Studies of chromaffin granule functioning by flow cytometry: Transport of fluorescent epsilon-ATP and granular size increase induced by ATP

Flow cytometry techniques, usually employed to characterize cellular popula tions, are reported here to be a valuable tool to approach the study of sub cellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detec table by flow cytometry. Moreover, these storage granules are able to trans port the fluorescent ATP analogue, epsilon-ATP (1,N-6-ethenoadenosine 5'-tr iphosphate), and the resulting granular fluorescence increase can also be f ollowed by this technique. The saturation studies show a non-hyperbolic kin etic behaviour, with a two step curve. The K-0.5 values were 0.26 and 2.5 m M and Hill numbers 1 and 6 respectively. In addition, an unexpected granula r size increase, which was dependent on the epsilon-ATP concentration, occu rred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable a nalogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect , which exhibited sigmoidal saturation curves with K-0.5 values of 1.8 and 3.1 mM for ATP and ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence o f ATP. Extragranular addition of noradrenaline has no effect by itself on t he granular size, but significantly reduced the granular size increase indu ced by ATP. This effect was reversed by the amine transport inhibitor reser pine. The granular size increase induced by ATP was more effective in the p resence of Cl- than Br- or I-. Moreover, no increase occurred in the presen ce of F- or acetate. The Cl- channel blockers were poorly effective, and on ly 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induce d granular size increase.