Saturation kinetics of the adrenergic neurone uptake system in the perfused rabbit heart. A new method for determination of initial rates of amine uptake

Summary

• 1. Hearts were obtained from normal or reserpine-pretreated rabbits and perfused at a constant rate (3.6 ml·g⁻¹·min⁻¹) with Tyrode's solution containing ¹⁴C- or ³H-sorbitol and various concentrations of ³H-(−)noradrenaline (NA), ¹⁴C-(+)NA or ³H-(±)metaraminol; when NA was used, monoamine oxidase and catechol-O-methyl transferase were inhibited. During perfusion for 2 min the arterio-venous difference for ³H and ¹⁴C activity (and in this way the removal of amine and sorbitol from the perfusion fluid) was determined at intervals of 5 s. The uptake of amine into intracellular spaces of the heart was obtained by subtraction of the removal of sorbitol from that of amine; it was cumulatively added and plotted against time (uptake curve). Uptake was overwhelmingly neuronal.

• 2. The uptake curves were sigmoidal: after a brief initial lag period, uptake curves became linear; there-after, the slope of the curves decreased. The last phase of divergence from linearity occurred the earlier and was the more pronounced, the higher the amine concentration. It was interpreted to indicate that neuronal efflux of amine then began to reduce net uptake.

• 3. From the slope of the linear phase of the uptake curves initial rates of amine transport were obtained. They were saturable with increasing amine concentrations and obeyed Michaelis-Menten kinetics. The apparent K _m values of the three amines were similar in magnitude and ranged from 2.9 to 5.9 μM. Uptake was stereoselective in that the V _max of (+)NA was significantly lower than that of (−)NA. Pretreatment with reserpine affected neither the K _m nor the V _max for uptake. Cocaine was a potent competitive inhibitor of amine transport (K _i=0.5–1.0 μM).

• 4. The intercept of the linear phase of the uptake curves on the time axis (t _lag) (corrected for the time necessary for transit through the dead space) was taken as a measure of the lag period. It declined when uptake was progressively saturated (or inhibited) by increasing substrate (or cocaine) concentrations. Moreover, t _lag was always linearly correlated with the fraction of amine removed from the perfusion fluid. These findings indicate that the equilibration of the uptake sites with the substrate concentration in the perfusion fluid is delayed by the uptake process itself, especially under low saturation conditions (i.e., when S<K _m).