Pantoprazole and riluzole target H+/K+-ATPases and pH-sensitive K+ channels in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains the most lethal cancer type. PDAC is characterized by fibrotic, hypoxic, and presumably acidic tumor microenvironment (TME). Acidic TME is an important player in tumor development, progression, aggressiveness, and chemoresistance. The dysregulation of ductal ion transporters/channels might contribute to extracellular pH (pHe) acidification and PDAC progression. Our aim was to test whether H+/K+-ATPases and pH-sensitive K+ channels contribute to these processes and could be targeted by clinically approved drugs. We used human pancreatic cancer cells adapted to various pHe conditions and grown in monolayers and spheroids. First, we created cells expressing pHoran4 at the outer plasma membrane and showed that pantoprazole, the H+/K+-ATPase inhibitor, alkalinized pHe. Second, we used FluoVolt to monitor the membrane voltage (Vm) and showed that riluzole hyperpolarized Vm, most likely by opening of pH-sensitive K+ channels such as TREK-1. Third, we show that pantoprazole and riluzole inhibited cell proliferation and viability of monolayers and spheroids of cancer cells adapted to various pHe conditions. Most importantly, combination of the two drugs had significantly larger inhibitory effects on PDAC cell survival. We propose that co-targeting H+/K+-ATPases and pH-sensitive K+ channels by re-purposing of pantoprazole and riluzole could provide novel acidosis-targeted therapies of PDAC.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer.

BACKGROUND: The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC. MATERIALS AND METHODS: Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay. RESULTS: Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1ß, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects. CONCLUSION: In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1.

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats.

AIM/HYPOTHESIS: Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. METHODS: We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. RESULTS: In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3's cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.

A New, MWCNT-Based, Solid-State Thiabendazole-Selective Sensor.

Direct potentiometric measurements using solid-state sensors have a great potential for thiabendazole (TBZ) determination, considering simplicity, accuracy, and low cost. Modifying the sensing material of the sensor with multi-walled carbon nanotubes (MWCNTs) leads to improved analytical properties of the sensor. In this study, a new potentiometric solid-state sensor for TBZ determination, based on MWCNTs modified with a sulfate group, and TBZ ion as sensing material was developed. The sensor exhibited a Nernstian response for TBZ (60.4 mV/decade of activity) in a working range between 8.6 × 10-7 and 1.0 × 10-3 M. The detection limit for TBZ was 6.2 × 10-7 M. The response time of the sensor for TBZ was 8 s, and its signal drift was only 1.7 mV/h. The new sensor is applicable for direct potentiometric determination of TBZ in complex real samples, such as fruit peel. The accuracy of TBZ determination is confirmed using the standard addition method.

Deconstructing the principles of ductal network formation in the pancreas.

The mammalian pancreas is a branched organ that does not exhibit stereotypic branching patterns, similarly to most other glands. Inside branches, it contains a network of ducts that undergo a transition from unconnected microlumen to a mesh of interconnected ducts and finally to a treelike structure. This ductal remodeling is poorly understood, both on a microscopic and macroscopic level. In this article, we quantify the network properties at different developmental stages. We find that the pancreatic network exhibits stereotypic traits at each stage and that the network properties change with time toward the most economical and optimized delivery of exocrine products into the duodenum. Using in silico modeling, we show how steps of pancreatic network development can be deconstructed into two simple rules likely to be conserved for many other glands. The early stage of the network is explained by noisy, redundant duct connection as new microlumens form. The later transition is attributed to pruning of the network based on the flux of fluid running through the pancreatic network into the duodenum.

Purinergic Signaling in Pancreas-From Physiology to Therapeutic Strategies in Pancreatic Cancer.

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.

Assessments of attrition bias in Cochrane systematic reviews are highly inconsistent and thus hindering trial comparability.

BACKGROUND: An important part of the systematic review methodology is appraisal of the risk of bias in included studies. Cochrane systematic reviews are considered golden standard regarding systematic review methodology, but Cochrane's instructions for assessing risk of attrition bias are vague, which may lead to inconsistencies in authors' assessments. The aim of this study was to analyze consistency of judgments and support for judgments of attrition bias in Cochrane reviews of interventions published in the Cochrane Database of Systematic Reviews (CDSR). METHODS: We analyzed Cochrane reviews published from July 2015 to June 2016 in the CDSR. We extracted data on number of included trials, judgment of attrition risk of bias for each included trial (low, unclear or high) and accompanying support for the judgment (supporting explanation). We also assessed how many Cochrane reviews had different judgments for the same supporting explanations. RESULTS: In the main analysis we included 10,292 judgments and supporting explanations for attrition bias from 729 Cochrane reviews. We categorized supporting explanations for those judgments into four categories and we found that most of the supporting explanations were unclear. Numerical indicators for percent of attrition, as well as statistics related to attrition were judged very differently. One third of Cochrane review authors had more than one category of supporting explanation; some had up to four different categories. Inconsistencies were found even with the number of judgments, names of risk of bias domains and different judgments for the same supporting explanations in the same Cochrane review. CONCLUSION: We found very high inconsistency in methods of appraising risk of attrition bias in recent Cochrane reviews. Systematic review authors need clear guidance about different categories they should assess and judgments for those explanations. Clear instructions about appraising risk of attrition bias will improve reliability of the Cochrane's risk of bias tool, help authors in making decisions about risk of bias and help in making reliable decisions in healthcare.

Alternating pH landscapes shape epithelial cancer initiation and progression: Focus on pancreatic cancer.

We present here the hypothesis that the unique microenvironmental pH landscape of acid-base transporting epithelia is an important factor in development of epithelial cancers, by rendering the epithelial and stromal cells pre-adapted to the heterogeneous extracellular pH (pHe ) in the tumor microenvironment. Cells residing in organs with net acid-base transporting epithelia such as the pancreatic ductal and gastric epithelia are exposed to very different, temporally highly variable pHe values apically and basolaterally. This translates into spatially and temporally non-uniform intracellular pH (pHi ) patterns. Disturbed pHe - and pHi -homeostasis contributes to essentially all hallmarks of cancer. Our hypothesis, that the physiological pHe microenvironment in acid-base secreting epithelia shapes cancers arising in these tissues, can be tested using novel imaging tools. The acidic tumor pHe in turn might be exploited therapeutically. Pancreatic cancers are used as our prime example, but we propose that this concept is also relevant for other cancers of acid-base transporting epithelia.

pH-sensitive K(+) channel TREK-1 is a novel target in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and new therapeutic targets are urgently needed. One of the hallmarks of cancer is changed pH-homeostasis and potentially pH-sensors may play an important role in cancer cell behavior. Two-pore potassium channels (K2P) are pH-regulated channels that conduct a background K(+) current, which is involved in setting the plasma membrane potential (Vm). Some members of the K2P superfamily were reported as crucial players in driving tumor progression. The aim of this study was to investigate pH-regulated K(+) currents in PDAC cells and determine possible effects on their pathological phenotype. Using a planar high-throughput patch-clamp system (SyncroPatch 384PE) we identified a pH-regulated K(+) current in the PDAC cell line BxPC-3. The current was inhibited by extracellular acidification and intracellular alkalization. Exposure to a set of different K(+) channel inhibitors, and the TREK-1 (K2P2.1)-specific activator BL1249, TREK-1 was identified as the main component of pH-regulated current. A voltage-sensor dye (VF2.1.Cl) was used to monitor effects of pH and BL1249 on Vm in more physiological conditions and TREK-1-mediated current was found as critical player in setting Vm. We assessed a possible role of TREK-1 in PDAC progression using cell proliferation and migration assays and observed similar trends with attenuated proliferation/migration rates in acidic (pH<7.0) and alkaline (pH>7.4) conditions. Notably, BL1249 inhibited both PDAC cell proliferation and migration indicating that hyperpolarization of Vm attenuates cancer cell behavior. TREK-1 may therefore be a promising novel target for PDAC therapy.

A role for ubiquitin in selective autophagy.

Ubiquitination is the hallmark of protein degradation by the 26S proteasome. However, the proteasome is limited in its capacity to degrade oligomeric and aggregated proteins. Removal of harmful protein aggregates is mediated by autophagy, a mechanism by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Identification of autophagy receptors, such as p62/SQSTM1 and NBR1, which simultaneously bind both ubiquitin and autophagy-specific ubiquitin-like modifiers, LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy. This review explores the hypothesis that ubiquitin represents a selective degradation signal suitable for targeting various types of cargo, ranging from protein aggregates to membrane-bound organelles and microbes.

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

The ATP-gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu-1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu-1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7(-/-) animals. PancTu-1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120-treated mice showed reduced bioluminescence compared to saline-treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120-treated tumours.

Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour. METHODS: We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays). RESULTS: We found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R-pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion. CONCLUSIONS: PDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.

Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells.

BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling. RESULTS: Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.03(YEMK). In duct cells, CDCA caused reversible increase in the intracellular Ca(2+) concentration [Ca(2 +)]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca(2+) signals, but could stimulate Na(+)/Ca(2+) exchange in some conditions. CONCLUSIONS: CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis.

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x(L) positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

Acid-base transport in pancreatic cancer: molecular mechanisms and clinical potential.

Solid tumors are characterized by a microenvironment that is highly acidic, while intracellular pH (pHi) is normal or even elevated. This is the result of elevated metabolic rates in the highly proliferative cancer cells, in conjunction with often greatly increased rates of net cellular acid extrusion. Studies in various cancers have suggested that while the acid extrusion mechanisms employed are generally the same as those in healthy cells, the specific transporters upregulated vary with the cancer type. The main such transporters include Na(+)/H(+) exchangers, various HCO3(-) transporters, H(+) pumps, and lactate-H(+) cotransporters. The mechanisms leading to their dysregulation in cancer are incompletely understood but include changes in transporter expression levels, trafficking and membrane localization, and posttranslational modifications. In turn, accumulating evidence has revealed that in addition to supporting their elevated metabolic rate, their increased acid efflux capacity endows the cancer cells with increased capacity for invasiveness, proliferation, and chemotherapy resistance. The pancreatic duct exhibits an enormous capacity for acid-base transport, rendering pHi dysregulation a potentially very important topic in pancreatic ductal adenocarcinoma (PDAC). PDAC - accounting for about 90% of all pancreatic cancers - has one of the highest cancer mortality rates known, and new diagnostic and treatment options are highly needed. However, very little is known about whether pH regulation is altered in PDAC and, if so, the possible role of this in cancer development. Here, we review current models for pancreatic acid-base transport and pH homeostasis and summarize current views on acid-base dysregulation in cancer, focusing where possible on the few studies to date in PDAC. Finally, we present new data-mining analyses of acid-base transporter expression changes in PDAC and discuss essential directions for future work.

UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes.

Osteocytes reside as a cellular network throughout the mineralised matrix of bone and are considered the primary mechanosensors of this tissue. They sense mechanical stimulation such as fluid flow and are able to regulate osteoblast and osteoclast functions on the bone surface. Previously, we found that ATP is released load-dependently from osteocytes from the onset of mechanical stimulation. Therefore, the aim of the present study was to investigate whether and how ATP release can be evoked in osteocytes via purinergic receptor activation. ATP release was quantified by real-time determination using the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium responses to P2 receptor agonists. These investigations demonstrated that MLO-Y4 osteocytes express functional P2Y(2), P2Y(4), P2Y(12) and P2Y(13) receptors in addition to the previously reported P2X receptors. Further, we found that osteocytes respond to nucleotides such as ATP, UTP and ADP by increasing the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1-10 µM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP signals may be propagated by P2 receptor activation and further ATP release in the osteocyte network and implicate purinergic signalling as a central signalling pathway in osteocyte mechanotransduction.

Imaging of extracellular and intracellular ATP in pancreatic beta cells reveals correlation between glucose metabolism and purinergic signalling.

Adenosine triphosphate (ATP) is a universal energy molecule and yet cells release it and extracellular ATP is an important signalling molecule between cells. Monitoring of ATP levels outside of cells is important for our understanding of physiological and pathophysiological processes in cells/tissues. Here, we focus on pancreatic beta cells (INS-1E) and test the hypothesis that there is an association between intra- and extracellular ATP levels which depends on glucose provision. We imaged real-time changes in extracellular ATP in pancreatic beta cells using two sensors tethered to extracellular aspects of the plasma membrane (eATeam3.10, iATPSnFR1.0). Increase in glucose induced fast micromolar ATP release to the cell surface, depending on glucose concentrations. Chronic pre-treatment with glucose increased the basal ATP signal. In addition, we co-expressed intracellular ATP sensors (ATeam1.30, PercevalHR) in the same cultures and showed that glucose induced fast increases in extracellular and intracellular ATP. Glucose and extracellular ATP stimulated glucose transport monitored by the glucose sensor (FLII12Pglu-700uDelta6). In conclusion, we propose that in beta cells there is a dynamic relation between intra- and extracellular ATP that depends on glucose transport and metabolism and these processes may be tuned by purinergic signalling. Future development of ATP sensors for imaging may aid development of novel approaches to target extracellular ATP in, for example, type 2 diabetes mellitus therapy.

Purinergic regulation of CFTR and Ca(2+)-activated Cl(-) channels and K(+) channels in human pancreatic duct epithelium.

Purinergic agonists have been considered for the treatment of respiratory epithelia in cystic fibrosis (CF) patients. The pancreas, one of the most seriously affected organs in CF, expresses various purinergic receptors. Studies on the rodent pancreas show that purinergic signaling regulates pancreatic secretion. In the present study we aim to identify Cl(-) and K(+) channels in human pancreatic ducts and their regulation by purinergic receptors. Human pancreatic duct epithelia formed by Capan-1 or CFPAC-1 cells were studied in open-circuit Ussing chambers. In Capan-1 cells, ATP/UTP effects were dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3.1). The apical effects of ATP/UTP were greatly potentiated by the IK channel opener DC-EBIO. Determination of RNA and protein levels revealed that Capan-1 cells have high expression of TMEM16A (ANO1), a likely CaCC candidate. We conclude that in human pancreatic duct cells ATP/UTP regulates via purinergic receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct function should consider sidedness of purinergic signaling and the essential role of K(+) channels.