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.
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) approximately 70 microM) 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) approximately 5 microM) attaining a value of six-fold that of 'resting' cells at maximum stimulation; achieved at approximately 100 microM (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.
Sinclair
Colin
C
Neurochemistry Group, BioMolecular Sciences Centre, School of Biology, University of St Andrews, Fife KY16 9ST, Scotland, UK.
Reavy
Helen
H
Grieve
Angus
A
Schousboe
Arne
A
Morelli
Elena
E
Novellino
Ettore
E
Campiani
Giuseppe
G
Griffiths
Roger
R
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D011744:Pyrimidinones / Q000494:pharmacology*; D018091:Receptors, AMPA / Q000819:agonists*
C443679:CPW 399; D018690:Excitatory Amino Acid Agonists; D011744:Pyrimidinones; D018091:Receptors, AMPA; D007770:L-Lactate Dehydrogenase; D000409:Alanine; D002118:Calcium