Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 139 - 144 (1977) Naunyn-Schmiedeberg's

Archives of Pharmacology �9 by Springer-Verlag 1977

Adrenergic Nerve Degeneration Induced by Condensation Products of Adrenaline and Acetaldehyde*

ISABEL AZEVEDO and WALTER OSSWALD

with the technical assistance of M. LUISA VASQUES

Laborat6rio de Farmacologia, Faculdade de Medicina, Porto, Portugal

Summary. Adult and new-born rats were treated with MA 3 or MA4 (tetrahydroisoquinoline alkaloids re- sulting from the condensation of adrenaline with acetaldehyde) to investigate the action of these al- kaloids on the adrenergic innervation.

The irides, right heart auricles and superior cer- vical ganglia of control and treated rats were pro- cessed for microscopy and spectrofluorimetrical de- termination of noradrenaline (NA).

Marked ultrastructural alterations of the adren- ergic nerve terminals were observed in the irides and auricles of the treated adult and new-born rats. The superior cervical ganglia of the adult rats exhibited only minor ultrastructural alterations while those of the new-born animals presented anomalies even at the light microscopy level.

Absence of significant alterations of the NA con- tent in treated adult rats is attributed to the accumu- lation of fluorescing alkaloid derivatives.

We conclude that MA3 and MA4 can induce selective degeneration of the adrenergic nerve ter- minals of the adult rats and of the whole adrenergic neurone of new-born rats. They are, in this respect, less potent than 6-OHDA.

As evidence has been put forward that these alkaloids may be formed in vivo during ethanol intoxication, our results may constitute a contribution to the understanding of some of the phenomena re- lated to ethanol intoxication and/or the alcohol withdrawal syndrome.

Key words: Condensation of adrenaline with acetal- dehyde - Degeneration of adrenergic nerve ter-

Send offprint requests to I. Azevedo at the above address * Part of the results was presented to the 6th International Congress of Pharmacology, Helsinki 1975 (Abstr, 1513) and to the 6th Annual Meeting of the Portuguese Pharmacological Society (Lisbon, I976). This work was supported by a grant from Instituto Nacional de Investigag~o Cientifica (PMC-2)

minals - Morphologic changes in ganglion cells of new-born rats - 6-Hydroxydopamine like actions of tetrahydroisoquinolines.

INTRODUCTION

Osswald et al. (1975) have recently described the preparation and pharmacological activity of the amor- phous condensation product of adrenaline with acetal- dehyde (MA 3) and of one of its components, MA 4 (1,2- dimethyl-4,6,7- trihydroxy- 1,2,3,4- tetrahydroiso- quinoline). In these experiments it was shown that the tetrahydroisoquinoline alkaloids (TIQs) thus formed exhibit sympathomimetic activity and are able to release noradrenaline (NA); it was further described that they are taken up by adrenergic nerve terminals and may act as false transmitters. Similar suggestions had been advanced, for other TIQs, by Greenberg and Cohen (1973) and Mytilineou et al. (1974).

The evident similarity of some of these character- istics with some of those exhibited by 6-hydroxy- dopamine (6-OHDA) (Thoenen and Tranzer, 1973) led us to the present study, in order to investigate a possible 6-OHDA-like action of these TIQs.

METHODS

Experimental. Sixteen adult male rats were injected i.p. with MA 3 (10 mg/kg) or MA4 (50 mg/kg) and this dose was repeated after 7 days. Twenty four hours after the last administration the treated as well as the control rats (injected with the same volume of saline) were killed by decapitation and the irides, hearts and superior cervical ganglia removed immediately.

Twelve new-born rats from three litters were injected within 12 h after birth with MA3 (10 mg/kg, Lp.); the same dose was thereafter injected 5 days a week, during a fortnight. Eight rats were killed 24 h after the last administration of MA 3 and the other

140 Naunyn-Schmiedeberg's Arch, Pharmacol. 300 (1977)

4 rats were killed when 8 weeks had elapsed after the last injection of MA 3. Of each litter, one half of the rats were injected while the other half served as untreated controls. The superior cervical ganglia and hearts were removed immediately after sacrifice.

Determination of Noradrenaline (NA). After removal of the right auricle for morphologic study, the remaining heart tissue was rapidly washed with ice-cold Krebs solution, blotted, weighed and homogenized in 5 ml of ice-cold 0.4 N perchloric acid, with added EDTA (1.0 mg/ml) and sodium metabisulphite (1.25 mg/ml). After standing for at least 2 h, the extract was centrifuged during 10 rain at 8000 g in a refrigerated centrifuge. An aliquot of 2 ml of the supernatant was transferred to an alumina column and the NA determined spectrophotofluorimetrically with the trihydroxy- indole method of yon Euler and Lishajko (1961).

Light and Electron Microscopy. Small pieces of the ganglia, right heart auricles and irides were cut with a razor blade and fixed by immersion in 3 ~ glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, at 4 ~ C for 90 rain. The tissues were then washed overnight in the cacodylate buffer with 0.6 M sucrose at 4~ post-fixed in a 2~ solution of osmium tetroxide in 0.1 M cacodylate buffer, pH 7.3, at 4 ~ C for i h, dehydrated and embedded in Epon 812. Semithin sections were obtained from the ganglia with a LKB Ultrotome, stained with 1 ~ toluidine blue in 1 ~ borax and examined by light microscopy.

Thin sections were cut from the irides and right heart auricle blocks as well as the ganglia blocks after retrimming according to the information obtained by light microscopy. The thin sections were stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 101 at 80 kV.

R E S U L T S

Morphological Data. In the irides and right heart auricles of the rats treated with M A 3 or M A 4 the adrenergic nerve terminals presented marked ultra-

s t ructural al terations. Some appeared swollen and empty ; other terminals were packed with lamellar

and non- lamel la r very dense bodies (Fig. 1). Other morphologica l a l terat ions inc luded relative or total absence of vesicles, d i s rupt ion of neurotubules , ano- malous appearance of mi tochondr ia , deposi t ion of glycogen and big lamellar format ions (Fig. 2). In some cases dense degenerat ion of the whole terminal was observed. Adrenergic terminals in var ious stages of degenera t ion as well as n o r m a l appear ing ones coexisted in the same organ (Fig. 3). The cholinergic terminals had a no rma l appearance. The Schwann cells, ma in ly in the auricle, showed marked reactive changes: presence of inden ta t ions of the nuclear surface, mult ipl ic i ty of the r ibosomes and very pro- minen t rough endoplasmic re t iculum (RER) and Golgi appara tus . The Schwann cell cytoplasm emitted processes toward the degenerat ing nerves, engulfed them and apparent ly digested them (Fig. 3).

The general s t ructure of the iris was otherwise normal . In the auricle a decrease of the glycogen con-

Fig. 1. Electron micrograph of the right heart auricle of a rat in- jected with MA 3 (10 mg/kg) twice in a week. Observation 24 h after the last administration: part of an adrenergic nerve terminal packed with irregularly shaped dense bodies close to a cholinergic terminal of normal appearance (C). Calibration: 1 gm x 23 750

I. Azevedo and W. Osswald: Adrenergic Nerve Degeneration Induced by TIQs 141

Fig. 2 A large lamellar formation occupying most of the volume of a degenerating terminal in the right heart auricle of a rat treated as the one referred to in Figure 1. Calibration : 1 ~tm x 45 600

Fig. 3. Process of Schwann cell cytoplasm (S) with dilated rough endoplasmic reticulum and an inhomogeneous dense formation. In close vicinity there are some cholinergic (C) and adrenergic (A) nerve profiles. One of the latter has lost the vesicles and exhibits a dense body. Calibration: 1 pm x 32000

142 Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

Fig. 4. Light micrograph of a superior cervical ganglion from a new-born rat treated with MA 3 (10 mg/kg) for 2 weeks and sacrificed 24 h after the last administration. There are some normal appearing ganglion cells (N); others present a much more dense aspect showing an irregular nucleus and cytoplasm (~). Glial cells can be seen between the ganglion cells (arrows). Calibration: 120 gm x 10 x 900

tent and an apparent increase in the amount of lipid droplets and in the number of atrial granules were noted. There was also some mitochondrial damage in the myocardial cells.

No difference was detected between the results obtained after treatment with MA 3 or MA 4.

In the treated new-born rats the results observed in the irides and heart auricles were similar to those described above for the adult animals. Eight weeks after the end of the treatment, adrenergic terminals with abnormal aspect were rarer but still present. Norma l appearing terminals appeared to be more frequent than 24 h after the treatment. Macrophage- like cells were abundant at 8 weeks in contrast with what happened immediately after the treatment. The other structural anomalies described above did not disappear in this period of time.

A sharp contrast between adult and new-born treated animals was found in the results obtained in the ganglia. In the adult rats there were no changes at the light microscopy level, but some ultrastructural changes were noted, mostly in the axons: discrete swelling with disruption of the neurotubules. The superior cervical ganglia of the 15 days old rats treated with MA 3 presented marked alterations, even at the light microscopy level: irregularity of the nucleus, increased density of the nucleoplasm, and, less fre- quently, clumping of the chromatin beneath the nuclear membrane (Fig. 4). The cytoplasm of these

cells showed also an increased density and an irre- gular shape. Electron microscopy revealed a large increase in the number of free ribosomes; in some cases clumped free ribosomes were separated by clear spaces representing the cisternae of the R E R which were more or less dilated. Dense bodies and auto- phagic vacuoles appeared somewhat more frequently than in the controls.

Satellite cells, somewhat increased in number, had dilated the R E R that contained a floccular ma- terial. At places these cells were seen surrounding degenerating cells and clumps of debris, probably representing cells or axons in the final stages of disintegration.

Lipid droplets of small dimensions were seen at places in the interstitial space of the ganglionic tissue.

The endothelial cells of most capillaries observed in the different tissues of the treated rats were packed with micropinocytotic vesicles, some of which had large dimensions, like cisternae. The luminal mem- brane was folded, giving origin to prolongations which looked like vilosities. The volume of some of these cells was increased.

Noradrenaline Determination. The "noradrenal ine" content of the hearts of MA 3- or MA 4-treated adult rats (1.16 _ 0.18 ~tg/g; n = 8 and 1.21 + 0.10 gg/g; n = 8, respectively) was apparently not different from

I. Azevedo and W. Osswald: Adrenergic Nerve Degeneration Induced by TIQs 143

the control value (1.10 • 0.15 lag/g; n = 11). How- ever we found evidence that these values probably correspond to accumulated TIQs which exhibit fluo- rescence under the same conditions as those required for determination of noradrenaline (see Discussion).

In the new-born rats, 24 h after the last administra- tion of MA3, we found 0.56 +_ 0.06 lag/g of "nor- adrenaline" (control: 0.74 _+ 0.09 lag/g). Eight weeks later noradrenaline depletion caused by MA 3 was more evident (0.39 + 0.05 vs 0.96 • 0.06 lag/g; n = 6; P < 0.001).

General Observations. The general appearance of the adult rats was not modified by the administration of MA 3 or MA 4 nor were any differences observed in their gross behaviour or spontaneous activity. However, after completion of the dosage schedule, the new-born rats weighed about 30 % less than their untreated litter controls. After each injection of MA 3 or MA 4 they became extremely pale, prostrated and showed respiratory disturbances. After a period of about 30 min they returned to a normal appearance.

The size and aspect of the superior cervical ganglia of the adult rats was not modified by the treatment with MA3 or MA4. In the new-born rats the ad- ministration of MA3 led to an increase in the size of the ganglia which presented a yellowish colour and a less elastic consistency than the control ones. Eight weeks after treatment their aspect was still abnormal.

Marked macroscopic and microscopic alterations were found in the liver of the treated rats. These re- sults have been presented elsewhere (Moura et al., 1977).

DISCUSSION

The similarity of some of the pharmacological ac- tions of the condensation product of adrenaline and acetaldehyde (MA3) with those of 6-OHDA led us to look for morphologic alterations in the adrenergic nerve terminals. We have chosen the iris and heart auricle because it is known that these organs possess an abundant adrenergic innervation and it is possible to compare the reaction of adrenergic and cholinergic nerves since they appear in close vicinity (Tranzer and Thoenen, 1968). In the superior cervical ganglia we were able to study the neuronal cell bodies. The schedule of administration of MA3 and MA4 was the same as that described for 6-OHDA to give optimal results of induced nerve degeneration in rats (Finch et al., 1973).

We have observed that MA3 and MA4 induced marked ultrastructural alterations in most of the adrenergic nerve terminals of the iris and heart

auricles. In a given preparation the adrenergic nerve terminals were in various stages of degeneration but there were always some normal appearing ones. This is not different from what happens after surgical denervation where only rarely a number of axons are seen in the same stage of degeneration (Roth and Richardson, 1969; Bray et al., 1972; Thomas and King, 1974).

The neuronal cells of the superior cervical ganglion of newborn rats were much more sensitive to the effects of MA 3 than the corresponding cells of adult animals. The same difference has been described for 6-OHDA (Finch et al., 1973). The ultrastructural changes affected both the cytoplasm and nucleus. It is necessary to study earlier phases of the lesions in order to discover where they begin or if they are simultaneously initiated in the nucleus and in the cytoplasm. This could help to understand the mecha- nism of the damage of the ceil body (Pilar and Land- messer, 1976; Landmesser and Pilar, 1976).

The NA content of the hearts of the MA3- or MA 4-treated adult rats was not diminished, although extensive adrenergic nerve degeneration was observed at the ultrastructural level. Even in the new-born rats, 24 h after the last administration of MA 3 the NA content of the heart largely exceeded what we expected to find. Only in the animals sacrificed 8 weeks after the end of the MA 3 treatment we observed a net NA depletion (41 ~ of the control values). These facts led us to consider the hypothesis that the TIQs are or give rise to fluorescent compounds when in contact with the tissues. We performed a few ex- perinaents to test our hypothesis and verified that in- cubation of heart tissue in Krebs solution, at 37 ~ C, with MA 3 for 30 rain, increased the apparent "NA content" proportionally to the MA 3 concentrations in the incubation fluid. We think this explains our results and deserves a future investigation to clarify the situation.

We conclude that the condensation product of adrenaline and acetaldehyde can induce a selective degeneration of the adrenergic nerve terminals of adult rats and of the whole adrenergic neurones of newborn rats; however, the TIQs are, in this respect, less potent than 6-OHDA.

The doses of MA 3 and MA 4 used here were not very different from the maximally tolerated doses (unpublished observations) and it is possible that with a carefully chosen schedule of administration the selec- tivity of the neuronal damage may be improved.

Evidence has been put forward that TIQ alkaloids may be formed in vivo during ethanol intoxication. As a matter of fact, they have been obtained by per- fusing adrenal glands with acetaldehyde (Cohen and Collins, 1970; Cohen, 1971; Greenberg and Cohen,

144 Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

1973; Schneider, 1974a, b) as had already been sug- gested by Toscano-Rico and Malafaya-Baptista (1935); ethanol intoxication in patients on L-dopa therapy led to the appearance of salsolinol (the con- densation product of dopamine and acetaldehyde) in their urine (Sandler et al., 1973); salsolinol has also been formed in vivo in rats after ethanol administra- tion (Collins and Bigdeli, 1975); ethanol ingestion by chronic alcoholic subjects leads to much higher blood levels of acetaldehyde than in non-alcoholic controls (Korsten et al., 1975).

Moreover, these TIQs have a molecular structure similar to plant alkaloids with a high addictive po- tency and the hypothesis has been advanced that these alkaloids may be linked to the addictive properties of ethanol (Mendelson, 1970; Walsh et al., 1970).

Finally, chronic ethanol intoxication induces changes in central monoamine levels (Griffiths et al., 1974) and in central and peripheral sympathetic func- tion (Pohorecky, 1974) by a yet unknown mechanism.

We think that our results may constitute a contri- bution to the understanding of some of the phenomena related to ethanol intoxication and the alcohol with- drawal syndrome.

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Received May 9/Accepted July 8, 1977