A refined method for the photoaffinity labelling of the nitrobenzylthioinosine-sensitive nucleoside transport protein: application to cell membranes of calf lung tissue.

A refined method for the photoaffinity labelling of the NBI-sensitive nucleoside transport protein is described. It involves the use of low concentrations of the photolabile probe [3H]nitrobenzylthioinosine ([3H]NBI), whereas the usual inclusion of dithiothreitol in the protocol is omitted. The method was successfully applied to cell membranes of calf lung tissue, which was shown to be a rich source of this physiologically important protein with all the characteristics (both in membrane bound and solubilized form) known from similar proteins on other cell types. Specific covalent incorporation of radioactivity appeared to be pH independent. SDS-polyacrylamide gel electrophoresis revealed a specifically labelled protein with an apparent molecular weight of 55 kDa.

A comparative analysis of the neuroprotective properties of competitive and uncompetitive N-methyl-D-aspartate receptor antagonists in vivo: implications for the process of excitotoxic degeneration and its therapy.

Injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, into the rat striatum in vivo results in the degeneration of cholinergic and GABAergic neurons, as determined seven days later using the marker enzymes, choline acetyltransferase and glutamate decarboxylase, respectively. Such damage was dose-dependently prevented by CGP 37849 or MK-801 (competitive and uncompetitive N-methyl-D-aspartate receptor antagonists, respectively) administered systemically or intrastriatally at the same time as quinolinic acid. The neuroprotective activity of CGP 37849 was associated with the D-enantiomer, CGP 40116 (ED50 7.5 mg/kg i.p.), which was approximately 1.5-fold and 3.5-fold more potent than the related compounds, D-CPPene and CGS 19755, respectively. CGP 37849 was a weaker neuroprotectant than MK-801 (ED50 0.8 mg/kg i.p) when administered systemically, but was dramatically more potent following coinjection with quinolinic acid (ED50's 0.2 and 117 nmol, respectively). When injected intrastriatally 0.5-2 h post-quinolinic acid, CGP 37849 was protective over the entire period studied, whereas MK-801 was less effective at all post-quinolinic acid injection times. The finding that CGP 37849 is neuroprotective when administered intrastriatally 1-2 h post-quinolinic acid supports the hypothesis that a period exists following excitotoxic insult in which neurons are not committed to die, and can be rescued by blockade of ongoing N-methyl-D-aspartate receptor activation. Competition studies indicated that, when coinjected with 100-400 nmol quinolinic acid into the striatum, CGP 37849 exhibited kinetics predicted of a competitive N-methyl-D-aspartate receptor antagonist (declining neuroprotective potency with increasing doses of agonist), whereas MK-801 displayed a complex picture, with weak protective activity at low doses of quinolinic acid. Following systemic administration, neither antagonist was markedly affected by the dose of excitotoxin. When given i.p. at up to 6 h post-quinolinic acid, CGP 37849 and MK-801 showed essentially identical profiles of post-insult protection; degeneration of cholinergic neurons was reduced significantly throughout the entire post-insult period, whereas GABAergic neurons were protected only when drugs were administered 2 h or earlier post-quinolinic acid. The data indicate that competitive and uncompetitive N-methyl-D-aspartate receptor antagonists are effective neuroprotectants in vivo, and that parameters such as drug lipophilicity or mechanism of action at the receptor do not impinge upon their properties as systemically active cerebroprotectants.

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity.

1. The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2. Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-((+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nM, and was 4, 5 and 7 fold more potent than the antagonists [+/-)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3. CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4. In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CA1 pyramidal cell firing rate. In slices bathed in medium containing low Mg2+ levels, concentrations of CGP 37849 up to 10 microM suppressed burst firing evoked in CAl neurones by stimulation of Schaffer collateral-commissural fibres without affecting the magnitude of the initial population spike; CGP 39551 exerted the same effect but was weaker. In vivo, oral administration to rats of either CGP 37849 or CGP 39551 selectively blocked firing in hippocampal neurones induced by ionophoreticallyapplied NMDA, without affecting the responses to quisqualate or kainate. 5. CGP 37849 and CGP 39551 suppressed maximal electroshock-induced seizures in mice with ED50 s of 21 and 4 mg kg'- p.o., respectively. 6. CGP 37849 and CGP 39551 are potent and competitive NMDA receptor antagonists which show significant central effects following oral administration to animals. As such, they may find value as tools to elucidate the roles of NMDA receptors in brain function, and potentially as therapeutic agents for the treatment of neurological disorders such as epilepsy and ischaemic brain damage in man.

Evaluation of quinolinic acid induced excitotoxic neurodegeneration in rat striatum by quantitative magnetic resonance imaging in vivo.

Excitotoxic neurodegeneration in the rat striatum was induced by direct injection of quinolinic acid. The degree of damage was evaluated in vivo 1 day later by quantitative magnetic resonance imaging (MRI) and 7 days later in the same animals by measuring the activities of the neuronal marker enzymes choline acetyltransferase and glutamic acid decarboxylase. Striatal damage assessed using the two approaches was highly correlated. Moreover the cerebroprotective efficacy of the N-methyl-D-aspartate receptor antagonist CGP 40116 was indistinguishable based on all analytical parameters. MRI, however, was more reproducible than the enzymatic methods and was faster and simpler for routine analyses of excitotoxic damage and cerebroprotection in vivo.

Inhibition of nucleoside transport by a new series of compounds related to lidoflazine and mioflazine.

A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed.

The N-methyl-D-aspartate (NMDA) receptor complex: a stoichiometric analysis of radioligand binding domains.

A stoichiometric analysis of pharmacological domains within the N-methyl-D-aspartate (NMDA) receptor complex was made by evaluating the binding of L-[3H]glutamate, [3H]CPP, [3H]glycine and [3H]MK-801 to purified synaptic membranes isolated from rat telencephalon. The binding of all radioligands exhibited pharmacological and kinetic properties consistent with the labeling of homogeneous populations of sites associated with the NMDA receptor. However, strychnine-insensitive [3H]glycine binding sites were present at close to 2-fold the density of the other sites examined. These data, together with recent electrophysiological and receptor autoradiographic findings, are utilized as a basis for hypotheses regarding the ratio of transmitter recognition, allosteric and channel binding sites within the NMDA receptor complex.

CGP 31358 binds to a site on the NMDA receptor that is coupled to both the transmitter recognition site and the channel domain.

CGP 31358, a novel triazole, inhibited the binding of L-[3H]glutamate and [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex in rat brain synaptic membrane fractions, and showed anticonvulsant activity in mice. It had no effect on the strychnine-insensitive binding of [3H]glycine. Saturation and Hill analyses indicated that CGP 31358 binds to a site on the NMDA receptor which is separate from, but coupled to, both the transmitter recognition site and the channel domain. Available data indicate that this site is distinct from those with which tricyclic antidepressants and ifenprodil interact. CGP 31358 is a new chemical entity with a novel mechanism of action at the NMDA receptor, and as such may form a tool for understanding the molecular pharmacology of this receptor-channel complex.