The mechanism of nitrous oxide (N20) analgesia remains uncertain, although research in mice has implicated an involvement of both kappa-opioid receptors and nitric oxide (N0). Differential responsiveness inbred C57BL/6 and DBA/2 mice to N20 antinociception has focused attention on the role of N0 in N20 action, and results of preliminary studies suggest a difference in N0 function between these strains. C57BL/6 mice are responsive to N20 antinociception and show increased N0 synthase (NOS) activity after N20 exposure, but DBA/2 mice are poorly responsive and show no such increase in NOS activity. The reduced sensitivity to N20 antinociception persists in certain BXD recombinant inbred (Rl) strains derived from the C57BL/6 and DBA/2 progenitors. Analysis of the BXD Rl data has identified provisional quantitative trait loci (QTL) or chromosome sites containing alleles that influence antinociceptive responsiveness to N20.
The specific aims of this research are to: (1) compare the role of NO in N20 antinociception in the progenitors by determining how the N20 response is affected by selective inhibition of neuronal vs. endothelial NOS in C57BL/6 mice and by NO loading in DBA/2 mice; (2) compare NOS function in C57BL/6 and DBA/2 mice by measuring conversion of [14C]L-arginine to [14C]L-citrulline in brain regions and spinal cords of naive and N20-exposed C57BL/6 and DBA/2 mice; (3) compare the role of NO in N20-induced neuronal release of opioid peptides by determining the influence of NOS inhibition on N20 protection against alkylation of opioid receptors; (4) identify QTL associated with N20 antinociception by determining responsiveness of B6D2F2 mice to N20 and genotyping using a whole genome scan; and (5) ascertain the correlation between antinociceptive responsiveness of B6D2F2 mice and their regional brain and spinal cord NOS activity levels. Methods to be utilized will include the following: antinociceptive testing; NOS assay; opioid radioligand binding; and PCR amplification of polymorphic genetic markers. The results of this research will determine why C57BL/6 and DBA/2 mice respond differently to N20 and help to identify specific genes and the roles played by NOS and NO in analgesic and anesthetic drug effects. This work will also contribute better understanding of the variability in human responsiveness to N20.
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