CHARACTERIZING CATECHOLAMINE TRANSPORT AND THE CATECHOLAMINE TRANSPORTER OF THE CHROMAFFIN GRANULE MEMBRANE

Catecholamines (CA)--dopamine (DA), norepinephrine (NE) and epinephrine (E)--are taken up and stored by organelles in the chromaffin cell of the adrenal medulla. The energy for accumulating the high concentration of CA in the granule is derived from the protonmotive force established by proton pumping ATPase in the granule membrane. Active transport of CA is accomplished by electrogenic exchange of two protons for each CA(\u27+) via a reserpine sensitive CA transporter in the granule membrane. In experiments to further probe the role of ATP in CA uptake and release we have found that ATP can stimulate net uptake, not just exchange, of CA in freshly isolated granules and in lysed resealed vesicles (ghosts). We have also found that ATP limits release of CA from granules or ghosts by a process other than reuptake. This process depends primarily on hydrolysis of ATP and activity of the proton pump, but is reserpine insensitive and therefore does not depend on CA transporter function. It is proposed that release of CA occurs by diffusion across the granule membrane and that the rate of diffusion is controlled by the effective concentration of CA(\u27o). CA(\u27o) concentration is decreased at lowered pH, and in the presence of ATP as a consequence of the formation of weak electrostatic CA:ATP complexes. Experiments to test the ability of several CA receptor ligands to bind to the CA transporter have revealed that (alpha)(,1) adrenergic receptor ligands, prazosin, and phenoxybenzamine are the most effective inhibitors of CA transport, followed closely by (beta) adrenergic and dopamine receptor ligands, propranolol, alprenolol, isoproterenol, NPA and ADTN. The (alpha)(,2) agonist clonidine, and the serotonin receptor antagonist methysergide were an order of magnitude less effective. Antagonists were generally more effective in their inhibition of NE uptake than agonists. These results suggest that the binding site of the transporter shares similarities with the binding site of CA receptors. Covalent labeling of granule membrane proteins, and separation on SDS PAGE gels revealed 3 bands labeled in common by ((\u273)H) phenoxybenzamine and ((\u273)H)NE. These were at molecular weights about 20K, 32K and 45K