Journal: The Journal of Physiology

Article Title: Intracellular rupture, exocytosis and actin interaction of endocytic vacuoles in pancreatic acinar cells: initiating events in acute pancreatitis

doi: 10.1113/JP275879

Figure Lengend Snippet: A , cells were stimulated by 10 n m CCK for 2 h at 35 °C in the presence of diS‐Cy5 and dextran Texas Red 3000 MW neutral (TRD). Cells were then washed by perfusion with standard extracellular solution. Following the wash, cells were immersed in the extracellular solution containing FITCD and imaged. Upper left: transmitted light image of the cells. Scale bar = 10 μm. Images of the fluorescence of the cells and extracellular milieu recorded using excitation/emission corresponding to the specified probes shown elsewhere. Note the EV in the right cell and increased cytosolic fluorescence on diS‐Cy5 and TRD images of the left cell. The FITCD image indicates that the plasma membrane of the cell is intact. B , representative fluorescence emission spectra recorded from cells stimulated as in ( A ) but in the presence of diS‐Cy5 only. The fluorescence was excited by a 595 nm laser line. The graphs show fluorescence spectra recorded from an EV (green), cytosol of a cell that satisfies criteria for detecting the EV's rupture/leakage (red; see Methods and C ), cytosol of a cell that did not satisfy criteria for detecting rupture/leakage (black; see Methods and C ). The blue trace in the insert is produced by subtracting black trace (mainly determined by cellular autofluorescence) from the cytosolic fluorescence of a cell that satisfies the criteria for detecting rupture/leakage. The residual trace shows a spectrum that is similar to the spectrum of diS‐Cy5 recorded from an EV. The red and black traces were recorded from different cells and shown on the same graph for illustration. C , left: intensities of cytosolic fluorescence in cells that were immersed in the indicator‐containing diS‐Cy5 solution for 30 min but were not stimulated. Only a very few small vacuoles are expected to form during this period of time (Fig. B and C ) and the distribution should therefore reflect cytosolic fluorescence in the cells that did not have ruptured EVs. The blue trace represents a single Gaussian approximation of the distribution. Right: frequency histogram of cells after two hours of incubation with diS‐Cy5. The CCK concentration was 10 n m . The first two Gaussian peaks of the approximation are shown by blue and magenta lines. Cells with cytosolic fluorescence above threshold (central value of the first peak plus 3 sigma) are classified as the cells that experienced rupture/leakage of EV(s). D , the method illustrated in ( A ) to ( C ) was used to evaluate the proportions of cells with ruptured vacuoles. CCK concentration was 10 n m (in specified experiments). Neither inhibition of serine protease with benzamidine (1 m m ), nor inhibition of cathepsin B with combination of CA074 (10 μ m ) and CA074‐Me (1 μ m ) (abbreviated as CA074/Me) produced a significant difference in the proportion of cells with increased cytosolic fluorescence from control. Inhibition of V‐ATPase with 100 n m of bafilomycin A1 (Baf) also did not produce a statistically significant change in the proportion of cells with increased cytosolic fluorescence. The number of experiments in each condition was: n = 20 experiments for control (unstimulated cells) and CCK; n = 9 for CA074/Me and CA074/Me + CCK; n = 8 for benzamidine and benzamidine + CCK; n = 6 for bafilomycin A1 and bafilomycin A1 + CCK. Each of the individual experiments involved acquisition and analysis of a fluorescence distribution similar to that shown on the right of ( C ).

Article Snippet: CA‐074 and bafilomycin A1 were obtained from Tocris Bioscience (Bristol, UK) and CA‐074Me was obtained from Calbiochem.

Techniques: Fluorescence, Clinical Proteomics, Membrane, Produced, Incubation, Concentration Assay, Inhibition, Control