Inherent desensitisation-preventing properties of a novel, subtype-selective AMPA receptor agonist, ( S)-CPW 399, as a possible explanation for its excitotoxic action in cultured cerebellar granule cells

Abstract

The synthesis and pharmacological characterisation of ( S)-CPW 399 as a novel, potent and subtype-selective agonist of the AMPA receptor was recently reported. Studies have been extended to investigate its excitotoxic action in primary cultures of mouse cerebellar granule cells. ( S)-CPW 399 induced neuronal cell death in a time- and concentration-dependent manner (EC 50 ∼70 μM) at 24-h exposure. ( S)-CPW-induced neuronal death could be prevented by co-administration with either of the AMPA/kainate selective receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[ f]quinoxaline-2,3-dione (NBQX) or by the 2,3-benzodiazepine, GYKI 53655 (a selective AMPA receptor antagonist); while no protection was afforded by either the NMDA receptor antagonist d,l(±)-2-amino-5-phosphonopentanoate (APV) or by nifedipine (an L-type calcium channel antagonist) when used alone or in combination. Cyclothiazide, which blocks AMPA receptor desensitisation, caused minimal potentiation of ( S)-CPW 399-induced neuronal death, supporting accumulating evidence that ( S)-CPW 399 is a full AMPA receptor agonist that markedly prevents a receptor desensitised conformation. ( S)-AMPA, ( S)-willardiine (a naturally-occurring heterocyclic excitatory amino acid) and its halogenated derivative, ( S)-5-fluorowillardiine, had no deleterious effect on neuronal viability when used alone but each, in the presence of cyclothiazide, induced a concentration-dependent excitotoxic cell death with a rank order of potency (fluorowillardiine⪢AMPA=willardiine). ( S)-CPW 399 stimulated an increase in intracellular free-calcium levels ([Ca 2+] i ) in a concentration-dependent fashion (EC 50 ∼5 μM) attaining a value of six-fold that of ‘resting’ cells at maximum stimulation; achieved at ∼100 μM ( S)-CPW 399. The ( S)-CPW 399-stimulated increase in [Ca 2+] i was virtually abolished by GYKI 53655, NBQX, CNQX and by cobalt ions; markedly inhibited by nifedipine and marginally affected by d-APV. These results suggest that ( S)-CPW 399 may be used as a pharmacological tool to aid in the investigation of the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitisation.