l-701324 and Inflammation

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

1. NMDA receptor antagonists have previously been shown to have antinociceptive effects in behavioural experiments, but controversy remains as to the role of NMDA receptors in mechanical hyperalgesia. We have studied the effects on mechanical nociceptive thresholds in rats with carrageenin-induced paw inflammation of L-687,414, a low efficacy partial agonist which acts as a functional antagonist at the glycine modulatory site of the NMDA receptor and of L-701,324, a structurally novel, highly selective, full antagonist at this site. 2. Mechanical thresholds were measured for both hind paws 1 h before and 3 h after carrageenin or saline was injected into 1 hind paw. Dose-response curves were constructed for each test compound in separate experiments, with test compound or vehicle being given i.p. 1 h before the final test. 3. Both compounds produced selective dose-dependent and statistically significant reversal of mechanical hyperalgesia, with minimum effective doses of 100 mg kg-1 L-687,414 and 3 mg kg-1 L-701,324. Neither L-687,414 nor L-701,324 affected the response threshold of the contralateral non-inflamed paw over the dose-range producing reversal of carrageenin-induced hyperalgesia. Neither compound had any effect on the paw oedema produced by carrageenin injection. 4. These results show that both a full antagonist and a low efficacy partial agonist at the glycine modulatory site of the NMDA receptor complex reverse inflammation-induced mechanical hyperalgesia, thus supporting the argument that maximal activation of the glycine site is required for transmission via NMDA receptors, and showing that NMDA receptor-mediated actions are important in mechanical hyperalgesia induced by inflammation.

Topics: Animals; Binding Sites; Carrageenan; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Hyperalgesia; Inflammation; Male; Pyrrolidinones; Quinolones; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate