Figure 1.

cAMP induces pRII immunoreactivity in intact sensory neurons, but not cell lysates. (A) Representative HCS microscopy images of control (Ctrl; 0.1% DMSO) and 10 µM Fsk-stimulated (15 min) rat sensory neurons. Cultures were stained with UCHL1 to identify the neurons and pRII to quantify PKA-II signaling. Green/red encircled neurons indicate automatically selected/rejected objects. Bar, 100 µm. (B) Time course of pRII intensity in sensory neurons after stimulation with Ctrl (0.1% DMSO), 10 µM Fsk, 10 µM Sp-8-Br-cAMPS-AM, or 200 nM 5-HT. (C) Time course of pCREB (Ser133) intensity. (D) Immunoblot of sensory neuron lysates probed with pRII and RIIβ antibodies (left) and densitometry results (right). The pRII antibody recognized phosphorylated RIIα and RIIβ. Stimulation with 10/100 µM Fsk/IBMX or 10 µM Sp-8-Br-cAMPS-AM for 15 min did not increase the pRII density. (E) Immunoblot and densitometry results of the same cell lysates probed with the pCREB antibody. (F and G) Time course of pRII intensity after stimulation with different doses of 1–10 µM Fsk or 5–200 nM 5-HT. (H and I) Pretreatment of sensory neurons with 4–25 µM H89 (30 min) did not inhibit the pRII response to 3 µM Fsk (H) or 200 nM 5-HT (I). (J and K) Pretreatment with 25 µM H89 (30 min) significantly inhibited the induction of CREB phosphorylation by 10 µM Fsk (H) or 200 nM 5-HT (K). (L and M) Pretreatment with H89 also inhibited the induction of ERK1/2 phosphorylation. HCS results are means ± SEM; n = 3–4; >1,000 neurons/condition; two-way ANOVA with Bonferroni’s test. Immunoblot results are means ± SEM; n = 6; one-way ANOVA with Dunnett’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Kinetic experiments are plotted in nonlinear scale.