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

Abstract

Isolated rabbit hearts were perfused according to a modified Langendorff method for 1 h (unstimulated hearts). In different hearts, release of dopamine β-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°C. In homogenates from unstimulated hearts, the soluble dopamine β-hydroxylase (determined in the supernatant) accounted for 17% of the total dopamine β-hydroxylase (determined in the homogenate). In stimulated hearts the soluble fraction of dopamine β-hydroxylase was reduced by 65%. The dopamine β-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 β-hydroxylase determined from the supernatant represents the releasable pool. Gadolinium ions (Gd 3+) added to the homogenization medium of unstimulated hearts reduced the soluble fraction of dopamine β-hydroxylase up to 63%, with the maximum effect at 200 μM. Similarly, when neurohypophyses were homogenized and spun at 0–4°C, the fraction of vasopressin in the soluble phase was about 50% of the total. Gd 3+ reduced this fraction by maximally 60%, an effect which was accompanied by an increase of vasopressin in the sedimentable fraction. When cytochalasin B (10 μM) was present during the homogenization of the hearts the soluble fraction of dopamine β-hydroxylase was reduced to the same extent as in the presence of Gd 3+. 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 μM, reduced the soluble fraction of dopamine β-hydroxylase by only 40%. However, the electrically evoked noradrenaline release from perfused hearts was completely blocked at 100–300 μM gallopamil. When neurohypophyses were homogenized and fractionated at room temperature only 13% of the total vasopressin was found in the soluble fraction and Gd 3+ did not further reduce this fraction. When unstimulated hearts were homogenized and fractionated at room temperature the fraction of soluble dopamine β-hydroxylase was reduced by 40% compared to the experiments at 0–4°C. However, the dopamine β-hydroxylase still retained in the vesicles could be released by subsequent homogenization in the cold. The present findings demonstrate on two different organs that during tissue homogenization at 0–4°C an extensive exocytotic release of soluble vesicular components occurs which can be modified by agents acting beyond the calcium signal in the process of exocytosis. The low temperature rather than the homogenization procedure has been identified as the real stimulus for the release.