Anita Gemignani

Noradrenaline inhibits central serotonin release through alpha2-adrenoceptors located on serotonergic nerve terminals

SummaryThe effect of noradrenaline on the depolarization-evoked release of 3H-5-hydroxytryptamine was investigated in superfused synaptosomes prepared from rat cortex and hippocampus and prelabelled with the radioactive indoleamine. Noradrenaline reduced in a concentration-dependent way the release of 3H-5-hydroxytryptamine elicited by 15 mM KCl. The inhibition was counteracted by the alpha-adrenoceptor antagonists phentolamine or yohimbine, but not by prazosin. The results indicate that, in rat brain, the inhibition of 5-hydroxytryptamine release by noradrenaline is mediated by adrenoceptors of the alpha2-type localized on the terminal serotonergic fibres.

CGP 52432 : a novel potent and selective GABAB autoreceptor antagonist in rat cerebral cortex

As previously reported GABAB receptors are heterogeneous. Three pharmacologically distinct receptor subtypes mediating inhibition of gamma-aminobutyric acid (GABA), glutamate or somatostatin release, respectively, exist on axon terminals of rat cerebral cortex. We investigated the novel GABAB receptor antagonist, [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxy-methyl) phosphinic acid (CGP 52432), on the above receptor subtypes. The effects of (-)-baclofen on the K(+)-evoked release of GABA, glutamate or somatostatin from rat cortical synaptosomes were antagonized by CGP 52432. The IC50 of the drug at GABA autoreceptors (0.085 microM) was 35- and 100-fold lower than at the receptors regulating somatostatin and glutamate overflow, respectively. At the autoreceptor the calculated pA2 for CGP 52432 amounted to 7.70, which makes the drug about 1000-fold more potent than phaclofen at this receptor. The potency and selectivity characteristics of CGP 52432 indicate that the drug is by far the most appropriate tool to investigate the terminal GABAB autoreceptors of the rat cerebral cortex.

α2-Adrenoceptors in rat hypothalamus and cerebral cortex: functional evidence for pharmacologically distinct subpopulations

The presynaptic alpha 2-adrenoceptors regulating, respectively, [3H]noradrenaline and [3H]5-hydroxytryptamine release were compared in experiments with noradrenaline and clonidine as agonists and the two enantiomers of mianserin as antagonists in rat hypothalamic and cortical synaptosomes depolarized with 15 mM KCl. The affinity of clonidine was 10 times higher at the alpha 2-autoreceptors than at the alpha 2-heteroreceptors. (-)Mianserin antagonized noradrenaline at the heteroreceptors but not at the autoreceptors. In contrast, (+)mianserin did not discriminate between the two receptors. The results support the existence in the rat brain of subtypes of alpha 2-adrenoceptors having different neuronal location, function and pharmacological properties.

Evidence for functional native NMDA receptors activated by glycine or d‐serine alone in the absence of glutamatergic coagonist

In this study we have examined the effects of N‐methyl‐d‐aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin‐like immunoreactivity (CCK‐LI) and of somatostatin‐like immunoreactivity (SRIF‐LI) elicited by 12 mm K+ from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration‐dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration‐dependent manner the depolarization‐evoked release of both CCK‐LI and SRIF‐LI. This effect of glycine was strychnine‐insensitive and could be mimicked by d‐serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/d‐serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by d‐serine was strongly inhibited by ifenprodil (0.3 μm) and by Zn2+ ions (50 nm), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK‐ and SRIF‐releasing neurons possess non‐conventional NMDA receptors whose channels can be operated by glycine or d‐serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B.

The HIV-1 coat protein gp120 and some of its fragments potently activate native cerebral NMDA receptors mediating neuropeptide release

The objective of this study was to investigate the effects of the HIV‐1 envelope protein gp120 and its peptide fragments on the function of N‐methyl‐ d‐aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or d‐serine ‘only’. The release of cholecystokinin‐like immunoreactivity (CCK‐LI) and of somatostatin‐like immunoreactivity (SRIF‐LI) elicited by 12 mm K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU‐C‐34‐A, but not by RP‐135 (a central portion of BRU‐C‐34‐A). The EC50 values of gp120 were 0.02 nm (CCK‐LI release) and 0.01 nm (SRIF‐LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK‐ and SRIF‐releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near‐maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.

Differential blockade by (−)mianserin of the alpha2-adrenoceptors mediating inhibition of noradrenaline and serotonin release from rat brain synaptosomes

SummaryMianserin and its two enantiomers were studied as antagonists of the alpha2-adrenoceptors mediating inhibition of noradrenaline and 5-hydroxytryptamine release in rat brain cortex. The inhibitory effect of exogenous noradrenaline on the release of 3H-noradrenaline evoked by 15 mM KCl from superfused cortical synaptosomes was antagonized by racemic mianserin and by (+)mianserin; the (−)enatiomer was ineffective. In contrast, both (+)mianserin and (−)mianserin antagonized the inhibitory effect of noradrenaline on the release of 3H-5-hydroxytryptamine. The results suggest that the alpha2-autoreceptors on noradrenergic nerve endings differ from the alpha2-adrenoceptors located on serotoninergic terminals.

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l‐Glutamate, NMDA, dl‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin‐like immunoreactivity (SRIF‐LI) from primary cultures of rat hippocampal neurons. In Mg2+‐containing medium, the maximal effects (reached at ∼100 µM) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µM (AMPA), 39 µM (glutamate), 41 µM (KA), and 70 µM (NMDA). The metabotropic receptor agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylate did not affect SRIF‐LI release. The release evoked by glutamate (100 µM) was abolished by 10 µM dizocilpine (MK‐801) plus 30 µM 1‐aminophenyl‐4‐methyl‐7,8‐methylenedioxy‐5H‐2,3‐benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK‐801 but insensitive to GYKI 52466. The AMPA‐ and KA‐evoked releases were blocked by 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) or by GYKI 52466 but were insensitive to MK‐801. The release of SRIF‐LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA‐evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF‐LI release, an effect abolished by MK‐801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.

Human brain somatostatin release from isolated cortical nerve endings and its modulation through GABAB receptors.

1 The release of somatostatin‐like immunoreactivity (SRIF‐LI) in the human brain was studied in synaptosomal preparations from fresh neocortical specimens obtained from patients undergoing neurosurgery to remove deeply sited tumours. 2 The basal outflow of SRIF‐LI from superfused synaptosomes was increased about 3 fold during exposure to a depolarizing medium containing 15 mM KCl. The K+‐evoked overflow of SRIF‐LI was almost totally dependent on the presence of Ca2+ in the superfusion medium. 3 The GABAB receptor agonist, (—)‐baclofen (0.3–100 μm), inhibited the overflow of SRIF‐LI in a concentration‐dependent manner (EC50 = 1.84 ± 0.20 μm; maximal effect: about 50%). The novel GABAB receptor ligand, 3‐aminopropyl(difluoromethyl)phosphinic acid (CGP 47656) mimicked (—)‐baclofen in inhibiting the SRIF‐LI overflow (EC50 = 3.06 ± 0.52 μm; maximal effect: about 50%), whereas the GABAA receptor agonist, muscimol, was ineffective up to 100 μm. 4 The inhibition by 10 μm (—)‐baclofen of the K+‐evoked SRIF‐LI overflow was concentration‐dependently prevented by two selective GABAB receptor antagonists, 3‐amino‐propyl (diethoxymethyl)‐phosphinic acid (CGP 35348) (IC50 = 24.40 ± 2.52 μm) and [3‐[[(3,4‐dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) (IC50 = 0.06 ± 0.005 μm). 5 The inhibition of SRIF‐LI overflow caused by 10 μm CGP 47656 was abolished by 1 μm CGP 52432. 6 When human synaptosomes were labelled with [3H]‐GABA and depolarized in superfusion with 15 mM KCl, the inhibition by 10 μm (—)‐baclofen of the depolarization‐evoked [3H]‐GABA overflow was largely prevented by 10 μm CGP 47656 which therefore behaved as an autoreceptor antagonist. 7 In conclusion: (a) the characteristics of SRIF‐LI release from synaptosomal preparations of human neocortex are compatible with a neuronal origin; (b) the nerve terminals releasing the neuropeptide possess inhibitory receptors of the GABAB type; (c) these receptors differ pharmacologically from the GABAB autoreceptors present on human neocortex nerve terminals since the latter have been shown to be CGP 35348‐insensitive but can be blocked by CGP 47656.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings.

It was previously reported that the K+-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or D-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or D-serine on the K+-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K+-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca2+-free medium and with voltage-sensitive Ca2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30-50%) dependent on external Ca2+ and Ca2+ channel activation. In contrast, the alkalinization-evoked release of [3H]noradrenaline was highly sensitive to external Ca2+ removal and to blockade of Ca2+ channels with omega-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.

Presynaptic Receptors Modulating Transmitter Release: Physiological and Pharmacological Aspects

Abstract The mammalian brain contains a myriad of receptors localized on neuronal axon terminals (presynaptic receptors) the function of which is to modulate neurotransmitter release. Similarly to postsynaptic receptors, presynaptic receptors exist as pharmacologically distinct types and subtypes. Inasmuch as modulation of release is the functional response that follows their activation, transmitter release represents an excellent model to investigate receptors in general as well as to test potentially novel and more selective drugs. Types and subtypes of presynaptic receptors regulating the release of various transmitters will be described and their potential involvement in different pathophysiological conditions will be discussed.