Catecholamine transport by isolated chromaffin granules. Influence of MgATP and a disulfonic stilbene on (R)-norepinephrine/epinephrine exchange and spontaneous epinephrine efflux

Isolated chromaffin granules were found to accumulate exogenous (R)-[³H]norepinephrine in the presence of Mg²⁺-ATP by exchange for endogenous epinephrine at nearly a 1:1 ratio. At low concentrations of (R)-norepinephrine (<200 μM), uptake followed Michaelis-Menten kinetics with regard to both MgATP and [(R)-norepinephrine] (K(m)=123 μM and 49 μM, respectively). At high concentrations of (R)-norepinephrine (e.g. 5.7 mM), substantial amounts of additional (R)-[³H]norepinephrine entered granules in a manner independent of [MgATP]. Incubation of granules without added MgATP or external (R)-norepinephrine resulted in the release of substantial amounts of endogenous epinephrine into the medium, and addition of ATP suppressed this epinephrine leakage by a saturable mechanism (K(m)=40 μM). However, while the MgATP-dependent (R)-[³H]norepinephrine/epinephrine exchange and ATPase activity were both inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), SITS did not affect either ATP-inhibited epinephrine leakage or ATP-independent (R)-norepinephrine uptake. We conclude from these data that ATP plays several independent roles in the function of intact granules. One role of ATP is to catalyze bidirectional catecholamine flux across the granule membrane rather than unidirectional net accumulation into intact granules. This flux depends on the ATPase activity. A second role, apparently independent of the ATPase activity, is to prevent spontaneous epinephrine leakage from granules. The fact that ATP is required for an otherwise isoenergetic exchange of catecholamines had led us to certain general conclusions about transport mechanism in this system, as well as a consideration of the possible relevance of the heterogeneous structure of the granule interior to transport properties.