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Evidence of cl-/h+ -exchange activity in secretory granules from rat peritoneal mast cells The nine members of the CLC gene family can be divided into three subbrances. One group functions mainly in the plasma membrane and the other two functions mainly in intracellular membranes. Vesicular Cl- channels (mainly CLC-3) are thought to provide neutralizing anion currents for the V-ATPases acidifying the lumen of the synaptic vesicles (Jentsch et al., 2005; Nelson et al., 1999). Recently, it was reported that ClC-4 and ClC-5 function as electrogenic Cl-/H+ exchangers (Scheel et al., 2005; Picollo et al., 2005). Their physiological role has still to be established. However, they might constitute a break to limit acidification by the V-ATPases or constitute a part of a postulated H+ leak of the synaptic membrane (Picollo et al., 2005). We have compared pH-changes over time in isolated granule from rat mast cells suspended in chloride containing and chloride-free solution with gluconate in order to study if the granule membrane exhibits Cl-/H+ exchange activity. Methods: Granules were isolated from pure populations of peritoneal mast cells from male Sprague-Dawley rat (260-490 g) by sonication. They were loaded with 5μM 2’,7´-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescin-acetoxymethyl (BCECF-AM). Measurements of pH were performed with a fluorescence spectrometer. Excitation was performed at 435nm and 490nm and emission was measured at 535nm. The membrane permeable dye (BCECF-AM) becomes pH-sensitive (excitation at 490nm) when the acetoxymethyl group is removed by hydrolysis with esterases. A measurement of fluorescence intensity at 535nm with excitation at 435nm (pH-insensitive) was used to quantify changes in BCECF concentration. Conversion of excitation ratio (490nm/ 435nm) to pH was performed by intragranular calibration using nigericin to equilibrate pH across the membrane. Results: The rate of appearance of BCECF in granule suspensions was enhanced compared to the spontaneous hydrolysis of BCECF-AM in salt solutions and pure water, which indicates esterase activity in the granules. Loading of isolated granules with BCECF was observed using imaging fluorescence technique. This was confirmed by measuring fluorescence intensity of the supernatant to be 19% (N=4) of the light emission from granules after resuspension. The pH of granules was 6.1-6.2 in potassium gluconate as well as in KCl. In chloride-free medium there was a pH-increase of the granules which was linear related to time of incubation. The rate of increase was 0.09 and 0.07 pH-units per 10 min incubation in two groups of experiments. In chloride medium there was an initial enhancement of the pH-increase to 0.25 pH-units in 10 min. The pH-increase following addition of FCCP was also 0.25 pH-units per 10 min. The pH-increase in chloride medium was enlarged when medium pH was increased to 7.4. Using one-way ANOVA for statistical analysis the pH-increase in chloride medium was significantly increased compared to the pH-increase in chloride-free medium. FCCP also caused a significantly increase of granular pH. Conclusion: Our results indicate that isolated granules from rat mast cells contain esterase activity, have a pH of 6.1-6.2, and show Cl-/H+ exchange activity.
Jentsch, T et al.. (2005). Ann. Rev. Physiol. 67:779-807 |
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