Evidence for stimulation of anion transport in ATP-evoked transmitter release from isolated secretory vesicles.

Permeable anions such as chloride are required for ATP-evoked release of epinephrine and protein from isolated secretory vesicles (chromaffin granules) of the adrenal medulla. This reaction has been studied as a model for the exocytosis process, and we have found that the release reaction is a saturable function of ATP concentration at a variety of chloride concentrations. However, at constant levels of ATP, the stimulation of release by increasing chloride concentrations showed sigmoid kinetics with release being minimal at [Cl] = 10 to 20 mn and saturated at [Cl] = 120 to 130 mn. The saturation of release rate at higher chloride levels led us to investigate the possible existence of an anion transport system in chromaffin granules. Chemical agents which block red cell anion transport were also found to block epinephrine release from granules. The agents included 4-acetamido-4’-isothiocyanostilbene2,2’-disulfonic acid, disodium (SITS, & = 40 PM); probenecid (Ki = 125 PM); pyridoxal5’-phosphate (Ki = 3.6 mu); and sodium isethionate (Ki = 35 mM). Sodium bisulfite (NaHS03) also inhibited release (Ki = 1.1 mn). We had previously shown (Pollard, H. B. Zinder, O., Hoffman, P. G., and Nikodijevik, 0. (1976) J. Biol. Chem. 251,4544; Pollard, H. B., Pazoles, C. J., Hoffman, P. G., Zinder, O., and Nikodijevik, 0. (1977) in CeZZuZur iVeurobiology (Hall, Z., Kelly, P., and Fox, F., eds) Vol. 15, p. 269, Alan R. Liss, N.Y.) that ATP induced anion uptake into granules by making the transmembrane potential relatively positive inside, and that release was a consequence of subsequent osmotic lysis. The present results suggest that anions such as chloride enter the granule via an anion transport site pharmacologically homologous to that in the human erythrocyte anion transport system. We suggest that these newly discovered granule anion transport sites may be intimately related to the mechanism of release by exocytosis.