Fig. 5.

norBNI treatment produces long-term inhibition of U50488-mediated inhibition of PGE₂-stimulated cAMP accumulation, but not U50488 stimulation of ERK. (A) Peripheral sensory neuron cultures were pretreated with norBNI (3 nM) or vehicle. After a 1-hour incubation, cells were washed thoroughly. Twenty-four hours later, cells were treated with PGE₂ (1 µM) with U50488 (100 nM), DPDPE (100 nM), DAMGO (1 µM), or vehicle, and cellular cAMP levels were measured after 15 minutes. Data represent the mean ± S.E.M. of cAMP levels expressed as the percentage of PGE₂-stimulated cAMP levels of three or four independent experiments. *P < 0.05 compared with vehicle-treated cells. (B) Cells were pretreated with SP600125 (1 µM) or vehicle (Veh) 30 minutes before addition of norBNI (3 nM) or vehicle. After a 1-hour incubation, cells were washed thoroughly and cellular cAMP was determined [as in (A)]. Data were analyzed with one-way ANOVA, followed by Dunnett’s post hoc test. ***P < 0.001, **P < 0.01 compared with vehicle-treated cells. (C) Cells were pretreated with norBNI (3 nM) or vehicle for 1 hour, and washed thoroughly [as in (A)]. Twenty-four hours later, levels of pERK were measured at the indicated time points following stimulation with U50488 (100 nM) in the absence or presence of norBNI (3 nM). pERK levels were measured using the pERK Surefire assay kit from PerkinElmer Life and Analytical Sciences, according to the manufacturer’s protocol. Data are expressed as the percentage increase in pERK over basal (no ligand) activity and represent the mean ± S.E.M. of four to six independent experiments. Data were analyzed with two-way ANOVA, followed by Bonferroni’s post hoc test. ***P < 0.001, **P < 0.01 compared with Veh-pretreated cells.