H. Thoenen

Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine

ZusammenfassungNachdem elektronenmikroskopische Untersuchungen gezeigt hatten, daß 6-OH-DA selektiv die peripheren adrenergen Nervenendigungen zerstört, wurde untersucht, welche Beziehungen zwischen der dosisabhängigen Senkung des Noradrenalingehaltes durch 6-OH-DA und dessen Aufnahme und Speicherung in sympathisch innervierten Organen besteht und ob 6-OH-DA zu einer generellen chemischen Sympathektomie benützt werden kann.Nach Verabreichung von 1 mg/kg 6-OH-DA intravenös war der Noradrenalingehalt in Herz und Milz der Ratte nur kurzfristig (Ausgangswert nach 24 Std wieder erreicht) gesenkt, nach 30 mg/kg dagegen zeigte die Senkung des Noradrenalingehaltes während einer Woche keine Rückbildungstendenz. Nach Verabreichung von 1 mg/kg 3H-6-OH-DA intravenös war das fehlende Noradrenalin mehr als stöchiometrisch durch 3H-Amine ersetzt, nach 30 mg/kg dagegen fanden sich bei stärkerer Senkung des Noradrenalingehaltes nach 2 Std nur noch geringe Mengen, nach 24 Std überhaupt keine 3H-Amine mehr. Bei Verabreichung kleiner Dosen von 6-OH-DA wird dieses offenbar in den sympathischen Nerven gespeichert und kann als falscher Transmitter freigesetzt werden (Nachweis an der isolierten Milz der Katze). Hohe Dosen dagegen zerstören die adrenergen Nervenendigungen und damit ihre eigenen Aminspeicher.Die vollständigste chemische Sympathektomie an Ratten wurde erzielt, wenn die Tiere innerhalb 24 Std mit 2×34 mg/kg und eine Woche später mit 2×68 mg/kg 6-OH-DA intravenös behandelt wurden. Eine Woche nach der letzten Dosis betrug der Noradrenalingehalt im Herzen 7,6%, in der Milz 5,1% und im Vas deferens 18,8% des Gehaltes entsprechender Kontrollen. Nach 2 Wochen war der Noradrenalingehalt nicht oder nur unbedeutend angestiegen, nach 4 Wochen hingegen fand sich in allen Organen eine eindeutige Anstiegstendenz.Die Aufnahme von 3H-Noradrenalin war ungefähr im gleichen Maße vermindert wie der Gehalt an endogenem Noradrenalin.Wurden Katzen mit 2×14 und 2×34 mg/kg 6-OH-DA intravenös behandelt, so betrug eine Woche nach Verabreichung der letzten Dosis der Noradrenalingehalt im Herzen 2,8%, in der Milz 3,1%, in der Nickhaut 8,3% und in der Iris 2,8% des Gehaltes unbehandelter Kontrollen.Die chemische Sympathektomie mit 6-OH-DA ist in ihrer Wirksamkeit der chirurgischen Denervation und der Immunosympathektomie ebenbürtig, z. T. auch überlegen, doch sind auch hier Organ- und Speziesunterschiede vorhanden.SummaryAfter electronmicroscope studies had shown that 6-hydroxydopamine (6-OH-DA) induced a selective destruction of adrenergic nerve terminals, we studied the correlation between the dose-dependent norepinephrine depletion by 6-OH-DA and its uptake and storage in sympathetically innervated organs. We also investigated whether 6-OH-DA can be used for general chemical sympathectomy.Whereas the intravenous administration of 1 mg/kg of 6-OH-DA caused only a temporary drop in the norepinephrine content of the rat heart and spleen (original values were regained after 24 hr), 30 mg/kg provoked a norepinephrine depletion which showed no signs of recovery for up to a week. Following the intravenous administration of 1 mg/kg of 3H-6-OH-DA, the norepinephrine missing in sympathetically innervated organs was more than stoichiometrically replaced by 3H-amines, whereas in the case of 30 mg/kg of 3H-6-OH-DA after 2 hr only very small quantities of 3H-amines could be detected, and after 24 hr none at all. It seems that after administration of low doses 6-OH-DA is stored in sympathetic nerves and can be liberated as a false transmitter (as shown in the isolated cat spleen). Higher doses, however, lead to a destruction of adrenergic nerve endings and consequently to a destruction of their own amine stores as well.The most complete chemical sympathectomy in rats could be achieved when animals were treated intravenously with 2×34 mg/kg within a 24 hr period and a week later with 2×68 mg/kg. A week after the last dose of 6-OH-DA the norepinephrine level in the heart amounted to 7.6%, in the spleen to 5.1% and in the vas deferens to 18.8% of the level in corresponding control animals. The uptake of intravenously injected 3H-norepinephrine was reduced to approximately the same extent as the endogenous norepinephrine content.In cats pretreated with 2×14 and 2×34 mg/kg of 6-OH-DA intravenously, one week after the last dose of 6-OH-DA the norepinephrine content in the heart amounted to 2.8%, in the spleen to 3.1%, in the nictitating membrane to 8.3% and in the iris to 2.8% of that of untreated controls.Chemical sympathectomy with 6-OH-DA compares favourably with surgical denervation or immunosympathectomy, but with this method, too, considerable differences between species and organs occur.

An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine

Le traitement de chats par la 6-hydroxydopamine (6-HO-DA) provoque une dégénérescence sélective et réversible des terminaisons nerveuses adrénergiques postganglionaires. Les terminaisons nerveuses cholinergiques ne sont pas affectées par ce traitement.

Central adrenergic neurones and the initiation and development of experimental hypertension

Zerstörung zentraler adrenerger Neurone durch Injektion von 6-Hydroxydopamin in einen Seitenventrikel verursachte bei normotonen und genetisch hypertonen Ratten lediglich eine geringe und vorübergehende Blutdrucksenkung und beeinflusste die DOCA- und renale Hypertonie überhaupt nicht. Im Gegensatz dazu verhinderte 6-Hydroxydopamin die Entwicklung der DOCA- und renalen Hypertonie, wenn es 7–10 Tage vor deren Induktion in einen Seitenventrikel injiziert wurde. Bei genetisch hypertonen Ratten, bei denen eine allmähliche Blutdrucksteigerung schon bald nach der Geburt einsetzt, unterbrach intraventrikulär injiziertes 6-Hydroxydopamin die weitere Entwicklung der Hypertonie. Die Resultate weisen auf die Bedeutung zentraler adrenerger Neurone für die Pathogenese verschiedener experimenteller Hypertonieformen hin.

Recent Developments on the Ultrastructural Aspect of Adrenergic Nerve Endings in Various Experimental Conditions

Publisher Summary This chapter reviews the work on the precise and direct ultrastructural localization of neurotransmitter substances in the vesiculated part of postganglionic adrenergic nerves in various organs of the cat and rat. Adrenergic vesiculated nerve endings contain both small dense-core vesicles and small empty-looking vesicles. The dual population of vesicles is observed when the tissues are prepared for electron microscopy by conventional methods. The dense core of the vesicles represents the physiological transmitter noradrenaline (NA). The empty vesicles in adrenergic nerve endings might contain acetylcholine (Ach) and represent the morphological correlate of a cholinergic link in postganglionic sympathetic transmission. In adrenergic-vesiculated nerve endings, biogenic amines are stored not only in the small but also in the large dense-core (LDC) vesicles. The direct and precise localization of false adrenergic transmitters with the electron microscope requires as a prerequisite that they be rendered insoluble and electron dense during the processing of the tissues for electron microscopy. The small vesicles of some nerves of the central nervous system (CNS) are able to take up and store amines in a way similar to that in the adrenergic nerves of the periphery. The possibility of a direct, visual localization of this neurotransmitter with a high degree of resolution opened a new field of investigation in the physiology and pharmacology of the adrenergic nervous system.

Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCl

SummaryThe isolated cat heart with intact sympathetic nerve supply was perfused with varying concentrations of acetylcholine, DMPP and KCl. The ensuing asynchronous discharge in cardiac sympathetic nerves was recorded and the noradrenaline liberated into the perfusate was measured.The infusion of acetylcholine for one minute at a relatively low concentration (5×10−5 to 8×10−5 g/ml) induced asynchronous firing over the entire period of infusion and an average liberation of 44 ng/min noradrenaline. Increasing the concentration of acetylcholine or adding atropine to the perfusion fluid greatly shortened the duration of the antidromic discharge but enhanced severalfold the amount of noradrenaline liberated.Within a narrow range of very low concentrations DMPP induced continuous firing. At higher concentrations antidromic discharges were restricted to the first few seconds of infusion. Muscarinic drugs such as pilocarpine and methacholine neither induced firing nor released noradrenaline. When added to acetylcholine, pilocarpine reduced the amplitude of acetylcholine-evoked firing and the amount of noradrenaline liberated.KCl in concentrations above 50 mM induced a very short-lasting firing and a considerable noradrenaline output which was concentration-dependent.It is concluded that the acetylcholine- and DMPP-induced action potentials by themselves contribute little to the noradrenaline-releasing activity of these drugs. As in the case of KCl, the sustained depolarization of the sympathetic nerve ending by acetylcholine and DMPP seems to enhance entry of Ca++ into the ending, an essential factor for the liberation of noradrenaline.ZusammenfassungAm isolierten, nach Langendorff perfundierten Katzenherzen wurden die auf Injektion oder Infusion von Acetylcholin, DMPP und KCl an den sympathischen Nervenendigungen asynchron auftretenden und antidrom fortgeleiteten Entladungen vom N. cardiacus medius oder inferior abgeleitet. Gleichzeitig wurde das Coronarperfusat gesammelt und sein Noradrenalingehalt fluorimetrisch gemessen.Eine 1minütige Infusion von Acetylcholin in verhältnismäßig niedriger Konzentration (5·10−5 bis 8·10−5 g/ml) verursachte asynchrone Entladungen, die praktisch unvermindert während der gesamten Infusionsdauer anhielten. Die Noradrenalinabgabe aus dem Herzen betrug dabei durchschnittlich 44 ng/min. Eine Steigerung der Acetylcholinkonzentration oder der Zusatz von Atropin zur Perfusionslösung verkürzte die Dauer der Entladungen auf die ersten Sekunden der Infusion, aber verstärkte die Freisetzung von Noradrenalin um das Mehrfache.Innerhalb eines sehr engen Konzentrationsbereiches führte auch die Infusion von DMPP zu anhaltenden antidromen Entladungen im kardialen Sympathicus. Jedoch bewirkte bereits eine geringe darüber hinausgehende Konzentrationserhöhung eine starke Verkürzung der Zeit, in der Entladungen registriert werden konnten.Unter der Infusion von muscarinartig wirkenden Substanzen wie Pilocarpin und Methacholin traten weder asynchrone Entladungen auf noch wurde Noradrenalin freigesetzt. Dem infundierten Acetylcholin zugesetztes Pilocarpin verringerte die Amplitude der Acetylcholin-bedingten Entladungen und reduzierte die Menge des abgegebenen Noradrenalins.In Konzentrationen über 50 mM verursachte KCl außerordentlich kurz dauernde Entladungen und eine während der gesamten Infusionsdauer anhaltende Noradrenalinausschüttung. Die Menge des abgegebenen Noradrenalins hing von der Konzentration des KCl ab.Aus der Gegenüberstellung des zeitlichen Verlaufs von elektrischer Aktivität und Noradrenalinfreisetzung sowie aus der Konzentrationsabhängigkeit beider Phänomene geht hervor, daß unter der Infusion von Acetylcholin, DMPP und KCl die Noradrenalinfreisetzung nur zu einem geringen Teil durch Auslösung von Aktionspotentialen erfolgt. Wesentlich mehr Noradrenalin wird während der permanenten Depolarisation der sympathischen Nervenendigungen abgegeben, deren Auftreten wahrscheinlich gemacht werden konnte. Dabei ist offenbar der während der Depolarisation erfolgende Ca++-Einstrom in die Nervenendigung entscheidend für die Einleitung der Noradrenalinausschüttung.

Neurally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit?

SummaryCold-exposure of rats for 1–4 days leads to a gradual increase in tyrosine hydroxylase and dopamine β-hydroxylase activity in superior cervical ganglia and adrenals. This increase is neurally mediated, since it can be abolished by decentralization of the ganglia and transsection of the splanchnic fibres supplying the adrenal medulla. Enzyme kinetic data suggest that the increased enzyme activity results from an increased synthesis of enzyme protein. The activity of dopa decarboxylase, the third enzyme involved in the synthesis of norepinephrine, remains unchanged both in ganglia and in adrenals. In the adrenals, the gradual rise in tyrosine hydroxylase and dopamine β-hydroxylase activity is virtually parallel and reaches more than twice the control values. In the superior cervical ganglion, however, there is a marked difference between the rise in activity of the two enzymes. The increase in tyrosine hydroxylase activity amounts to 217% after 4 days of cold-exposure, that of dopamine β-hydroxylase to 131% of controls (100%) only. The determination of enzyme turnovers by measuring the decay in activity after inhibition of protein synthesis by cycloheximide was attempted but the turnover of tyrosine hydroxylase both in adrenals and superior cervical ganglia was too slow to be determined by this method. The same was true for dopamine β-hydroxylase in the adrenal medulla. However, in the superior cervical ganglion the turnover of the latter enzyme could be determined; the half-life amounts to 13 h. The fact that the turnover of dopamine β-hydroxylase in the superior cervical ganglion is more rapid than that of tyrosine hydroxylase could explain the difference in the increase in enzyme activity in spite of a similar rise in the rate of synthesis. It is concluded that the enzymes involved in the synthesis of norepinephrine are not regulated as an operational unit, but that the results are compatible with the assumption that the synthesis of the two enzymes, tyrosine hydroxylase and dopamine β-hydroxylase, which are specifically located in adrenergic neurons, may be regulated by a common mechanism.

A comparison of the effects of chemical sympathectomy by 6‐hydroxydopamine in newborn and adult rats

1 The effects of chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA) on the cardiovascular system of the rat were compared in, (a) 10‐week‐old rats treated during the first 14 days after birth with 150 μg/g subcutaneously, and (b) adult rats injected intravenously with 2 × 50 mg/kg on day 1 and 2 × 100 mg/kg on day 7 and the experiments performed on day 8. 2 Intravenous administration of 6‐OHDA to adult rats almost completely abolished the pressor responses to stimulation of the entire sympathetic outflow in the pithed rat, the contractions of the lower eyelid to stimulation of the cervical sympathetic trunk and the vasconstrictor responses produced by periarterial nerve stimulation of the isolated renal artery preparation. Pressor responses to physostigmine and to tyramine were markedly reduced or abolished in anaesthetized and pithed rat preparations, respectively. 3 In corresponding experiments, 10‐week‐old rats treated as newborns with 6‐OHDA showed a marked reduction in the stimulation‐induced pressor responses and contractions of the lower eyelid, but completely normal vasoconstrictor responses to periarterial nerve stimulation of the isolated perfused renal artery were obtained. The pressor responses to physostigmine were slightly reduced but the tyramine responses were unchanged. 4 Treatment with 6‐OHDA at birth caused an almost complete and long‐lasting noradrenaline depletion in the heart, spleen, salivary glands and ileum but only a partial depletion in the mesentery from 10‐week‐old rats. These low noradrenaline levels showed no recovery in rats up to an age of 4 months. The tyrosine hydroxylase activity in both the cervical and stellate ganglia from 10‐week‐old rats was markedly reduced by treatment with 6‐OHDA after birth. 5 Injections of 6‐OHDA after birth produce an almost complete and permanent sympathectomy of various adrenergically innervated organs in the rat. The vascular system represents a major exception, exhibiting a surprisingly high resistance to this type of chemical adrenergic denervation.

Mechanism of amphetamine accumulation in the isolated perfused heart of the rat

Rat hearts were perfused with 10 to 1,000 ng/ml of (±)‐[3H]amphetamine. The time course of accumulation and the maximal tissue/medium ratio (T/M) were identical for all concentrations studied. The maximal T/M varied between 5 and 6 and was reached after 5 min of perfusion. The accumulation of amphetamine was not inhibited by cocaine or noradrenaline. It was not impaired by combined inhibition of aerobic and anaerobic energy metabolism and it was not dependent on the intactness of sympathetic nerve endings, indicating that the greater amount of amphetamine was located extraneuronally. The diminished accumulation of amphetamine following reduction of temperature or sodium concentration of the perfusion fluid most probably reflects impaired tissue perfusion resulting from vascular constriction. The time course of accumulation and decay of amphetamine is compatible with a rapidly reversible phase‐distribution of this amine possibly related to its relatively high lipophilic properties. The possible significance of phenolic hydroxyl groups in membrane transport and diffusion of phenethylamines is discussed.

Genetically hypertensive rats: relationship between the development of hypertension and the changes in norepinephrine turnover of peripheral and central adrenergic neurons.

SummaryIn genetically hypertensive rats, the norepinephrine turnover of peripheral and central adrenergic neurons was determined either by the decline in endogenous norepinephrine after inhibition of tyrosine hydroxylase or by the decay in the specific activity of norepinephrine after labelling the stores by intravenous or intraventricular injection of3H-norepinephrine.In the periphery (heart and submaxillary gland), the norepinephrine turnover of genetically hypertensive rats was reduced in proportion to the rise in systolic blood pressure. In the hypothalamus, medulla-pons and the residual parts of the brain, the turnover was unchanged both in the prehypertensive and the hypertensive state. The results indicate that the central adrenergic neurons, involved in the control of blood pressure, may act independently from the activity of peripheral baroreceptors. The elevated blood pressure resulting from an enhanced peripheral vascular reactivity to the physiological neurotransmitter norepinephrine may induce a compensatory inhibition of the activity of the peripheral adrenergic neurons. In the genetically hypertensive rats, neither the peripheral nor the central adrenergic nervous system seems to play a primary role in the development of hypertension.

Rapid recovery of vascular adrenergic nerves in the rat after chemical sympathectomy with 6-hydroxydopamine

1 Twenty‐four hours after the last of 4 intravenous doses of 6‐hydroxydopamine (2×50 mg/kg on day 1 and 2×100 mg/kg on day 7) a complete impairment of adrenergic nerve function was observed in various organs of the rat. 2 A complete recovery of adrenergic nerve function in vascular smooth muscle was observed 7 days after the last dose of 6‐hydroxydopamine whilst in non‐vascular smooth muscle recovery took between 14 and 21 days. 3 On day 8, noradrenaline depletion produced by 6‐hydroxydopamine was not as great in vascular tissues, such as the mesentery and renal artery, as in other tissues, such as the heart and salivary glands. Noradrenaline concentrations recovered much more rapidly in vascular than in other tissues. 4 Electron microscope studies of small mesenteric arteries showed a complete destruction of adrenergic nerve terminals 24 h after 6‐hydroxydopamine (2×100 mg/kg). However, there was a reappearance of growing terminals within 7 days and after 28 days the regrowth of adrenergic nerve terminals seemed complete. 5 From the morphological and functional data it is concluded that 6‐hydroxydopamine does produce complete destruction of vascular adrenergic nerve terminals. However, these terminals regenerate more rapidly than those in other tissues. This could explain the failure of intravenously administered 6‐hydroxydopamine to prevent the development of experimental hypertension in the rat.