ATP-generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/ phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release.Extracellular ATP and other purines mediate signaling between cells by means of neural transmission, paracrine, and autocrine effects. There are several important steps in this purinergic cascade including release of ATP, interaction with specific P2 purinergic receptors on the target cells, and not the least, ATP-degrading and -converting enzymes, which determine the balance between nucleotides and nucleosides and thus activation of P2 versus P1 (adenosine) receptors.Extracellular nucleotides can be hydrolyzed by a number of enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases from the NTPDase 2 (CD39) family that hydrolyze nucleoside 5Ј-tri-and diphosphates (1). Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPP) have broad substrate specificity and can convert ATP to AMP and PP i (2). Ecto-5Ј-nucleotidases (CD73) further hydrolyze AMP to adenosine, which can be taken up into the cell by specific Na ϩ -dependent nucleoside transporters or converted into inosine by adenosine deaminase. Ecto-alkaline phosphatases have broad action and dephosphorylate 5Ј-tri-, di-, and monophosphates. In addition to these degradation and inactivation pathways, there are also counteracting ATP-generating pathways, which include reverse nucleotide transphosphorylation by adenylate kinase and nucleoside diphosphate kinase (NDPK). These kinases, which play roles in intracellular signaling and in DNA and RNA synthesis among others, are also expressed on surfaces of different cell types and can contribute to nucleotide balance (3).In cardiovascular system and in respiratory epithelia, a number of enzymes handling nucleotides are well characterized. In human blood, soluble NPP, 5Ј-nucleotidase, and adenosine deaminase, as well as adenylate and NDP kinases, c...

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…However, one should recall that we monitor ATP concentrations that depend on a balance between ATP release and ATP hydrolysis. In pancreatic acini, agonists and hormones also stimulate release/activation of ecto-nucleotidases (CD39 and CD73) that contribute to rapid breakdown of ATP [5,7]. Whether mechanical stimuli bypass these processes in acini is not clear.…”

“…ATP is released from most cells, and the release mechanism is currently an intensive research field, where the focus is on identifying whether ATP is released via ion channels/ transporters, pannexin or connexins, or via vesicular transport [2,3]. In the exocrine pancreas, it has been well documented that acini release ATP in response to physiological signals, such as cholinergic agonists, cholecystokinin, and neurotensin [4][5][6]. Acinar cells also secrete digestive enzymes and nucleotideconverting enzymes [5,7], and our earlier studies indicated that zymogen granules and fluorescent markers for ATP stores are co-localized [4].…”

“…In the exocrine pancreas, it has been well documented that acini release ATP in response to physiological signals, such as cholinergic agonists, cholecystokinin, and neurotensin [4][5][6]. Acinar cells also secrete digestive enzymes and nucleotideconverting enzymes [5,7], and our earlier studies indicated that zymogen granules and fluorescent markers for ATP stores are co-localized [4]. Sawada and coworkers published their discovery of a putative ATP transporter (SLC17A9), named vesicular nucleotide transporter (VNUT), which was expressed in enterochromaffin granules [8].…”

Cellular function and molecular structure of ecto-nucleotidases

“…There is evidence that catalytically active NTPDase1 can be shed in membrane-bound form from plasma membranes of NTPDase1-expressing cells. The hydrophobic form of NTPDase1 has been found in particulate secretions of rat pancreas under resting conditions [155] and as a result of stimulation with cholecystokininoctapeptide-8 where it is thought to regulate intraluminal ATP concentrations within the ductal tree [129,156]. However, ecto-ATPase appears to be absent from guinea pig and human pancreatic secretions, implicating species-specific differences [157].…”

“…It can also be released into pancreatic juice [156]. Binding of monoclonal antibodies to lymphocyte eN but not to endothelial eN caused shedding of the protein [249].…”