Kinetic analyses of the mechanisms of action of inhibitors of the extraneuronal uptake of adrenaline in smooth muscle cells of guinea-pig trachea.

1. Tracheal segments from guinea-pigs pretreated with 6-hydroxydopamine were incubated with adrenaline (50--300 microM) at 37 degrees C for 5 min in the absence or presence of an extraneuronal uptake inhibitor (normetanephrine, corticosterone or phenoxybenzamine). Catechol-O-methyl transferase and monoamine oxidase were inhibited by 100 microM U-0521 and pargyline, respectively. Tissues were prepared for fluorescence histochemistry, and accumulated adrenaline in trachealis smooth muscle cells was measured by fluorescence microphotometry. Initial rates of adrenaline uptake were calculated. 2. In the absence of an extraneuronal uptake inhibitor, adrenaline uptake obeyed Michaelis-Menten saturation kinetics. The mean Km value for adrenaline from a total of 25 guinea-pigs was 157 microM. 3. The mechanisms of action of three extraneuronal uptake inhibitors were determined by analysis of initial rate kinetic data for adrenaline uptake. The results showed that normetanephrine and corticosterone were reversible, competitive inhibitors and phenoxybenzamine was an irreversible inhibitor of extraneuronal uptake in the smooth muscle cells. 4. Two methods of analysis were used to obtain estimates of the affinities of the reversible inhibitors for extraneuronal uptake. The Ki values calculated by Marquardt non-linear least squares regression analysis and Hunter-Downs method, respectively, were normetanephrine: 2.75 microM and 3.47 microM, and corticosterone: 1.52 microM and 1.44 microM. 5. Normetanephrine and corticosterone had higher affinities than adrenaline for extraneuronal uptake in the trachealis smooth muscle cells.