W.P. De Potter

Release of dopamine β-hydroxylase and chromogranin A upon stimulation of the splenic nerve

Two proteins present in noradrenergic vesicles of the splenic nerve (dopamine beta-hydroxylase and chromogranin A) are released into the perfusate from the spleen when the splenic nerve is stimulated. Experiments in which drugs were added to the perfusion fluid showed that the proteins were released from terminals of the splenic nerve. There was a correlation between the amounts of the proteins released and the quantity of noradrenaline released; and the release process was dependent upon calcium. It is suggested that the proteins are released from the large dense-cored vesicles present in the terminals of the splenic nerve, and that secretion from these vesicles occurs by exocytosis.

Subcellular fractionation of splenic nerve: ATP, chromogranin A and dopamine β-hydroxylase in noradrenergic vesicles

The subcellular particles in axons of the splenic nerve have been studied by centifrugation techniques. By differential centifrugation, five different types of particle could be distinguished and partly separated: noradrenaline-containing particles (noradrenergic vesicles), large and small lysosomes, mitochondria, and microsomal particles. In density gradient centrifugation, only one type of noradrenergic vesicle could he demonstrated. The noradrenergic vesicles and the mitochondria contain ATP. Two proteins (chromogranin A and dopamine beta-hydroxylase) are present in the noradrenergic vesicles.

Biochemical observations on the formation of small noradrenergic vesicles in the splenic nerve of the dog

Abstract Splenic nerve terminals contain noradrenergic vesicles which can be classified as small and large on the basis of both morphological and biochemical experiments. The origin of the large vesicles is likely to be the cell body and they are probably supplied to the nerve terminals by axonal transport. The origin of the small noradrenergic vesicles is still obscure so we have tested, using centrifugation techniques, the hypothesis that they are formed from the large noradrenergic vesicles during secretory activity in the nerve endings. Normally the small noradrenergic vesicles in resting splenic nerve terminals contain very little dopamine β-hydroxylase activity, as distinct from the large vesicles. However, after stimulation in vitro of the perfused spleen there is an appreciable amount of the enzyme associated with the small particles. These newly formed small vesicles are able to concentrate exogenous noradrenaline and their formation is dependent upon release of secretory products from the large vesicles as shown by their absence if the splenic nerve of a spleen perfused with a Ca 2+ -free solution is stimulated. A balance sheet has been drawn up which suggests that membrane-bound dopamine β-hydroxylase lost from the large noradrenergic vesicles upon stimulation is almost quantitatively recovered in the small noradrenergic vesicles. It is suggested that at least three types of noradrenaline storage particles exist in splenic nerve terminals (i) large vesicles which contain noradrenaline and dopamine β-hydroxylase and that can give rise to (ii) small vesicles which also contain both constituents and, (iii) vesicles which are virtually devoid of dopamine β-hydroxylase activity but which can store noradrenaline and have an origin quite different from those of the other two types.

The presence of dopamine β-hydroxylase in the cerebrospinal fluid of rabbits and its increased concentration after stimulation of peripheral nerves and cold stress

The presence of dopamine beta-hydroxylase activity in the cerebrospinal fluid of rabbits has been shown using a sensitive radiochemical assay; the identity of the reaction product was confirmed by using thin layer chromatographic techniques. The enzyme found in this fluid has some properties (sedimentation and electrophoretic migration) in common with the best characterized preparation of dopamine beta-hydroxylase, that prepared from bovine adrenal chromaffin granules. It also has these properties in common with the enzyme present in the high-speed supernatants obtained from osmotically disrupted synaptosomes prepared from rabbit brain. When the sciatic nerves of rabbits under urethane anaesthesia were stimulated, or when shaved rabbits were subjected to cold stress, the level of dopamine beta-hydroxylase in the cerebrospinal fluid increased. The increase in response to nerve stimulation was gradual, starting within 90 min of stimulation and remained high for at least 3 h after the stimulation had ended, at which time it was 280% of the normal value. There was no equivalent increase in the protein concentration of the cerebrospinal fluid nor was there a change in the enzyme activity when sciatic nerves were exposed but not stimulated. The enzyme present in the cerebrospinal fluid during this period of high activity is identical in its sedimentation and electrophoretic properties to that present in normal fluid. It is suggested that the dopamine beta-hydroxylase activity in cerebrospinal fluid may be derived from noradrenergic neurons within the brain and that the enzyme is released together with noradrenaline.

Tyramine does not release noradrenaline from splenic nerve by exocytosis.

SummaryThe release of noradrenaline by tyramine from bovine splenic nerves was found to be dose dependent. No dopamine-β-hydroxylase could be detected in perfusates from tyramine stimulated spleens in contrast to results obtained by electrical stimulation. It is concluded that the releasing action of tyramine differs fundamentally from that of electrical stimulation in that exocytosis is not involved.

Thin layer chromatography of catecholamines and their metabolites.

Eine einfache, schnelle und empfindliche Methode zur dünnschichtchromatographischen Trennung von 0,2–1µg Menge von Noradrenalin, Adrenalin, Normetanephrin, Metanephrin, 3,4-Dihydroxy-mandelsäure und 3-Methoxy-4-hydroxymandelsäure wird beschrieben.

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit.

SummaryHypothalamic slices of the rabbit brain were incubated with 10−7 M of 3H-serotonin (3H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca2+-free solution or by the 5HT-antagonist metitepin (10−5 M), but was augmented by chlorimipramine (10−5 M) and by unlabelled 5HT (10−6 M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10−5 M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K+ (50 mM) induced an overflow of 3H. This overflow was nearly abolished in Ca2+-free solution. In presence of chlorimipramine (10−5 M) both the tritium overflow evoked by electrical stimulation and that evoked by high K+ were augmented by metitepin (10−5 M) and decreased in a concentration dependent manner by unlabelled serotonin (10−8–10−6 M); the latter effect was antagonized by metitepin (10−6 M and 10−5 M). These experiments suggest that in rabbit hypothalamic slices, the release of 3H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.

The effect of drugs on the concentration of dopamine β-hydroxylase in the cerebrospinal fluid of rabbits

Abstract Systemic administration of each of the drugs studied (phenoxybenzamine, yohimbine, phentolamine, nicotine, physostigmine and pentylenetetrazol) caused an increase in the dopamine β-hydroxylase activity per unit volume of cerebrospinal fluid. The increase was gradual starting within 90 min after injection of the drug and remained high for at least 3 h. At that time the dopamine β-hydroxylase levels after treatment with phenoxybenzamine and yohimbine reached values of 600% and 580%, respectively, of the control. The increase in dopamine β-hydroxylase activity upon phenoxybenzamine treatment was dose dependent. Injection of pentylenetetrazol caused an increase in dopamine β-hydroxylase to 230% of the control value. Pretreatment of rabbits with an intracisternal injection of 6-hydroxydopamine not only caused a 68% decrease in the noradrenaline content and a 69% decrease in the dopamine β-hydroxylase levels in the brain, but also led to a 52% fall in the dopamine β-hydroxylase level of the cerebrospinal fluid. Upon injection of pentylenetetrazol into rabbits pretreated with 6-hydroxydopamine, the dopamine β-hydroxylase levels in the cerebrospinal fluid only reached a mean value of 0.98 units/ml compared with 2.62 units/ ml for control rabbits injected with pentylenetetrazol; the increase in the blood level of dopamine β-hydroxylase activity was, however, unaffected. Data derived from normal and 6-hydroxydopamine pretreated rabbits revealed a fairly good correlation between dopamine β-hydroxylase levels in the cerebrospinal fluid and the brain. From these experiments it is concluded that dopamine β-hydroxylase activity in the cerebrospinal fluid is derived from central noradrenergic neurons and that drugs which increase central noradrenergic activity also increase dopamine β-hydroxylase levels in the cerebrospinal fluid. It is further suggested that determination of dopamine β-hydroxylase activity in the cerebrospinal fluid may be a sensitive and relatively easy method to assess central noradrenergic activity.

RELEASE OF AMINES FROM SYMPATHETIC NERVES

Publisher Summary This chapter discusses release of amines from sympathetic nerves. Biochemical studies from several laboratories have established that, at least for the adrenal gland, the release of catecholamines occurs by exocytosis. Evidence for this secretion mechanism comes from studies which showed that: (1) granule specific proteins (chromogranins)—which occur in a soluble state within the particle—are released stoichiometrically with ATP and catecholamines, (2) enzymes which are located in the cell sap are not released and (3) specific components of the storage particle membrane were retained within the cell. That this mechanism holds for adrenergic nerves has been more difficult to establish. Considerable evidence has, however, accumulated which strongly indicates that exocytosis does occur in adrenergic nerve terminals. Axons and ganglia show a unimodal distribution of the noradrenaline on density gradients. In the ganglia the noradrenaline peak is partially paralleled by dopamine-β-hydroxylase, the rest of the enzyme activity being attributable to broken or immature vesicles. The distribution of axonal particles is paralleled quite closely by dopamine-β-hydroxylase and chromogranin A.

Biochemical evidence for two types of noradrenaline storage particles in rabbit iris

Centrifugation techniques were used to determine the subcellular distribution of noradrenaline (NA) and dopamine β-hydroxylase (DβH) in the rabbit iris. By application of isopycnic and differential gradient centrifugation methods 2 types of NA vesicles could be demonstrated. Of the total particle bound NA about 70% is associated with ‘light’ and about 30% with ‘heavy’ vesicles. For both types of vesicles the distribution of DβH reflected that of NA.