Chemical evidence that catecholamines are transported across the chromaffin granule membrane as noncationic species.
Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process under conditions in which the substrate exists primarily as a positively charged or neutral species. In order to distinguish between these two states, we studied the transport properties of a permanently charged quaternary analogue of epinephrine, (R,S)-dimethylepinephrine. We found that this compound was a classical competitive inhibitor of (R)-[3H]norepinephrine uptake, with a Ki of 3.8 mM for the racemic form, or 1.9 mM for the R form. However, the [3H]dimethylepinephrine was not transported at all into granules. Our control for steric hindrance as an explanation for deficient translocation was analysis of the transport properties of isoproterenol, a secondary catecholamine with an isopropyl group around the amine residue. (R)-Isoproterenol was an effective competitive inhibitor of (R)-[3H]norepinephrine transport, with a Ki of 91 microM. In contrast to dimethylepinephrine, (R,S)-[3H]isoproterenol was clearly translocated across the granule membrane, with a Km of 123 microM, or 61.5 microM for the R isomer. Thus, the positive charge on dimethylepinephrine and not the size of the amine moiety appeared to be responsible for the lack of translocation. We interpret these data to indicate that, although the positively charged species can interact with the transport site, an uncharged species is the one actually transported.
D000818:Animals; D001667:Binding, Competitive; D001693:Biological Transport, Active; D002395:Catecholamines / Q000378:metabolism*; D002412:Cations; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D004837:Epinephrine / Q000031:analogs & derivatives*; D007545:Isoproterenol / Q000378:metabolism; D007700:Kinetics; D009638:Norepinephrine / Q000378:metabolism; D013329:Structure-Activity Relationship
D002395:Catecholamines; D002412:Cations; C037919:N,N-dimethylepinephrine; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine