Potentiation by cocaine of relaxations of the guinea-pig colon caused by noradrenaline and by stimulation of adrenergic nerves.

The distal colon of the guinea-pig is relaxed by noradrenaline, by isoprenaline and by the stimulation of fibres running with the colonic nerves or intramurally. The relaxations in response to stimulation of the colonic nerves have a guanethidine-sensitive (adrenergic) and a guanethidine-insensitive (non-adrenergic) component. Cocaine causes a three-fold sensitization of the muscle to noradrenaline but no sensitization to isoprenaline. Cocaine increases the duration, but does not affect the amplitude, of the relaxation observed when adrenergic nerves are stimulated, and affects neither duration nor amplitude of the non-adrenergic response. The adrenergic nerve terminals lie in Auerbach's plexus, not in the longitudinal muscle. It is concluded that the sensitization to noradrenaline and the increases in durations of responses to adrenergic nerve stimulation are due to inhibition of catecholamine uptake into adrenergic nerves by cocaine. It appears that, even where the neuromuscular separation is large as it is in the colon, the concentration of exogenous noradrenaline at the receptors can be decreased by neuronal uptake, and the uptake mechanism can modify responses to nerve stimulation in vitro.     

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.     

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.     

Effect of an inhibitor of noradrenaline uptake,desipramine, on cell proliferation in the intestinal crypt epithelium

The intestinal mucosa receives an adrenergic innervation for which there is no commonly accepted function. However, in recent years, cell kinetic studies have raised the possibility that this innervation may be an important regulator of crypt cell proliferation. The effects of noradrenaline released from adrenergic nerves is terminated principally by re-uptake of the amine into the nerve and this process can be inhibited by the antidepressant drug, desipramine. In this report desipramine is shown to accelerate crypt cell proliferation in intact, but not in chemically sympathectomized rats, thus adding support to the notion that regulation of crypt cell division is an important function of the sympathetic nervous system.     

6-AMINODOPAMINE-INDUCED DEGENERATION OF CATECHOLAMINE NEURONS

the effects of 6-aminodopamine on central and peripheral catecholamine neurons using fluorescence histochemical and isotope techniques have been investigated. Systematic administration of 6-aminodopamine (20 mg/kg intraveneously) produced a rapid (within 1 h) and long-lasting depletion of endogenous noradrenaline in adrenergic nerves of mouse atrium and iris with a concomitant loss of [³H]noradrenaline uptake. The effects were dosedependent. Accumulations of noradrenaline in non-terminal axons were observed histochemically, indicating that 6-aminodopamine induces neuronal damage. Desipramine completely blocked the 6-aminodopamine induced noradrenaline depletion and reduction in [³H]noradrenaline uptake, indicating that 6-aminodopamine has to be taken up by the axonal ‘membrane pump’ to produce its effects. Themonoamine oxidase inhibitor, nialamide, potentiated the effect of 6-aminodopamine on [³H]noradrenaline uptake. 6-Aminodopamine did not affect the cell bodies of the adrenergic neurons and there was a reappearance of adrenergic nerves and recovery of [³H]noradrenaline uptake. 6-Aminodopamine does not seem to pass the blood-brain barrier after systemic injection. Intraventricular injection of 6-aminodopamine in rats led to a considerable reduction in endogenous whole brain noradrenaline and [³H]noradrenaline uptake in slices from cerebral cortex and hypothalamus. Similar, but less pronounced effects were observed on dopamine neurons in the caudate nucleus. Histochemically, pronounced accumulations of transmitter were observed in the axons of the catecholamine neurons. The results obtained favour the view that 6-aminodopamine is able to produce an acute and selective degeneration of catecholamine neurons similar to that seen after the neurotoxicagent, 6-hydroxydopamine. Both compounds seemed to be approximately equally potent in their neurotoxicity, although 6-aminodopamine seemed to be more generally toxic.     

Adrenergic nerves to the dermal melanophores of the rainbow trout,Salmo Gairdneri

Summary The stellate processes and cell bodies of the dermal melanophores in the rainbow trout are intimately enclosed by a plexus of thin varicose nerves which display a specific catecholamine fluorescence. The nerves contain probably small amounts of noradrenaline and have the ability to take up and concentrate this amine. Denervation of the skin leading to dispersion of the melanophores causes the nerves to disappear. The findings leave little doubt that the dermal chromatic motor nerves are adrenergic.This study was supported by a grant from the Swedish Research Council for Natural Sciences (99-35) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B69-14X-56-05C and No. B69-14X-712-04C).     

The mechanism of [3H]noradrenaline release by histamine and its analogs from the rat vas deferens

In this study the mechanism by which histamine and H1 and H2 agonists evoked an overflow of radioactivity from rat vasa deferentia preloaded with [3H]noradrenaline was investigated. The overflow evoked by the various agonists was unaffected by the presence of such receptor antagonists as propranolol, phentolamine, cimetidine, or scopolamine. On the other hand, the overflow evoked by all agonists except dimaprit was inhibited by mepyramine and by two well-known neuronal uptake inhibitors, cocaine and desipramine. The inhibition by mepyramine has been attributed to its effect on the neuronal uptake process. Metabolic profile studies showed that 3,4-dihydroxyphenylglycol (DOPEG) was the major constituent in the evoked overflow caused by histamine, 2-methylhistamine, 4-methylhistamine, and dimaprit and that the overflow evoked by 2-pyridylethylamine and 2-thiazolylethylamine consisted predominantly of unchanged noradrenaline. Based on these findings, it is concluded that all of the agonists tested evoke noradrenaline release intraneuronally by entering the adrenergic nerve terminals. While dimaprit might enter by passively diffusing into the adrenergic nerves, other agonists seem to use the neuronal uptake process. Noradrenaline released intraneuronally is subsequently degraded by neuronal monoamine oxidase to form DOPEG. However, there are qualitative and quantitative differences in the metabolic profile of the overflow evoked by various agonists. It is suggested that these differences could arise from their additional properties, such as their effect on the neuronal uptake process and (or) their ability to act as substrate for neuronal monoamine oxidase.     

Adrenergic component in the mechanism of action of sodium oxybutyrate]

The influence of sodium oxybutyrate on the adrenergic neurotransmitter content in the nerve fibers and the synaptic vesicles and the uptake of exogenous noradrenaline (NA) by the sympathetic nerves of rat Vas deferens was studied by spectrofluorimetry, fluorescent-histochemistry and cytochemical electron microscopy. Sodium oxybutirate failed to influence the stores of the adrenergic mediator, but was capable of blocking the uptake and accumulation of the exogenous NA.     

Chemical sympathectomy induced by 5.6-dihydroxytryptamine

Summary The effect of different doses of 5.6-dihydroxytryptamine—a serotonin analogue which produces a degeneration of serotonin containing nerve terminals in the rat brain—on the noradrenaline (NA) content and—storage sites of peripheral sympathetic nerves in the mouse and rat heart, spleen, rectum and vas deferens has been investigated by fluorescence—, electron microscopical and chemical methods. Moderate doses of 5.6-dihydroxytryptamine (5.6-DHT) (10–45 mg/kg ip.) cause a temporary, reversible displacement of noradrenaline from the adrenergic nerves concomitant with a significant increase in the number and opacity of small and especially large granular vesicles. The recovery of the neuronal NA concentration is, however, retarded after doses higher than 45 mg/kg (60 or 100 mg/kg ip.); a partial degeneration of varicose NA terminals is verified fluorescence- and electron microscopically. A combined treatment of animals with tyrosine hydroxylase inhibitors (-methyl-paratyrosine or -propyl-dopacetamide) and 5.6-DHT, in some instances also followed by reserpine, potentiates the destructive properties of 5.6-DHT; a similar potentiation is accomplished by reserpine posttreatment or by an additional pretreatment of animals with reserpine and nialamide.The results suggest that 5.6-DHT when given in moderate doses (up to 45 mg/kg) may be handled by sympathetic adrenergic nerves like a false neurotransmitter which displaces noradrenaline from the stores, but that it causes a chemical degeneration of noradrenaline containing nerve terminals when applied either in single high doses (60 or 100 mg/kg ip.), or when administered in moderate non-degenerative doses together with drugs that impair the neuronal inactivation mechanisms for 5.6-DHT (granular uptake and storage mechanism and/or monoamine oxidase activity) and thus provoke a temporary increase in the amount of free 5.6-DHT in the neuron's cytoplasm.The molar efficiency of 5.6-DHT in causing a chemical sympathectomy is clearly inferior to that caused by 6-hydroxydopamine. The differences are probably mainly due to differences in the affinity of both drugs to the amine uptake system located at the cell membrane and the membrane of the intraneuronal storage vesicles of the adrenergic nerve terminals.Supported by grants from the Deutsche Forschungsgemeinschaft.     

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.     

A method for the demonstration of adrenergic nerve fibres in peripheral nerves

Summary The adrenergic nerve fibres running from the ganglia to the innervated tissues usually have too low a content of noradrenaline to be clearly visualized with the histochemical fluorescence method of Falck and Hillarp. They can easily be demonstrated, however, as early as 24 hours after axotomy (crushing or constriction of the nerves) due to the rapid accumulation of what is probably noradrenaline taking place proximally to the lesion. The fibres can be visualized even more clearly if axotomy is combined with the administration of l-dopa and with monoamine oxidase inhibition. In this way the presence, distribution and direction of adrenergic fibres can be directly studied in peripheral nerves.For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine) and Swedish Pfizer, Stockholm (nialamide). The investigation has been supported by research grants from the United States Public Health Service (NB 02854-04), the Swedish Medical Research Council, and Knut and Alice Wallenbergs Foundation.     

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.     

Effect of an inhibitor of noradrenaline uptake, desipramine, on cell proliferation in the intestinal crypt epithelium

The intestinal mucosa receives an adrenergic innervation for which there is no commonly accepted function. However, in recent years, cell kinetic studies have raised the possibility that this innervation may be an important regulator of crypt cell proliferation. The effects of noradrenaline released from adrenergic nerves is terminated principally by re-uptake of the amine into the nerve and this process can be inhibited by the antidepressant drug, desipramine. In this report desipramine is shown to accelerate crypt cell proliferation in intact, but not in chemically sympathectomized rats, thus adding support to the notion that regulation of crypt cell division is an important function of the sympathetic nervous system.     

Mechanism of Neurotransmitter Release Elicited by the Preferential α1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.     

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.     

Uptake and accumulation in vitro of 3H-noradrenaline in adrenergic nerves of human atrium

Summary The adrenergic innervation and the in vitro uptake of ³H-noradrenaline has been investigated in human atrial tissue slices from patients undergoing thoracic surgery. The atrial appendage was richly innervated, but the density of the adrenergic nerve plexus varied considerably between different tissues examined. The nerve terminals were of characteristic varicose appearance, running singly or in bundles along the long axis of the muscle fibers. The nerve fibers seemed to penetrate in between the muscle cells. The distribution and appearance of the adrenergic nerves were quite similar to those described in earlier investigations of heart tissue from other species. The uptake and accumulation of ³H-noradrenaline in vitro increased with increasing concentration in the medium and with time, and the uptake process could efficiently be blocked by desmethylimipramine (DMI), a potent inhibitor of the uptake mechanism located at the axonal membrane, the so called membrane pump. There was a true accumulation of ³H-noradrenaline in the atrial tissue during the incubation, compared to the medium. The metabolism of ³H-noradrenaline during the incubation has also been studied. The data presented speak in favour of the view that the adrenergic nerves of human atria possess an efficient uptake accumulation mechanism for noradrenaline.Abbreviations used DMI desmethylimipramine - NA noradrenaline - NM normetanephrine     

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.     

Influence of nonakhlazin on adrenergic neurotransmission in the vas deferens of rats]

The effect of a new antianginal drug--nonachlazine on the adrenergic neurotransmission in the isolated rat vas deferens was studied by examining the vas deferens contractions in response to the transmural electric stimulation of the postganglionic sympathetic nerves and addition of noradrenaline (NA) or BaCl2. After the nonachlazine treatment the NA content in the vas deferens was also studied by the spectrofluorometric method. Besides, the effect of the drug on the uptake of the exogenous NA was investigated. Nonachlazine was found to possess some sympatholytic and spasmolytic effect and could block the uptake of the exogenous NA greatly.     

The effect of decentralisation or chronic hypogastric nerve stimulation in vivo on the innervation and responses of the guinea-pig vas deferens

Summary The physiological, pharmacological and morphological characteristics of guinea-pig vas deferens supplied by hypogastric nerves rendered inactive by decentralisation were compared with those of vas deferens in which the nerve supply had been chronically stimulated for 3–9 days using implanted electrodes. No change was seen in decentralised preparations prior to 7 days, but from 8–15 days, increased sensitivity to application of noradrenaline in vitro was observed, which was shown to be related to reduced transmitter uptake by nerve terminals as well as to an increase in postjunctional sensitivity; there was also increased fatigability 7–14 days following decentralisation. Continuous stimulation of hypogastric nerves at 2 Hz for 4–8 h daily for 4–8 days resulted in enhanced transmitter uptake and reduced responses to noradrenaline; this was associated with a slight increase in noradrenaline content and a faster adrenergic neuromuscular response with a shorter latency. No appreciable changes in nerve or muscle structure studied by electron microscopy were observed following decentralisation, but there was an increase of between 12.5 and 29.6% in the number of close (< 100 nm) neuromuscular junctions following chronic stimulation for 8 days.     

The effect of caesium on adrenergic transmission in rabbit vas deferens.

The effect of caesium on the responses of rabbit vas deferens to transmural stimulation was investigated. The tissue responded to transmural stimulation with a phasic spike contraction followed bya sustained contractile response. The sustained response was inhibited by phentolamine and guanethidine and thus apparently results from noradrenaline release from adrenergic nerves. Addition of 2-5mM Cs+ greatly potentiated this secondary response without altering the sensitivity of the tissue to added (minus)-noradrenaline. This potentiation was not due to Cs+ decreasing the neuronal uptake of noradrenaline, or by Cs+ altering prostaglandin synthesis. Addition of 2mM Cs+ significantly increased the amount of (plus or minus)-[3-H] metaraminol released from tissues in response to transmural stimulation (5 Hz). It is suggested that caesium potentiated responses of rabbit vas deferens to transmural stimulation by increasing the amount of transmitter released per nerve impulse, possibly as a result of prolongation of the action potential.