Figure 3.

The effect of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) on HIV-1 replication is dependent on protein kinases A (PKA) and C (PKC). (A,B) Uninfected macrophages were treated with VIP or PACAP (10 nM) and levels of PKA and pPKA (A), PKCα and pPKCα (B) were analyzed by western blot at different time-points; figures show the ratios between band densitometry normalized based on β-actin intensity (n = 3). (C–E) Infected macrophages were treated with VIP or PACAP (10 nM) in the presence or not of signaling inhibitors (PKAi, H89; PKCi, Gö 6983; Pan-protein kinase inhibitor, H7, 50 nM each one) for 30 min; cells were washed before neuropeptide addition [in (C), results are shown normalized to medium to rule out any possible inhibitor interference on HIV-1 replication]. (D,E) Treatment with VIP (D) or PACAP (E) plus inhibitors; results are shown normalized to respective controls [shown in (C)], to compare the individual inhibitory effects. Supernatants were collected after 12 days and viral replication was measured (n = 5). (F,G) Infected macrophages were treated with VIP or PACAP (10 nM) in the presence or not of signaling inhibitors as above, for 30 min; cells were washed before neuropeptide addition. After 48 h, supernatants were collected and production of MIP-1α (F) and IL-10 (G) were analyzed by ELISA (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA, with Dunnett post-test.