Figure 3.

Western blots and graphs illustrating the effect of i.t. treatment with Sig-1 R agonists, PRE-084 (PRE, a and c) or carbetapentane (CAR, b and d) on PKA-dependent pNR1 (122 kDa) in the spinal dorsal horn. Expression of pNR1 (Ser⁸⁹⁷) in the spinal cord dorsal horn was found to increase as early as 10 min after i.t. injection of either PRE-084(3 nmol, a) or carbetapentane (10 nmol, b), and peaked between 30–60 min post-injection. ^*P<0.05 and ^**P<0.01 as compared with those of non-treated mice; indicated by the ‘0' time point in panels (a) and (b). Protein expression of pNR1 before treatment with Sig-1 R agonists was calculated as 100%. I.t. injection of PRE-084 (0.3, 1 and 3 nmol, c) or carbetapentane (1, 3 and 10 nmol, d) dose dependently increased the expression of the pNR1 (Ser⁸⁹⁷) subunit in the spinal cord dorsal horn. I.t. pretreatment with 100 nmol BD-1047 (BD, a selective Sig-1 R antagonist) significantly reduced the increase in pNR1 (Ser⁸⁹⁷) expression induced by i.t. injection of PRE-084or CAR. Protein expression of pNR1 in response to the lowest dose of Sig-1 R agonists was calculated as 100%. ^*P<0.05 and ^**P<0.01 as compared with pNR1 (Ser⁸⁹⁷) expression in response to the lowest dose of PRE-084(0.3 nmol) or carbetapentane (3 nmol). All immunoblots were normalized for analysis using β-actin (as a loading control) in each group (see text). The number of mice was five in each group. I.t., intrathecal; PKA, protein kinase A; pNR1, phosphorylation of the NMDA receptor subunit NR1; Sig-1 R, sigma-1 receptor.