Tat±morphine-induced alterations in cytoplasmic-nuclear shuttling of the p65 subunit of NF-κB regulate cytokine production by astrocytes.

<p>Cells were pretreated with vehicle or leptomycin B (LMB) followed by 30 min, 2 or 6 h treatment (only data at 2 h are shown) with Tat±morphine (Morph) and analyzed by immunofluorescence microscopy to determine the proportion of GFAP (red fluorescence) cells with p65(RelA; green fluorescence) subunit present within their nuclei (labeled with Hoechst 33258; blue fluorescence) (A). GFAP⁺ cells containing p65 in the nucleus were counted and divided by the total number of GFAP⁺ cells (B). Tat alone or in combination with morphine increased the trafficking of the p65(RelA) subunit of NF-κB into the nucleus (A–B), while blocking nuclear export with LMB increases the accumulation of p65(RelA) subunits in the nucleus (A–B; *<i>P</i><0.05 vs. vehicle-or LMB-treated controls; ^§<i>P</i><0.05 vs. vehicle-treated controls; ^‡<i>P</i><0.05 vs. Tat±morphine treatment without LMB). (C–E): LMB attenuates cytokine release caused by Tat±morphine. Analysis of astrocyte conditioned medium by ELISA showed a significant decrease in MCP-1, IL-6 and TNF-α production in astrocytes pretreated with LMB (*<i>P</i><0.05 vs. vehicle or LMB-treated controls lacking Tat±morphine; ^#<i>P</i><0.05 vs. Tat+morphine treated astrocytes without LMB). Data represent the mean±SEM of 3 separate experiments at 6 h.