Kinetic constants for uptake and metabolism of 3H-(-)noradrenaline in rabbit aorta. Possible falsification of the constants by diffusion barriers within the vessel wall

Abstract

1. The neuronal uptake of 3H-(-)noradrenaline into aortic rings from reserpine-pretreated animals was a saturable process with a Km of 2.3 mumol X l-1 and a Vmax of 0.5 nmol X g-1 X min-1. Similar constants were obtained when the neuronal deamination of noradrenaline was taken as an index for neuronal uptake. When the tissue was incubated in the usual way, i.e., when the amine was allowed to enter the aortic rings via both the intimal and the adventitial surface, then there was no initial delay (tlag) for the neuronal uptake of noradrenaline (MAO and COMT inhibited). On the other hand, when the amine entry was restricted to the intimal surface, there was a tlag of 2 min, probably due to the slow equilibration of the extracellular space of the media with the incubation medium. Furthermore, for low amine concentrations the rate of uptake in the latter situation was about 10 times lower than that in the former one. Thus, the rate of uptake clearly depended on the way the amine entered the tissue. 2. The corticosterone-sensitive extraneural uptake of 3H-(-)noradrenaline determined in nerve-free aortic rings was characterised by a high Km (490 mumol X l-1) and Vmax (35 nmol X g-1 X min-1). For uptake2 a tlag of 1 min was observed. 3. The analysis of the extraneuronal O-methylating system in nerve-free aortic rings yielded a Km of 3.6 mumol X l-1 and a Vmax of 0.6 nmol X g-1 X min-1. The tlag for the O-methylation of noradrenaline was in the same order of magnitude as that for uptake2. At low amine concentrations the corticosterone-sensitive accumulation of noradrenaline was prevented by COMT, but not by MAO. The latter enzyme reduced the steady-state accumulation of noradrenaline by about 50%. When the amine entry was restricted to one surface only, the results indicated that the extraneuronal O-methylation of noradrenaline generated a steep concentration gradient of the parent amine within the extracellular space of the aorta. 4. All saturable processes fitted the Michaelis-Menten equation. However, kinetic constants determined in incubated organs may be falsified by poor diffusion of the substrate through the extracellular space.