Additional file 1: Figure S1. of The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells

Expression of P2X7R in PDACs and HPDE cells. Real time PCR analysis of P2X and P2Y receptors expression in HPDE and PDAC cells. The data were normalized with respect to the three housekeeping genes: β-actin, β glucuronidase (GUSB) and glutaminyl-tRNA synthetase (QARS) and expression in HPDE was set to be 1. The bargraph shows data for four experiments (mean ± SEM). The relative amount of mRNA was calculated from a standard curve run on each plate. Significant difference of expression in PDAC in comparison to HPDE cells is indicated P < 0.05 (*) and P < 0.001 (**). (PDF 201 kb

Additional file 4: Figure S3. of The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells

Absence of cytotoxicity of AZ10606120 on PDAC cells. Representative bright field images of A. Panc-1 and B. BxPC-3 at 20, 40 and 60 h incubation with AZ10606120 (10 μM) and control. Cells were stained with Trypan Blue at the time points given above. In every experiment pictures were taken from eight to twelve fields (n = 3). All bars are 50 μm. (PDF 1214 kb

Expression of H⁺/K⁺-ATPases in human pancreatic duct cell lines Capan-1 (CA), CFPAC-1 (CF) and PANC-1 (PA).

<p><b>A:</b> RT-PCR analysis of gastric H^<b>+</b>/K^<b>+</b>-ATPase α (HKα1, 200 bp), non-gastric H^<b>+</b>/K^<b>+</b>-ATPase α (HKα2, 339 bp) and β (HKβ, 136 bp) subunits. Representative gels for at least three independent experiments. Real time PCR was used to evaluate the relative expression (2^{<b>-ΔΔCt</b>}). The house keeping genes 18S ribosomal RNA (18SrRNA), β-actin, β glucuronidase (GUSB) and glutaminyl-tRNA synthetase (QARS) were used and expression in Capan-1 cells was set to be 1. The graph shows data for three experiments (mean±SEM). Significance of expression was tested by one-way ANOVA using the value 2^<b>-ΔCt</b>. <b>B:</b> Western blot on cell lysates from duct cell lines, as well as control tissues—mouse stomach (S) and colon (C). Antibodies against gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunits (HKα1, Abcam EPR12251), non-gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunits (HKα2, Sigma, HPA039526) and gastric H^<b>+</b>/K^<b>+</b>-ATPase β subunits (HKβ, Sigma A274) were used. Loading control was β-actin detected at 43 kDa. All lanes were loaded with 60 μg of protein. Stomach and colon gels were run separately. Lower bargraphs show expression of the subunits normalized to actin: the bands at 115 kDa (HKα1); 100 kDa (HKα2) and 45 kDa (HKβ) were used. Data is from 3–4 independent experiments and * indicates <i>P</i><0.05 and ** <i>P</i><0.001 compared to Capan-1.</p

Effect of H⁺/K⁺-ATPase inhibitors on pH_i recovery.

<p><b>A:</b> Representative recording of a pH_<b>i</b> measurement in Capan-1 cells challenged with ammonium pulse in Na^<b>+</b>-containing physiological buffer, then in a Na^<b>+</b>-free buffer with or without Na^<b>+</b> with omeprazole (10 μM). Dotted lines show the slope of the pH_<b>i</b> recovery from acidosis, i.e. dpH/dt. The pHi recovery was determined when cells were returned to control conditions (periods I, II and III) and in periods with Na^<b>+</b>-free buffer +/- proton inhibitor. <b>B:</b> Summary of recovery rates expressed as dpH/dt upon return to Na^<b>+</b>-containing buffer (first three bars) and in Na^<b>+</b>-free buffer (next two bars) (n = 6). <b>C, D</b>: similar representative recording and summary for SCH28080 (10 μM, n = 5). Cells were stimulated with secretin (10^<b>–9</b> M) and buffers were HCO_<b>3</b>^<b>—</b>free in order to eliminate contribution of HCO_<b>3</b>^<b>-</b> transporters. Bars show paired measurements as means ± SEM. Asterisks indicates P < 0.05.</p

Composition of pancreatic secretions.

<p>Relation between pancreatic secretory rates and concentrations of HCO_<b>3</b>^<b>-</b> (<b>A</b>), Cl^<b>-</b> (<b>B</b>), Na^<b>+</b> (<b>C</b>) and K^<b>+</b> (<b>D</b>). Data for control experiments are represented blue, acute omeprazole treatment experiments in red and long-term omeprazole treatment experiments in green. Data for effect of SCH28080 on HCO_<b>3</b>^<b>-</b> concentrations are also included and marked in yellow symbols. Data are represented as mean value±SEM for 3–15 samples that were binned in 1 µl/min-kg secretion rate intervals.</p

Proton Pump Inhibitors Inhibit Pancreatic Secretion: Role of Gastric and Non-Gastric H⁺/K⁺-ATPases

<div><p>The mechanism by which pancreas secretes high HCO₃^- has not been fully resolved. This alkaline secretion, formed in pancreatic ducts, can be achieved by transporting HCO₃^- from serosa to mucosa or by moving H⁺ in the opposite direction. The aim of the present study was to determine whether H⁺/K⁺-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKβ subunits (<i>ATP4A</i>; <i>ATP4B</i>) and non-gastric HKα2 subunits (<i>ATP12A</i>) of H⁺/K⁺-ATPases are expressed in human pancreatic cells. Pumps have similar localizations in duct cell monolayers (Capan-1) and human pancreas, and notably the gastric pumps are localized on the luminal membranes. In Capan-1 cells, PPIs inhibited recovery of intracellular pH from acidosis. Furthermore, in rats treated with PPIs, pancreatic secretion was inhibited but concentrations of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO₃^-, pancreas also secretes K⁺. In conclusion, this study calls for a revision of the basic model for HCO₃^- secretion. We propose that proton transport is driving secretion, and that in addition it may provide a protective pH buffer zone and K⁺ recirculation. Furthermore, it seems relevant to re-evaluate whether PPIs should be used in treatment therapies where pancreatic functions are already compromised.</p></div

Immunolocalization of H⁺/K⁺-ATPases in sections of human pancreas.

<p><b>A</b>: The gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunit (HKα1) was labeled with Calbiochem 119101 (polyclonal, against HKα1C-terminal) (image 1 and 2) and Calbiochem 119102 (polyclonal, against HKα1 N-terminal) and Alexa 488 (green); <b>B</b>: The gastric H^<b>+</b>/K^<b>+</b>-ATPase β subunit (HKβ) was stained with Sigma A-274 (2G11, anti HKβ, monoclonal) and Alexa 488 (green). <b>C:</b> The non-gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunit (HKα2) was stained with non-gastric HKα2 antibody (C384-M79) and Alexa 488 (green). <b>D</b>: Example of a control image without primary antibodies. DAPI was used to stain the nucleus (blue). All bars are 25 μm, and images are from 3 independent experiments and show localization in ducts of various sizes.</p

Immunolocalization of H⁺/K⁺-ATPases in Capan-1 cells grown on permeable membranes.

<p><b>A</b>: The gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunit (HKα1) was labeled with Calbiochem 119101 (polyclonal, against HKα1C-terminal) and Alexa 488 (green). <b>B</b>: The gastric H^<b>+</b>/K^<b>+</b>-ATPase β subunit (HKβ) was detected with Sigma A-274 (2G11, anti HKβ, monoclonal) and Alexa 488 (green). <b>C</b>: The non-gastric H^<b>+</b>/K^<b>+</b>-ATPase α subunit (HKα2) was stained with non-gastric HKα2 antibody (C384-M79) and Alexa 488 (green) Three pairs of images are shown. <b>D</b>: Example of a control image without primary antibodies but with F-actin marker (phalloidin Texas red). DAPI was used to stain the nucleus (blue). All bars are 10 μm, and images from at least 10 independent experiments are presented as both x-y and x-z scans. In x-y scan the left images are taken mid-way through the monolayer, the right images are taken close to the apical membrane. Dotted lines in x-y scans indicate where the x-z scan was taken.</p

Primer sets used for human pancreatic duct cell lines in RT-PCR and real-time analysis.

<p>Primer sets used for human pancreatic duct cell lines in RT-PCR and real-time analysis.</p

Effects of proton pump inhibitors on pancreatic secretion rates in rats.

<p>Controls are represented as blue line and in each graph n is 6, 4, 7 and 4 respectively. <b>A</b>: Effects of 5 mg/kg (red line, n = 4) and 20 mg/kg omeprazole (green line, n = 6) on non-fasted rats. <b>B</b>: Effects of 5 mg/kg (red line, n = 4) and 20 mg/kg omeprazole (green line, n = 4) on fasted rats. <b>C</b>: Effect of 10 mg/kg SCH28080 (yellow line, n = 6) on fasted rats. <b>D</b>: Effects of long term (30 days) daily administration of 5 mg/kg omeprazole (red line, n = 4). Secretory rates are represented as mean values ± SEM. * indicates <i>P</i><0.05, ** P<0.01.</p