Glycine site antagonist attenuates infarct size in experimental focal ischemia. Postmortem and diffusion mapping studies.

BACKGROUND AND PURPOSE: The glycine site on the N-methyl-D-aspartate (NMDA) receptor complex offers a therapeutic target for acute focal ischemia, potentially devoid of most side effects associated with competitive and noncompetitive NMDA antagonists. METHODS: A novel glycine receptor antagonist, ZD9379, was studied in 70 Sprague-Dawley rats using the suture occlusion model of permanent middle cerebral artery occlusion (MCAO). In the first experiment, 20 rats received an initial bolus of vehicle or 10 mg/kg ZD9379 (n = 10 in each group) 30 minutes after MCAO, followed by a continuous infusion of the same dose per hour for 4 hours. Diffusion-weighted MRI with echo-planar acquisition was used to generate maps of the apparent diffusion coefficient (ADC) of water. In a second experiment, 50 rats were assigned to five groups: vehicle and 10, 5, 2.5, and 1 mg/kg ZD9379 (n = 10 in each group) with the same dosing protocol but no imaging. In both experiments, infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining. RESULTS: In the first experiment, before therapy was begun, there was no significant difference in ADC-derived ischemic lesion volume between the two groups. Over time, the 10-mg/kg ZD9379-treated rats had a significant delayed regional recovery of reduced ADC values in the peripheral parietal cortex (P = .0156). Postmortem corrected infarct volume at 24 hours after MCAO was significantly smaller in the group treated with 10 mg/kg ZD9379 than in the vehicle group (119.2 +/- 52.2 versus 211.2 +/- 50.0 mm3 [mean +/- SD]; P = .0008; a reduction of 43.6%). In the second experiment, postmortem corrected infarct volumes in rats receiving 10, 5, and 2.5 mg/kg ZD9379 were significantly smaller than in those receiving vehicle, a reduction of 42.6%, 51.4%, and 42.9%, respectively (P = .0001). CONCLUSIONS: This study demonstrates that 2.5- to 10-mg/kg doses of ZD9379 initiated 30 minutes after MCAO significantly reduced infarct size. Diffusion mapping disclosed a delayed treatment effect of this glycine antagonist in focal ischemia, confirmed by the postmortem study.

Centrally-administered glycine antagonists increase locomotion in monoamine-depleted mice.

It was shown in the present study that several structurally diverse antagonists of the glycine site of the NMDA receptor, including (R)-HA-966, L689,560, 5,7-dichlorokynurenic acid, 7-chlorokynurenic acid, and two of ZENECA's novel pyridazinoindole glycine antagonists, caused marked reversal of akinesia when administered intrastriatally to monoamine depleted mice. Coinjection of the glycine agonist D-serine antagonized this locomotor stimulation. In addition, all glycine antagonists tested did not cause significant locomotor stimulation when intrastriatally administered to normal mice. These data suggest that glycine antagonists may offer therapeutic utility in the treatment of idiopathic Parkinson's disease.

Lack of involvement of nitric oxide in NMDA-induced neuronal cell death in cortical culture.

In primary cultures of rat cerebral cortex, N-methyl-D-aspartate causes widespread neurotoxicity. Inhibitors of the nitric oxide generating the enzyme nitric oxide synthase has been shown to attenuate the effects of N-methyl-D-aspartate in a number of neuronal systems both in vivo and in vitro. In our experiments, the nitric oxide synthase inhibitor N-nitroarginine was ineffective at blocking neurotoxicity induced by N-methyl-D-aspartate. Cyclic guanine monophosphate, known to be synthesized in response to nitric oxide was demonstrably inhibited by identical treatments with N-nitroarginine in sister cultures. We conclude that although nitric oxide is produced in response to N-methyl-D-aspartate, it is neither necessary nor sufficient for neurotoxicity.

Neomycin is an agonist at a polyamine site on the N-methyl-D-aspartate receptor.

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.

Comparison of binding at strychnine-sensitive (inhibitory glycine receptor) and strychnine-insensitive (N-methyl-D-aspartate receptor) glycine binding sites.

We compared, for a number of ligands to the two receptors, the displacement of [3H]strychnine binding to the glycine-gated chloride channel of spinal cord and brainstem synaptic membranes to the displacement of [3H]glycine binding to the NMDA receptor complex of hippocampal and cortex synaptic membranes. Glycine and beta-alanine are recognized by both receptors. In the NMDA receptor glycine antagonists, the kynurenic acids, most of the quinoxalinediones, and the (R)-enantiomer of HA-966 had little affinity at the strychnine-sensitive site. Surprisingly, the quinoxalinedione widely used as an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor antagonist, NBQX (2,3-dihydro-6-nitro-sulfamoylbenzo[f]quinoxaline-2,3-dione) displaced [3H]strychnine binding (IC50 = 11 microM) and to a lesser extent [3H]glycine binding (IC50 = 119 microM). Of the compounds tested, only strychnine, brucine, taurine and (S)-HA-966 were more potent displacers of [3H]strychnine than of glycine binding. Generally, the two glycine recognition sites appear to have remarkably different structural requirements.

An antagonist/partial agonist at the polyamine recognition site of the N-methyl-D-aspartate receptor that alters the properties of the glutamate recognition site.

The effects of N-(3-aminopropyl)-1,10-diaminodecane (APDA10) on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex were investigated. In the presence of 100 microM glutamate and 100 microM glycine, APDA10 had biphasic effects on the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imin e (MK-801) to NMDA receptors on well washed synaptic plasma membranes. The maximal stimulation of binding by APDA10 was less than that seen with spermine. In the presence of glutamate and glycine, APDA10 attenuated the stimulatory effect of spermine and the inhibitory effect of 1,10-diaminodecane. In the nominal absence of glutamate and glycine, APDA10 had no effect on the binding of [3H]MK-801, but antagonized the stimulatory effect of spermine on the binding of [3H] MK-801. These data suggest that APDA10 acts as a mixed antagonist/partial agonist at the polyamine recognition site, and that the partial agonist properties of APDA10 are dependent on the activation state of the receptor complex. An increase in the potency of the glutamate site antagonists D-2-amino-5-phosphonovaleric acid and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid for inhibiting the binding of [3H]MK-801 was seen in the presence of APDA10. APDA10 also increased the affinity of binding of [3H]3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid to the NMDA receptor complex but had no effect on the binding of [3H]glycine. These data suggest that the polyamine APDA10 may alter the properties of the glutamate recognition site on the NMDA receptor complex.

Agonist-like character of the (R)-enantiomer of 1-hydroxy-3-amino-pyrrolid-2-one (HA-966).

HA-966 (1-hydroxy-3-amino-pyrrolid-2-one), an antagonist at the strychnine-insensitive glycine site on the N-methyl-D-aspartate (NMDA) receptor complex, only partially inhibits the binding of noncompetitive antagonists to the NMDA receptor but enhances the binding of the NMDA competitive antagonist CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid). Here we report that the IC50 of the active (R)-enantiomer of HA-966 for displacement of [3H]glycine binding is decreased in the presence of spermine, suggesting that spermine increases the affinity of (R)-HA-966 at the [3H]glycine binding site. The IC50 values of the agonist glycine and the partial agonist 1-aminocyclopropane-1-carboxylate are also decreased. The IC50 values of glycine antagonists 6,7-dinitroquinoxalin-2,3-dione and 7-chlorokynurenic acid are not significantly altered. The spermine shift represents the first demonstration of the agonist-like character of the (R)-enantiomer of HA-966 at the glycine site.

The inhibition of [125I]omega-conotoxin GVIA binding to neuronal membranes by neomycin may be mediated by a GTP-binding protein.

omega-Conotoxin GVIA (omega-CT) has been reported to block calcium currents at the L- and N-type calcium channels. In neuronal membranes omega-CT, and the aminoglycoside antibiotic neomycin, have been shown to inhibit [125I]omega-CT binding, presumably acting at the N-type calcium channel. We demonstrate here that the concentration curve for neomycin sulfate inhibition of [125I]omega-CT binding is shifted to the right by GTP analogues or fluoride, increasing the IC50 for neomycin. [125I]omega-CT binding is unaffected by these agents and in competition studies the potency of omega-CT, Ca2+, or La3+ is not modulated by GTP analogues or fluoride. These results indicate that the inhibition of [125I]omega-CT binding by neomycin may be mediated by a GTP binding protein.

Stereoselectivity for the (R)-enantiomer of HA-966 (1-hydroxy-3-aminopyrrolidone-2) at the glycine site of the N-methyl-D-aspartate receptor complex.

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.

Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex.

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.

The polyamine spermine affects omega-conotoxin binding and function at N-type voltage-sensitive calcium channels.

1. The effects of the polyamines, spermine and spermidine on neuronal N-type voltage-sensitive calcium channels were investigated using the binding and function of the ligand omega-conotoxin GVIA (omega-CT). 2. Spermine and spermidine enhanced (EC50 approximately 0.16 and 0.45 microM) and, at higher concentrations, inhibited (IC50 of 9 and 240 microM) the binding of [125I]omega-CT to rat hippocampal synaptosomes. 3. Spermine and, less potently, spermidine inhibited the neurotransmitter-mediated, omega-CT-sensitive, electrical-field-stimulated contractile responses of the rat vas deferens. 4. The polyamines also inhibited the phenylephrine-evoked contractile responses of the vas deferens with the same rank order, consistent with a postsynaptic mechanism of inhibition. 5. However, pre-exposure to spermine prevented the irreversible inhibition of vas deferens twitch responses by omega-CT (previously found to be presynaptic). The prevention of inhibition by omega-CT demonstrates that the neuronal binding of spermine and omega-CT is mutually exclusive. Thus spermine (and presumably spermidine at higher concentrations) appears to modulate the actions of omega-CT at N-type voltage-sensitive calcium channels.

Schild plot analysis of glycine and kynurenic acid at the N-methyl-D-aspartate excitatory amino acid receptor.

Glycine enhanced the N-methyl-D-aspartate (NMDA)-stimulated sodium flux in rat hippocampal slides in a concentration-dependent manner. The potentiation by glycine was apparently not competitive to NMDA with a maximal effect of about 50% enhancement at approximately 150 microM glycine. Glycine also reversed the kynurenic acid inhibition of NMDA-stimulated sodium flux, increasing flux two-fold. In the absence of glycine, the kynurenic acid inhibition of NMDA-stimulated sodium flux appeared not competitive to NMDA, with a Schild plot slope of 0.6 +/- 0.1, significantly less than 1 (P less than 0.05). Addition of 100 microM glycine gave a Schild plot slope of 1.0 +/- 0.3, classically defining competitive inhibition. However, the pKb of 4.5 obtained from the x intercept gives an inaccurate estimate of the affinity of kynurenic acid for the NMDA recognition site since glycine is present. Addition of 400 microM glycine resulted in a Schild plot slope of 1.9 +/- 0.2. By varying the concentrations of glycine, we have apparently reproduced a portion of the theoretical set of curves for various ratios of affinities of the antagonist for the agonist recognition site and a second site. These results support a model where glycine modulates NMDA receptor function and where kynurenic acid acts both as a competitive antagonist to NMDA and at a second site from which it is displaced by glycine.

Receptor specific inhibition of N-methyl-D-aspartate stimulated 22Na flux from rat hippocampal slices by phencyclidine and other drugs.

Phencyclidine inhibited the flux of 22Na from hippocampal slices of the rat stimulated by N-methyl-D-aspartate (NMDA) in a concentration-dependent manner and consistent with the subclass of excitatory amino acid receptors, with a close interaction between NMDA and phencyclidine receptors. The inhibition by phencyclidine of the NMDA-stimulated flux of 22Na was non-competitive, in contrast to that produced by D-2-amino-5-phosphonovalerate. Other compounds which produce stereotyped behavior in vivo also inhibited the NMDA-stimulated flux of 22Na and the rank of the percentage inhibition of the NMDA-stimulated flux correlated with the affinities at the phencyclidine receptor with 97% confidence. The structural diversity and selectivity for the phencyclidine receptor of those compounds which inhibited the NMDA-stimulated flux of 22Na argue that the interaction was at the phencyclidine receptor. The presence of magnesium did not alter the relative magnitude of inhibition by phencyclidine, suggesting that the phencyclidine site is separate from the pressumably, channel-blocking magnesium site. Thus, the data of the present study support a model where the phencyclidine site is separate from the NMDA recognition site and from the presumed channel-blocking site of magnesium within the NMDA-associated ion channel.

Excitatory amino acid receptor potency and subclass specificity of sulfur-containing amino acids.

The sulfur-containing amino acids, L- and D-cysteate, L-cysteine, L- and D-cysteine sulfinate, L- and D-cysteine-S-sulfate, L-cystine, L- and D-homocysteate, L- and D-homocysteine sulfinate, L-homocysteine, L-serine-O-sulfate, and taurine were tested in two excitatory amino acid receptor functional assays and in receptor binding assays designed to label specifically the AA1/N-methyl-D-aspartate (NMDA), AA2/quisqualate, and AA3/kainate receptor recognition sites, as well as a CaCl2-dependent L-2-amino-4-phosphonobutanoate site, and a putative glutamate uptake site. Agonist efficacies were determined by chick retinal excitotoxicity and stimulated sodium efflux from rat brain slices. D-Homocysteine sulfinate, L-homocysteate, and L-serine-O-sulfate had affinities most selective for the NMDA binding site, whereas the binding affinities of D-cysteate, D-cysteine sulfinate, D-homocysteate, and L-homocysteine sulfinate were less selective. However, the correlation of agonist activity sensitive to blockade by D-2-amino-7-phosphonoheptanoate or D-2-amino-5-phosphonopentanoate in the functional assays with affinity in the NMDA binding assay (r = 0.87, p less than 0.005 and r = 0.98, p less than 0.005 for excitotoxicity and sodium efflux, respectively) allows characterization of these sulfur-containing amino acids as acting at NMDA subclass receptors. L-Homocysteate, which has been found in the brain, and L-serine-O-sulfate are selective agonists and could serve as endogenous neurotransmitters at the NMDA receptor.

Phencyclidine. Physiological actions, interactions with excitatory amino acids and endogenous ligands.

Phenycyclidine (PCP) produces many profound effects in the central nervous system. PCP has numerous behavioral and neurochemical effects such as inhibiting the uptake and facilitating the release of dopamine, serotonin, and norepinephrine. PCP also interacts with sigma, mu opioid, muscarinic, and nicotinic receptors. However, the psychotomimetic effects induced by PCP are believed to be mediated by specific PCP receptors, where PCP binds with greater potency than sigma compounds. Electrophysiological, behavioral, and neuro-chemical evidence strongly suggests that at least some of the many PCP actions result from antagonism of excitatory amino acid-induced responses via PCP receptors. The recent isolation and partial characterization of the alpha and beta endopsychosins and the identification of other endogenous ligands for the PCP and sigma receptors, is another promising area of research in the elucidation of the physiological role of an endogenous PCP and sigma system.

Purification and properties of pig muscle carbonic anhydrase III.

Pig muscle carbonic anhydrase III (carbonate hydro-lyase, EC 4.2.1.1) has been isolated and purified to homogeneity with chromatographic techniques. It has been found to be a 30 kDa protein displaying the same three activities (CO2 hydratase, acetate esterase, p-nitrophenyl phosphatase) previously described for the rabbit muscle isoenzyme, including the phosphatase activity not seen in the erythrocyte isoenzymes. The turnover numbers of the three activities are of the same order of magnitude as previously reported for rabbit muscle carbonic anhydrase III. Km and Vmax for the pig muscle CO2 hydratase activity were found to be 83 mM and 6000 s-1, respectively. The extinction coefficient at 280 nm (1 cm light path) is 22.2 for a 1% solution. Five half-cystine residues determined by performic acid oxidation are free for reaction with p-mercuribenzoate but only four are accessible to titration with dithiobisnitrobenzene. The amino acid composition of the pig muscle isoenzyme III has a high level of homology compared with that of rabbit and bovine muscle carbonic anhydrases III.

Specific arginine modification at the phosphatase site of muscle carbonic anhydrase.

Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.