Alessandro Galli

Presynaptic kynurenate-sensitive receptors inhibit glutamate release

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and α7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30–100 nm) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3–10 nm), a selective α7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nm), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mm)‐induced release of transmitter or previously taken‐up [3H]‐D‐aspartate. This inhibition was not modified by glycine, N‐methyl‐d‐aspartate or subtype‐selective kainate receptor agents, while CNQX or DNQX (10 µm), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]‐kainate (high and low affinity) or [3H]‐AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1–100 nm) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate‐induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

Antinociception induced by systemic administration of local anaesthetics depends on a central cholinergic mechanism.

1 The antinociceptive effects of systemically‐administered procaine, lignocaine and bupivacaine were examined in mice and rats by using the hot‐plate, writhing and tail flick tests. 2 In both species all three local anaesthetics produced significant antinociception which was prevented by atropine (5 mg kg−1, i.p.) and by hemicholinium‐3 (1 μg per mouse, i.c.v.), but not by naloxone (3 mg kg−1, i.p.), α‐methyl‐p‐tyrosine (100 mg kg−1, s.c.), reserpine (2 mg kg−1, i.p.) or atropine methylbromide (5.5 mg kg−1, i.p.). 3 Atropine (5 mg kg−1, i.p.) which totally antagonized oxotremorine (40 μg kg−1, s.c.) antinociception did not modify morphine (5 mg kg−1, s.c.) or baclofen (4 mg kg−1, s.c.) antinociception. On the other hand, hemicholinium, which antagonized local anaesthetic antinociception, did not prevent oxotremorine, morphine or baclofen antinociception. 4 Intracerebroventricular injection in mice of procaine (200 μg), lignocaine (150 μg) and bupivacaine (25 μg), doses which were largely ineffective by parenteral routes, induced an antinociception whose intensity equalled that obtainable subcutaneously. Moreover, the i.c.v. injection of antinociceptive doses did not impair performance on the rota‐rod test. 5 Concentrations below 10−10 m of procaine, lignocaine and bupivacaine did not evoke any response on the isolated longitudinal muscle strip of guinea‐pig ileum, or modify acetylcholine (ACh)‐induced contractions. On the other hand, they always increased electrically‐evoked twitches. 6 The same concentrations of local anaesthetics which induced antinociception did not inhibit acetylcholinesterase (AChE) in vitro. 7 On the basis of the above findings and the existing literature, a facilitation of cholinergic transmission by the local anaesthetics is postulated; this could be due to blockade of presynaptic muscarinic receptors.

Thiokynurenates: a new group of antagonists of the glycine moduiatory site of the NMBA receptor

Several substituted derivatives of kynurenic acid were tested on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex present in the guinea pig myenteric plexus, on the binding of [3H]glycine and of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat cortical membranes and on the depolarization of mice cortical wedges induced by NMDA or quisqualic acid (QA). Kynurenic acid derivatives, having a chlorine (CI) or a fluorine atom in position 5 or 7 but not in position 6 or 8 had significantly lower IC50s than the parent compound when tested on the antagonism of glutamate-induced ileal contraction and in the glycine binding assay. A further significant increase in potency was obtained by substituting a thio group for the hydroxy group in position 4 of kynurenic acid: the IC50 was 160 +/- 20 microM of kynurenic acid and 70 +/- 15 microM of thiokynurenic acid in the myenteric plexus whereas these IC50s for glycine binding were 25 +/- 3 and 9 +/- 2 microM respectively. Several thiokynurenic acid derivatives were synthetized and showed an increased affinity for the glycine recognition site over the corresponding kynurenic acid derivatives. Glycine competitively antagonized the actions of the thiokynurenates in the ileum, in cortical wedges and on [3H]TCP binding. In this preparation, 7-Cl-thiokynurenic acid had an IC50 of 8 microM for antagonizing 10 microM NMDA-induced depolarization while 50% of the 10 microM QA depolarization was antagonized at 300 microM. Thus thiokynurenic acid derivatives seem to be a new group of potent and selective antagonists of strychnine-insensitive glycine receptors.

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

Quinoxalines interact with the glycine recognition site of NMDA receptors: studies in guinea‐pig myenteric plexus and in rat cortical membranes

1 The effects of several quinoxalines, including 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7,dinitroquinoxaline‐2,3‐dione (DNQX), and of two kynurenates, kynurenate (KYNA) and 7‐Cl‐kynurenate (7‐Cl‐KYNA), have been evaluated on the N‐methyl‐D‐aspartate (NMDA) receptors present in the guinea‐pig ileum myenteric plexus preparation and on the strychnine‐insensitive [3H]‐glycine binding sites of cortical membranes. 2 Quinoxalines and kynurenates antagonized in a non‐competitive manner L‐glutamate‐induced contraction. Their IC50s were (in μm): 5 for 7‐Cl‐KYNA, 7.5 for 6,7‐Cl‐3‐hydroxy‐2‐quinoxaline carboxylate (6,7‐Cl‐HQCA), 20 for DNQX, 50 for CNQX, 76 for KYNA and 125 for 3‐hydroxy‐2‐quinoxaline carboxylate (HQCA). 3 Glycine (5–50 μm) completely reversed the antagonism displayed by both quinoxalines and kynurenates. The interaction between glycine and the tested compounds appeared to be competitive in nature. 4 Quinoxalines and kynurenates displaced, in a concentration‐dependent manner, [3H]‐glycine from its strychnine‐insensitive binding sites present in rat cortical membranes. Their IC50s for this action were (in μm): 0.45 for 7‐Cl‐KYNA, 0.6 for 6,7‐Cl‐HQCA, 2.4 for DNQX, 3.5 for CNQX, 20 for KYNA and 40 for HQCA. 5 When the IC50s for the displacement effect of [3H]‐glycine binding were plotted against the IC50s obtained in the myenteric plexus, a significant correlation was found. 6 These data show that quinoxalines and kynurenates may antagonize the responses to L‐glutamate by interacting with the glycine recognition sites of the NMDA receptor ion channel complex.

A facilitatory effect of bicuculline on the enteric neurones in the guinea-pig isolated colon

1 Changes in the efficiency of the peristaltic reflex, acetylcholine (ACh) output and motor responses to transmural and periarterial nerve stimulation produced by bicuculline and y‐aminobutyric acid (GABA) receptor desensitization were investigated in the guinea‐pig isolated colon. 2 Bicuculline, at concentrations unable to affect spontaneous colonic motility and lacking anticholinesterase activity, produced a dose‐dependent increase of both the efficiency of the peristaltic reflex and the stimulated ACh output. Such effects could not be observed in GABA‐desensitized preparations. 3 A frequency‐dependent potentiation of the cholinergic excitatory and non‐adrenergic non‐cholinergic (NANC) inhibitory responses to transmural stimulation was also observed in the presence of bicuculline. Conversely bicuculline exhibited an inhibitory effect on the relaxation induced by periarterial nerve stimulation. 4 Acute GABA‐desensitization was unable to affect the contractile responses to transmural stimulation, the ACh output and the efficiency of the peristaltic reflex. On the contrary, desensitization was able to mimic the effects of bicuculline on the inhibitory responses to both transmural and periarterial nerve stimulation. 5 Our results are consistent with a significant role played by an intrinsic GABAergic pathway in the modulation of both cholinergic excitatory and NANC inhibitory neurones. The hypothesis is advanced that a feed‐back modulation carried out through bicuculline‐sensitive GABAergic synapses could operate during the propagation of peristaltic motor activity.

Inhibition of cholinesterase-associated aryl acylamidase activity by anticholinesterase agents : Focus on drugs potentially effective in Alzheimer's disease

The potency of a series of anticholinesterase (anti-ChE) agents and serotonin-related amines as inhibitors of the aryl acylamidase (AAA) activity associated with electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) and horse serum butyrylcholinesterase (BuChE) (EC 3.1.1.8) was examined and compared with the potency of the same compounds as ChE inhibitors. Neostigmine, physostigmine, BW 284C51, (+/-)-huperzine A, E2020, tacrine, edrophonium and heptyl-physostigmine were, in that order, the most potent in inhibiting eel AChE-associated AAA activity, their inhibitor constant (Ki) values being in the range 0.02-0.37 microM. The rank order of the same compounds as AChE inhibitors basically paralleled that of AAA, although they were in general stronger on AChE (Ki = 0.001-0.05). The peripheral anionic site inhibitors propidium and gallamine were inactive on AChE-associated AAA. Serotonin and its derivatives were slightly stronger on AAA (Ki = 7.5-30 microM) than on AChE (Ki = 20-140 microM). Tacrine (IC50 = 0.03 microM), diisopropylfluorophosphate (IC50 = 0.04 microM), heptyl-physostigmine (IC50 = 0.11 microM), physostigmine (IC50 = 0.15 microM) and tetra-iso-propylpyrophosphoramide (iso-OMPA) (IC50 = 0.75 microM) were the most potent in inhibiting horse serum BuChE-associated AAA activity. Serotonin and related amines were very weak on BuChE-associated AAA activity. These results indicate that the inhibitory potencies of the active site anti-ChE agents on the AAA activity associated with eel AChE and horse serum BuChE are closely correlated with their action on the respective ChE. In addition, the efficacy of tacrine, E2020, heptyl-physostigmine and (+/-)-huperzine A in the treatment of Alzheimers disease is unlikely to be related to the action of these drugs on ChE-associated AAA.

EFFECTS OF MORPHINE, PHYSOSTIGMINE AND RAPHE NUCLEI STIMULATION ON 5‐HYDROXYTRYPTAMINE RELEASE FROM THE CEREBRAL CORTEX OF THE CAT

1 The release of 5‐hydroxytryptamine (5‐HT) from the cerebral cortex and caudate nucleus of brainstem‐transected cats and from the cerebral cortex of rats anaesthetized with urethane was determined by radioenzymatic and biological assay. 2 The stimulation of nucleus linearis intermedius of raphe doubles the basal 5‐HT release in the caudate nucleus and increases it 3 fold in the cerebral cortex. The effects of the electrical stimulation of the raphe are potentiated by chlorimipramine. 3 Brain 5‐HT release is greatly increased by morphine hydrochloride (6 mg/kg i.v.) and by physostigmine (100 μg/kg i.v.), but not by dl‐DOPA (50 mg/kg i.v.). 4 It is suggested that the 5‐HT releasing action of physostigmine can contribute to some of its pharmacological effects such as the analgesic effect so far attributed exclusively to its indirect cholinomimetic activity. 5 The 5‐HT releasing action of physostigmine seems unrelated to its anticholinesterase activity.

Glycine and kynurenate modulate the glutamate receptors in the myenteric plexus and in cortical membranes.

The responses evoked by stimulation of the N-methyl-D-aspartate receptors in the guinea-pig myenteric plexus were potentiated by micromolar concentrations of glycine and were non-competitively antagonized by kynurenate (IC50: 60 microM). The effects of kynurenate were competitively prevented by glycine. Furthermore, kynurenate displaced [3H]glycine from its binding sites on rat cortical membranes (IC50: 20 microM). Kynurenate and glycine, therefore, probably act at the same site, evoking opposite effects on the function of the ion channel complex of the N-methyl-D-aspartate receptor.

Synthesis of a set of ethyl 1-carbamoyl-3-oxoquinoxaline-2-carboxylates and of their constrained analogue imidazo[1,5-a]quinoxaline-1,3,4-triones as glycine/NMDA receptor antagonists

The synthesis and glycine/NMDA and AMPA receptor affinities of a set of ethyl (+/-) 1-N-carbamoyl-1,2,3,4-tetrahydro-3-oxoquinoxaline-2-carboxylates 1-11 and those of their constrained analogue (+/-) 1,2,3,3a,4,5-hexahydroimidazo[1,5-a]quinoxaline-1,3,4-triones 12-24 are reported. Compounds 1-11 bear a side-chain at position 1 which has been spatially constrained in compounds 12-24. Most of the reported tricyclic derivatives 12-24 showed glycine/NMDA binding activity comparable to that of their corresponding bicyclic analogues 1-11 providing further evidence that the spatial orientation of the side-chain is an important structural requirement for glycine/NMDA receptor antagonists.