Enhanced Release of Dopamine-β-Hydroxylase from Sympathetic Nerves by Calcium and Phenoxybenzamine and Its Reversal by Prostaglandins

Abstract

Dopamine-j8-hydroxylase (EC 1.14.2.1), an enzyme localized in sympathetic synaptic vesicles, was released together with norepinephrine during in vitro stimulation of the hypogastric nerve that innervates the vas deferens of the guinea pig. Stimulation of the nerve in the presence of high concentrations of calcium (7.5 mM) or phenoxybenzamine caused a marked increase in the amounts of the enzyme and norepinephrine released into the bath. The augmentation of release of dopamine-,0-hydroxylase by 7.5 mM calcium or phenoxybenzamine was reversed by prostaglandin E2. These findings suggest that the release of the sympathetic neurotransmitter, norepinephrine, occurs by a process of exocytosis in which the vesicular content of soluble dopamine-j6-hydroxylase is also released. The depolarization-induced exocytosis, which is stimulated by calcium, may be affected by prostaglandin E2 or phenoxybenzamine through inhibition or enhancement of the actions of calcium in the release process.Release of epinephrine from the adrenal medulla in response to splanchnic-nerve stimulation or to acetylcholine is accompanied by release of ATP (1), soluble proteinst (2), and dopamine-,B-hydroxylase (DOHase) that are stored in the vesicles of adrenergic neurons (3). These findings, together with electron microscopic evidence (4), have suggested that the secretion of the adrenal medulla occurs by a process of exocytosis in which the storage vesicles open at the cell membrane and release their contents into the extracellular space. DOHase, the enzyme that converts dopamine to norepinephrine, is highly localized in synaptic vesicles (5) and is released simultaneously with norepinephrine into the perfusate of isolated organs during electrical stimulation of sympathetic nerves4 (6)(7). This has been taken as evidence that sympathetic neurosecretion might occur by a similar process of exocytosis.By the use of a sensitive assay for measuring DOHase activity (8, 9), we have investigated the in vitro release of the enzyme during stimulation of the hypogastric nerve to the vas deferens of the guinea pig. The effects of calcium, which is required for both release of epinephrine from the adrenal gland (10) and norepinephrine from sympathetic nerves (11, 12), and phenoxybenzamine, an a-blocking adrenergic Abbreviations: DOHase, dopamine-j3-hydroxylase; PG, prostaglandins.t Banks, P., and K. Helle, Biochem. J., 97, 40c (1965 (17) or phenoxybenzamine (18), its effect on release of DOHase was also examined. MATERIALS AND METHODS Preparation of organsMale albino guinea pigs, weighing 500-800 g, were killed by a blow on the head and exsanguinated. The vasa deferentia with attached hypogastric nerves were excised and put into 10-ml organ baths (19) at 37°C, containing medium of the following composition per liter. NaCl, 8.06 g; KCl, 0.35 g; CaCl2 -2H20, 0.3 g; MgSO4 -7H20, 294 mg; KH2P04, 162 mg; glucose, 2.07 g, adjusted to pH 7.4 and aerated with 5% CO2 in 95% 02. The bath fluid was changed 4 times and then replaced by fresh medium containing 0.25...

Effect of Parathyroidectomy on Cardiovascular Reactivity in Rats With Mineralocorticoid‐induced Hypertension

“…The activity of the autonomic nervous system increased during TXAPT-C: thyroidectomized and autografted parathyroid control rats; TXAPT-D: thyroidectomized and autografted parathyroid DOCA-treated rats; TXPTX-C: thyroparathyroidectomized control rats; TXPTX-D: thyroparathyroidectomized DOCA-treated rats; TXPTX-CT4; thyroparathyroidectomized control rats treated with L-thyroxine alone; TXPTX-DT4; thyroparathyroidectomized rats DOCA-treated rats given a thyroxine supple- Reid et al (1975) have reported increased serum catecholamines in DOCA-treated rats. Hypocalcemia after parathyroid removal probably diminished noradrenaline release from sympathetic nerves, since variations in calcium concentration produced this phenomenon (Johnson, Thoa, Weinshilboum, Axelrod & Kopin, 1971;George & Leach, 1975). Parathyroidectomy may protect against development of hypertension by partially inactivating the autonomic nervous system.…”

Inhibition of Release of Dopamine-β-Hydroxylase and Norepinephrine from Sympathetic Nerves by Colchicine, Vinblastine, or Cytochalasin-B

“…Both electron microscopic (13)(14)(15) and biochemical evidence (9,11,12) indicate that the adrenergic nerve discharges the neurotransmitter by a process of exocytosis. Exocytosis presunmably involves a fusion of norel)in el)phrine storage-vesicle membrane with the neuronal membrane, followed by an extrusion of both the neurotransmitter and the soluble contents of the vesicles, including the high-molecular-weight protein (300,000), dopaminie-f-hydroxylase.…”

“…Observation of release of norepinephrine and dopamine-fl-hydroxylase from the adrenal gland that is stimulated by acetylcholine (11) suggests that exocytosis is the process by which norepinephrine release occurs. Because of the similarity of the ratios of norepinephrine to dopamine-,B-hydroxylase in tissue and in the amounts of norepinephrine and dopamine-,B-hydroxylase released (9,12), dopamine-,3-hyroxylase may be considered a reliable index of exocytosis.…”

“…Dopamine-,B-hydroxylase and norepinephrine in the incubation medium were determined as described (9,12 Electrical stimulation of the hypogastric nerve for 1 hr caused a marked release of norepinephrine and dopamine-f3-hydroxylase into the bath fluid. The ratio of dopamine-3-hydroxylase to norepinephrine was identical, whether release was spontaneous or electrically induced ( dopamine-0-hydroxylase or norep)inep)hrine (Fig.…”

Enhancement of noradrenaline depletion in the cat spleen by phenoxybenzamine and phentolamine

“…More recently, it has been shown that the overflow of dopainine-f3-hydroxylase, a vesicular protein which is released along with noradrenaline from nerve terminals (de Potter, de Schaepdryver, Moerman & Smith, 1969;Geffen, Livett & Rush, 1969), is also proportionally increased by phenoxybenzamine and by phentolamine in the saline-perfused spleen and vas deferens during nerve stimulation (de Potter, Chubb, Put & de Schaepdryver, 1971;Johnson, Thoa, Weinshilboum, Axelrod & Kopin, 1971).…”