Nitric oxide (NO) functions as a messenger throughout the central nervous system, where many of its actions are exerted through guanylyl cyclase activation, leading to cGMP formation. A putative alternative transduction pathway is the modification of protein function by nitrosation of thiol groups (Ahern et al. 2002). A prototypic protein considered to be regulated in this way is the NMDA receptor but, despite extensive research, there is little direct evidence that NO performs this function. To address this issue, we have investigated the effect of NO on native or cloned NMDA receptors (NMDARs) in hippocampal slices and HEK-293 cells, respectively.
Field EPSPs were recorded from the CA1 stratum radiatum of hippocampal slices obtained from 6- to 8-week-old rats killed humanely according to Home Office regulations (Bon & Garthwaite, 2001). EPSPs mediated through NMDARs were isolated pharmacologically. The responses were not significantly changed by bath application of the NO synthase substrate, L-arginine (100 µM), nor by inhibition of NO synthase using L-nitroarginine (100 µM). Diethylamine NO adduct (DEA/NO) was used to supply NO exogenously. Whilst DEA/NO at a concentration of 10 µM caused maximal cGMP accumulation in hippocampal slices, concentrations up to 30-fold higher produced no detectable change in the NMDAR-mediated synaptic responses. However, in accordance with a previous study (Murphy et al. 1994), photolysis of a caged NO derivative using a flash of UV light depressed NMDAR-mediated EPSPs by 94 ± 3 % (mean ± S.E.M.; n = 4).
As a further test, whole-cell membrane currents in response to 100 ms pulses of 100 µM glutamate were recorded at -60 mV from HEK-293 cells transfected with NMDAR subunits NR1 and NR2A (Köhr & Seeburg, 1996). Similar to findings in the slices, photolysis of the caged compound by UV light depressed the NMDAR currents by 55 ± 3% (n = 5), whereas 100 µM DEA/NO had no significant effect on the NMDAR current alone, but depressed the NMDAR currents by 35 ± 7% (n = 4) when in combination with UV light. Perfusion of 100 nM DEA/NO had no effect on NMDAR function.
The results suggest that NO released endogenously, or exogenous NO in concentrations well in excess of those needed to activate guanylyl cyclase, does not modify NMDAR function. It is possible that other factors, such as UV light, may have contributed to the inhibition of NMDAR responses observed previously (Murphy et al. 1994).
This work was supported by a BBSRC CASE studentship with Merck Sharp and Dohme (Harlow, UK) and The Wellcome Trust. We thank Dr B. Lancaster for expert advice.