Evidence for exocytotic release of dopamine beta-hydroxylase from rabbit heart and of vasopressin from rat neurohypophyses during homogenization and fractionation. Effects of gadolinium ions, cytochalasin B, gallopamil and different temperatures.

Isolated rabbit hearts were perfused according to a modified Langendorff method for 1 h (unstimulated hearts). In different hearts, release of dopamine beta-hydroxylase activity into the transmyocardial fluid draining the interstitium was evoked by electrical field stimulation for six periods of 1 min at 30 min intervals (stimulated hearts). The hearts were then homogenized and fractionated into 100,000 g supernatant and sedimented at 4 degrees C. In homogenates from unstimulated hearts, the soluble dopamine beta-hydroxylase (determined in the supernatant) accounted for 17% of the total dopamine beta-hydroxylase (determined in the homogenate). In stimulated hearts the soluble fraction of dopamine beta-hydroxylase was reduced by 65%. The dopamine beta-hydroxylase released into the transmyocardial fluid by electrical stimulation, expressed as fraction of the total activity, corresponded well to the loss of enzyme from the supernatant demonstrating that the soluble dopamine beta-hydroxylase determined from the supernatant represents the releasable pool. Gadolinium ions (Gd3+) added to the homogenization medium of unstimulated hearts reduced the soluble fraction of dopamine beta-hydroxylase up to 63%, with the maximum effect at 200 microM. Similarly, when neurohypophyses were homogenized and spun at 0-4 degrees C, the fraction of vasopressin in the soluble phase was about 50% of the total. Gd3+ reduced this fraction by maximally 60%, an effect which was accompanied by an increase of vasopressin in the sedimentable fraction. When cytochalasin B (10 microM) was present during the homogenization of the hearts the soluble fraction of dopamine beta-hydroxylase was reduced to the same extent as in the presence of Gd3+. However, cytochalasin B had no effect on the distribution of vasopressin in the soluble and sedimentable fractions of homogenates of neurohypophyses. Gallopamil, when present during the homogenization of the hearts at a maximum effective concentration of 1 microM, reduced the soluble fraction of dopamine beta-hydroxylase by only 40%. However, the electrically evoked noradrenaline release from perfused hearts was completely blocked at 100-300 microM gallopamil. When neurohypophyses were homogenized and fractionated at room temperature only 13% of the total vasopressin was found in the soluble fraction and Gd3+ did not further reduce this fraction. When unstimulated hearts were homogenized and fractionated at room temperature the fraction of soluble dopamine beta-hydroxylase was reduced by 40% compared to the experiments at 0-4 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic analysis of stimulation-evoked overflow of noradrenaline and dopamine beta-hydroxylase from the isolated rabbit heart. The effect of DOPA decarboxylase inhibition.

Rabbit isolated hearts were perfused according to a modified Langendorff procedure. Noradrenaline was measured in the venous effluents whereas dopamine beta-hydroxylase was determined in the transmyocardial fluid drained from the interstitium. Calcium-dependent release of noradrenaline and dopamine beta-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 beta-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 decarboxylase, transformed the biexponential decline of noradrenaline overflow into a monoexponential one with the same rate constant as that of dopamine beta-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 beta-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 beta-hydroxylase activity was in the soluble form. Stimulation depleted the hearts of noradrenaline and soluble dopamine beta-hydroxylase in a stoichiometric manner, provided that noradrenaline synthesis was blocked. The present work utilizes a novel method of measuring soluble dopamine beta-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 beta-hydroxylase release is the re-synthesis of noradrenaline.