Torbjörn Malmfors

Degeneration of adrenergic nerves produced by 6-hydroxydopamine

Abstract 6-Hydroxydopamine (6-OH-DA) has been shown to deplete adrenergic nerves very rapidly of endogenous noradrenaline (NA). Its action is probably more rapid than any other known drug. The depletion is more pronounced in the terminals than in the main axons and nerve cell bodies. The iris was found to be a very sensitive organ, while the vas deferens was less sensitive. The depletion may be due partially or solely to destruction of the nerves by 6-OH-DA or some metabolite, taken up by the axon membrane pump into the adrenergic nerves. The destruction of the nerves would thus make the nerves unable to retain endogenous NA. At the same time the nerves cannot take up and store α-methyl-NA. This inhibition of uptake is not due to direct interference by 6-OH-DA with the uptake mechanisms of the cell membrane, but the inhibition of uptake occurs simultaneously with the disappearance of endogenous NA. Therefore, our data indicate a degenerative destruction of the sympathetic nerves, and suggest that 6-OH-DA might be used in achieving a chemical sympathectomy.

STANDARDIZATION OF PARAFORMALDEHYDE AND OF CERTAIN PROCEDURES FOR THE HISTOCHEMICAL DEMONSTRATION OF CATECHOLAMINES

Adrenergic mechanisms can now be studied directly at cellular and subcellular levels with the help c)f the sensitive fluorescence method c)f Falck and Hillarp for the histochemica! demonstration of certain catecholamines, e.g. the adrenergic transmitter. Briefly this method inve)lves the treatment of freeze-dried or air-dried tissues with formaldehyde gas derived from paraformaldehyde at 80#{176}C(for details, see Dahlstr#{246}m and Fuxe, Ada Physiol. Scand. 62: Suppl. 232, 1964; Norberg and Hamberger, ibid. 63: Suppl. 238, 1964). During this treatment catecholaniines are converted to intensely fluorescent 3, 4-dihydro isoquinolines (Corrodi and Hil!arp, Helv. Chini. Ada 46: 2425, 1963; 47: 911, 1964). This reaction requires the presence of water, but if too much water is present the amines or their fluorescent products can diffuse. Water for the reaction is derived frem three main sources: the tissue, the air initially enclosed in the reaction vessels, and the paraformaldeiiyde used. It is of great importance to standardize the freeze-drying (or air-drying) in such a way that. the pieces will have a low and fairly constant content of water. The dried pieces readily adsorb water from the air and must be handled in a dry atmosphere. Paraformaldehyde under these condi tions becomes the most important source of water. This water comes partly from pyrolysis of the polymer, but of greater importance is adsorbed water, which varies considerably depending for example on the storage of the paraformaldehyde. The present work reports the principles of a simple procedure for standardization of paraformaldehyde based on the finding that this substance takes up or loses water to constant levels when incubated at room temperature (20-22#{176}C)in an atmosphere of constant relative humidity. Incubations were performed in closed vessels containing aqueous solutions of sulfuric acid of varying density. Any relative humidity between 10 amid 90% can be obtained in this way (Handbook of Chemistry and Physics. 44th ed. Chemical * Supported by USPHS Grant (NB 02854-04), National Institute of Neurological Diseases and Blindness, and a grant from the Swedmsh Medical Research Council. Rubber Pumbhishiing Co., Cleveland, 1963). Amounts of adsorbed water were deterniiuied by using the Karl Fischer reagent (Mitchell and South , 4 quametry, Interscience, New York , 1948). A full account of these experiments will be pubhished in a forthcoming paper (Hamnberger, to l)e

Uptake and accumulation of 3H-noradrenaline in adrenergic nerves of rat iris. Effect reserpine, monoamine oxidase and tyrosine hydroxylase inhibition

Abstract Isolated rat irides were incubated in Krebs-Ringer bicarbonate buffer containing 3 H-noradrenaline ( 3 H-NA) and the 3 H-NA taken up was determined. The extracellular space determined with 14 C-sorbitol was about 40% of the iris. The equilibration and clearing of the extracellular space occured within 15 min incubation time. Four pharmacologically different uptake models for NA were used: irides from untreated rats, nialamide pretreated rats, reserpine + nialamide pretreated rats and H44/68 (synthesis inhibitor) pretreated rats. Specific chemical determinations disclosed that very small amounts of metabolites are formed in the iris preparation after an in vitro incubation. It was concluded that there is an efficient uptake and accumulation of NA in adrenergic nerves of rat iris. The membrane pump seemed to be the dominant and most important mechanism for the initial uptake while the granular uptake mechanism was of importance for the total storage capacity and intraneuronal retention. Reserpine did not affect the axonal membrane uptake to any significant degree. After pretreatment with a synthesis inhibitor, H44/68, the uptake and accumulation of 3 H-NA in the iris was only increased 10–20% compared to the untreated iris, indicating that the depleted NA stores were not completely refilled. The extraneuronal uptake was very small provided that the medium concentration of NA was not too high (10 −6 M or lower). This uptake increased considerably in relation to the neuronal uptake when NA concentrations of 10 −5 M of higher were used, although there was no true accumulation of amine in the tissue compared with the medium.

Combined fluorescence histochemistry and 3h-noradrenaline measurements of adrenergic nerves

SummaryA method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA1 content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.

Histochemical studies on the uptake of noradrenaline and α-methyl-noradrenaline in the perfused rat heart

Abstract The cellular localization of noradrenaline and α-methyl-noradrenaline taken up by the rat isolated heart during perfusion was studied. After perfusion with low concentrations, 0.02–0.2 μg/ml, uptake of the catecholamines into the adrenergic nerves occurred but no evidence for an extraneuronal uptake was found. After perfusion with high concentrations, 5–20 μg/ml, there was a markedly increased flourescencein the heart muscle cells. In the atria numerous small intensely fluorescent connective tissue cells could be found. In both cases the fluorescence proved to be due to the presence of catecholamines. The extraneuronal uptake could be inhibited by normetanephrine but not desipramine. It is concluded that the catecholamines taken up in the rat heart during perfusion with high concentrations are located partly in the adrenenic nerves but also to a great extent extraneuronally.

The postnatal development of noradrenaline uptake in the adrenergic nerves of different tissues from the rat

Abstract The uptake and accumulation of 3 H-NA was studied in vitro in iris, atrium, submaxillary and sublingual glands, and the muscular and mucosal layers of the duodenum from rats at different ages (0–60 days). Most of the 3 H-NA found in the tissues was located in the adrenergic nerves. The uptake of 3 H-NA increased progressively with age in all the organs examined, when calculated per tissue piece. When the radioactivity was expressed per gram tissue, the concentration in the iris and atrium increased progressively as the animal grew older, but the concentration in the salivary glands and in the gut decreased. The variation in the uptake of 3 H-NA per gram wet weight is probably due to differences in growth rates between neuronal and extraneuronal components of the different organs, rather than to differences in the uptake ability of the adrenergic nerves, during the time of development.

The effect of drugs on accumulation of monoamines in tubero-infundibular dopamine neurons

Abstract Histochemical studies have been carried out on the effects drugs on the accumulation of monoamines in the tubero-infundibular dopamine neurons of rat after preceding depletion. It was found that (+)-amphetamine and cocaine blocked the reserpine-resistant accumulation of dopamine, noradrenaline and α-methyl-noradrenaline; desipramine, guanethidine and chlorpromazine did not block the accumulation of these amines. Very high doses of 5-hydroxytryptamine, given after depletion with reserpine caused an accumulation of 5-hydroxytryptamine which was blocked by (+)-amphetamine. The mechanism at the nerve cell membrane, which is able to take up and concentrate amines and which is resistant to reserpine in contrast to the reserpine-sensitive storage mechanism, was not stereospecific. The accumulation of catecholamines observed after their injection to reserpine pretreated rats could be released by (+)-amphetamine but not by cocaine. The present results indicate that the mechanism at the nerve cell membrane of the dopamine neurons in fundamentally different from that in the noradrenaline neurons, since it was not blocked by desipramine, chlorpromazine or guanethidine which block this mechanism in the NA neurons. Furthermore, cocaine may block this mechanism in the DA neurons.

Effect of a new series of bicyclic compounds with potential thymoleptic properties on the reserpine‐resistant uptake mechanism of central and peripheral monoamine neurones in vivo and in vitro

1 Bicyclic compounds with potential thymoleptic properties (Lu‐compounds) have recently become available, and their effects on the membrane pumps of the central and peripheral monoamine neurones have now been tested and compared with those of the tricyclic antidepressant drugs. 2 Biochemical and histochemical in vivo studies have been performed. The possible blocking action of Lu‐compounds on the noradrenaline (NA) and 5‐hydroxytryptamine (5‐HT) displacement caused by 4,α‐dimethyl‐meta‐tyramine (H 77/77) and 4‐methyl‐α‐ethyl‐meta‐tyramine (H 75/12), respectively, has been studied, and a positive result has been taken as evidence for membrane pump blocking activity. No certain effects were obtained on the 5‐HT displacement induced by H 75/12, whereas a partial blockade of the NA displacement by H 77/77 in central NA neurones was obtained after most of the Lu‐compounds (Lu‐3–010, 3–049, 3–092, 4–012) and especially after the thiophthalane derivative Lu 5–003. The ED50 of the latter drug was around 8 mg/kg, that is, somewhere between protriptyline (ED50 4 mg/kg) and desipramine (ED50 15 mg/kg) in potency. 3 Histochemical in vivo studies on the rat iris revealed that Lu 5–003 and especially the corresponding phthalane derivative Lu 3–010 were potent in blocking the uptake of α‐methyl‐NA in the adrenergic nerve terminals of the iris. The other Lu‐compounds were less active. The releasing effects of the Lu‐compounds on the extragranular accumulation of α‐methyl‐NA in the adrenergic terminals were weak compared with membrane blocking activity. 4 In vitro studies on the central and peripheral catecholamine (CA) neurones have also been performed. In the same way as, for example, protriptyline the Lu‐compounds only blocked accumulation of α‐methyl‐NA in the NA terminals but not in the dopamine (DA) nerve terminals. Lu 5–003 and Lu 3–010 were the most potent of the Lu‐drugs when added in vitro. The Lu‐drugs were also injected in vivo after which the effect on the α‐methyl‐NA accumulation was studied in vitro. In isotope experiments with labelled α‐methyl‐NA it was found that desipramine, Lu‐3–010, Lu 3–092 and Lu 4–012 were equally potent in blocking uptake in the central nervous system. 5 In general the results obtained with the various models are in agreement. The high activity of Lu 5–003 on the central NA neurones may be related to its high heptane/H2O distribution coefficient. 6 It is concluded that the Lu‐compounds have a selective action on the membrane pump mechanism of the central and peripheral NA neurones. In these respects they behave like desipramine and protriptyline.

The role of plasma protein binding in the inhibitory effect of nortriptyline on the neuronal uptake of norepinephrine

The inhibitory effect of nortriptyline (NT) on the neuronal uptake of radio labeled norepinephrine (NE) was studied with the use of the rat iris preparation in Krebs‐Ringer solution or human plasma. When added to the incubation medium, NT inhibited the uptake of NE approximately ten times as effectively in buffer as in human plasma within the tested concentration range of NT (10−8 to 10−6M). This result is in good agreement with the 94 per cent binding (at NT concentration of 1.1 10−6M) obtained by the ultrafiltration method. Plasma from patients treated with NT also inhibited the uptake of NE. Correlation between the inhibitory effect and the “endogenous” plasma level of NT in the 14 patients studied was significant (p < 0.001). When NT was added in different concentrations to control plasma the inhibitory effect observed was close to that obtained with patient plasma containing the same “endogenous” concentration of NT.

Release and depletion of the transmitter in adrenergic terminals produced by nerve impulses after the inhibition of noradrenaline synthesis or reabsorption

Abstract Changes in the content and intraneuronal distribution of noradrenaline in the adrenergic nerves of the rat iris were studied following sympathetic stimulation in combination with the inhibition of transmitter resynthesis or reabsorption. The synthesis inhibitors developed by Carlsson and Corrodi were found to be so efficient that a transmitter depletion can be readily demonstrated in activated adrenergic terminals. This provides an entirely new way of studying the state of activity of such terminals. Much less marked depletion was observed after inhibition of transmitter reabsorption by imipramine. Direct evidence was obtained for the view that the abundant varicosities present along the entire lenght of the terminals are precisely the structures specialized for the synthesis, storage and relase of the transmitter.