THE BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF 6-AMINODOPAMINE: SIMILARITY WITH 6-HYDROXYDOPAMINE

6-aminodopamine was injected intraperitoneally into male Swiss–Webster mice. At 72 h post injection 6-aminodopamine had caused a reduction in the endogenous content of heart norepinephrine, a decrease in the capacity of heart slices to accumulate [³H]-norepinephrine in vitro, and a virtual disappearance of the adrenergic plexus of the mouse iris as viewed by fluorescence histochemistry. Similar data were obtained with the same dose of 6-hydroxydopamine. These data suggest that 6-aminodopamine causes a destruction of sympathetic nerve terminals. Model experiments showed that 6-aminodopamine, like 6-hydroxydopamine, generated H₂o₂both in vitro and in vivo. 6-Aminodopamine, like 6-hydroxydopamine, also blocked the accumulation of [³H]dopamine into slices of rat brain in vitro.     

NEUROCHEMICAL PROPERTIES OF ADRENERGIC NERVES REGENERATED AFTER 6-HYDROXYDOPAMINE

Abstract— The uptake-storage properties and synthesis of noradrenaline, and fluorescence morphology of adrenergic nerves which have been allowed to regenerate for 4 weeks after a chemical sympathectomy produced by 6-hydroxydopamine have been investigated in mouse iris and atrium. The regenerated nerve terminals displayed a lower formaldehyde-induced fluorescence intensity whereas the non-terminal axons exhibited a stronger fluorescence intensity and a more beaded appearance compared with mature nerves. The endogenous noradrenaline concentration after 6-hydroxydopamine was 30% in iris and 45% in atrium compared to control values. Recovery of [³H]noradrenaline uptake was found to be more rapid than that of endogenous noradrenaline concentration after the 6-hydroxydopamine treatment. [³H]Noradrenaline uptake in regenerating and adult mature nerves both obeyed Michaelis-Menten kinetics having identical K_m values. There was a close correlation between [³H]noradrenaline uptake and nerve density of adrenergic nerves regenerated after 6-hydroxydopamine. These results show that [³H]noradrenaline uptake is a better index for the number of regenerated nerve terminals than is the endogenous noradrenaline concentration. The retention of [³H]noradrenaline taken up and accumulated in vitro was about the same in regenerated and mature nerves, although a slight tendency to less effective retention was observed in the regenerated nerves. Subcellular distribution studies showed that relatively less [³H]noradrenaline was recovered in the microsomal fraction after 6-hydroxydopamine treatment. The formation of ¹⁴C-labelled catecholamines from [¹⁴C]DOPA was higher in regenerating nerves than indicated by the endogenous noradrenaline concentration but lower than that indicated by the [³H]noradrenaline. It is concluded that the regenerating nerves contain less endogenous noradrenaline than adult mature nerves and that the uptake mechanism develops promptly, whereas the development of the storage mechanism lags behind.     

NORADRENALINE NERVE TERMINALS IN THE CEREBRAL CORTEX: EFFECTS ON NORADRENALINE UPTAKE AND STORAGE FOLLOWING AXONAL LESION WITH 6-HYDROXYDOPAMINE

The ascending noradrenaline-containing neuronal system from the locus coeruleus to the cerebral cortex was unilaterally lesioned by an intracerebral injection of 8 μg 6-hydroxydopamine in the dorsomedial reticular formation in the caudal mesencephalon. The 6-hydroxydopamine caused injury to axons of the dorsal catecholamine bundle associated with its specific neurotoxic action, while very limited unspecific tissue necrosis was observed. Following this treatment the endogenous noradrenaline in the ipsilateral cerebral cortex (neocortex) increased acutely (up to 2 days), as observed both with noradrenaline assay and fluorescence histochemistry. The noradrenaline concentration then gradually decreased to 15 per cent of the contralateral side 15 days after the lesion. At this time interval and up to at least 90 days no fluorescent catecholamine nerve terminals could be detected. The acute noradrenaline increase could be blocked partially by tyrosine hydroxylase inhibition produced by α-methyl-p-tyrosine. The disappearance of endogenous noradrenaline following tyrosine hydroxylase inhibition was also reduced after the 6-hydroxydopamine lesion. Studies on the in vitro uptake of [³H]noradrenaline (0.1 μM for 5 min) in slices from the neocortex after the 6-hydroxydopamine lesion showed a gradual decline in uptake reaching maximal reduction (35-40 per cent of the contralateral side) after 15 days. No recovery of [³H]noradrenaline uptake was seen up to 90 days after the lesion. The formation of [³H]noradrenaline from [³H]dopamine in vitro was reduced to 15 per cent of the contralateral side after a chronic lesion. The present results indicate that the disappearance of noradrenaline uptake-storage mechanisms in the neocortex is due to an anterograde degeneration of axons and nerve terminals of the dorsal catecholamine bundle. The data on endogenous noradrenaline and noradrenaline synthesis suggest that approx. 15 per cent of the noradrenaline nerve terminals in the neocortex remain intact following the lesion, while the [³H]noradrenaline uptake data reflect uptake in other tissue structures in addition to noradrenaline nerve terminals, e.g. dopamine nerve terminals, pericytes and/or glial cells.     

DEGENERATION OF CENTRAL AND PERIPHERAL NORADRENALINE NEURONS PRODUCED BY 6-HYDROXY-DOPA

—The effects of systemically administered 2,4,5-trihydroxyphenylalanine (6-OH-DOPA) on endogenous noradrenaline, [³H]amine uptake and fluorescence morphology has been investigated in mouse brain, heart and iris. 6-OH-DOPA in a dose of 100 mg/kg intraperitoneally caused practically no changes in these parameters. Pretreatment with a potent monoamine oxidase inhibitor (nialamide) led to a pronounced long-lasting 6-OH-DOPA induced reduction in endogenous noradrenaline, [ ³ H]amine uptake and nerve density of noradrenaline nerve terminals both in the central and peripheral nervous system. Histochemically accumulations of noradrenaline were observed in non-terminal axons. These results strongly support the view that 6-OH-DOPA can produce degeneration of both central and peripheral noradrenaline neurons. The degeneration is mediated by decarboxylation of 6-OH-DOPA to 6-OH-DA, since the effects could be abolished by decarboxylase inhibition. The effect of 6-OH-DOPA was selective on noradrenaline neurons in the brain, since neither 5-hydroxytryptamine nor dopamine neurons were affected, opening up new possibilities for studies on central noradrenaline transmitter mechanisms. In the brain there were pronounced accumulations of noradrenaline in the ascending noradrenaline axons making 6-OH-DOPA a powerful tool in the mapping of central noradrenaline pathways.     

Development of transmitter secretory mechanisms by adrenergic neurons in the embryonic chick heart ventricle

Developmental changes in the function of adrenergic axons within the right ventricle of the chick embryo were assessed by measuring the ability of these axons (1) to release endogenous transmitter, and (2) to transport, retain, and release tritiated norepinephrine ([³H]NE). The release of endogenous catecholamines was assayed indirectly by measuring the increase in the twitch tension of ventricular muscle evoked by electrical stimulation of intramural nerves. The release of endogenous transmitter, which acted via β-adrenergic receptors, was first detected by this method on the 16th embryonic day. A cocaine-sensitive uptake of [³H]NE was first observed on the 12th embryonic day. At this time, elevated potassium first evoked a calcium-sensitive release of [³H]NE. Electrical stimulation of intramural axons first evoked a tetrodotoxin-sensitive release of [³H]NE on the 14th embryonic day. It is concluded that the axons of developing adrenergic neurons are capable of releasing transmitter soon after they contact their target tissue.     

DEVELOPMENT OF THE BLOOD-BRAIN BARRIER FOR 6-HYDROXYDOPAMINE

The postnatal development of the blood-brain barrier for the neurotoxic action of 6-hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [³H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6-hydroxydopamine at different ages. The [³H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6-OH-DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6-OH-DA on [³H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood-brain barrier for 6-OH-DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time-course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood-brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6-OH-DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6-OH-DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6-OH-DA treatment.     

Actions of 6-hydroxydopamine quinones on catecholamine neurons.

—The effect of the para-(PQ) and the ortho-(OQ) quinones of 6-hydroxydopamine (6-OH-DA) on transmitter uptake-storage mechanisms of catecholamine neurons in mouse and rat has been investigated. After the administration of PQ and OQ there was a dose-dependent and long-lasting disappearance of noradrenaline (NA) nerve terminals as demonstrated by fluorescence histochemistry and a reduction of the in vitro uptake of [³H]NA in mouse atrium and iris. These effects could be completely counteracted by blockade of the ‘membrane pump’ transport mechanism with desipramine, while monoamine oxidase inhibition, by nialamide and administration of ascorbic acid potentiated the effects produced by the two quinones. The results obtained after PQ and OQ were largely identical with those seen after administration of 6-OH-DA, well-known for its neurotoxic action on catecholamine neurons. It is therefore concluded that PQ and OQ are able to produce an acute and selective degeneration of NA nerve terminals similar to that of 6-OH-DA. The results obtained after intraventricular injection of the quinones into rat brain were also in agreement with this view. Neonatal administration of PQ or OQ to mice caused a permanent and marked decrease in [³H]NA uptake in the cerebral cortex and the spinal cord, whereas the uptake was markedly increased in the pons-medulla, similar to that seen after 6-OH-DA. The PQ and the OQ were equally potent in most experiments although clearly less potent than 6-OH-DA itself. The quinones were also found to be equally or slightly less potent than 6-OH-DA in affecting [³H]NA uptake and retention in vitro in atrium and cerebral cortex from untreated mice. It may be concluded that PQ and OQ exert their neurotoxic action on NA neurons after transition to 6-OH-DA, after a rapid extraneuronal equilibration. 6-OH-DA thus formed can thereafter be taken up and accumulated intraneuronally by use of the ‘membrane pump’ and the specific degenerative action is elicited. The lower neurotoxic potency of the quinones may be attributed to their known ability to undergo covalent binding with proteins and/or formation of 5,6-dihydroxyindole.     

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [³H] dopa, [³H] dopamine, [³H] noradrenaline and [³H] adrenaline have been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of the innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

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.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles

The uptake and retrograde transport of noradrenaline (NA) within the axons of sympathetic neurons was investigated in an in vitro system. Dissociated neurons from the sympathetic ganglia of newborn rats were cultured for 3-6 wk in the absence of non-neuronal cells in a culture dish divided into three chambers. These allowed separate access to the axonal networks and to their cell bodies of origin. [3H]NA (0.5 X 10(-6) M), added to the axon chambers, was taken up by the desmethylimipramine- and cocaine-sensitive neuronal amine uptake mechanisms, and a substantial part was rapidly transported retrogradely along the axons to the nerve cell bodies. This transport was blocked by vinblastine or colchicine. In contrast with the storage of [3H]NA in the axonal varicosities, which was totally prevented by reserpine (a drug that selectively inactivates the uptake of NA into adrenergic storage vesicles), the retrograde transport of [3H]NA was only slightly diminished by reserpine pretreatment. Electron microscopic localization of the NA analogue 5-hydroxydopamine (5-OHDA) indicated that mainly large dense-core vesicles (700-1,200-A diam) are the transport compartment involved. Whereas the majority of small and large vesicles lost their amine dense-core and were resistant to this drug. It, therefore, seems that these vesicles maintained the amine uptake and storage mechanisms characteristic for adrenergic vesicles, but have lost the sensitivity of their amine carrier for reserpine. The retrograde transport of NA and 5-OHDA probably reflects the return of used synaptic vesicle membrane to the cell body in a form that is distinct from the membranous cisternae and prelysosomal structures involved in the retrograde axonal transport of extracellular tracers.     

IS THERE BIDIRECTIONAL TRANSPORT OF NORADRENALINE IN SYMPATHETIC NERVES?

The experiments were designed to detect somatopetal transport of [¹⁴C]noradrenaline in the postganglionic sympathetic nerves supplying the cat spleen and sheep eye. The animals were treated with nialamide to protect the radioactive noradrenaline, after uptake into the nerve terminals, from monoamine oxidase. In the spleen, the transmitter stores were labelled by infusion of [¹⁴C]noradrenaline into a branch of the splenic artery. The branches of the nerves to the infused and non-infused sides of the spleen were ligated in an attempt to arrest, distal to the constriction, any noradrenaline transported somatopetally in the axons from their terminals. After 24 hr, however, there was less radioactivity in the nerves distal compared to proximal to the constriction, despite heavier labelling of the terminal transmitter stores in the infused portion of the spleen. The proximal accumulation of radioactivity could be attributed to a somatofugal transport of [¹⁴C]noradrenaline. Experiments were also done on the intact sympathetic nerve supply of the sheep eye. The sympathetic nerve terminals in the smooth muscle of the left eye were heavily labelled 5 days after the injection of [¹⁴C]noradrenaline into the left vitreous humour. However, both superior cervical ganglia were only lightly labelled, and there was no significant difference in the radioactivity present in the two ganglia. The results provide no support for a bidirectional transport of noradrenaline in sympathetic nerves but are consistent with a somatofugal transport of the amine storage vesicles from their site of synthesis in the soma to the axon terminals.     

On the uptake and storage of 5-hydroxytryptamine, 5-hydroxytryptophan and catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiographs of the adrenal medulla at various intervals after the intravenous injection of [³H] 5-HTP, [³H] 5-HT, [³H] noradrenaline and [³H] adrenaline have been studied. The distribution of silver grains following [³H] 5-HTP uptake was found to be uniform over each of the two main cell populations, adrenaline-storing (A) cells and noradrenaline-storing (NA) cells in the adrenal medulla, but A cells were twice as active as NA cells in incorporating the isotope, a situation very similar to that found after [³H] dopa uptake. 5-HT administration resulted in a pattern resembling the distribution of [³H] noradrenaline uptake, with A cells being 4 or 5 times more active than NA cells and a gradient of activity from the periphery of the medulla inwards. However, the time-course for the loss of radioactivity was not the same for both amines: levels of 5-HT activity were not significantly reduced after one week whereas the degree of [³H] noradrenaline labelling after one week was less than 10% of that at one hour. Thus 5-HT may be bound to sites in the adrenal medulla normally occupied by noradrenaline but it would appear that the release mechanism is different. There was no evidence of 5-HT uptake by adrenal nerve endings.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Effect of hydrocortisone,reserpine, propranolol and phentolamine on in vivo uptake of exogenous amines by adrenal chromaffin cells

Summary An autoradiographic study was performed on the effects of hydrocortisone, reserpine, propranolol and phentolamine on the uptake of tritiated amines by adrenal medullary cells of the mouse. Oral feeding of hydrocortisone had no significant effect on the normal uptake pattern of dopamine, noradrenaline or adrenaline by medullary cells of different type (A cells or NA cells) or location (marginal or central), although the overall amounts taken up were markedly reduced. Handling the animals led to similar reductions in the uptake of all three amines and was thus clearly shown to be the important factor in this effect. Reserpine reduced the uptake of [³H] noradrenaline to 25 % of the control value although the relative distribution remained unchanged. Propranolol and phentolamine had no observed effect on [³H] noradrenaline uptake. These results are discussed in the light of the previously reported action of ACTH in reversing the effects of hypophysectomy on medullary amine uptake (Hirano and Kobayashi 1978), and it is concluded that ACTH must exert this effect directly on the adrenal medulla rather than through the secretion of adrenal corticosteroids. It is also suggested that reserpine acts, as in neurons, by blocking amine uptake into intracellular granules rather than by blocking uptake into the cell itself.     

[3H]Desipramine Binding to Rat Brain Tissue: Binding to Both Noradrenaline Uptake Sites and Sites Not Related to Noradrenaline Neurons

Abstract The pharmacological and biochemical characteristics of [³H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [³H]desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The [³H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 μM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of ～1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromo-benzylamine (DSP4). In contrast, when 10 μM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with K_D values of 0.5 and >100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (B_max) for binding defined by 0.1 μM nisoxetine varied between brain regions, with very low density in the striatum (B_max not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of [³H]noradrenaline uptake but not with 5-[³H]hydroxytryptamine uptake. The low-affinity sites did not correlate with the regional distribution of [³H]noradrenaline uptake. Drug inhibition studies showed that noradrenaline inhibits the binding defined by 0.1 μM nisoxetine in a competitive manner. Together, these findings suggest that only a small fraction of the [³H]desipramine binding can be regarded as “specific” binding, and this binding fraction may represent the substrate recognition site for noradrenaline uptake. Assuming that one molecule of desipramine binds to each carrier molecule, the turnover number for the noradrenaline carrier was calculated to be 20/min, i.e., the duration of one transport cycle was 3 s.     

Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.     

Effects of the parkinsonism-inducing neurotoxin MPTP and its metabolite MPP+ on sympathetic adrenergic nerves in mouse iris and atrium

The effect of systemic administration of the parkinsonism-inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenylpyridine) on sympathetic adrenergic nerves in mouse iris and atrium has been investigated employing histo- and neurochemical techniques. The results indicate that MPTP does not have any potent neurotoxic effects on sympathetic adrenergic nerves. The effects of MPTP noted appear mainly to be restricted to a noradrenaline (NA) -depleting action and an acutely transient impairment of the NA uptake mechanism. This latter effect could be counteracted by monoamine oxidase inhibition. MPP+ was found to have more potent neurotoxic actions than MPTP as reflected i.e. by a patchy loss of histochemically demonstrable adrenergic nerves in iris which persisted for at least 7 days. Pretreatment with the NA uptake blocker desipramine antagonised the effects of MPP+, indicating that neurotoxicity is mediated via the NA uptake mechanism. The difference in neurotoxic potency of MPTP between sympathetic adrenergic nerves and central catecholamine neurons might be related to differences in metabolism of MPTP in the CNS and the periphery and/or due to the sympathetic adrenergic nerves being more resistant towards the cytotoxic actions following MPTP administration.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.