Cell debris was removed by centrifugation at 15,000 g for 20 minutes at 4 C, 144l of cell lysates were transferred to a microplate, 6l of 7. 8 mM Ac DEVD pNA were added and the volume completed to 300l with the reaction buffer. Plates were covered and incubated at 37 C for two to six hours until a yellowish colour was observed. The amount of released p nitroaniline was measured spectrophotometrically at 405 nm in a microplate reader. cAMP levels, amylase activity and secretory profile Acinar cells were assayed for their functional ability to secrete salivary protein and stimulate cAMP levels in response to VIP by determining basal and VIP stimulated cAMP by RIA and amylase secretion as previously reported.
Acinar suspension was incubated for 15 minutes in the absence and presence of 100 nM VIP and amylase activity was determined at 30 minutes in the intracellular fraction and selelck kinase inhibitor in the superna tants. Percentage of secretion was calculated as the ratio of secretion over total amylase and normal ised per mg protein. Protein detection was performed using the Micro BCA Protein Assay in acinar suspension aliquots. Statistical analysis Statistical significance of differences was determined by the two tailed t test for independent populations. When multiple comparisons were necessary, the Student Newman Keuls test was used after analysis of variance. Differences between groups were considered significant at P 0. 05. Results Apoptosis pattern of acinar cells in resting conditions Figure 1 shows acinar cell suspension isolated from sub mandibular glands of NOD and control BALBc mice, both at 16 weeks of age, stained with acridine orange and propidium iodide.
Viable cells fluoresce green under dark field fluores cence microscopy, while nonviable cells fluoresce orange. We investigated further whether freshly isolated acinar cells from NOD mice presented signals of apoptotic events in resting unstimulated conditions. Control acini were obtained from age matched BALBc mice and from NOD mice at eight weeks. As shown in selleck inhibitor Figures 2a and 2b, an increased count of apoptotic acinar cells by Hoechst staining along with an increased expression of Bax at mRNA and protein levels in NOD mice acini compared with control mice was found. An over expression of TP53INP1 has been associated with Bax expression and apoptosis in acinar cells but not in ductal or Langerhans cells in the pancreas of a mouse model of pancre atitis, so we determined TP53INP1 and expression in NOD acinar cells.
Figure 2c shows TP53INP1 and mRNA and protein expression increased only in NOD mice acini com pared with BALBc acinar cells. A faint increase of the TP53INP1 isoform was detected at eight weeks in NOD mice only at the protein level. TNF induced apoptosis in NOD acinar cells With the knowledge that TNF TNF R interaction mediates apoptosis in pancreatic acinar cells and the observation that acinar cells isolated from NOD submandibular glands pre sented several signals of apoptotic events in resting condi tions shown above, we first analysed TNF R expression in NOD and control isolated acinar cells in basal conditions.