Table 1.
Primer sets used for human pancreatic duct cell lines in RT-PCR and real-time analysis.
Fig 1.
Expression of H+/K+-ATPases in human pancreatic duct cell lines Capan-1 (CA), CFPAC-1 (CF) and PANC-1 (PA).
A: RT-PCR analysis of gastric H+/K+-ATPase α (HKα1, 200 bp), non-gastric H+/K+-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-ΔΔCt). 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-ΔCt. 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+/K+-ATPase α subunits (HKα1, Abcam EPR12251), non-gastric H+/K+-ATPase α subunits (HKα2, Sigma, HPA039526) and gastric H+/K+-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 P<0.05 and ** P<0.001 compared to Capan-1.
Fig 2.
Immunolocalization of H+/K+-ATPases in Capan-1 cells grown on permeable membranes.
A: The gastric H+/K+-ATPase α subunit (HKα1) was labeled with Calbiochem 119101 (polyclonal, against HKα1C-terminal) and Alexa 488 (green). B: The gastric H+/K+-ATPase β subunit (HKβ) was detected with Sigma A-274 (2G11, anti HKβ, monoclonal) and Alexa 488 (green). C: The non-gastric H+/K+-ATPase α subunit (HKα2) was stained with non-gastric HKα2 antibody (C384-M79) and Alexa 488 (green) Three pairs of images are shown. D: 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.
Fig 3.
Immunolocalization of H+/K+-ATPases in sections of human pancreas.
A: The gastric H+/K+-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: The gastric H+/K+-ATPase β subunit (HKβ) was stained with Sigma A-274 (2G11, anti HKβ, monoclonal) and Alexa 488 (green). C: The non-gastric H+/K+-ATPase α subunit (HKα2) was stained with non-gastric HKα2 antibody (C384-M79) and Alexa 488 (green). D: 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.
Fig 4.
Effect of H+/K+-ATPase inhibitors on pHi recovery.
A: Representative recording of a pHi measurement in Capan-1 cells challenged with ammonium pulse in Na+-containing physiological buffer, then in a Na+-free buffer with or without Na+ with omeprazole (10 μM). Dotted lines show the slope of the pHi 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+-free buffer +/- proton inhibitor. B: Summary of recovery rates expressed as dpH/dt upon return to Na+-containing buffer (first three bars) and in Na+-free buffer (next two bars) (n = 6). C, D: similar representative recording and summary for SCH28080 (10 μM, n = 5). Cells were stimulated with secretin (10–9 M) and buffers were HCO3—free in order to eliminate contribution of HCO3- transporters. Bars show paired measurements as means ± SEM. Asterisks indicates P < 0.05.
Fig 5.
Effects of proton pump inhibitors on pancreatic secretion rates in rats.
Controls are represented as blue line and in each graph n is 6, 4, 7 and 4 respectively. A: Effects of 5 mg/kg (red line, n = 4) and 20 mg/kg omeprazole (green line, n = 6) on non-fasted rats. B: Effects of 5 mg/kg (red line, n = 4) and 20 mg/kg omeprazole (green line, n = 4) on fasted rats. C: Effect of 10 mg/kg SCH28080 (yellow line, n = 6) on fasted rats. D: 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 P<0.05, ** P<0.01.
Fig 6.
Composition of pancreatic secretions.
Relation between pancreatic secretory rates and concentrations of HCO3- (A), Cl- (B), Na+ (C) and K+ (D). 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 HCO3- 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.