Effect of Amine Uptake Inhibitors on the Uptake of 14C-Bretylium in Intact and Degenerating Sympathetic Nerves of the Rat

Abstract: The effect of different amine uptake inhibitors on the accumulation of ¹⁴C-bretylium in sympathetically denervated or decentralized salivary glands were studied in vivo rats 11–14 hours after the surgical intervention. The time period chosen is known to be critical for the delaying effect of bretylium on the degeneration transmitter release in sympathetically innervated organs. Cocaine, desmethylimipramine (DMI), protriptyline or reserpine all depressed the uptake of ¹⁴C-bretylium in both denervated and decentralized salivary glands, cocaine being the most efficient one. DMI and protriptyline, but not cocaine inhibit the degeneration delaying effect of bretylium, while all three agents inhibit amine uptake at level of the nerve cell membrane. Apparently, bretylium reaches the critical sites of its degeneration delaying action by the axonal amine pump but only a small fraction of the drug entering the degenerating adrenergic nerve terminal is needed at the critical sites to interact with the degeneration processes. The difference between the tricyclic antidepressants on one hand and cocaine on the other with respect to the effect on the degeneration delaying action of bretylium, must depend on some action different from the axonal membrane uptake inhibition. Reserpine which is known not to interfere with the delaying effect of bretylium on the denervation degeneration did reduce the uptake of, ¹⁴C-bretylium. This fact seems to indicate that the site of action of bretylium is located outside the adrenergic nerve granules.     

Ca2+ dependence of the release of noradrenaline during nerve degeneration in the parotid gland

Summary Amylase secretion in vitro was used as an indication of the degeneration activity of sympathetically denervated parotid glands. Seventeen and a half hours after sympathetic denervation, slices of parotid gland released amylase into the incubation medium at a rate higher than that observed for non-denervated glands. The time course of amylase release from denervated glands followed a bellshaped pattern similar to that observed in other denervated structures. Lowering extracellular Ca²⁺ to 0.25 mmol/l diminished significantly the release of amylase. Low Ca²⁺ however, did not decrease the amylase release in response to added noradrenaline.These results indicate that Ca²⁺ is required for the release of noradrenaline from degenerating nerve endings.     

Degeneration activity of the pineal gland after sympathetic denervation

Summary After removal of the superior cervical ganglia the pincal gland of the rat showed a period of transient increase in activity of serotonin-N-acetyltransferase. This degeneration activity was preceded by a decline of the levels of endogenous noradrenaline and an impairment of the ³H-metaraminol uptake. Twenty-seven hours after sympathetic denervation the levels of the endogenous neurotransmitter were almost completely depleted, while either 24 h or 21 days after denervation total uptake of the tritiated amine decreased to only about 50% of the control values. Previous chronic sympathetic decentralization elicited in the pineal gland postjunctional supersensitivity, as judged by the enhanced degeneration activity observed in decentralized glands after sympathetic denervation.The present findings confirm that, in endocrine glands and similarly to other autonomically innervated organs, acute denervation induces degeneration acitivity.This study was supported by grants from the CONICET and SUBCYT     

The effect of corticosterone on extraneuronal amine uptake and effector response in rat salivary glands

The effect of corticosterone on effector cell response to noradrenaline in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillary glands. When tissue slices were incubated with [³H]noradrenaline the level of extraneuronally retained radioactive material was found to be markedly reduced at a concentration of 10 μg ml^?1 of corticosterone after inhibition of neuronal uptake by protriptyline. Corticosterone in a dose of 10 mg kg^?1 was found to markedly potentiate the secretory response to noradrenaline in vivo, when the neuronal uptake of noradrenaline was blocked by protriptyline (10 mg kg^?1, i.p.). Inhibition of neuronal uptake alone by protriptyline or of the extraneuronal uptake alone by corticosterone in the doses used here did not affect the dose-response curve for noradrenaline, at least not in its lower part. The data thus clearly show that the extraneuronal amine uptake of rat salivary glands is blocked by corticosterone and that this extraneuronal uptake might be regarded as a mechanism of importance for the inactivation of the adrenergic transmitter.     

Electron microscopic evidence that bretylium and pargyline delay adrenergic nerve degeneration after sympathectomy of the pineal gland

Summary Seventeen and twenty four hours after sympathetic denervation, noradrenaline stores of the rat pineal gland were depleted to 50% and 10% of controls, respectively. Electron microscopic studies showed the coexistence of normal and altered nerve endings 17 h after denervation, while 24 h after denervation, only degenerated nerve terminals were observed.Treatment with pargyline (512 moles/kg) or bretylium (24 moles/kg) significantly delayed the loss of noradrenaline from denervated glands. In pargyline treated rats, 17 h after denervation, noradrenaline stores were 90% of control glands. After bretylium, values obtained 24 h after denervation, declined to 36% of innervated glands. Persistence of neurotransmitter coincided with the presence of normal nerve endings as observed electron microscopically.It is concluded that both, pargyline and bretylium, prolonged the survival of nerve endings severed from the cell body.Supported partially by grants N^o 9087/80-22, CONICET, and 1003011-242/80 SECYT     

Chronic sympathetic denervation increases muscarinic cholinoceptor binding in the rat submaxillary gland

Summary Superior cervical ganglionectomy was found to produce a large decrease in the cocaine-sensitive accumulation of ³H-noradrenaline in the rat sub-maxillary gland, indicating an effective sympathetic denervation. Six weeks after unilateral denervation the muscarinic cholinoceptor binding of ³H-QNB was increased by over 50% compared to the contralateral, innervated gland. There were no differences in the Kd values between the innervated and denervated glands. These results suggest that changes in muscarinic cholinoceptor density might be in part responsible for the postsynaptic supersensitivity to cholinoceptor agonists observed after chronic sympathetic denervation.     

Structural and functional alterations caused at the extraneuronal level by sympathetic denervation of blood vessels

Summary The lateral saphenous vein of the dog and the rabbit ear artery were surgically denervated, by clamping the vessel or by removal of the superior cervical ganglion, respectively. Both procedures resulted in denervation of the vessels.The denervated, lateral saphenous vein was supersensitive to exogenous noradrenaline and inactivation of the amine (in oil immersion experiments) was slower in denervated vein strips than in control strips treated with cocaine. Incubation experiments with ³H-noradrenaline confirmed that denervated strips formed considerably fewer metabolites than control ones (in the absence or presence of cocaine) and that O-methylation of noradrenaline was reduced by about 50%. When the strips were incubated with ³H-isoprenaline, the denervated ones accumulated and metabolized isoprenaline to a lesser degree than control strips. Hydrocortisone did not reduce the accumulation of isoprenaline in the denervated vein and had only minor effects on O-methylation. The metabolism of noradrenaline and isoprenaline gradually recovered with time.In the ear artery, denervation was accompanied by a marked reduction in O-methylation, but not in accumulation, of isoprenaline. In both vessels there was a highly significant positive correlation between noradrenaline content and O-methylating capacity; in the saphenous vein accumulation of isoprenaline was also positively correlated to noradrenaline content.Morphological changes observed in the denervated vessels consisted essentially in dedifferentiation of smooth muscle cells (which attained larger dimensions, had an indented, large nucleus, augmented euchromatin and an increased amount of ribosomes), abundance of extracellular material and fibroblasts. Mast cells were present in denervated veins (but not in controls) and the histamine content was increased in the former. Structural alterations were homogeneously distributed in the saphenous vein but restricted to the adventitio-medial area in the rabbit ear artery. Depletion of endogenous noradrenaline by reserpine pretreatment did not cause the alterations seen after denervation. On the other hand, continuous intravenous infusion of noradrenaline during 5 days did not prevent, and even worsened, the alterations caused by denervation. It was concluded that noradrenaline does not appear to be the factor the lack of which is exclusively responsible for the impairment of the extraneuronal system, in the denervated tissue.Taken together, the data show that the sympathetic innervation of blood vessels exerts a regulatory function on extraneuronal events; the disappearance of innervation results in marked impairment of the corticosteroid-sensitive O-methylating system and in morphological changes of both smooth muscle cells and fibroblasts.Part of the results were presented at 3rd Joint Meeting of the French and Spanish Pharmacological Societies in Toulouse, 1981 (Branco et al. 1982)     

The recovery of the capacity for uptake-retention of ( 3 H)noradrenaline in rat adrenergic nerves after reserpine

The uptake retention of [³H]noradrenaline (2.5 μg/kg, i.v., 30 min before death) in rat salivary glands was studied at different times after reserpine treatment (10 mg/kg, i.p.). The effect of removing the cervical superior ganglion 12 h before death on the recovery of the [³H]noradrenaline uptake-retention capacity after reserpine was also investigated. The ganglionectomy was unilateral, and the contralateral side was always preganglionically denervated. In glands with uninterrupted postganglionic adrenergic nerves the onset of recovery of the [³H]noradrenaline retention capacity occurred 24–36 h after reserpine. Normal contents were found on the second to third days. Between day 3 and 6 a possible overshoot of [³H]nor- adrenaline content, followed by normal and subnormal contents (7–21 days) were recorded. Ganglionectomy, 12 h before death, markedly delayed the recovery of [³H]noradrenaline retention capacity. Both the recovery curve for [³H]noradrenaline retention in glands with intact postganglionic nerves, and the effect of ganglionectomy on the [³H]noradrenaline retention capacity, were clearly related to the relative number of new functioning amine granules that are transported via the axons to the nerve terminals at different times after the reserpine-pretreatment. The results indicate that young amine granules, recently transported to the nerve terminals via the axons, have the greatest capacity to take up and retain [³H]noradrenaline. The half-life of this capacity in the young granules appears to be about 12 h. Since published results indicate that [³H]noradrenaline is initially taken up in the “small easily releasable pool” of transmitter, we suggest that young amine granules are of the greatest importance for adrenergic function, i.e. that they are particularly active in taking up recaptured noradrenaline, in the synthesis, and in the release of this transmitter.     

Effects of sympathectomy on the in vitro α and β-responses of the parotid gland

Summary Amylase and K⁺ release were measured in slices obtained from innervated and sympathetically denervated rat parotid glands. Amylase release induced by noradrenaline was found to be increased after chronic denervation. The postjunctional component of supersensitivity to noradrenaline was evidenced by an increase in the maximal response to this agonist. The maximal response of denervated glands was blocked by propranolol 10^–5 M. On the other hand, chronic denervation did not modify the -adrenoceptor-mediated response, K⁺ release, either in the presence or in the absence of ouabain. It is concluded that, after sympathetic denervation, postjunctional supersensitivity develops for the -adrenoceptor-mediated but not for the -adrenoceptors-mediated effects of noradrenaline.     

Electrical stimulation releases 3H-betaxolol from rat atrial slices: Possible role of noradrenergic nerves

Summary Under in vitro conditions, ³H-betaxolol was accumulated in rat atrial slices, reaching a tissue-medium ratio of 12.3±1.8 ml/g. This process was temperature-dependent, but ouabain-resistant. ³H-Betaxolol accumulated in atrial slices was subsequently released by electrical stimulation. The electrically-evoked release of ³H-betaxolol was abolished in the absence of calcium, and reduced in the presence of bretylium 10 M. After surgical sympathetic denervation by stellate ganglionectomy there was a marked reduction in the endogenous content of noradrenaline and in the retention of ³H-noradrenaline in atrial slices. The concomitant decrease in the amount of ³H-noradrenaline released by electrical stimulation following denervation was modest, although statistically significant. Following sympathetic denervation, the tissue retention of ³H-betaxolol was not significantly affected, but the release of ³H-betaxolol by electrical stimulation was considerably reduced. After pretreatment with reserpine the amount of radioactivity released by electrical stimulation from slices labelled with ³H-betaxolol or ³H-noradrenaline was markedly reduced. The tissue retention of ³H-noradrenaline was reduced to a larger extent than that of ³H-betaxolol following the administration of reserpine. The simultaneous release of noradrenaline and betaxolol by nerve stimulation observed under our experimental conditions may represent a mechanism through which betaxolol can reach selectively the postsynaptic ₁-adrenoceptors and reinforce -adrenoceptor blockade in the heart.     

Effects of neonatal sympathetic denervation on amylase secretion in the adult rat parotid gland: Difference in β1- and β2-adrenoceptor response

Changes in amylase secretion and cyclic AMP accumulation in response to various secretagogues were studied in parotid glands of adult rats subjected to neonatal sympathetic denervation by unilateral excision of the superior cervical ganglion. Denervation decreased the gland content of amylase and both basal and the stimulated levels of cyclic AMP were elevated. The secretory cells of neonatally devervated glands exhibited enhanced maximal enzyme discharge in response to β-adrenoceptor agonists. However, the selective β₁-agonist, prenalterol, was not effective in this respect whereas an enhanced maximal secretory response to the β₂-selective agonist, terbutaline, was particularly prominent. DBcAMP was also more efficient in inducing amylase release from the denervated gland. The results of the present study demonstrate that the usual dominance of the β₁-adrenoceptor subtype in eliciting amylase 3elease is lost, implying that the differentiation of the β-adrenoceptor into its subtypes is altered by neonatal sympathetic denervation.     

Phorbol ester,an activator of protein kinase C,enhances calcium-dependent release of sympathetic neurotransmitter

Summary The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from ³H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased to a maximum level (over 4-fold) at 30 nmol/l. The spontaneous overflow of radioactivity, however, was not affected by any concentration of phorbol ester. The facilitatory effect of phorbol ester on stimulation-evoked overflow was observed in the presence of inhibitors of neuronal and extraneuronal uptake as well as after removal of negative feedback inhibition of release by presynaptic alpha-adrenoceptors. Tyramine (7 mol/l for 10 min) caused a marked increase in the overflow of tritium in either the presence or absence of calcium. However, tyramine-induced overflow was not enhanced by phorbol ester. It is concluded that protein kinase C of sympathetic neurons is involved in an exocytotic release of the transmitter.     

Effect of N-hydroxyethylpromethazine (Aprobit®) on the distribution of 35S-chlorpromazine studied by autoradiography in cats and mice

Summary The effect of a quaternary phenothiazine compound, N-hydroxyethylpromethazine (Aprobit^®, NHP), on the distribution of ³⁵S-chlorpromazine (³⁶S-CPZ) was studied in mice and cats using an autoradiographic technique. NHP did not change the sedative action of CPZ or ether but it caused a strong relaxation of the nictitating membrane, mydriasis and shivering in all cats.A pronounced accumulation of ³⁵S-CPZ was recorded in the inferior cervical ganglion of the cat during the entire 16 h period of observation. In addition to the lung parenchyma a strong activity was concentrated in the bronchial mucosa and excretions. The uptake was very high in the brain, eye, liver, kidney, pancreas, oesophagic excretion, in the gastro-intestinal contents, salivary glands and especially in the sublingual gland.The NHP-pretreatment increased the radioactivity in the sympathetic ganglia, sublingual glands, lungs, liver and bone marrow. NHP delayed the penetration of ³⁵S-CPZ into the brain. At 16 h after the i.v. injection a stronger retention of the radioactivity in the cerebral tissue was observed in the NHP-pretreated animals. In pregnant mice NHP decreased the accumulation of ³⁵S-CPZ or its metabolites in the fetuses.The possible mechanism whereby NHP increases the retention of ³⁵S-CPZ or its metabolites in the brain is discussed.This work was supported by the Finnish Medical Research Council.     

Immunohistochemical study on GABAergic system in salivary glands

Gamma-aminobutyric acid (GABA) and its receptors are found in the central nervous system and several peripheral tissues. The purpose of this study was to determine the expression and distribution of GABA and glutamate decarboxylase (GAD), a GABA biosynthetic enzyme, in rat salivary gland. Western blot and real time quantitative RT-PCR revealed that GAD67 was the major isoform of GAD in the salivary glands. Furthermore, both GABA and GAD were detected around the acinar cells in the submandibular glands by immunohistochemical analysis. When both sympathetic and parasympathetic nerves related to the submandibular glands were denervated, the immunoreactivities of GABA and GAD were dramatically depressed, and levels of GAD67 and GABA significantly decreased. However, no morphological changes in the glands were observed after denervation. These results indicate that GAD67 is present around acinar cells in the salivary glands, and suggest that the GABAergic system in the glands is closely related to the autonomic nervous system.     

Functional significance of neuronal and extraneuronal transmitter uptake in rat salivary glands

Summary The effect of normetanephrine (NM) on effector cell response to noradrenaline (NA) in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillayr glands. Tissue slices were incubated with ³H-NA and the metabolic pattern of the radioactivity in the slices as well as of the efflux from the slices was analyzed. The level of extraneuronally retained radioactive material was found to be markedly reduced at a NM concentration of 20 g/ml after inhibition of neuronal uptake by protriptyline (PTP). In the presence of an intact neuronal membrane pump mechanism 200 g/ml of NM also reduced the level of radioactive material, indicating that NM has an inhibitory effect on extraneuronal and in higher concentration also on neuronal uptake and retention of NA and its metabolites. NM was found to markedly potentiate the secretory response to NA in vivo, when doses of NA larger than 1 g were used. At lower NA doses there was no difference between NM pretreated and control rats. When neuronal uptake was prevented by pretreatment with PTP, NM was found to potentiate the secretory response to NA over the entire dose range. Inhibition by PTP of neuronal uptake only did not affect the dose-response curve for NA and data are presented which indicate that the absence of such a potentiation is due to a decreased salivary gland blood flow induced by NA after PTP pretreatment. The increased responses recorded after NM could not be ascribed to an additive effect of this amine, since NM was found to be a very weak -receptor agonist in the rat submaxillary gland; doses of more than 1000 g i.v. were needed to detect a secretory response. The data indicate that the extraneuronal uptake of the transmitter has a functional significance in the rat submaxillary gland by reducing high transmitter concentrations in the vicinity of the receptors.     

On the role of the adrenergic receptors for extraneuronal amine uptake and retention in rat salivary glands in vitro

Summary The effect of an -receptor blocking agent, phenoxybenzamine, and a series of -receptor blocking agents on extraneuronal uptake and retention of radioactivity after incubation of rat salivary gland slices with ³H-noradrenaline or ³H-isoprenaline has been investigated. In some experiments with ³H-noradrenaline as substrate, neuronal uptake was prevented by adding protriptyline to the incubation medium. Phenoxybenzamine reduced extraneuronal accumulation of radioactivity after both ³H-noradrenaline and ³H-isoprenaline in a concentration-dependent manner, whereas after propranolol the levels of extraneuronally retained radioactive material were markedly increased, the efflux from the slices not being diminished. Some other nonselective and selective -receptor blocking agents with and without intrinsic activity were found to produce the same effect as propranolol upon the retention of radioactivity after incubation with ³H-noradrenaline and ³H-isoprenaline. When both phenoxybenzamine and propranolol were present in the incubation medium the effect of extraneuronally retained radioactivity after ³H-noradrenaline as well as after ³H-isoprenaline was the same as when only phenoxybenzamine was used. The metabolic pattern of the radioactivity in the slices revealed that the extraneuronally retained radioactive compounds consisted mainly of tritiated catabolites, especially 3-O-methylated ones. The relationship between the extraneuronal accumulation and adrenergic receptor mechanisms is discussed.     

Studies on the mode of action of bretylium and guanethidine in post-ganglionic sympathetic nerve fibres

Summary 1. The effects of bretylium and guanethidine on the nerve terminal impulse and transmitter release from sympathetic postganglionic nerve terminals in the guinea-pig vas deferens have been studied in vitro using focal extracellular recording. Excitatory junction currents (EJCs) were used as a measure of transmitter release. 2. Both bretylium and guanethidine altered the configuration of the nerve terminal impulse in a manner consistent with their being local anaesthetics. 3. Bretylium (1–3 M) only completely inhibited transmitter release when impulse propagation in the sympathetic nerve terminal was blocked. 4. In contrast, guanethidine (1–10 M) could block transmitter release with little effect on the configuration of the nerve terminal impulse. 5. The inhibitory effects of these agents on both the nerve terminal impulse and on transmitter release were reversed by the indirectly acting sympathomimetic agent, d-amphetamine (1–10 M). 6. Using this technique the mechanisms of action of drugs known to modify the transmitter release in sympathetic nerve terminals can be more precisely determined. Send offprint requests to T. C. Cunnane at the above address     

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells

BACKGROUND AND PURPOSE: Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands. EXPERIMENTAL APPROACH: Tissue specimens of the right submandibular gland taken from 18 Wistar rats after intraglandular injection of BoNT A, BoNT B, or a combination of both were examined. As a sham control, an equivalent volume of saline was injected into the left submandibular gland. Morphometric measurements, immunohistochemistry, electron microscopy and western blot analysis were used to analyse the morphological and functional changes of the denervated glands. KEY RESULTS: Morphological and ultrastructural analyses of the cell organelles and secretory granula showed a clear atrophy of the acini, which was more prominent in glands injected with the combination of BoNT/A and B. Morphometric measurements of the glandular acini revealed a significant reduction of the area of the acinar cells after injection of BoNT (P=0.031). The expression of amylase was significantly reduced in BoNT treated glands. CONCLUSIONS AND IMPLICATIONS: Intraglandular application of BoNT induces structural and functional changes of the salivary glands indicated by glandular atrophy. These effects may be due to glandular denervation induced by the inhibition of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) involved in acetylcholine release at the neuroglandular junction and also specially inhibition of those involved in exocytosis of the granula of the acinar cells.     

On the disposition of [3H]metaraminol in the rat salivary gland

The left salivary glands of rats were sympathetically denervated or decentralized. In some experiments the excretory ducts of the left submaxillary and sublingual glands were ligated to produce atrophy of the acinar cells. The rats received [³H]metaraminol (³H-MA) intravenously and were killed at various time intervals thereafter. The amount of ³H-MA in the salivary glands was determined. ³H-MA was taken up and retained in the intact gland, but disappeared rapidly from the denervated one, indicating that ³H-MA is taken up and stored in the adrenergic neuron. Decentralization resulted in a decreased turnover of the amine, especially during the first 18 hr, which supports the view that metaraminol is released by nerve activity. The ability of the salivary gland to take up ³H-MA was diminished by glandular atrophy, and the disappearance of the ³H-MA so taken up was delayed.     

Is neuropeptide Y co-released with catecholamines in experimental arterial hypertension following sinoaortic denervation?

Summary The release of catecholamines and their co-neurotransmitter neuropeptide Y (NPY) was investigated in conscious dogs with neurogenic arterial hypertension elicited by sinoaortic denervation. One month after denervation, an elevation of catecholamine levels (measured by HPLC) without elevation of NPY-like immunoreactivity (NPY-LI) levels in plasma (evaluated by RIA) has been found. This dissociation could be explained by 1) a transient release of NPY during the first weeks after surgery, 2) a depletion of neuronal NPY due to the permanent sympathetic stimulation, or 3) an insufficient increase in sympathetic tone.To test these hypotheses, we investigated the time courses of catecholamine and NPY-LI levels in arterial plasma during the first five weeks after sinoaortic denervation and responses to yohimbine (an alpha2 antagonist which enhances transmitter release). Resting NPY-LI levels in plasma remained normal during the first five weeks after sinoaortic denervation. In normal dogs, a high dose of yohimbine (0.5 mg/kg i. v.) elevated both catecholamine (6-fold) and NPY-LI levels (1.5-fold), whereas a lower dose (0.05 mg/kg i. v.) induced a two fold elevation of catecholamine levels without changing NPY-LI concentrations. In sinoaortically denervated dogs, yohimbine elicited elevation of both catecholamines and NPY-LI whatever the dose used.Thus, neurogenic arterial hypertension in dogs seems to involve catecholamines but not NPY. Moreover, the present work suggests that a high level of sympathetic stimulation is required for a co-release of catecholamines and NPY. Send offprint requests to J.-L. Montastruc at the above address