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)
D000818:Animals; D002458:Cell Fractionation; D003571:Cytochalasin B / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004558:Electric Stimulation; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D005682:Gadolinium / Q000494:pharmacology; D005711:Gallopamil / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010904:Pituitary Gland, Posterior / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013696:Temperature; D013779:Tetrodotoxin / Q000494:pharmacology
D005711:Gallopamil; D003571:Cytochalasin B; D013779:Tetrodotoxin; D005682:Gadolinium; D004299:Dopamine beta-Hydroxylase