F. Lishajko

Localization of different steps in noradrenaline synthesis to different fractions of a bovine splenic nerve homogenate.

Abstract Experiments were carried out to determine to what extent the synthesis of noradrenaline in bovine splenic nerve tissue can proceed in the high-speed sedimentable particulate fraction that contains the specific noradrenaline storage vesicles. Various fractions of a homogenate of this tissue were incubated in a potassium phosphate medium together with radioactively labeled precursors of noradrenaline. The first two steps in the synthesis, the ring hydroxylation of tyrosine and the decarboxylation of dopa, were shown not to require the presence of this particulate fraction. On the other hand, this fraction was an absolute prerequisite for the last step, the β-hydroxylation of dopamine. No significant formation of catechol compounds from tyrosine could be demonstrated on incubation with this particulate fraction after it had been washed and resuspended in potassium phosphate. Some dopa decarboxylase activity remained in the particulate fraction even after washing. The results support the view that in this tissue only one enzyme involved in noradrenaline synthesis, dopamine β-hydroxylase, is truly located in the above-mentioned particles, possibly inside the membrane of the noradrenaline-storing vesicles. Tyrosine hydroxylase and dopa decarboxylase appear to be located outside these particles. Dopa decarboxylase shows some association with the particulate fraction studied, and might be, to some extent, bound to the outer aspect of the same particles which contain the β-hydroxylase, or could just be accumulated in the perivesicular parts of the probably highly organized axoplasm.

Noradrenaline formation from dopamine in isolated subcellular particles from bovine splenic nerve

Abstract The process of β-hydroxylation of dopamine (DA) and the fate of the newly formed noradrenaline (NA) were studied in high-speed sedimentable particles from bovine splenic nerves, after resuspension in potassium phosphate. The NA formation was found to be a linear function of the DA concentration in the medium. However, only a fixed amount of the newly formed NA was retained in the particulate fraction; the remainder appeared in the supernatant. Addition of NA to the medium reduced NA formation from DA, but hardly affected binding of the newly formed NA, while severely depressing DA retention. On the other hand, DA was shown to have about the same affinity as NA for the amine uptake mechanism of the particles. Reserpine added to the medium inhibited the formation of NA from DA, as well as the binding of NA and of DA, to an extent dependent on the drug/DA concentration ratio. The inhibition of NA formation was much less pronounced than that of firm amine binding. Although the results suggest that the membranes of the particles are permeable to NA and to DA, they do not rule out a certain inhibitory effect of reserpine on amine passage across the membranes. Washing was found to release loosely bound amines or amine derivatives from the particles. Loose binding appeared to be blocked by reserpine. The results indicate that deamination proceeds in the high-speed sedimentable particles. Inhibition of monoamine oxidase did not affect the NA formation from DA.

Effect of reserpine on the uptake of catecholamines in isolated nerve storage granules

Abstract The uptake of added adrenaline or noradrenaline from a suspension of partially depleted adrenergic nerve granules is not inhibited by reserpine about 10 −5 M. On the other hand pretreatment of adrenergic nerve granules with reserpine in phosphate buffer for 1 hour prevents the amine uptake. Undepleted nerve granules which normally take up added adrenaline through exchange with noradrenaline fail to do so in the presence of reserpine owing to the concomitant block of noradrenaline release.

A specific kind of noradrenaline granules in the vesicular gland and the vas deferens of the bull

Abstract Noradrenaline containing granules from the vas deferens and the vesicular gland of the adult bull show a spontaneous NA release rate which is about 3 times lower than that in granules from bovine splenic nerves. The granules also differ from splenic nerve granules with regard to amine uptake and in response to various drugs. It is suggested that the appearance of a special kind of NA granules in the accessory male genital organs studied is related to the special function and to the occurrence of short adrenergic neurons characteristic of these organs.

CATECHOLAMINES AND NUCLEOTIDES IN PHAEOCHROMOCYTOMA.

Abstract Isolated catecholamine (CA) storage granules from a phaeochromocytoma were studied in vitro . The tumour granules had a high NA content, while A amounted to only a few per cent of the total CA. Most of the amines were particle bound. The storage granules had a lower density and adenine nucleotide content than bovine adrenal medullary granules. Thus only a minor part of the CA in this tumour could be stored by a mechanism requiring stoichiometric relationship between amines and adenine nucleotides. On incubation in vitro ATP was lost at a slightly higher rate than the CA, leading to an increase in the molar CA/ATP ratio. The ATP lost was quantitatively recovered as AMP in the extragranular medium. ATP-splitting activity occurred to some extent in the particle-free supernatant but was more evident in the high speed sediment. When the tumour was stored in a refrigerator at +2° a gradual loss of CA occurred while the ATP remained unchanged. Thus from the third day and onwards a CA/ATP ratio giving an equivalence ratio close to unity was established. On incubation of such granules the rate of CA loss was found to be slightly increased while that of ATP loss was unchanged. ATP or ADP and magnesium addition to the incubation medium partly prevented the spontaneous decrease of the storage granule CA. Phenoxybenzamine caused a profound fall in the CA and ATP content, while reserpine did not have any clearcut effects on the granules, in the concentrations used.

The uptake of isoprenaline in nerve granules

Abstract Isolated bovine splenic nerve granules take up isoprenaline to the same extent as noradrenaline or adrenaline. The uptake is enhanced by addition of ATP to the incubation medium. The results indicate that the low uptake of isoprenaline in tissues depends on an uptake barrier in the axon membrane rather than in the storage granules. This appears also to be true for adrenaline which is less readily taken up by the tissue than noradrenaline. It is concluded that the granules are protected from certain catecholamines by a discriminating mechanism located in the axon membrane.

FREE AND BOUND NORADRENALINE IN THE RABBIT HEART.

SUB-CELLULAR storage particles containing noradrenaline (NA) have been demonstrated in noradrenergic nerves and in organs supplied with such nerves1,2. The nerve vesicles, which have a diameter of 0.03–0.1µ (ref. 3), have been subjected to a number of investigations regarding stability in various media, release and uptake of NA and other amines, and action of drugs4,5.

Stereospecific catecholamine uptake in rabbit hearts depleted by decaborane.

Abstract After 80–90 per cent depletion of its noradrenaline (NA) content following decaborane, the rabbit heart may take up both NA and A to the normal capacity upon injection of amines. The amines taken up are well retained for several hours, but gradually disappear in the course of about 20 hr. The high speed sediment and supernatant of a homogenate of the heart contain approximately constant proportions of NA, irrespective of the total amount, indicating a dynamic equilibrium between these pools. When the d - or the l -isomer of A are given together with l -NA, simultaneously and in equal amounts, the uptake of l -A is 2–3 times larger than that of d -A, relative to the uptake of l -Na. This ratio is unchanged over a period of from 1 min-4 hr after the injection of the amine mixture. It is suggested that the uptake is partly stereospeeific but that once taken up in the specific stores, the l - and d -isomer are released at approximately the same rate.

Uptake of catecholamines in the rabbit heart after depletion with decaborane

Abstract After 80–90 per cent depletion of the rabbit heart with decaborane, injection of noradrenaline (NA) causes an increase of the amine content to near normal values. The amines taken up show normal distribution between the high speed sediment and supernatant, indicating a dynamic equilibrium between the particulate and the supernatant fractions. The amines taken up leave the heart gradually in some 20 hours. When either the 1- or the d-isomer of adrenaline (A) is injected together with 1-NA, 2–3 times more of 1-A than of dA is taken up for the same amount of 1-NA. The two isomers appear to leave the stores at a similar rate.

Differentiated storage mechanisms for noradrenaline and dopamine in the rabbit heart

Abstract Uptake and binding of dopamine and noradrenaline were studied in rabbit heart and brain after partial depletion of the amine stores with decaborane and inhibition of β-hydroxylation with disulfiram. Intravenous or intramuscular injection of noradrenaline and dopamine increased the noradrenaline content in partially noradrenaline-depleted hearts. After decaborane + disulfiram, 50 μg/kg noradrenaline intravenously still increased the noradrenaline content from 0.28 to 0.77 μg/g in 1 hr (P =