A MULTISUBSTRATE MECHANISM OF STRIATAL DOPAMINE UPTAKE AND ITS INHIBITION BY COCAINE

A study of Na+ and Cl- as co-substrates in dopamine uptake into striatal suspensions and inhibition of dopamine uptake by cocaine was made by monitoring the initial velocity of the uptake of exogenously added non-radioactively labeled dopamine using a rotating disk electroanalytical technique with 50 msec resolution. Dopamine, in the concentration range of 0.025 to 4.00-mu-M, was found to be taken up rapidly into the tissue phase of striatal suspensions following the apparent zero order rate law for the first 25 sec. The observed, dopamine concentration-dependent, initial velocity data were first analyzed graphically using the Eadie-Hofstee transformation of the Michaelis-Menten kinetic equation and, subsequently, using all of the velocity data and the results of the graphical analyses, by non-linear curve fitting. Dopamine uptake was found to be first order in dopamine with a V(max) of 582 pmol/sec/g wet weight and a K(m) of 1.2-mu-M. The results of experiments in which choline and isethionate were substituted for Na+ and Cl-, respectively, suggested that the uptake process is second order in Na+ and first order in Cl-. Multisubstrate analyses of the initial velocities of uptake over the concentration range of 0.025 to 1.5-mu-M dopamine suggested that the mechanism of binding of dopamine to the uptake carrier is a partially random, sequential mechanism where dopamine or Na+ binds first with the uptake carrier and Cl- binds last. Cocaine was found to uncompetitively inhibit dopamine uptake and competitively inhibit both Na+ and Cl- binding (apparent K(m) values: 131 and 51 mM, respectively), suggesting that the mechanism of cocaine inhibition may be to bind to the dopamine occupied uptake carrier complex at the Na+ binding site.