Catecholamines Are Present in a Synaptic-Like Microvesicle-Enriched Fraction from Bovine Adrenal Medulla

Abstract: "Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b₅₆₁. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [³H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b₅₆₁ was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

Changes in the development of central noradrenaline neurones following neonatal administration of 6-hydroxydopamine

Abstract— The effects of the neurotoxic compound 6-hydroxydopamine on central noradrenaline (NA) neurones have been investigated in the adult rat after systemic administration of the drug at birth. This treatment produced a permanent and selective reduction in endogenous noradrenaline, [³H]noradrenaline uptake in vitro and the number of histochemically demonstrable noradrenaline nerve terminals in the forebrain, certainly related to neuroneal degeneration. The fluorescence morphology of the noradrenaline perikarya in the locus coeruleus was not notably affected. In the pons-medulla region, the 6-hydroxydopamine treatment led to an almost two-fold increase in endogenous noradrenaline with a similar increase in [³H]noradrenaline uptake and formation of ³H-catecholamines from [³H]tyrosine. Fluorescence histochemistry revealed an increased number of noradrenaline nerve terminals which in addition showed an increased fluorescence intensity. Subcellular distribution studies of endogenous noradrenaline in pons—medulla disclosed the highest relative noradrenaline increase in the microsomal fraction after 6-hydroxydopamine at birth. Sucrose gradient centrifugations disclosed that the pons-medulla synaptosomes from 6-OH-DA treated rats sedimented at a higher sucrose concentration than those from untreated controls. It is concluded that treatment of neonate rats with 6-hydroxydopamine produces a selective degeneration of noradrenaline nerve terminals in the forebrain, especially in the cerebral cortex, whereas in the pons-medulla this treatment leads to an increased intraneuronal noradrenaline concentration due to accumulation of noradrenaline in collateral systems not affected by 6-hydroxydopamine and probably also to an increased outgrowth of noradrenaline nerve terminals.     

Effect of carbidine on the content and storage of adrenergic neurotransmitter in the synaptic vesicles]

The influence of carbidine, an original psychotropic drug, on the adrenergic neurotransmitter content and storage in the sympathetic nerves was studied with the use of cytochemical electron microscopy. The influence of carbidine on the uptake of the exogenous noradrenaline (NA) in the synaptic vesicles was also studied. Carbidine was found to be capable to decreasing the NA storage in the synaptic vesicles and failed to block the accumulation of the exogenous NA in the synaptic vesicles.     

In Vitro Synthesis of Dopamine and Noradrenaline in the Isolated Rat Pineal Gland: Day-Night Variations and Effects of Electrical Stimulation

Isolated rat pineal glands were incubated in vitro in a medium containing [14C]dopamine or [14C]tyrosine, and the tissue contents of 14C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [14C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [14C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [14C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine beta-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [14C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [14C]dopamine enhanced the accumulation of [14C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [14C]dopamine during incubation with [14C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine beta-hydroxylase, the two enzymes required for the synthesis of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and effect of prostaglandin H2 in adipose tissue.

Prostaglandin H2 (PGH2) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC50 was 10-25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH2 increased with time of incubation. A stable PGH2 analog did not inhibit cyclic AMP accumulation. PGH2 was rapidly converted by isolated fat cells to PGD2, PGE2 and PGF2alpha' but no formation of thromboxane B2 was found either in vitro or in vivo. PGE2 was a more potent inhibitor than PGH2 of noradrenaline induced cyclic AMP accumulation. PGD2 enhanced cyclic AMP accumulation in a limited concentration interval, while PGF2alpha was essentially uneffective. Our results suggest that PGH2 is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE2. A physiological role for PGH2 as a modulator of lipolysis is considered unlikely.     

Accumulation of intra-arterially administered [3H]adrenaline and [3H]noradrenaline in various tissues of the Atlantic cod, Gadus morhua

The accumulation of intra-arterially administered radiolabelled adrenaline and noradrenaline was studied in various tissues of the Atlantic cod, Gadus morhua. The largest uptake was seen in the posterior cardinal vein (chromaffin tissue), head kidney, kidney, heart and gill filaments. All these tissues, except the heart, also accumulated noradrenaline to a greater extent than adrenaline. The heart, spleen, gas gland and muscularis mucosae of the swimbladder instead favoured adrenaline accumulation. Small amounts of the injected label (both adrenaline and noradrenaline) were also recovered in the intestine, liver and hypothalamus. The lowest detectable amine accumulation was seen in the rest of the brain and in the skeletal muscle. It is suggested that innervation density, blood flow to the tissue and the concentration of circulating and endogenously stored amine, as well as the affinity of the amine for the degrading enzymes and a possible stereospecificity of the uptake mechanisms, determine the rate and preference of accumulation between the amines.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Some evidence for the maturity of peripheral adrenergic nerves in new-born guinea-pigs.

The fluorescence histochemical technique of Falck and Hillarp revealed a similar distribution and density of peripheral adrenergic nerves in new-born and adult guinea-pigs. The accumulation of tritiated noradrenaline by tracheae from new-born guinea-pigs, assumed to be uptake into adrenergic nerves, was not less than the accumulation by tracheae from adult animals. There was equal potentiation by cocaine (1 x 10(-5)M) of responses to noradrenaline on tracheal chain preparations taken from new-born and adult guinea-pigs. The evidence supports the hypothesis that the guinea-pig has a functional, well differentiated peripheral adrenergic nervous system at birth. This would account for the apparent inability to produce a long-lasting sympathectomy by administration of 6-hydroxydopamine to new-born guinea-pigs.     

Noradrenaline Stimulation Unbalances the Phosphoinositide Cycle in Rat Cerebral Cortical Slices

Abstract: Muscarinic cholinergic and α₁-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'- O -(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of ³H-inositol phosphates and [³²P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [³H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [³H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 m M ) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the α_1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single α_1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain α₁ adrenoceptors involves an increase in intracellular calcium concentration.     

Impaired orthograde axonal transport in acute hypoglycaemia, an effect mediated via hypothermia

The effect of hypoglycaemia (blood glucose 1 mmol/l) on the axonal transport of acetylcholinesterase activity and noradrenaline was examined in non-diabetic rats. Rats were made hypoglycaemic over a 6-h period during which acetylcholinesterase and noradrenaline accumulated proximal to a tight ligature applied to the left sciatic nerve. The hypoglycaemic rats were either kept at room temperature, when they became profoundly hypothermic, or kept in a 31 degrees C incubator to maintain body temperature as close to normal as possible. Hypoglycaemia without temperature control caused marked reductions in the accumulation of acetylcholinesterase activity and of noradrenaline proximal to the ligature. These accumulation deficits were obviated by body heating. The findings indicate that hypoglycaemia impairs fast orthograde axonal transport, but that this effect is a consequence of hypothermia rather than glucopenia.     

Functional molecular mass of binding sites for [3H]dihydrotetrabenazine and [3H]reserpine and of dopamine beta-hydroxylase and cytochrome b561 from chromaffin granule membrane as determined by radiation inactivation

The monoamine transporter of chromaffin granule membrane has two distinct high-affinity binding sites for tetrabenazine and reserpine, which can be assayed by [3H]dihydrotetrabenazine and [3H]reserpine binding, respectively. The functional molecular mass of the components bearing these sites has been investigated by the radiation inactivation technique. The decline of [3H]dihydrotetrabenazine binding activity with increasing radiation doses followed a single exponential, from which a functional molecular mass of 68 kDa was derived for tetrabenazine binding sites. [3H]Reserpine binding activity declined in a more complex way; however, under conditions where high-affinity reserpine binding sites were specifically assayed, the decline was also exponential, corresponding to a functional molecular mass of 37 kDa for these sites. The figures obtained for high-affinity tetrabenazine and reserpine binding sites are consistent with previous values obtained by photoaffinity of tetrabenazine and serotonin binding sites, respectively. It is thus concluded that the monoamine transporter has an oligomeric structure. By the radiation inactivation technique, cytochrome b561 and dopamine beta-hydroxylase have functional molecular masses of 25 and 123 kDa, respectively. The latter value might be attributed to the dimeric form of the enzyme.     

On the mechanism by which antiadrenergic drugs increase survival of critical skin flaps

In order to asses the possibility that degeneration release of noradrenaline influences the survival of critical skin flaps, we studied the effect of various antiadrenergic drugs on skin-flap levels of noradrenaline, ATP, and cyclic AMP. Reserpine treatment depleted the skin flaps of noradrenaline and counteracted the fall in ATP and the cyclic AMP accumulation. Guanethidine had similar but less pronounced effects. Propranolol did not affect noradrenaline levels or depletion rate, but reduced the metabolic stimulation, as assessed by cyclic AMP levels in the flap. Phentolamine had no effect on basal noradrenaline levels, but tended to accelerate its disappearance and reduce lactate accumulation, a measure of hypoxia. All these drugs are known to increase skin-flap survival. It is suggested that they do so by, respectively, depleting the flap of its content of noradrenaline prior to operation or preventing the vasoconstriction and metabolic stimulation caused by released noradrenaline.     

Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells)

The dependence on Na+, K+, and Cl- of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km = 0.14 microM) and inhibited by a series of substrates and inhibitors of "uptake". The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation of [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and Cl-; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for Cl-. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium-carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.     

Spiperone: a receptor ligand and/or a granular uptake tracer?

The accumulation of [³H]spiperone and [³H]dopamine was measured in striatum and pituitary gland slices of rat. Contrary to [³H]dopamine, [³H]spiperone storage was similar in striatum and pituitary gland. In addition, [³H]spiperone accumulation was not diminished by reserpine and tetrabenazine. These data show that spiperone is not subject to the granular uptake/storage mechanism and suggest that spiperone and its derivatives are specific ligands for dopamine receptors only.     

Effect of Tetrabenazine on Extrapyramidal Movement Disorders

Thirty patients with various extrapyramidal movement disorders were treated for prolonged periods with 75 to 225 mg. daily of tetrabenazine. In patients with choreiform and hemiballistic motor activity the involuntary movements were diminished or abolished. In patients with cerebellar or Parkinsonian tremor the tremor was aggravated in moderately severe cases, but was uninfluenced in severe cases. In all cases the dyskinesia returned when- the drug was stopped.Side-effects were inconsiderable and disappeared on reducing the dose slightly. Hence the drug may be an important alternative to neurosurgical treatment of hyperkinesias and especially suitable for severely disabled patients.     

Uptake of3H-adrenaline and14C-noradrenaline into neuronal and extraneuronal tissue compartments in the perfused gas gland of the swimbladder of the Atlantic cod,Gadus morhua

Summary The neuronal and extraneuronal accumulation of radiolabelledl-adrenaline andl-noradrenaline was studied in the gas gland of the swimbladder of the Atlantic cod,Gadus morhua. Both amines are taken up into the tissue compartments, and a preference for noradrenaline for both uptake processes was demonstrated. A relatively high neuronal accumulation compared to earlier results (cod spleen; Ungell and Nilsson 1984) was seen and this is probably due to the more dense innervation of the swimbladder gas gland. A higher extraneuronal accumulation may be due to the presence of arterial smooth muscle.It is concluded that the adrenergic nerves of the swimbladder gas gland of the cod preferentially accumulate noradrenaline, a situation similar to that in mammals.Abbreviations A/NA adrenaline/noradrenaline ratio - PBA phenoxybenzamine - PNMT phenylethanolamine-N-methyl tranferase     

Noradrenaline dynamics in prolonged ischemia after ischemic preconditioning

AIM OF THE STUDY: To determine the effects of two-staged ischemic preconditioning on myocardial noradrenaline in prolonged ischemia and reperfusion. METHODS: Thirty-two male Wistar rats anesthetised with urethane randomly divided into 2 groups: group 1 (ischemic preconditioning group, n = 16), and group 2 (control, n = 16). Myocardial interstitial noradrenaline levels were measured using a microdialysis technique. Ischemic preconditioning was elicited by two episodes: 5 min of ischemia and 10 min of reperfusion. The intermittent occlusions were followed by prolonged occlusion (60 min) and reperfusion (60 min). RESULTS: An increase in interstitial noradrenaline was observed in 10 min of prolonged ischemia in group 2, and in 20 min in group 1. After 20 min of myocardial ischemia there was a significant difference between groups (p < 0.05) in interstitial noradrenaline levels. In control group, it was 60% higher. In reperfusion, noradrenaline levels decreased markedly in group 1. CONCLUSION: We suggest that ischemic preconditioning by two episodes: 5-min ischemia and 10-min reperfusion prevents excessive noradrenaline interstitial accumulation, perhaps, through protection of physiological uptake I carrier.     

Neurochemical Characterization of the Alterations in the Noradrenergic Afferents to the Cerebellum of Adult Rats Exposed to X-Irradiation at Birth

Abstract: A single dose of x-irradiation was applied on the cephalic end of newborn rats, and the alterations in the noradrenergic afferents to the cerebellum were studied 180 days later. A net increase in the noradrenaline content of cerebellum was found (122% of nonirradiated controls). The response of noradrenaline content to reserpine injection (0.9 mg/kg, i.p.) was similar in exposed and control rats. Likewise, the ³H release induced by Ro 4-1284 from cerebellar cortex slices labeled with [³H]noradrenaline was unmodified by x-rays, although a mild increase in the spontaneous efflux of ³H was found. The retention of ³H by the slices was reduced in exposed animals (58% of controls). Both the in vitro activity of tyrosine hydroxylase and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) were not significantly different between x-treated rats and controls. In contrast, monoamine oxidase activity was markedly reduced in x-irradiated cerebellum (38% of controls). The x-ray-induced decrease in cerebellar weight (—60%) resulted in marked increases in noradrenaline concentration (223%), tyrosine hydroxylase activity per milligram of protein (206%), and ³H retention (50%). The accumulation of L-DOPA per gram of tissue was also increased at every time considered. These data indicate that x-irradiation at birth produces a cerebellar loss not completely shared by the noradrenergic afferents, and a permanent imbalance between the noradrenergic afferent input and its target cells might eventually result. In spite of the enhanced noradrenaline content, the lack of increase in maximal tyrosine hydroxylase activity and ³H retention seems to indicate that a long-term sprouting of the noradrenergic terminals in the cerebellum induced by the ionizing treatment is unlikely.