Blood-brain barrier and neuroendocrine regulations.
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Animals↗
Biomedical subjects
F Héry
Publications and source records attributed to F Héry.
Effect of serotonin on alpha-melanocyte-stimulating hormone secretion from perifused frog neurointermediate lobe: evidence for the presence of serotonin-containing cells in the frog pars intermedia.
We have examined the presence of 5-hydroxytryptamine (serotonin; 5-HT) in the intermediate lobe of the frog pituitary and investigated the effect of exogenous 5-HT on alpha-melanocyte-stimulating hormone (alpha-MSH) release from the perifused neurointermediate lobe (NIL). Using a specific antiserum against 5-HT, the indirect immunofluorescence technique revealed the presence of 5-HT-like immunoreactivity (5-HT-LI) in discrete cells, generally gathered in small clusters among parenchymal cells, and in numerous neurites surrounding melanotrophic cells. At the electron microscopic level, using a silver-gold intensification procedure, 5-HT-LI was localized in dense-core secretory vesicles within specific pituitary cells which appear to be different from pituitary melanotrophs. Dense accumulation of gold particles was also observed in nerve fibres running between parenchymal cells. A combination of high-performance liquid chromatography analysis and electrochemical detection showed the presence of both 5-HT and its metabolite 5-hydroxyindol acetic acid (5-HIAA) in frog NIL extracts (534 +/- 40 and 1245 +/- 65 (S.E.M.) pg/mg wet tissue respectively). Administration of graded doses of 5-HT (from 1 to 30 mumol/l) to perifused frog NIL induced a dose-dependent inhibition of alpha-MSH release. Repeated pulses of 5-HT (10 mumol/l each) induced a reproducible inhibition of alpha-MSH without any desensitization phenomena. The inhibitory effect of 5-HT was partially blocked by the serotonergic antagonists methysergide and ICS-205-930 (10 mumol/l each). Concomitant administration of methysergide and ICS-205-930 (10 mumol/l each) totally abolished 5-HT-evoked inhibition of alpha-MSH. Fenfluramine, a releaser of 5-HT, induced a slight but significant reduction of alpha-MSH secretion. While 5-HT caused a marked inhibition of alpha-MSH release from intact NIL, 5-HT was devoid of effect on acutely dispersed pars intermedia cells suggesting that 5-HT does not exert a direct action on pituitary melanotrophs. We have examined the effect of specific dopaminergic, GABAergic and alpha-adrenergic antagonists on 5-HT-induced alpha-MSH inhibition. We observed that sulpiride and SR 95531 (10 mumol/l each) did not affect the response of NIL to 5-HT while yohimbine (10 mumol/l) suppressed the inhibitory action of 5-HT. Taken together, our results indicate that discrete cells of the frog pars intermedia contain the neurotransmitter 5-HT which may act locally to inhibit alpha-MSH release.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Immunohistochemical and biochemical evidence for the presence of serotonin in amphibian adrenal chromaffin cells.
The presence of serotonin (5-HT) in chromaffin cells of the frog adrenal (inter-renal) gland has been demonstrated both by immunocytochemical and biochemical techniques. Using antisera against 5-HT and tyrosine hydroxylase (TH) on consecutive sections, we found by means of the indirect immunofluorescence technique that a majority of chromaffin cells were also immunopositive for 5-HT. When antibodies to 5-HT and phenylethanolamine-N-methyltransferase (PNMT) were applied on consecutive sections, 5-HT-like immunoreactivity was observed in almost all epinephrine-producing cells which represented about 90% of the total chromaffin cells. No 5-HT-containing fibres could be detected. At the ultrastructural level, using a pre-embedding procedure associated with gold-silver intensification of the immunoperoxidase reaction, 5-HT-immunoreactivity was visualized in secretory vesicles essentially located in the periphery of epinephrine cells. Combination of high performance liquid chromatography and electrochemical detection showed the presence of both 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in frog adrenal extracts. Transection of the splanchnic nerve enhanced 5-HT immunoreactivity and augmented the amount of 5-HT in adrenal extracts. Taken together, these results indicate that epinephrine-producing cells of the frog adrenal contain significant amounts of serotonin. The observation of the storage of 5-HT in secretory vesicles of epinephrine cells suggests that serotonin may be released with catecholamines under stress conditions.
Adrenal Medulla↗
Daily variations in serotonin metabolism in the suprachiasmatic nucleus of the rat: influence of oestradiol impregnation.
In order to determine the temporal relationships between variations in 5-hydroxy-tryptamine (5-HT, serotonin) metabolism in the suprachiasmatic nucleus (SCN) and the cyclic LH surge, and also to check whether implantation of oestradiol capsules might modulate 5-HT metabolism in the SCN, we carried out a parallel study of 5-HT content in the SCN and median eminence, and 5-HT metabolism in the SCN and supraoptic region in vitro. These experiments were performed on intact male rats, ovariectomized females and ovariectomized females implanted with oestradiol. It was only in ovariectomized rats implanted with oestradiol, in which we have described the existence of a clear-cut circadian rhythm of LH secretion, that we found fluctuations in the content, synthesis and utilization of 5-HT. The content and synthesis were characterized by a peak between 12.00 and 15.00 h, whereas utilization was 50% higher at 09.00 and 19.00 h than at 15.00 h. These fluctuations in 5-HT content and metabolism were specific to the SCN; the median eminence and the supraoptic region did not show such variations. They were also specific to ovariectomized rats implanted with oestradiol, since the patterns of 5-HT content and metabolism in the SCN were the same in males and ovariectomized females and did not differ from those in the median eminence, the supraoptic region or the whole hypothalamus. These results suggest that 5-HT terminals in the SCN play an important role in the control of cyclic LH secretion at a critical period. Moreover, oestradiol seems to be partly responsible for the fluctuations of 5-HT metabolism in the SCN of ovariectomized rats implanted with oestradiol.
Animals↗
5-HT metabolism in the intestinal wall of the rabbit.
The levels of TRP, 5-HTP, 5-HT and 5-HIAA were measured in the proximal colon of the rabbit using ion exchange chromatography and spectrofluorimetry. Assays were performed on fragments of the intact colonic wall, on the muscle layers containing nerve plexuses, and on the mucosa-containing enterochromaffin cells. In spite of identical TRP concentration, 5-HT levels were higher in the mucosa than in the muscle containing nerve plexus. In the muscle, the ratio 5-HIAA/5-HT was about 1, i.e., comparable to that of the brain, whereas the ratio was very low in the mucosa, suggesting a weak 5-HT catabolism in the enterochromaffin cells. When the mucosa was severed from the muscle, 5-HT synthesis was increased. In this case, 5-HT and 5-HIAA concentrations in the reconstituted wall (muscles + mucosa) were higher than in the intact wall. In in vitro studies, levels of newly synthesized (3H)5-HT from (3H)TRP were 3 times higher in the reconstituted wall than in the intact one, and the conversion index of TRP to 5-HT was higher in the mucosa and in the reconstituted wall respectively. These results suggest that enterochromaffin cells do synthesize 5-HT from TRP when the mucosa is divided from the muscle. (3H)5-HT was taken up by both nerve plexuses and mucosa, with a high affinity mechanism. Results from tryptophan loading experiments indicated that 5-HTP decarboxylase in the gut might be involved in the regulation of 5-HT synthesis. The mucosa severed from the muscles might represent a good model for the study of carcinoid tumour development.
5-Hydroxytryptophan↗
[Kinetic study of serotonin metabolism in the suprachiasmatic nucleus of the rat: neuroendocrine incidence (author's transl)].
The suprachiasmatic nucleus (SCN), which is implicated in the regulation of several cyclic neuroendocrine rhythms, displays a conspicuous and apparently specific serotoninergic innervation. Our study was intended to establish more precise correlations between the incidence of serotonin (5 HT) metabolism in the activity of the SCN, and neuroendocrine rhythms. For this purpose, castrated female rats, having subcutaneous implants of estradiol, were studied. These animals display very marked circadian fluctuations in plasma levels of the pituitary hormones ACTH, LH and PRL; a relatively well-synchronized increment in all these hormones occurs between 11.00 and 15.00. Punches were obtained to determine the endogenous content of 5 HT, measured by a radioenzymatic technique, simultaneously in SCN and median eminence (ME). An index of SCN activity was determined from in vivo SCN 2-deoxy (1-14C) glucose (DG) uptake; the retention was estimated on radioautographs. Endogenous level of 5 HT displayed a marked circadian rhythm with a peak between 12.00 and 15.00 in the SCN; 5 HT levels were constant throughout the day in the ME. 14C-DG uptake was greater at 15.00 than at 9.00. However, after PCPA treatment or raphe lesions, the uptake of 14C-DG was the same at 9.00 and 15.00. Taken together, these results strongly suggest that in our model: (1) SCN displays a rhythm of activity in the light period; (2) SCN displays specific rhythms in the content of 5 HT; (3) the SCN rhythm of activity must be under an inhibitory control of 5 HT, since the lowest metabolic level is increased at 9.00 by inactivation of 5 HT system; (4) the close relationships between the initial release phase of several pituitary hormones, the increase of metabolic activity in the SCN and the higher level of the 5 HT in the nucleus suggest that 5 HT terminals in the SCN play an important part in the control of cyclic hormonal secretion.
Adrenocorticotropic Hormone↗
Regulation of release processes in central serotoninergic neurons.
Different technical, physiological and biochemical aspects concerning the study of the release of 5-HT are discussed herein. Isotopic methods are the most suitable techniques since these allow the release of 3H-5-HT to be measured after having determined the identity of the labelled compounds formed from 3H-tryptophan by co-chromatography. Under these conditions, the 3H-amine released in the superfusates comes from serotoninergic nerve endings, since tryptophan hydroxylase is exclusively localized in serotoninergic neurons. Moreover, it appears that newly synthesized 5-HT is preferentially released. The release of 5-HT is dependent on neuronal activity, but is not always linked to the synthesis of 5-HT. The increase in the firing rate of serotoninergic cell bodies by a local application of glutamate in the area of the nucleus raphe dorsalis induces a marked increase n the release of 5-HT in the caudate nucleus; an opposite effect is observed after cooling this region. The local depolarization of serotoninergic terminals located in the caudate nucleus increases the release of this amine. This effect is blocked by TTX. LSD reduces the stimulating effect of KCl, thus indicating that the release of 5-HT can be controlled at a presynaptic level. In addition, the release of the amine is dependent on the presence of calcium. Serotoninergic neuronal activity can be controlled at the preterminal or at the cell body levels by the activity of other neuronal systems. The effects of the release of dopamine from dendrites, and that of GABA in the substantia nigra are reported herein. Furthermore, changes in the activity of the dopaminergic, gabaergic and serotoninergic systems innervating the nucleus raphe dorsalis modulate the release of 5-HT, measured both in the caudate nucleus and in the nucleus raphe magnus. Finally, it has been reported that the release of 5-HT can be estimated in the raphe nuclei dorsalis and magnus. It has been shown that the amounts of 3H-5-HT continuously formed from 3H-TRP and released in the nucleus raphe dorsalis are much greater than those estimated in the caudate nucleus or in the substantia nigra. Although the quantities of endogenous 5-HT measured in the nucleus raphe dorsalis are the highest in the brain, this structure presents only a few serotoninergic nerve endings. This raises the question of the origin of the 5-HT released in serotoninergic nuclei. A possible dendritic release of 5-HT is discussed.
Acetylcholine↗
Effect of nerve activity on the in vivo release of [3H]serotonin continuously formed from L-[3H]tryptophan in the caudate nucleus of the cat.
A new isotopic approach has been developed to study the in vivo release of serotonin (5-HT). 'Encéphale isolé' cats were implanted with a push-pull cannula in the ventrocaudal part of the head of the caudate nucleus to estimate the release of [3H]5-HT continuously synthesized from L-[3H]tryptophan. Both [3H]5-HT and [3H]tryptamine were found in superfusates. Resting steady state in the release of [3H]indoleamines was observed as soon as 20 min after the beginning of the superfusion with L-[3H]tryptophan; the levels of [3H]5-HT in superfusates were 2.5 times those of [3H]tryptamine and about 6 times the blank value. They were markedly enhanced in the presence of fluoxetine (5 x 10(-6)M), a blocker of the 5-HT uptake process. A marked increase in the release of [3H]5-HT was seen during the local depolarization of 5-HT terminals with potassium chloride (60 mM) or batrachotoxin (10(-6)M) or during the stimulation of 5-HT cell bodies in the nucleus raphe dorsalis with L-glutamic acid (5 x 10(-5)M). These treatments did not enhance the efflux of [3H]tryptamine. The potassium-evoked release of [3H]5-HT was reduced by LSD (10(-5)M). LSD added alone in the superfusing fluid was without effect. The batrachotoxin-evoked release of [3H]5-HT was inhibited in the presence of tetrodotoxin (9 x 10(-6)M). The spontaneous release of [3H]5-HT and [3H]tryptamine was markedly reduced in the presence of a calcium-free medium containing cobalt (10 mM). A transient slight reduction in the spontaneous release of [3H]5-HT was observed in the presence of tetrodotoxin (9 x 10(-6)M). The local cooling of 5-HT cell bodies with a cryoelectrode induced a slight reversible decrease in [3H]5-HT release. These last two treatments were without significant effect on [3H]tryptamine efflux in superfusates. These results indicate that the release of [3H]5-HT endogenously formed from [3H]tryptophan is dependent on nerve activity and that this is not the case for [3H]tryptamine. The advantages of the isotopic approach for in vivo studies on the release of 5-HT are discussed.
Animals↗
Paradoxical decrease of brain 5-HT turnover by metergoline, a central 5-HT receptor blocker.
Since metergoline (1-methyl-8-beta-carbobenzyloxy-aminomethyl-10-alpha-ergoline) is a potent 5-HT antagonist in peripheral organs, its possible blocking effects on 5-HT receptors in the rat brain were investigated. In vitro, metergoline inhibited both the specific high affinity binding of 3-H-5-HT onto synaptosomal membranes (IC 50 = 18 nM) and the stimulating effect of 10 micron 5-HT on the adenylate cyclase activity in colliculi homogenates from newborn rats (IC 50 = 12 micron. In vivo, the administration of metergoline (10 mg/kg i.p., 60 min before death) resulted in a significant decrease in the 3-H-5-HT binding capacity of synaptosomal membranes from the forebrain of adult rats. Taken together, these data clearly indicated that metergoline is a potent blocker of some serotoninergic receptors in the rat brain. Surprisingly, the changes in 5-HT turnover occurring in the brainstem and in the forebrain 1 h after metergoline (2-10 mg/kg) treatment were similar to those normally induced by a central 5-HT agonist: both the rate of 5-HT utilisation and that of 5-HT synthesis were significantly decreased. These changes were in contrast to the acceleration of 5-HT turnover induced by the administration of another potent central 5-HT antagonist, methiothepin. These results are discussed in relation to the possible existence of several types of serotoninergic receptors in the rat brain. It is possible that the positive feedback regulation of 5-HT turnover is triggered by the blockade of serotoninergic receptors sensitive to methiothepin, but not to metergoline.
Adenylyl Cyclases↗
Characteristics of the activation by dithiothreitol and Fe(2+) of tryptophan hydroxylase from the rat brain.
The preincubation of tryptophan hydroxylase extracted from various areas of the central nervous system of the rat with 30 mM dithiothreitol and 50 muM ferrous ammonium sulfate under nitrogen atmosphere resulted in a persistent increase of its activity. Studies on the enzyme characteristics indicated that this activation was associated with a doubling in its Vmax and a shift (from 7.6 to 7.2) of the optimal pH for its activity. In contrast, the molecular weight and the apparent affinities of tryptophan hydroxylase for its pterin cofactor and for tryptophan were not significantly altered by the preincubation with dithiothreitol and ferrous ammonium sulfate. Since this treatment did not prevent the stimulatory effects of various compounds (phosphatidylserine, ATP and MG(2+), Ca(2+)) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.
Animals↗
Activation of tryptophan hydroxylase by adenosine triphosphate, magnesium, and calcium.
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Adenosine Triphosphate↗
Midbrain raphe lesion in the newborn rat: II. Biochemical alterations in serotoninergic innervation.
Electrolytic raphe lesion was performed in 4-6-day-old rats and the resulting changes of 5HT metabolism within the central nervous system were analyzed up to 9 months later. As soon as the 2nd day following the selective destruction of B7 and B8 nuclei, forebrain 5HT levels were decreased by more than 75%. This reduction persisted for at least 9 months with no sign of recovery. The time course of 5-HIAA decrease was parallel to that of the indoleamine so that the ratio of 5-HIAA over 5-HT levels in the forebrain of lesioned rats was similar to that estimated in controls, whatever their age. This result would suggest that the remaining serotoninergic neurons in the lesioned rats did not develop a compensatory hyperactivity. The raphe lesion induced no change in MAO activity and synaptosomal tryptophan uptake but a pronounce decrease in the Vmax of synaptosomal KHT uptake process in various forebrain areas occurred. The serotonin sensitive adenylate cyclase activity in colliculi homogenate was not altered by the lesion suggesting that this enzyme was probably located in postsynaptic membranes. In addition, this observation would indicate that 5-HT receptors which are linked to this adenylate cyclase did not become supersensitive following the selective degeneration of serotoninergic neurons. Animals without forebrain serotoninergic innervation might be of great interest to analyse the role of serotoninergic neurons in various functions (sleep, analgesia, thermoregulation).
Adenylyl Cyclases↗
Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices.
The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized 3H-5-HT were examined in rat hypothalamic slices. 3H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with L-3H-tryptophan in the presence of the various drugs tested. ACh (10(-5) M) in the presence of eserine (2 X 10(-4) M), and carbachol (10(-5) M) stimulated the release of 3H-5-HT. In contrast, oxotremorine (10(-5) M) reduced the 3H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10(-6) M) and d-tubocurarine (10(-6) M). It was not reduced by the muscarinic antagonists, atropine (10(-6) M) and scopolamine (10(-6) M). In fact, each of two antagonists added alone to the incubating medium enhanced 3H-5-HT release. The scopolamine (10(-6) M) stimulating effect on 3H-5-HT release was suppressed by d-tubocurarine (10(-6) M). Finally, the inhibiting effect of oxotremorine on 3H-5-HT release was not prevented by d-tubocurarine (10(-6) M) but was in the presence of atropine (10(-6) M) or scopolamine (10(-6) M). In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of 3H-5-HT and 3H-5-HIAA from L-3H-tryptophan and on the accumulation of L-3H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous 3H-5-HT in hypothalamic synaptosomes (P2 fraction). These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of 3H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of 3H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.
Animals↗
The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat.
The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat was established owing to the combined use of microdissection techniques and biochemical microassays. The density of 5-HT terminals in various areas of both structures was quantified first by measuring 5-HT levels in microdiscs of frozen tissue. Since the high affinity uptake process for 5-HT appeared undamaged in isotonic homogenates of previously frozen (--5 degrees C) tissues, it was possible to confirm the findings obtained with the measurement of 5-HT levels by also determining 5-HT uptake activity in these microdiscs. However, in the rat neostriatum, but not in the cat caudate nucleus, [3H]5-HT even at a very low extracellular concentration (4.4 -x 10(-8) M) was taken up not only by serotoninergic terminals but also to a significant extent by dopaminergic terminals. In presence of benztropine, this second component was suppressed and [3H]5-HT uptake activity could then be considered as a specific marker of serotoninergic terminals also in the neostriatum of the rat. In both species, 5-HT terminals were mainly localized in the ventrocaudal area of the structure. In this area, 5-HT levels were among the highest values found in the brain (17 ng/mg protein). The density of 5-HT terminals decreased progressively from the acudal to the rostral planes of the neostriatum in rats or the caudate nucleus in cats. The poorest area, i.e. the dorsorostral zone, contained about 4 times less 5-HT than the ventrocaudal zone of the structure. Electrolytic lesion of the dorsalis (B7) and centralis superior (B8) raphe nuclei during early life resulted in a large decrease of 5-HT levels (--90%) in various parts of the neostriatum of adult rats. The present findings might be of interest to further analyze the role of serotoninergic neurons in extrapyramidal functions.
Animals↗
Rat brain stem tryptophan hydroxylase: mechanism of activation by calcium.
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Animals↗
Acute changes in central serotonin metabolism induced by the blockade or stimulation of serotoninergic receptors during ontogenesis in the rat.
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Adenylyl Cyclases↗
Effects of halothane and nitrous oxide anaesthesia on 5-HT turn-over in the rat brain.
The effects of anaesthesia with halothane and nitrous oxide on the turn-over of 5-HT in the rat brain were analysed. The rate of 5-HT synthesis was estimated on the basis of 5-HTP accumulation following 5-HTP decarboxylase inhibition. Inhalation anaesthetics reduced the rate of 5-HT synthesis in the whole brain, this effect being particularly marked in the forebrain. In addition, the reduction of the rate of 5-HT synthesis was still detected after a large tryptophan loading indicating that halothane and nitrous oxide anaesthesia reduced the activity of tryptophan hydroxylase in vivo. The utilization of 5-HT was also delayed during anaesthesia since the rate of 5-HT disappearance following the inhibition of its synthesis at the decarboxylation step was decreased in the whole brain. In spite of this reduced turnover of 5-HT in the brain of rats exposed to inhalation anaesthetics, the 5-HIAA levels were permanently elevated. This was the result of a partial inhibition of the active elimination of the acidic metabolite from the brain during this kind of anaesthesia.
5-Hydroxytryptophan↗
Effects of benzodiazepines on the binding of tryptophan in serum. Consequences on 5-hydroxyindoles concentrations in the rat brain.
Benzodiazepines (diazepam, oxazepam, chlordiazepoxide) inhibited competitively the binding of tryptophan onto rat serum albumin. As a result, in vivo treatment with one of these compounds (chlordiazepoxide for instance) induced a significant increase in the concentration of free tryptophan in serum. These observations suggest that the high levels of both 5-hydroxyindoles and tryptophan which occurred in the rat brain after any benzodiazepine administration could be at least partly the consequence of its effect on tryptophan binding in blood.
Amino Acids↗