Kinetic analysis of stimulation-evoked overflow of noradrenaline and dopamine β-hydroxylase from the isolated rabbit heart. The effect of dopa decar☐ylase inibition

Abstract

Rabbit isolated hearts were perfused according to a modified Langendorff procedure. Noradrenaline was measured in the venous effluents whereas dopamine β-hydroxylase was determined in the transmyocardial fluid drained from the interstitium. Calcium-dependent release of noradrenaline and dopamine β-hydroxylase was evoked by electrical stimulation of the heart for 6 periods of 1 min duration at 30 min intervals. Tetraethylammonium was added to enhance release. As a result of repeated stimulation, the dopamine β-hydroxylase overflow from the heart declined monoexponentially, whereas the decline of noradrenaline was biexponential. The two phases of decline were unaltered by cocaine, an inhibitor of noradrenaline uptake. However, pretreatment of the rabbits with monofluoromethyldopa, an irreversible inhibitor of DOPA decar☐ylase, transformed the biexponential decline of noradrenaline overflow into a monoexponential one with the same rate constant as that of dopamine β-hydroxylase overflow. The sum of noradrenaline released plus that remaining in the heart was perfectly matched by the area under the curve of noradrenaline overflow values. Using the dopamine β-hydroxylase overflow curve, the pool size of the soluble enzyme was calculated which, conversely, agreed with the enzyme contained in the 100,000 g supernatant of strongly homogenized unstimulated hearts. Before stimulation, 18% of the total dopamine β-hydroxylase activity was in the soluble form. Stimulation depleted the hearts of noradrenaline and soluble dopamine β-hydroxylase in a stoichiometric manner, provided that noradrenaline synthesis was blocked. The present work utilizes a novel method of measuring soluble dopamine β-hydroxylase that is available for exocytotic release. Under the conditions of repeated stimulation and prolonged action potentials, the only factor interfering with the stoichiometry of noradrenaline and dopamine β-hydroxylase release is the re-synthesis of noradrenaline.