Pharmacological Evidence for the Modulation of Monoamine Release by Adenosine in the Invertebrate Nervous System

An in vitro preparation from the pedal ganglia of the marine bivalve, Mytilus edulis, was used to examine the modulation of transmitter release by adenosine and its analogs from invertebrate nervous tissue. The ganglia of this organism contain the monoamines dopamine (DA), serotonin (5-HT), and norepinephrine (NE), and the presynaptic release of these substances is known to be calcium-dependent. This organism also contains a DA-sensitive adenylate cyclase system which resembles that seen in mammals. Neural tissue from the pedal ganglia was incubated with labeled monoamines, and release studies were then conducted in superfusion chambers; release of monoamines was evoked by the addition of 50 mM KCl. Addition to the superfusion medium of the adenosine analog, 5'-N-ethylcarboxamidoadenosine (NECA; 10 nM), inhibited the release of 5-HT and DA, and to a lesser extent NE, whereas 100-fold higher concentrations of adenosine itself and the adenosine analog, R-N6-phenylisopropyladenosine, were required to achieve comparable levels of inhibition. The inhibitory effects of NECA on neurotransmitter release were blocked by the adenosine receptor antagonist, theophylline (IC50 = 10-14 microM). The results from this study indicate for the first time the possible role of adenosine as a modulator of neurotransmitter release in the invertebrate nervous system.     

EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND CATECHOLAMINES

—Intraperitoneal injections of GABA (5 mg/kg) to rats lowered the level of norepinephrine in brain, heart and spleen but not suprarenals and raised that of serotonin in brain. Changes of these monoamines were most pronounced in the hypothalamic region after 20min. A reduction of hypothalamic norepinephrine was also observed 15min following the intracarotid administration of 0·5 mg/kg of GABA. In these experiments there was a concomitant increase in the level of free GABA in the anterior portion of the ventral hypothalamus. Brain dopamine level and 5-hydroxytryptophan decarboxylase, dihydroxyphenylalanine decarboxylase and monoamine oxidase activities were not affected. The 20 per cent increase of endogenous GABA observed in the midbrain 30 min following the administration of amino-oxyacetic acid was accompanied by a sharp fall in norepinephrine level (39 per cent) and an increase in serotonin (20 per cent). In in vitro studies 10–300 μg/ml of GABA were shown to release norepinephrine from cortical and hypothalamic slices, and to inhibit serotonin release without affecting 5-hydroxytryptophan uptake and to have no effect on the release of dopamine from slices of the region of the corpus striatum nor on the activity of the enzymes mentioned. Subcellular studies showed that the particulate:supernatant ratio for norepinephrine was reduced from a control value of 2·04 to 1·75 and that of serotonin was raised from 2·8 to 3·5. Following pretreatment with iproniazid, GABA reduced the raised level of brain norepinephrine to a greater extent than reserpine but not as intensively as amphetamine. The results obtained suggest that these monoamines may be involved in the mechanisms underlying the action of GABA in brain and that the effect of GABA on brain monoamines may be of certain significance in synaptic events.     

ON THE INVOLVEMENT OF INORGANIC IONS IN THE EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND NOREPINEPHRINE

Abstract— The loss of GABA, norepinephrine and serotonin and the uptake of GABA (in the presence of 1 mM-GABA) and the effect of GABA on the loss of norepinephrine and serotonin were investigated in rat midbrain slices incubated in media of various compositions. In a medium of low Na⁺ concentration the loss of serotonin from incubated slices was markedly inhibited while that of norepinephrine and GABA was significantly increased. Conversely the most pronounced loss of serotonin from slices was observed on the addition of ouabain to a medium of a balanced ionic composition. Whereas the loss of serotonin from slices increased in a medium of high K⁺ content, it was significantly reduced after 45 min incubation in a high K⁺-low Na⁺ medium. In all the modified media used, a significant loss of norepinephrine was observed while that of GABA was not affected by the omission of Ca2⁺ and was slightly reduced in the absence of K⁺. GABA enhanced the loss of norepinephrine and inhibited that of serotonin in a high-K⁺ medium and in one with a balanced ionic composition. A deficiency of Na⁺ in the medium had a differential effect on the loss of norepinephrine and serotonin similar to that observed with 1 mM-GABA. These results suggest that Na⁺ may be of crucial importance in the release of serotonin from midbrain slices and that an enhancement of the Na+ extrusion mechanism at the synaptosomal level may be involved in the effect of GABA on brain monoamines.     

Mutual modulating effects of serotonin and dopamine on neurons of rat spinal ganglia

It was established in experiments on isolated rat spinal ganglia that the introduction of dopoamine (0.01–1.0 µM) into a superfusate potentiates the depolarizing responses of the neurons evoked by the action of serotonin, which is delivered from a micropipette under pressure, while the addition of serotonin in the same concentrations potentiates the depolarizing responses of the neurons evoked by the action of dopamine. The mutual potentiation of the effects of dopamine and serotonin depends on the concentration of the monoamines and is eliminated by blockers of the D₁- (but not D₂-dopamine) and type 2 serotonin (but not IA) receptors. The mutual potentiation of the effects of monoamines is of a postsynaptic nature and is associated with a change in the intracellular concentration of second messengers (Ca²⁺ and cAMP).A. M. Gor'kii Donetsk Medical Institute, Ministry of Health of the Ukrainian SSR. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 168–173, March–April, 1991.     

Release of dopamine and serotonin from Limax ganglia in vitro

1. We wish to establish the kinetics of serotonin and dopamine release from Limax cerebral and buccal ganglia and find selective treatments to modify their release kinetics.2. The release of dopamine and serotonin from isolated ganglia was stimulated by high potassium exposure with and without prior treatment of ganglia with 6-hydroxydopamine (6-OHDA).3. Single ganglia release significant quantities of monoamines during a single 5 min high K⁺ exposure. Multiple high K⁺ exposures deplete a readily releasable transmitter store with little effect on storage pools.4. 6-OHDA exposure depletes readily releasable DA with little effect on total ganglion DA content or on serotonin.5. Feeding motor program responsiveness is suppressed reversibly by whole ganglion high K⁺ treatment.     

Effect of noradrenalin and serotonin on spontaneous unit activity in hippocampal area CA3 in vitro

The effect of noradrenalin and serotonin on spontaneous unit activity in hippocampal slices (area CA₃)in vitro was studied by the addition of mediators to the incubation medium. Both drugs were found to have both an inhibitory (32 and 49%, respectively) and an activating (52 and 35%, respectively) action. The effect of noradrenalin correlated with the type of spontaneous unit activity. Mainly cells without spontaneous discharges or with single irregular discharges were activated Cells with "complex discharges" were inhibited. Unlike the effect of serotonin, the activating action of noradrenalin was stimulated by the agonist phenylephrine and inhibited by the -adrenoblocker phentolamine. The action of the monoamines persisted in medium with an increased Mg⁺⁺ concentration.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 482–490, September–October, 1981.     

Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata

Metabolism of the major monoamines and their functions were studied in the freshwater snail Biomphalaria glabrata. In both juvenile and adult snails, the plasma (cell-free hemolymph) appears to act as a reservoir for most of these monoamines and their metabolites including among others, L-dopa and dopamine as major constituents. Significant quantities of L-tryptophan, precursor of indoleamines, also was found in the plasma. L-dopa, serotonin, homovanillic acid and dopamine were prominently represented in the central nervous system of the snail, while serotonin and its metabolites, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were found in the ovotestis. Catecholamines such as L-dopa, dopamine and homovanillic acid were identified in the albumen gland. Functional aspects of both dopamine and serotonin were studied using in vitro cultures of albumen glands, the site of perivitelline fluid and galactogen synthesis in B. glabrata. Dopamine was found to stimulate the release of secretory proteins when exogenously added to gland cultures and this process was inhibited by chlorpromazine, a dopamine receptor antagonist. Similarly, exogenous serotonin stimulated in vitro protein secretion by albumen glands. Thus, these results suggest that monoamines may play important roles in regulating reproductive activity of this snail and provides an excellent model for studying neurotransmitter function and metabolism in molluscs.     

The calcium-dependent neuronal release of serotonin and its antagonism by lithium

The cilio-excitatory serotonergic innervation of lateral gill cilia of Mytilus edulis was studied in vivo. Peripheral serotonin release was dependent on the external calcium concentration. Serotonin release was inhibited by autodialyzing calcium from the tissue or by increasing the calcium concentration in the medium, as determined by measuring ciliary activity stroboscopically and by biochemical and radioassays of serotonin. Lithium also inhibited serotonin release when added to the external bathing medium. Concomitantly, altering calcium concentrations altered the degree of inhibition of serotonin release caused by lithium. The study demonstrates that the terminal release of the monoamine serotonin is a calcium-dependent mechanism. The pharmacological effects of lithium in this system appear to be interrelated with the calcium-dependent releasing mechanism.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

The influence of chronic neurotization on the monoaminergic systems of various brain structures in rats with various typological characteristics

Typological behavioral features of Wistar rats were tested in the open field and in Porsolt test. Rats were assigned to groups with high (HAct), medium (MAct), and low (LAct) behavioral activities. The same rats were assigned to high (HDep), medium (MDep) and low depressive (LDep) groups. The release of norepinephrine, dopamine, serotonin and their metabolites in homogenates obtained from the hypothalamus, hippocampus, frontal cortex and amygdala was assessed by microdialysis and HPLC. In these groups, the monoamine concentrations were different: the level of serotonin was higher in the hypothalamus and norepinephrine and 5-HIAA levels were lower in the hippocampus of MAct - MDep rats as compared to LAct - HDep. Chronic neurotization caused changes in monoamine concentrations in the hypothalamus and amygdala in rats of all groups, whereas in the hippocampus and frontal cortex monoamine changes were observed in HAct - LDep and LAct -HDep rats. The most prominent changes in monoamines levels in neurotized rats with different types of behavior were found in the frontal cortex, amygdala and hippocampus. The results show a correlation between the typological of behavioral characteristics and the reaction to stress of monoaminergic systems of the hypothalamus, hippocampus, frontal cortex and amygdala.     

Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.     

Neurochemical changes following the administration of depleters of biogenic monoamines.

A variety of drugs which were thought to selectively effect the synthesis, release or reuptake of the biogenic monoamines, and thereby deplete their stores in brain, were administered to rats. The animals were given intraperitoneal injections of reserpine, α-methyl-meta-tyrosine, α-methyl-para-tyrosine, para-cholorophenylalanine and tetrabenazine, and were killed at intervals known to cause maximum depletion of serotonin, dopamine and norepinephrine. When compared to saline controls, the drugs depleted the respective monoamines, as expected, but changes in the levels of acetylcholine, alanine, glycine, γ-aminobutyric acid, and aspartate and glutamate were also observed.     

Venom effects on monoaminergic systems

The monoamines, dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin and tyramine serve many functions in animals. Many different venoms have evolved to manipulate monoaminergic systems via a variety of cellular mechanisms, for both offensive and defensive purposes. One common function of monoamines present in venoms is to produce pain. Some monoamines in venoms cause immobilizing hyperexcitation which precedes venom-induced paralysis or hypokinesia. A common function of venom components that affect monoaminergic systems is to facilitate distribution of other venom components by causing vasodilation at the site of injection or by increasing heart rate. Venoms of some scorpions, spiders, fish and jellyfish contain adrenergic agonists or cause massive release of catecholamines with serious effects on the cardiovascular system, including increased heart rate. Other venom components act as agonists, antagonists or modulators at monoaminergic receptors, or affect release, reuptake or synthesis of monoamines. Most arthropod venoms have insect targets, yet, little attention has been paid to possible effects of these venoms on monoaminergic systems in insects. Further research into this area may reveal novel effects of venom components on monoaminergic systems at the cellular, systems and behavioral levels.     

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.     

The activity of the monoaminergic systems of the rat hypothalamus in acute stress after chronic stressing]

The influence of chronic stress (footshock combined with randomized light flashes) on acute stress-induced (immobilization) release of noradrenaline, dopamine and serotonin in rat lateral hypothalamus was assessed by microdialysis. The chronic stress resulted in an increase and prolongation of the acute stress-induced release of noradrenaline but not of dopamine and serotonin. The increased rate of accumulation of dioxyphenylacetic acid and unchanged accumulation of homovanillic acid (dopamine metabolites) and dopamine during and after the acute stress in chronically stressed animals reflect a rise of synthetic activity of catecholaminergic systems in response to acute stress and reuptake increase. Marked stress-induced increase in hydroxyindoleacetic acid in chronically stressed rats without any changes in the ST dynamics may be regarded in a similar way. A significant increase in potassium-stimulated release of all the studied monoamines was found while their basal level remained unchanged. The conclusions was made that the hyperergic release of neurotransmitters may be the basis of an inadequate response of animals to acute stress, i.e., one of the neurotic symptoms.     

Effects of chronic exposure to ammonia concentrations on brain monoamines and ATPases of Nile tilapia (Oreochromis niloticus)

The effects of chronic exposure to total ammonia nitrogen (TAN) concentrations on the brain monoamines and ATPases of Nile tilapia, Oreochromis niloticus fingerlings, were studied. The period of exposure was 70 consecutive days, and the initial weight of the fingerlings was 18 ± 2.1 g. In addition to the control, three treatment groups exposed to 2.5 (low), 5 (medium), and 10 (high) mg TAN L^?1 concentrations were tested. The unionized ammonia nitrogen (NH₃) levels calculated in mg L^?1 were 0.059, 0.185, and 0.575 in aquaria at 26 °C. The brain monoamines were serotonin (5-HT), dopamine (DA), and norepinephrine (NE), as well as their derivatives, 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC). Compared with the controls, the levels of brain monoamines and Na⁺/K⁺- and Ca²⁺-ATPase activities were not significantly altered in fish exposed to low TAN concentration. However, there was a significant decrease in 5-HT, DA, and NE levels, and a significant increase in both serotonergic (5-HIAA/5-HT) and dopaminergic (DOPAC/DA) activities of fish exposed to medium TAN and high TAN concentrations. The activities of brain Na⁺/K⁺- and Ca²⁺-ATPases of fish exposed to medium TAN and high TAN concentrations significantly increased, while Mg²⁺-ATPase did not significantly change compared with that of the controls. The quantity of the detected alterations increased in fish exposed to high TAN concentration.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure

 Since definitive morphological studies showing the luminal release of serotonin have not been reported, we used a perfused system which allows physiological monitoring and biochemical as well as morphological evidence indicating release of serotonin from enterochromaffin cells. Isolated vascularly and luminally perfused rat duodenums exposed to 5–35 cmH₂O of luminal pressure were measured for release of serotonin into the blood vessels and intestinal lumen. Immediately after raising the luminal pressure, the duodenum was fixed for immunoelectron microscopic localization of serotonin. Peristaltic contraction and serotonin content of the perfusates were continuously measured. The luminal release of serotonin increased with elevated intraluminal pressure, but the vascular release of serotonin was not altered. Tetrodotoxin had no effect on the pressure-stimulated luminal serotonin release. Enterochromaffin cells in control animals without increased luminal pressure contained immunogold-labeled secretory granules in the apical and basal cytoplasm. After intraluminal pressure increased, many apical secretory granules were no longer dense and immunogold particles were localized over the cytoplasmic matrix and microvilli. These findings indicate that luminal serotonin release is increased after raising the intraluminal pressure and serotonin, normally stored in the secretory granules of enterochromaffin cells, appears to be released into the cytoplasmic matrix and then diffuses or is transported into the intestinal lumen. Accepted: 15 January 1997     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro

The effect of serotonin and adrenaline antagonists was tested on the early embryos of mice of three lines. All the substances tested produced an arrest or inhibition of cleavage division and the appearance of anomalies. Serotonin introduced in the incubation medium was effective against some serotoninolytics. We were unable to test the protective effect of adrenaline, as in the concentrations used it has its own effect on the development. From the data obtained, a conclusion is made of the existence in early mouse embryos of the structures sensitive to serotonin and adrenaline antagonists. The assumptions is made from the previously obtained data on the presence of biogenic monoamines in early mouse embryos, of functional activity of prospective mediators of the nervous system at the earliest stages of embryonic development of mammals.