Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

Homomeric and heteromeric -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1_o and GluR3_o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99–109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[ ]AMPA binding experiments, high expression was seen (B_max=0.8–3.8 pmol/mg protein) along with high affinity binding to a single site (K_d, nM±S.D.): GluR1_o, 32.5±2.7; GluR3_o, 23.7±2.4; GluR1_o+GluR3_o, 18.1±2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues> -glutamate>quinoxaline-2,3-diones>kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3_o vs. GluR1_o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3_o and heteromeric receptors than at homomeric GluR1_o, suggesting that its efficacy and/or desensitisation properties are different at GluR1_o vs. GluR3_o.     

The effects of cyclopentane and cyclopentene analogues of GABA at recombinant GABA(C) receptors.

The pharmacological effects of the enantiomers of cis-3-aminocyclopentanecarboxylic acids ((+)- and (−)-CACP), the enantiomers of trans-3-aminocyclopentanecarboxylic acids ((+)- and (−)-TACP), and the enantiomers of 4-aminocyclopent-1-ene-1-carboxylic acids ((+)- and (−)-4-ACPCA) were studied on human homomeric ρ₁ and ρ₂ GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp methods. These compounds are conformationally restricted analogues of γ-aminobutyric acid (GABA) held in a five-membered ring. (+)-TACP (EC₅₀ (ρ₁)=2.7±0.2 μM; EC₅₀ (ρ₂)=1.45±0.22 μM), (+)-CACP (EC₅₀ (ρ₁)=26.1±1.1 μM; EC₅₀ (ρ₂)=20.1±2.1 μM) and (−)-CACP (EC₅₀ (ρ₁)=78.5±3.5 μM; EC₅₀ (ρ₂)=63.8±23.3 μM) were moderately potent partial agonists at ρ₁ and ρ₂ GABA_C receptors, while (−)-TACP (100 μM inhibited 56% and 62% of the current produced by 1 μM GABA at ρ₁ and ρ₂ receptors, respectively) was a weak partial agonist with low intrinsic activity at these receptors. In contrast, (+)-4-ACPCA (K_i (ρ₁)=6.0±0.1 μM; K_i (ρ₂)=4.7±0.3 μM) did not activate GABA_C ρ₁ and ρ₂ receptors but potently inhibited the action of GABA at these receptors, while (−)-4-ACPCA had little effect as either an agonist or an antagonist. The affinity order at both GABA_C ρ₁ and ρ₂ receptors was (+)-TACP>(+)-4-ACPCA(+)-CACP>(−)-CACP(−)-TACP(−)-4-ACPCA. This study shows that the cyclopentane and cyclopentene analogues of GABA affect GABA_C receptors in a unique manner, defining a preferred stereochemical orientation of the amine and carboxylic acid groups when binding to GABA_C receptors. This is exemplified by the partial agonist, (+)-TACP, and the antagonist, (+)-4-ACPCA.     

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

Antagonists, but not agonists, of the 5-HT₃ receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT_3A and 5-HT_3AB receptors with an R_max (I_max / I_max5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC₅₀ = 16 and 27 μM, and K_i for displacement of [³H]granisetron binding = 0.8 and 1.8 μM for 5-HT_3A and 5-HT_3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT_3A receptors tryptamine is a weak (R_max = 0.15), low affinity (EC₅₀ = 113 μM; K_i = 4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC_50 = 8.1 μM; K_i = 2.7 μM) but is a very weak partial agonist (R_max = 0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.     

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1–12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu_{1–5, 7,8}). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu₁ or mGlu₅ receptors with K_i-values of 7.0 and 7.6 μM, respectively. When tested against 10 μM -glutamate-stimulated Ca²⁺ mobilisation in rat mGlu₅ expressing CHO cells, it produced substantial or complete block at a concentration of 100 μM. In rat hippocampal slices, LY341495 eliminated 30 μM DHPG-stimulated PI hydrolysis and 100 μM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 μM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.     

Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 μM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC₅₀ values of 1.77 μM (GluR1_i), 0.22 μM (GluR2_i), 0.56 μM (GluR2_o), 1.89 μM (GluR3_i) and 0.20 μM (GluR4_i) for LY392098 and EC₅₀ values of 5.65 μM (GluR1_i), 0.15 μM (GluR2_i), 1.44 μM (GluR2_o), 1.66 μM (GluR3_i) and 0.21 μM (GluR4_i) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3–10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro.     

Pharmacology of ACEA-1416: a potent systemically active NMDA receptor glycine site antagonist

Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1,4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites (K_b = 7.9 nM in oocytes, K_b = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower (K_b = 3.5 μM in oocytes, K_b = 1.6 μM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites (K_b > 5.9 μM) and metabotropic glutamate receptors (K_b > 57 μM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at 2 min and the biological half-time of protection was 60 min. The ED₅₀ measured at peak efficacy was 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.     

S 14297, a novel selective ligand at cloned human dopamine D3 receptors, blocks 7-OH-DPAT-induced hypothermia in rats

The selective dopamine D₃ receptor agonist, 7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and the novel naphthofurane, S 14297 ((+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho,2,3-furanel]) bound with high affinity and selectivity to recombinant, human dopamine D₃ versus D₂ receptors stably transfected into Chinese hamster ovary cells: K_i values = 2 versus 103 nM for 7-OH-DPAT and 13 versus 297 nM for S 14297. In contrast, the putative dopamine D₃ receptor antagonist, AJ 76 (cis-(+)-5-methoxyl-1-methyl-2- (n-propylamino)tetralin), displayed low affinity and selectivity for dopamine D₃ versus D₂ sites (70 versus 154 nM). 7-OH-DPAT (0.01–0.16 mg/kg s.c.) provoked hypothermia in rats, and action abolished by S 14297 (0.04–0.63 mg/kg s.c.) and, less potently, by AJ 76 (0.16–2.5 mg/kg s.c.) S 14297 (20.0 mg/kg s.c.) did not modify prolactin secretion. These data suggest that dopamine D₃ receptors mediate hypothermia in the rat and that S 14297 acts as a selective antagonist at these sites.     

Evidence that (-)-7-hydroxy-4'-dimethylheptyl-cannabidiol activates a non-CB(1), non-CB(2), non-TRPV1 target in the mouse vas deferens

Previous experiments showed that R-(+)-WIN55212-induced inhibition of electrically-evoked contractions of mouse vasa deferentia could be antagonized by cannabidiol in a manner that appeared to be competitive but not to involve direct competition for established cannabinoid receptors. We have now discovered that (−)-7-hydroxy-4′-dimethylheptyl-cannabidiol (7-OH-DMH-CBD) inhibits electrically-evoked contractions of the vas deferens (EC₅₀ = 13.3 nM). This it appeared to do by acting on prejunctional neurones as 100 nM 7-OH-DMH-CBD did not attenuate contractile responses to phenylephrine or β,γ-methylene-ATP. Although 7-OH-DMH-CBD was antagonized by SR141716A, it was less susceptible to antagonism by this CB₁ receptor antagonist than R-(+)-WIN55212. 7-OH-DMH-CBD was also antagonized by cannabidiol (1 μM; apparent K_B = 222.2 nM) but not by the CB₂ receptor antagonist, SR144528 (32 nM), or by naloxone (300 nM), ruthenium red (1 μM) or capsazepine (10 μM). Yohimbine (100 nM) enhanced the ability of 7-OH-DMH-CBD to inhibit electrically-evoked contractions. R-(+)-WIN55212 was also potentiated by 100 nM yohimbine, possibly reflecting ongoing sequestration of G_i/o proteins from CB₁ receptors by ₂-adrenoceptors. Our results suggest that 7-OH-DMH-CBD may activate a neuronal target in the vas deferens that is not a CB₁, CB₂, TRPV1, opioid or ₂-adrenergic receptor but do not exclude the possibility that it also activates CB₁ receptors.     

4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion

4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8.3 mM glucose, was compared to that of (1) its minor isomer (2R,3R,4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 μM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 μM for (2S,3R,4S) 4-hydroxyisoleucine, 500 μM for (2S,4R) and (2S,4S) γ-hydroxynorvalines as well as (2S,3S) and (2S,3R) γ-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon in S-configuration, full methylation and carbon γ-hydroxylation.     

5-HT3 receptors augment neuronal, cholinergic contractions in guinea pig trachea European Journal of Pharmacology, 234 (1993) 109–112

Serotonin (5-HT) and the selective 5-HT₃ receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC₅₀ = 3.5 μM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC₅₀ = 7.4 μM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT₃ receptor antagonist, zacopride (apparent pA₂ = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 μM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 μM) but contractions were blocked by atropine (1 μM). These results suggest that excitatory 5-HT₃ receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.     

Activity of 2,3-benzodiazepines at Native Rat and Recombinant Human Glutamate Receptors In Vitro: Stereospecificity and Selectivity Profiles

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYK152466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 μM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate ₅₀ values of 18 μM, 24 μM and 6 μM, respectively and AMPA (10 μM) responses in recombinant human GluR4 expressing HEK293 cells with approximate ₅₀ values of 22 μM, 28 μM and 5 μM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate ₅₀ values of 10 μM, 8 μM and 1.5 μM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (−) isomer of each compound. At a concentration of 100 μM, GYKI52466, LY293606 and LY300168 produced <30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 μM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 μM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate ₅₀ value of 1 μM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor. © 1997 Elsevier Science Ltd. All rights reserved.     

Blockade of 5-HT(3) receptor with MDL 72222 and Y 25130 reduces beta-amyloid protein (25--35)-induced neurotoxicity in cultured rat cortical neurons

The present study was performed to examine neuroprotective effects of 5-hydroxytryptamine (5-HT)₃ receptor antagonists against β-amyloid protein (25–35)-, a synthetic 25–35 amyloid peptide, induced neurotoxicity using cultured rat cortical neurons. β-Amyloid protein (25–35) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca²⁺ channel blocker, and N^G-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor. The 5-HT₃ receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1–10 μM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.05–5 μM), decreased the β-amyloid protein (25–35) (10 μM)-induced neuronal cell death as assessed by a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. MDL72222 and Y25130 inhibited the β-amyloid protein (25–35) (10 μM)-induced elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and glutamate release, generation of reactive oxygen species, and caspase-3 activity. These neuroprotective effects of MDL72222 (10 μM) and Y25130 (5 μM) were completely blocked by the simultaneous treatment with 100 μM 1-phenylbiguanide, a 5-HT₃ receptor agonist, indicating that the protective effects of these compounds were due to 5-HT₃ receptor blockade. These results suggest that the activation of the 5-HT₃ receptor may be partially involved in β-amyloid protein-induced neurotoxicity, by membrane depolarization for Ca²⁺ influx. Therefore, the blockade of 5-HT₃ receptor with MDL72222 and Y25130, may ameliorate the β-amyloid protein-induced neurotoxicity by interfering with the increase of [Ca²⁺]_c, and then by inhibiting glutamate release, generation of reactive oxygen species and caspase-3 activity.     

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABA_A receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABA_A (₁β₂γ_2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABA_A receptors. The GABA enhancing action of E-6375 on recombinant GABA_A receptors was unaffected by the subtype of the isoform (i.e. _xβ₂γ_2L; x=1–3) within the receptor, but was increased by the omission of the γ_2L subunit. Receptors incorporating β₂, or β₃, subunits were more sensitive to modulation by E-6375 than those containing the β₁ subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the β-subunit. The various in vivo actions of general anaesthetics may be mediated by GABA_A receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABA_A receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.     

γ-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices

γ-Hydroxybutyrate (GHB) has been shown to mimic different central actions of ethanol, to suppress alcohol withdrawal syndrome, and to reduce alcohol consumption both in rats and in humans. The aim of the present study was to determine if GHB shared with alcohol the ability to inhibit glutamate action at both NMDA and AMPA/kainate receptors. The NMDA or the AMPA/kainate receptors-mediated postsynaptic potentials were evoked in CA1 pyramidal neurons by stimulation of Schaffer-collateral commissural fibers in the presence of CGP 35348, bicuculline to block the GABA_B and GABA_A receptors, and 10 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 30 μM -2-amino-5-phosphonovalerate (d-APV) to block AMPA/kainate or NMDA receptors, respectively. GHB (600 μM) produced a depression of both NMDA and AMPA/kainate receptors-mediated excitatory postsynaptic potentials with recovery on washout. The GHB receptors antagonist, NCS-382, at the concentration of 500 μM had no effect per se on these responses but prevented the depressant effect of GHB (600 μM) on the NMDA and AMPA/kainate-mediated responses. In the paired-pulse experiments, GHB (600 μM) depressed the amplitude of the first and the second evoked AMPA/kainate excitatory postsynaptic potentials, and significantly increased the paired-pulse facilitation (PPF). These results suggest that GHB inhibits excitatory synaptic transmission at Schaffer-collateral commissural–pyramidal neurons synapses by decreasing the probability of release of glutamate.     

The role of NMDA receptors in regulating group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex

Previous work has shown that brief application of group II metabotropic glutamate receptor (mGluR) agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) can induce long-term depression (LTD) of excitatory transmission on layer V pyramidal neurons of rat medial prefrontal cortex (mPFC). An unusual feature of this LTD is that it relies on activation of both group II mGluRs and N-methyl-d-aspartate receptors (NMDARs). However, it is not known whether other specific group II mGluR agonists also induce LTD and whether they depend on the conjoint activation of group II mGluRs and NMDARs. We show here that the ability of DCG-IV to induce LTD was mimicked by a more selective group II mGluR agonist, LY379268. The induction of LTD by a lower concentration of DCG-IV (0.2 μM) or LY379268 (0.03 μM) was blocked by the NMDAR antagonist APV or the interruption of synaptic stimulation during drug application. In contrast, application of a higher concentration of DCG-IV (1 μM) or LY379268 (0.1 μM) can induce LTD that was independent of synaptic NMDAR activation. These results suggest that although molecular cooperation between group II mGluRs and synaptic NMDARs may facilitate the induction of group II mGluR-mediated LTD at excitatory synapses onto mPFC layer V pyramidal neurons, enhancing group II mGluR activation may remove NMDAR involvement in this form of synaptic plasticity.     

Studies of the antagonist actions of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) on non-NMDA receptors in cultured rat neurones

1. Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors.
2. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (K_i=16 μM) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 μM), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors.
3. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (K_i values: 27 and 23 μM, respectively).
4. Responses to MeGlu were unaffected by CTZ (100 μM), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 μg ml⁻¹). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A.
5. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 μM). The remaining component was further reduced by ATPO with a K_i of 36 μM, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA.
6. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors.

     

The Novel Metabotropic Glutamate Receptor Agonist 2R,4R-APDC Potentiates Stimulation of Phosphoinositide Hydrolysis in the Rat Hippocampus by 3,5-dihydroxyphenylglycine: Evidence for a Synergistic Interaction Between Group 1 and Group 2 Receptors

The mGlu receptor subtypes and second messenger pathways that mediate 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) responses in brain tissues are not fully understood. 1S,3R-ACPD differs from 3,5-dihydroxyphenylglycine (DHPG) or quisqualate in that 1S,3R-ACPD also activates group 2 mGlu receptors (mGlu2 and mGlu3) that are negatively linked to cAMP formation. To investigate the contribution of group 2 mGlu receptor activity of 1S,3R-ACPD to the phosphoinositide response in the rat hippocampus, we examined the effects of the novel group 2 mGlu receptor agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC). 2R,4R-APDC did not activate or inhibit group 1 mGlu receptors (human mGlu 1 and mGlu5a) or group 3 mGlu receptors (human mGlu4 and mGlu7), but potently decreased forskolin-stimulated cAMP formation in human mGlu2- and mGlu3-expressing cells. In slices of the adult rat hippocampus 2R,4R-APDC had no effect on basal phosphoinositide hydrolysis; however, it was found to greatly enhance phosphoinositide hydrolysis to DHPG or quisqualate. In the neonatal rat hippocampus, 2R,4R-APDC enhanced the potency of DHPG, while not affecting the maximal response to group 1 mGlu receptor agonists. Thus, the phosphoinositide response in the rat hippocampus to 1S,3R-ACPD is mediated by a synergistic interaction between group 1 and group 2 mGlu receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

Effects of β-ODAP and its biosynthetic precursor on the electrophysiological activity of cloned glutamate receptors

3-N-Oxalyl- -2,3-diaminopropanoic acid (β-ODAP) induces neurolathyrism, a motor neuron disease. To elucidate the pathogenic mechanism of this process, the action of β-ODAP on the excitatory amino acid (EAA) receptor-mediated currents was examined using cloned EAA receptors expressed in Xenopus oocytes. On the voltage-clamp recordings of an AMPA receptor (₁/₂ heterooligomer), β-ODAP was a strong agonist on this receptor, the potency being almost the same as -glutamate. On the other hand, β-ODAP had little effect on the glutamate-evoked currents through the expressed NMDA receptor (NR1_A/NR2A), but showed a weak inhibitory effect on the glycine-modulatory site. β-ODAP may cause the neurodegenerative disease, neurolathyrism, mainly through the excitotoxic interaction with AMPA receptors.     

Temporary and partial inhibition of platelets by SM-20302 prevents coronary artery thrombosis in a chronic canine model

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G₁₆ was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

Roles of metabotropic glutamate receptor subtypes in modulation of pentylenetetrazole-induced seizure activity in mice

The anticonvulsant or proconvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in a chemoconvulsant model using pentylenetetrazole (PTZ). Mice received mGluR ligands by intracerebroventricular (i.c.v.) infusion prior to a subcutaneous injection of PTZ and the latency to onset of tonic convulsions was recorded. The group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA) dose-dependently antagonised PTZ-induced seizures with a mean ED₅₀ value of 465 nmol. In contrast, the selective group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine [(S)-DHPG], was proconvulsive and decreased the PTZ-induced seizure latency (ED₅₀=60 nmol i.c.v.). A selective agonist of group II mGluRs, (1S,3S)-1-aminocyclopentane dicarboxylic acid [(1S,3S)-ACPD], was proconvulsive but did not affect PTZ-induced seizure latency. Moreover, the proconvulsant effect of (1S,3S)-ACPD was not blocked by the mGluR2 antagonist, -methylserine-O-phosphate monophenyl ester but was blocked by AIDA suggesting the involvement of group I mGluRs. (2S,1′S,2′S,3′R)-2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG-IV), which is a potent mGluR2 antagonist and a group III mGluR agonist at higher doses, increased the PTZ-induced seizure latency (ED₅₀=51 nmol) and this effect was fully reversed by the group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4). Similarly, the group III mGluR agonist 1-amino-3-(phosphonomethylene)cyclobutanecarboxylate (cyclobutylene-AP5) increased the PTZ-induced seizure latency (ED₅₀=12 nmol) in a MAP4-sensitive manner. Collectively, these data suggest that mGluR ligands modulate PTZ-induced seizure activity in mice by either antagonizing group I mGluRs or activating group III mGluRs.