Figure 4. DPDPE and SNC-80 differ in their ability to induce desensitization and acute analgesic tolerance. HEK293 cells were incubated with 10 μm cycloheximide and treated or not with monensin (50 μm) before exposing them to vehicle (DMSO 0.01%), DPDPE, or SNC-80 (1 μm; 60 min). At the end of treatment, cells were washed and either immediately used to monitor cAMP accumulation after agonist-induced desensitization (A) or were first allowed to recover in the absence of ligand so as to evaluate resensitization (B). Results are expressed as percentage of maximal inhibition obtained in corresponding untreated controls, and correspond to 5–6 independent experiments performed in duplicates. A, Desensitization data were analyzed by means of three-way ANOVA (agonist × monensin × concentration). Interaction for agonist × monensin groups (F(2,188) = 7.20, p < 0.01). Bonferroni post hoc for multiple comparisons: control versus desensitization by DPDPE p < 0.001; control versus desensitization by SNC-80 p < 0.001; desensitization by DPDPE versus desensitization by SNC-80 p < 0.001; desensitization by DPDPE versus desensitization by DPDPE + monensin p < 0.001; desensitization by SNC-80 versus desensitization by SNC-80 + monensin p > 0.05. B, Resensitization data were analyzed as above. Interaction for agonist × monensin groups was nonsignificant (F(2,235) = 1.7, p > 0.05). Effect of agonist factor: F(2,235) = 267, p < 0.001; effect of monensin factor: F(1,235) = 0.75, p > 0.05. Bonferroni for post hoc comparisons showed resensitization following DPDPE versus resensitization following SNC-80, p < 0.001. C, One month following induction of the spared nerve injury model of neuropathic pain, calibrated von Frey filaments were used to evaluate the reversal of mechanical hypersensitivity following administration of an intrathecal injection of either vehicle, DPDPE or SNC-80. The 50% withdrawal thresholds (g) were then evaluated three times every 20 min, before administering a second identical dose of the corresponding treatment followed by similar assessment. Results are expressed as mean ± SEM, n = 10–20/treatment group. Data were analyzed using three-way ANCOVA (treatment × testing over time × injection) using basal thresholds (t = 0) as coregressor (F(1,231) = 22.0, p < 0.001; heterogeneity of regression slope: F(17,214) = 1.15, p > 0.05). Treatment × injection interaction (F(1,231) = 5.3, p < 0.05) allowed the following comparisons: effect of first DPDPE injection versus effect of first SNC-80 injection: p > 0.05; effect of second DPDPE injection versus effect of second SNC-80 injection: p < 0.01; effect of first versus second DPDPE injection: p > 0.05; effect of first versus second SNC-80 injection: p < 0.05.