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Uptake and retention of 3H-noradrenaline in adrenergic nerve terminals after reserpine and axotomy
EUROPEAN JOURNAL OF PHARMACOLOGY 10 (1970) 4 1 1 - 4 1 5 . NORTH-HOLLAND PUBLISHING COMPANY
Short communication
U P T A K E AND R E T E N T I O N O F 3 H - N O R A D R E N A L I N E IN A D R E N E R G I C N E R V E T E R M I N A L S A F T E R R E S E R P I N E AND A X O T O M Y Jan HAGGENDAL Department of Pharmacology, University o f Gdteborg, GOteborg, Sweden
and Annica DAHLSTROM Institute o f Neurobiology, University of GOteborg, Goteborg, Sweden
Received 13 April 1970
Accepted 20 April 1970
1. H~,GGENDAL and A. DAHLSTROM, Uptake and retention of 3tl-noradrenaline in adrenergk: nerve terminals after reserpine and axotomy, European J. Pharmacol. 10 (1970) 411-415. The onset of recovery of endogenous noradrenaline levels and ot the capacity of the adrenergic neuron to take up and retain 3H-noradrenaline both occurred between 24 and 36 hr after reserpine treatment. Axotomy delayed the increase in 3H-noradrenaline retention. The importance of axonal transport, in particular of amine storage granules, for recovery after reserpine is discussed. Reserpine 3H-Noradrenaline
Endogenous noradrenaline Recovery
1. INTRODUCTION
Axoplasmic transport New amine granules
ules formed in the cell bodies to the neurons via the axons.
After large single dose reserpine, the adrenergic neuron is depleted of noradrenaline (NA) and adrenergic transmission is blocked. The NA depletion is probably due to a long-term blockage of the storage mechanism in amine storage granules (cf. Carlsson, 1965). The storage granules in the adrenergic nerve terminals are probably formed in the perikarya and transported down the axons at a higher rate (several mm/hr) to the nerve terminals where they appear to have a life-span of several weeks (Dahlstr6m and Hiiggendal, 1966a, 1970). After reserpine treatment, the capacity of the tissue to take up and retain a H-NA is lost (Muscholl, 1960). The recovery of nerve function, endogenous NA levels, and of the capacity of take up and retain exogenous NA (e.g. aH.NA) after reserpine is probably due either to the recovery of the "old" granules present in the nerve terminals at the time of the reserpine injection, or to transport of "young" gran-
The onset of recovery of the tissue capacity to retain small amounts of 3H-NA coincided with the return of response to nerve stimulation (between 3 0 - 4 8 hr after reserpine) when the endogenous NA levels were still very low (Anddn et al., 1964; Anddn and Henning, 1966, 1968). A recovery of the "old" granules in the nerve terminals is therefore discussed; the endogenous NA level would then be expected to increase as a result of the imported capacity of the old granules to take up and retain small amounts of NA. Normal levels of endogenous NA are not found until several weeks after administration of a single large dose of reserpine. The daily increase in the NA levels may correspond to the amount of NA present in the storage granules, transported to the nerve terminals via the axons (Dahlstr~Sm and H~/ggendal, 1966b, 1970; H~iggendal and Dahlstr~Sm, 1970). Axonal transport from the perikarya of fresh NA-
412
J.Hiiggendal, A.Dahlstrdm, 3H-noradrenaline retention after reserpine and axotom y
storing granules appears to start earlier than 18 hr after one large dose o f reserpine (Dahlstrbm, 1967). It was therefore suggested that the appearance of newly synthesized amine granules in the adrenergic nerve terminals was mainly responsible for the recovery of endogenous NA, nerve function, and the capacity of the tissue to take up and retain 3H-NA (DahlstrSm and H~iggendal, 1966 b, 1970). The ability of the adrenergic nerve terminals to store both endogenous and exogenous NA should thus be linked to one factor, namely a functioning storage mechanism, regardless of whether the storage occurs in a large "less mobile" pool or a small " m o b i l e " pool. Reserpine apparently affects the storage in b o t h pools. If the new granules, transported to the nerve terminals after the reserpine treatment, are responsible not only for the recovery of the endogenous NA (cf. H~iggendal and Dahlstr6m, 1970) but also for the reappearance of the ability to store exogenous NA, the onset of recovery of 3H-NA retention should appear at the same time as endo-
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Gastrocnemic muscle
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o
2. METHODS Male albino rats o f the Sprague-Dawley strain ( 2 0 0 - 2 5 0 g) were used. Reserpine (Serpasil ® 10 rag/ kg i.p.) was given 18, 24, 36 and 48 hr before killing. Twelve hours before sacrifice two types of operations were performed under ether anaesthesia; 1)unilateral ligation of the sciatic nerve or 2) unilateral removal of the cervical superior ganglion, and preganglionic denervation o f this ganglion to also deprive the tissue on this side from nerve impulses on the contralateral side. Non-reserpinized, operated animals served as controls. One unoperated group o f reserpine treated rats was used. The gastrocnemic muscles were excized and
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genous NA levels start to increase. Furthermore, the recovery of 3H-NA retention after reserpine should be delayed after inhibition of the supply o f new granules to the nerve terminals, by e.g. axotomy.
/~/4
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Submaxilay_gl r and ( A Ganolionect.~3H_NA(~ 0 Decentra'izedJ •
~
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Fig. 1. The effect of axotomy 12 hr before death on the levels of aH-NA in gastrocnemic muscles and submaxillary glands of reserpinized rats (10 mg/kg i.p.) after the injection of 1-3H-NA (2.5 ~tg/kg i.v. 30 min before death). Mean values and S.E.M. (vertical bars) are given, expressed in percent of the 3H-NA levels in controls on every experimental occasion. a) Gastrocnemic muscle: Reserpinized rats, operated (unilateral sciatic nerve ligation) (n = 3 except for at 48 hr where n = 4); controls, non-reserpinized, operated (intact side), 100 percent corresponds to 16, 784 + 2.022 counts/g and 10 min, n --- 13). Reserpinized rats, unoperated (t3----~ n = 4); controls, non-reserpinized rats, unoperated (100 percent corresponds to 22,588 +_ 1,240 counts/g and 10 rain, n = 18). b) Submaxillary gland: Reserpinized rats, operated (unilateral ganglionectomy and contralateral preganglionic denervation) (n = 3 except for at 18 hr where n = 4); controls, non-reserpinized, operated (decentralized side), (100 percent corresponds to 799,864 _+ 201,659 counts/g and 10 min, n = 12). The levels of endogenous NA in gastrocnemic muscle (a) and submaxillary gland (b) of unoperated rats after reserpine treatment are indicated (e---e, mean + S.E.M., n = 4), in percent of the levels in normal control (non-reserpinized, unoperated) rats (100 percent corresponds to 65.0 +_5.14 ng/g and 1,134.8 + 49.29 ng/g, respectively (uncorrected for recovery).
J.Hdggendal, A.Dahlstr6m, 3H-noradrenalineretention after reserpineand axotomy their 3H-NA content compared to that of muscles from unoperated, non-reserpinized controls. Either 1-3H-NA (spec.act. 2.34 Ci/mmole, Amersham, 2.5~g/kg) or dl-3H-NA (spec.act. 6.88Ci/ mmole, Amersham, 5/ag/kg) were given i.v. 30 min before death. 3 H-NA in the gastrocnemic muscles and the submaxillary glands was measured by liquid scintillation counting after chromatographical separation of the NA on columns according to Carlsson and Waldeck (1963). The endogenous NA levels in gastrocnemic muscles and submaxillary glands were estimated according to H/iggendal (1963), in unoperated rats with and without reserpine treatment as described above.
3. RESULTS * 3.1. Levels of 3H-NA 3.1.1. Gastrocnemic muscle After 1-3H-NA injection, the levels of 3H-NA in muscles on both sides of the operated (sciatic nerve ligation) reserpine-treated rats were rather low 1 8 24 hr (fig. l a). These levels had increased by 36 hr but to a greater extent on the intact side. After 48 hr, the levels on the intact side were considerably higher than those on the ligated side (i.e., the muscles to which the sciatic nerve had been ligated). Similar results were obtained from an experiment performed with dl-3H-NA (fig. 2). Taking these results together (figs. la and 2), the differences in NA levels between the intact and the ligated side were significant at 36 hr (p < 0.025) and highly significant at 48 hr (p < 0.005 ; t-test, matched pairs). The 3 H-NA levels (as a percentage of their respective controls) in unoperated reserpine-treated rats and on the intact side of operated rats were about the same (fig. la). 3.1.2. Submaxillary gland After 1-aH-NA injection (fig. lb) the results obtained were very similar to those in gastrocnemic muscles (fig. 1a).
* Presented in part at the Bayer Symposium on New Aspects of Storage and Release Mechanisms of Catecholamines, 912 October 1969, General Discussion (in press, 1970).
§
413
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48
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Fig. 2. The effect of axotomy 12hr before death on the levels of 3H-NA in gastrocnemic muscle of reserpine treated (10 mg/kg i.p.) rats after injection of dl-3H-NA (5 ug/kg i.v. 30 min before death). Mean + S.E.M. (vertical bars, n = 5 in all cases) are given in percent of the 3H-NA levels in controls (non-reserpinized, operated, intact side) on every experimental occasion (100 percent corresponds to 62,189 + 7,796 counts/g and 10 min, n = 7). 3.2. Levels of endogenous NA As seen from fig. l a and b, the endogenous NA levels, both in muscles and salivary glands, started to increase between 24 and 36 hr after reserpine treatment. This increase was statistically highly significant (p < 0.005 for muscles and p < 0.001 for salivary glands).
4. DISCUSSION The onset of recovery of the capacity of the tissue to take up and retain small amounts of 3 H-NA, which probably occurs predominantly in adrenergic nerves (cf. Iversen, 1967), occurred between 24 and 36 hr after reserpine treatment. This is in agreement with earlier observations (And~n et al., 1964; Iversen et al., 1965). The onset of increase of endogenous NA levels also occurred within the same time period (fig. 1). The coinciding onset of these two functions appear to be correlated in time with the transport of new, functioning amine storage granules, detectable in the axons 1 5 - 1 8 hr after the reserpine injection (DahlstrOm, 1967). There is an obvious quantitative difference between the rates of recovery of endogenous NA and of a H-NA uptake after the coincident onset. In a forthcoming publication, we will discuss this difference and relate it to the functions of granules of differing ages.
414
J.Hilggendal, A.Dahlstr6m, 3H.noradrenaline retention after reserpine and axotomy
The results obtained with both 1-3H-NA and dl3H-NA show that sciatic nerve ligation lowered the amount of 3H_NA retained in the gastrocnemic muscle 36 and 48 hr after reserpine (figs. l a, 2). Ganglionectomy (represented axotomy in this study) had the same effect in the salivary glands (fig. lb). This observation may have at least three different explanations: 1)The nerve terminals may have degenerated. 2) The tissues are deprived of their impulse activity. 3) The axoplasmic flow of material from the cell bodies to the terminals is interrupted. The effect of operational trauma was negated by the nature of the controls. Ad 1) In order to check if the nerve terminals were intact l 2 hr after ligation, a histochemical and chemical study on nerve terminal degeneration was carried out in the gastrocnemic muscle at different times after sciatic nerve ligation. No signs of nerve terminal degeneration were observed at 12 hr after ligation. At 24 hr fewer terminals were observed while no terminals and very little NA was found at 72 hr after ligation (Dahlstr6m and Hiiggendal, unpubl.). This is in accordance with an earlier study on rat irides (Malmfors and Sachs, 1965) which also showed an intact amine uptake mechanism at the nerve terminal membrane 12 hr after removal of the superior cervical ganglion. Ad 2) As regards the salivary glands, the side with intact post-ganglio:aic adrenergic neurons was preganglionically dene::vated and thus, like the ganglionectomized side, was deprived of normal impulse activity. The difference in 3H-NA content between the two sides is thus unlikely to be due to differences in impulse activity. Similar differences in 3H-NA levels were observed in the gastrocnemic muscles between the unligated and the ligated side. The adrenergic nerves (vasoconstrictor nerves) in the muscles on the unligated side received nerve impulses, whilst those on the ligated side probably did not. The unligated side was not preganglionically denervated in this tissue for technical reasons. Consequently, theoretically there may be a supra-normal impulse flow on the intact side of an operated animal. However, as seen in fig. l a, the 3H-NA levels in muscles of unoperated rats were similar to those observed in the intact (unligated) side of operated rats. This may indicate that nerve impulse activity was not responsible for the differences in a H-NA uptake-retention between the ligated and intact side of operated
reserpinized rats. As further supporting indirect evidence, the results from gastrocnemic muscles were similar to those obtained from submaxillary glands, which were preganglionicaUy denervated on the "intact" side to eliminate nerve impulses. Thus, the nerve impulse activity was probably of little or no importance in this study. Ad 3) The presence of functioning amine granules in the nerve terminals is essential for the retention of 3H-NA taken up through the nerve membrane (e.g. Jonsson et al., 1969). The retention of this 3H-NA probably occurs predominantly in these granules (cf. Iversen, 1967). With respect to the nerve terminals, axotomy for the rather short time period used in this study is likely to have the largest effects on the amine granules, since they are transported at much higher rates than other known substances in adrenergic axons (discussed in DahlstriSm and H~iggendal. 1970). Thus, the delayed increase of the capacity of the tissues to retain 3H-NA after axotomy at 36 and 48 hr after reserpine is most probably due to the interrupted axonal down-transport of axoplasmic material, particularly the amine storage granules. The small increase in 3H-NA content occurring on the axotomized sides between 24 and 36 hr after reserpine may be due to the arrival in the nerve terminals of new granules in the axonal parts distal to the lesion at the time of axotomy. The present study demonstrates that an uninterrupted axoplasmic-down-flow, probably of newly formed amine storages granules, is of major importance for the recovery after reserpine, of the tissue capacity to take up and retain exogenous NA. This mechanism is also probably essential for the recovery, both of endogenous levels of NA and of adrenergic transmission (cf. Hiiggendal and DahlstriSm, 1970).
ACKNOWLEDGEMENTS This work has been supported by grants from the Swedish State Medical Research Council (B70-14X-166-06B, B7014X.-2207-04), from the Medical Faculty, University of G6teborg, and from Gustav and Majen Lindgrens Foundation. We are indebted to the Swedish CIBA, Stockholm for generous supply of reserpine (Serpasil(~)). The technical assistance of Mr P~ir-Anders Larsson, Miss Mildred Pettersson and Miss Elisabeth Skugghall is gratefully acknowledged. We are grateful to Tor Magnusson, Research Engineer, for preparing the figures.
J.Hdggendal, A.DahlstrOm, 3H-noradrenaline retention after reserpine and axotom y REFERENCES And6n, N.-E. and M. Henning, 1966, Adrenergic nerve function, noradrenaline level and noradrenaline uptake in cat nictitating membrane after reserpine treatment, Acta Physiol. Scand. 67,498. And~n, N.-E. and M. Henning, 1968, Effect of reserpine on the urinary excretion and the tissue levels of noradrenaline in the rat, Acta Physiol. Scand. 72, 134. And6n, N.-E., T. Magnusson and B. Waldeck, 1964, Correlation between noradrenaline uptake and adrenergic nerve function after reserpine treatment, Life Sci. 3, 19. Carlsson, A., 1965, Drugs which block the storage of 5-hydroxytryptamine and related amines, in: Handbuch der exp. Pharmacol. (Springer-Verlag, Berlin, Heidelberg, New York) pp. 5 2 9 - 5 9 2 . Carlsson, A. and B. Waldeck, 1963, On the role of the liver catechol-O-methyl transferase in the metabolism of circulating catecholamines, Acta Pharmacol. Toxicol. 20, 47. Dahlstr6m, A., 1967, The effect of reserpine and tetrabenazine on the accumulation of noradrenaline in the rat sciatic nerve after ligation, Acta Physiol. Scand. 69, 167. DahlstrOm, A. and J. H~/ggendal, 1966a, Studies on the transport and lifespan of amine storage granules in a peripheral adrenergic neuron system, Acta Physiol. Scand. 67,278. Dahlstr6m, A. and J. H~iggendal, 1966b, Recovery of noradrenaline levels after reserpine compared with the lifespan of amine storage granules in rat and rabbit, J. Pharm. Pharmacol. 18,750.
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Dahlstr6m, A. and J. H~iggendal, 1970, Axonal transport of amine storage granules in sympathetic adrenergic neurons. IIIrd Internat. Congress of Neurochemistry, Milan Sept. 1969, in; Biochemistry of Simple Neuronal Models (Raven Press, New York) in press. H~gendal, J., 1963, An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues, Acta Physiol. Scand. 59, 242. H/iggendal, J. and A. Dahlstr6m, 1970, The recovery of noradrenaline in adrenergic nerve terminals after reserpine treatment. J. Pharm. Pharmacol., in press. Iversen, L.L., 1967, The uptake and storage of noradrenaline in sympathetic nerves (Cambridge University Press). Iversen, L.L., J. Glowinski and J. Axelrod, 1965, The uptake and storage of 3H-norepinephrine in the reserpinepretreated rat heart, J. Pharmacol. Exptl. Therap. 150, 173. Jonsson, G., B. Hamberger, T. Malmfors and Ch. Sachs, 1969. Uptake and accumulation of 3H-noradrenaline in adrenergic nerves of rat iris. Effect of reserpine, monomamine oxidase and tyrosine hydroxylase inhibition, European J. Pharmacol. 8, 58. Malmfors, T. and Ch. Sachs, 1965, Direct studies on the disappearance of the transmitter and the changes in the uptake-storage mechanisms of degenerating adrenergic nerves, Acta Physiol. Scand. 64, 211. Muscholl, E., 1960, Die Hemmung der NoradrenalinAufnahme des Herzens durch Reserpin und die Wirkung von Tyramin, Arch. Exptl. Pathol. Pharmakol. 240, 234.
Short communication
U P T A K E AND R E T E N T I O N O F 3 H - N O R A D R E N A L I N E IN A D R E N E R G I C N E R V E T E R M I N A L S A F T E R R E S E R P I N E AND A X O T O M Y Jan HAGGENDAL Department of Pharmacology, University o f Gdteborg, GOteborg, Sweden
and Annica DAHLSTROM Institute o f Neurobiology, University of GOteborg, Goteborg, Sweden
Received 13 April 1970
Accepted 20 April 1970
1. H~,GGENDAL and A. DAHLSTROM, Uptake and retention of 3tl-noradrenaline in adrenergk: nerve terminals after reserpine and axotomy, European J. Pharmacol. 10 (1970) 411-415. The onset of recovery of endogenous noradrenaline levels and ot the capacity of the adrenergic neuron to take up and retain 3H-noradrenaline both occurred between 24 and 36 hr after reserpine treatment. Axotomy delayed the increase in 3H-noradrenaline retention. The importance of axonal transport, in particular of amine storage granules, for recovery after reserpine is discussed. Reserpine 3H-Noradrenaline
Endogenous noradrenaline Recovery
1. INTRODUCTION
Axoplasmic transport New amine granules
ules formed in the cell bodies to the neurons via the axons.
After large single dose reserpine, the adrenergic neuron is depleted of noradrenaline (NA) and adrenergic transmission is blocked. The NA depletion is probably due to a long-term blockage of the storage mechanism in amine storage granules (cf. Carlsson, 1965). The storage granules in the adrenergic nerve terminals are probably formed in the perikarya and transported down the axons at a higher rate (several mm/hr) to the nerve terminals where they appear to have a life-span of several weeks (Dahlstr6m and Hiiggendal, 1966a, 1970). After reserpine treatment, the capacity of the tissue to take up and retain a H-NA is lost (Muscholl, 1960). The recovery of nerve function, endogenous NA levels, and of the capacity of take up and retain exogenous NA (e.g. aH.NA) after reserpine is probably due either to the recovery of the "old" granules present in the nerve terminals at the time of the reserpine injection, or to transport of "young" gran-
The onset of recovery of the tissue capacity to retain small amounts of 3H-NA coincided with the return of response to nerve stimulation (between 3 0 - 4 8 hr after reserpine) when the endogenous NA levels were still very low (Anddn et al., 1964; Anddn and Henning, 1966, 1968). A recovery of the "old" granules in the nerve terminals is therefore discussed; the endogenous NA level would then be expected to increase as a result of the imported capacity of the old granules to take up and retain small amounts of NA. Normal levels of endogenous NA are not found until several weeks after administration of a single large dose of reserpine. The daily increase in the NA levels may correspond to the amount of NA present in the storage granules, transported to the nerve terminals via the axons (Dahlstr~Sm and H~/ggendal, 1966b, 1970; H~iggendal and Dahlstr~Sm, 1970). Axonal transport from the perikarya of fresh NA-
412
J.Hiiggendal, A.Dahlstrdm, 3H-noradrenaline retention after reserpine and axotom y
storing granules appears to start earlier than 18 hr after one large dose o f reserpine (Dahlstrbm, 1967). It was therefore suggested that the appearance of newly synthesized amine granules in the adrenergic nerve terminals was mainly responsible for the recovery of endogenous NA, nerve function, and the capacity of the tissue to take up and retain 3H-NA (DahlstrSm and H~iggendal, 1966 b, 1970). The ability of the adrenergic nerve terminals to store both endogenous and exogenous NA should thus be linked to one factor, namely a functioning storage mechanism, regardless of whether the storage occurs in a large "less mobile" pool or a small " m o b i l e " pool. Reserpine apparently affects the storage in b o t h pools. If the new granules, transported to the nerve terminals after the reserpine treatment, are responsible not only for the recovery of the endogenous NA (cf. H~iggendal and Dahlstr6m, 1970) but also for the reappearance of the ability to store exogenous NA, the onset of recovery of 3H-NA retention should appear at the same time as endo-
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6o
o
2. METHODS Male albino rats o f the Sprague-Dawley strain ( 2 0 0 - 2 5 0 g) were used. Reserpine (Serpasil ® 10 rag/ kg i.p.) was given 18, 24, 36 and 48 hr before killing. Twelve hours before sacrifice two types of operations were performed under ether anaesthesia; 1)unilateral ligation of the sciatic nerve or 2) unilateral removal of the cervical superior ganglion, and preganglionic denervation o f this ganglion to also deprive the tissue on this side from nerve impulses on the contralateral side. Non-reserpinized, operated animals served as controls. One unoperated group o f reserpine treated rats was used. The gastrocnemic muscles were excized and
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Z~ Ligoted nerve] I 0 Intact nerve ~'3H'NAJ Q Unoperoted J
80
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genous NA levels start to increase. Furthermore, the recovery of 3H-NA retention after reserpine should be delayed after inhibition of the supply o f new granules to the nerve terminals, by e.g. axotomy.
/~/4
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24
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Submaxilay_gl r and ( A Ganolionect.~3H_NA(~ 0 Decentra'izedJ •
~
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40
00
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Fig. 1. The effect of axotomy 12 hr before death on the levels of aH-NA in gastrocnemic muscles and submaxillary glands of reserpinized rats (10 mg/kg i.p.) after the injection of 1-3H-NA (2.5 ~tg/kg i.v. 30 min before death). Mean values and S.E.M. (vertical bars) are given, expressed in percent of the 3H-NA levels in controls on every experimental occasion. a) Gastrocnemic muscle: Reserpinized rats, operated (unilateral sciatic nerve ligation) (n = 3 except for at 48 hr where n = 4); controls, non-reserpinized, operated (intact side), 100 percent corresponds to 16, 784 + 2.022 counts/g and 10 min, n --- 13). Reserpinized rats, unoperated (t3----~ n = 4); controls, non-reserpinized rats, unoperated (100 percent corresponds to 22,588 +_ 1,240 counts/g and 10 rain, n = 18). b) Submaxillary gland: Reserpinized rats, operated (unilateral ganglionectomy and contralateral preganglionic denervation) (n = 3 except for at 18 hr where n = 4); controls, non-reserpinized, operated (decentralized side), (100 percent corresponds to 799,864 _+ 201,659 counts/g and 10 min, n = 12). The levels of endogenous NA in gastrocnemic muscle (a) and submaxillary gland (b) of unoperated rats after reserpine treatment are indicated (e---e, mean + S.E.M., n = 4), in percent of the levels in normal control (non-reserpinized, unoperated) rats (100 percent corresponds to 65.0 +_5.14 ng/g and 1,134.8 + 49.29 ng/g, respectively (uncorrected for recovery).
J.Hdggendal, A.Dahlstr6m, 3H-noradrenalineretention after reserpineand axotomy their 3H-NA content compared to that of muscles from unoperated, non-reserpinized controls. Either 1-3H-NA (spec.act. 2.34 Ci/mmole, Amersham, 2.5~g/kg) or dl-3H-NA (spec.act. 6.88Ci/ mmole, Amersham, 5/ag/kg) were given i.v. 30 min before death. 3 H-NA in the gastrocnemic muscles and the submaxillary glands was measured by liquid scintillation counting after chromatographical separation of the NA on columns according to Carlsson and Waldeck (1963). The endogenous NA levels in gastrocnemic muscles and submaxillary glands were estimated according to H/iggendal (1963), in unoperated rats with and without reserpine treatment as described above.
3. RESULTS * 3.1. Levels of 3H-NA 3.1.1. Gastrocnemic muscle After 1-3H-NA injection, the levels of 3H-NA in muscles on both sides of the operated (sciatic nerve ligation) reserpine-treated rats were rather low 1 8 24 hr (fig. l a). These levels had increased by 36 hr but to a greater extent on the intact side. After 48 hr, the levels on the intact side were considerably higher than those on the ligated side (i.e., the muscles to which the sciatic nerve had been ligated). Similar results were obtained from an experiment performed with dl-3H-NA (fig. 2). Taking these results together (figs. la and 2), the differences in NA levels between the intact and the ligated side were significant at 36 hr (p < 0.025) and highly significant at 48 hr (p < 0.005 ; t-test, matched pairs). The 3 H-NA levels (as a percentage of their respective controls) in unoperated reserpine-treated rats and on the intact side of operated rats were about the same (fig. la). 3.1.2. Submaxillary gland After 1-aH-NA injection (fig. lb) the results obtained were very similar to those in gastrocnemic muscles (fig. 1a).
* Presented in part at the Bayer Symposium on New Aspects of Storage and Release Mechanisms of Catecholamines, 912 October 1969, General Discussion (in press, 1970).
§
413
Gostrocnemicmuscle 60
A I_igateclnerve
0 Intact nerve
/
/ q )
.~ 40
~ , ~ 20
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24
48
HOUrsoffer reserpine
Fig. 2. The effect of axotomy 12hr before death on the levels of 3H-NA in gastrocnemic muscle of reserpine treated (10 mg/kg i.p.) rats after injection of dl-3H-NA (5 ug/kg i.v. 30 min before death). Mean + S.E.M. (vertical bars, n = 5 in all cases) are given in percent of the 3H-NA levels in controls (non-reserpinized, operated, intact side) on every experimental occasion (100 percent corresponds to 62,189 + 7,796 counts/g and 10 min, n = 7). 3.2. Levels of endogenous NA As seen from fig. l a and b, the endogenous NA levels, both in muscles and salivary glands, started to increase between 24 and 36 hr after reserpine treatment. This increase was statistically highly significant (p < 0.005 for muscles and p < 0.001 for salivary glands).
4. DISCUSSION The onset of recovery of the capacity of the tissue to take up and retain small amounts of 3 H-NA, which probably occurs predominantly in adrenergic nerves (cf. Iversen, 1967), occurred between 24 and 36 hr after reserpine treatment. This is in agreement with earlier observations (And~n et al., 1964; Iversen et al., 1965). The onset of increase of endogenous NA levels also occurred within the same time period (fig. 1). The coinciding onset of these two functions appear to be correlated in time with the transport of new, functioning amine storage granules, detectable in the axons 1 5 - 1 8 hr after the reserpine injection (DahlstrOm, 1967). There is an obvious quantitative difference between the rates of recovery of endogenous NA and of a H-NA uptake after the coincident onset. In a forthcoming publication, we will discuss this difference and relate it to the functions of granules of differing ages.
414
J.Hilggendal, A.Dahlstr6m, 3H.noradrenaline retention after reserpine and axotomy
The results obtained with both 1-3H-NA and dl3H-NA show that sciatic nerve ligation lowered the amount of 3H_NA retained in the gastrocnemic muscle 36 and 48 hr after reserpine (figs. l a, 2). Ganglionectomy (represented axotomy in this study) had the same effect in the salivary glands (fig. lb). This observation may have at least three different explanations: 1)The nerve terminals may have degenerated. 2) The tissues are deprived of their impulse activity. 3) The axoplasmic flow of material from the cell bodies to the terminals is interrupted. The effect of operational trauma was negated by the nature of the controls. Ad 1) In order to check if the nerve terminals were intact l 2 hr after ligation, a histochemical and chemical study on nerve terminal degeneration was carried out in the gastrocnemic muscle at different times after sciatic nerve ligation. No signs of nerve terminal degeneration were observed at 12 hr after ligation. At 24 hr fewer terminals were observed while no terminals and very little NA was found at 72 hr after ligation (Dahlstr6m and Hiiggendal, unpubl.). This is in accordance with an earlier study on rat irides (Malmfors and Sachs, 1965) which also showed an intact amine uptake mechanism at the nerve terminal membrane 12 hr after removal of the superior cervical ganglion. Ad 2) As regards the salivary glands, the side with intact post-ganglio:aic adrenergic neurons was preganglionically dene::vated and thus, like the ganglionectomized side, was deprived of normal impulse activity. The difference in 3H-NA content between the two sides is thus unlikely to be due to differences in impulse activity. Similar differences in 3H-NA levels were observed in the gastrocnemic muscles between the unligated and the ligated side. The adrenergic nerves (vasoconstrictor nerves) in the muscles on the unligated side received nerve impulses, whilst those on the ligated side probably did not. The unligated side was not preganglionically denervated in this tissue for technical reasons. Consequently, theoretically there may be a supra-normal impulse flow on the intact side of an operated animal. However, as seen in fig. l a, the 3H-NA levels in muscles of unoperated rats were similar to those observed in the intact (unligated) side of operated rats. This may indicate that nerve impulse activity was not responsible for the differences in a H-NA uptake-retention between the ligated and intact side of operated
reserpinized rats. As further supporting indirect evidence, the results from gastrocnemic muscles were similar to those obtained from submaxillary glands, which were preganglionicaUy denervated on the "intact" side to eliminate nerve impulses. Thus, the nerve impulse activity was probably of little or no importance in this study. Ad 3) The presence of functioning amine granules in the nerve terminals is essential for the retention of 3H-NA taken up through the nerve membrane (e.g. Jonsson et al., 1969). The retention of this 3H-NA probably occurs predominantly in these granules (cf. Iversen, 1967). With respect to the nerve terminals, axotomy for the rather short time period used in this study is likely to have the largest effects on the amine granules, since they are transported at much higher rates than other known substances in adrenergic axons (discussed in DahlstriSm and H~iggendal. 1970). Thus, the delayed increase of the capacity of the tissues to retain 3H-NA after axotomy at 36 and 48 hr after reserpine is most probably due to the interrupted axonal down-transport of axoplasmic material, particularly the amine storage granules. The small increase in 3H-NA content occurring on the axotomized sides between 24 and 36 hr after reserpine may be due to the arrival in the nerve terminals of new granules in the axonal parts distal to the lesion at the time of axotomy. The present study demonstrates that an uninterrupted axoplasmic-down-flow, probably of newly formed amine storages granules, is of major importance for the recovery after reserpine, of the tissue capacity to take up and retain exogenous NA. This mechanism is also probably essential for the recovery, both of endogenous levels of NA and of adrenergic transmission (cf. Hiiggendal and DahlstriSm, 1970).
ACKNOWLEDGEMENTS This work has been supported by grants from the Swedish State Medical Research Council (B70-14X-166-06B, B7014X.-2207-04), from the Medical Faculty, University of G6teborg, and from Gustav and Majen Lindgrens Foundation. We are indebted to the Swedish CIBA, Stockholm for generous supply of reserpine (Serpasil(~)). The technical assistance of Mr P~ir-Anders Larsson, Miss Mildred Pettersson and Miss Elisabeth Skugghall is gratefully acknowledged. We are grateful to Tor Magnusson, Research Engineer, for preparing the figures.
J.Hdggendal, A.DahlstrOm, 3H-noradrenaline retention after reserpine and axotom y REFERENCES And6n, N.-E. and M. Henning, 1966, Adrenergic nerve function, noradrenaline level and noradrenaline uptake in cat nictitating membrane after reserpine treatment, Acta Physiol. Scand. 67,498. And~n, N.-E. and M. Henning, 1968, Effect of reserpine on the urinary excretion and the tissue levels of noradrenaline in the rat, Acta Physiol. Scand. 72, 134. And6n, N.-E., T. Magnusson and B. Waldeck, 1964, Correlation between noradrenaline uptake and adrenergic nerve function after reserpine treatment, Life Sci. 3, 19. Carlsson, A., 1965, Drugs which block the storage of 5-hydroxytryptamine and related amines, in: Handbuch der exp. Pharmacol. (Springer-Verlag, Berlin, Heidelberg, New York) pp. 5 2 9 - 5 9 2 . Carlsson, A. and B. Waldeck, 1963, On the role of the liver catechol-O-methyl transferase in the metabolism of circulating catecholamines, Acta Pharmacol. Toxicol. 20, 47. Dahlstr6m, A., 1967, The effect of reserpine and tetrabenazine on the accumulation of noradrenaline in the rat sciatic nerve after ligation, Acta Physiol. Scand. 69, 167. DahlstrOm, A. and J. H~/ggendal, 1966a, Studies on the transport and lifespan of amine storage granules in a peripheral adrenergic neuron system, Acta Physiol. Scand. 67,278. Dahlstr6m, A. and J. H~iggendal, 1966b, Recovery of noradrenaline levels after reserpine compared with the lifespan of amine storage granules in rat and rabbit, J. Pharm. Pharmacol. 18,750.
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