Figure 7
GSNO increases the number of M2/M4 AChR interacting high-affinity [35S]GTPγS binding sites in rat forebrain membranes. Membranes were preincubated for 30 min in control conditions or in the presence of 0.5 mM GSNO, and incubated thereafter for 90 min with 0.15 nM [35S]GTPγS, 10-5 M GDP and indicated concentrations of unlabeled GTPγS in the presence and absence of CCh (10-4 M), as detailed in the Methods section. Statistical comparison of one- versus two-site competition curves (nonlinear regression) indicated that the one-site model best described GTPγS displacement in agonist-stimulated conditions. Note that GSNO significantly increased the number of high-affinity [35S]GTPγS binding sites in CCh-treated membranes (CCh-control 1.49 ± 0.02; CCh-GSNO 1.75 ± 0.02 pmol/mg protein, P < 0.001, unpaired T-test). There were no statistical differences in the potency for GTPγS in displacing radioligand in any condition [log (EC50): basal-control -8.10 ± 0.07; basal-GSNO -8.11 ± 0.12; CCh-control -8.06 ± 0.06; CCh-GSNO -8.10 ± 0.04). Values represent specific binding (mean ± SE) from three independent experiments performed in duplicate.