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 populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.
Gualix
J
J
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Alvarez
A M
AM
Pintor
J
J
Miras-Portugal
M T
MT
D000311:Adrenal Glands; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002414:Cations, Monovalent / Q000494:pharmacology; D002417:Cattle; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism*; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D009638:Norepinephrine / Q000494:pharmacology; D010316:Particle Size; D012110:Reserpine / Q000494:pharmacology; D016708:Synaptophysin / Q000302:isolation & purification
D002414:Cations, Monovalent; D018118:Chloride Channels; D004985:Ethenoadenosine Triphosphate; D016708:Synaptophysin; D012110:Reserpine; D009638:Norepinephrine