Figure 6. Induction of SGK-1 by glucocorticoid enhances ATP release from astrocytes via pannexin-1 hemichannels.

(a) Negligible effects of CORT (100 ng ml⁻¹) on the expression of pannexin-1 in the membrane fraction of astrocytes (left) and release of ATP (right). Cells are treated with 100 ng ml⁻¹ for 4 h. CORT *P<0.05 significant difference between two groups (n=5, means±s.e.m.; t₈=−5.445, unpaired t-test, two-sided). Full-size images are presented in Supplementary Fig. 14. (b) The downregulation of pannexin-1 in the membrane fraction of astrocytes. Cells are transfected with 150 pmol of siRNA against pannexin-1 or scrambled (Control) siRNA. Protein levels were assessed at 72 h after transfection. Full-size images are presented in Supplementary Fig. 15. (c) Effects of the downregulation of pannexin-1 on the CORT-induced release of ATP from astrocytes (means±s.e.m.; n=5 per group). Astrocytic ATP release was assessed 4 h after the CORT treatment. **P<0.01 significant difference between two groups (F_3,16=19.130, P<0.001, ANOVA with Tukey–Kramer's PHT). (d) Tracing of the fluo-8 fluorescence intensity during and after Ca²⁺ depletion in astrocytes treated with 100 ng ml⁻¹ CORT in the presence or absence of 10 μM Cpd5J-4 an Orai1 inhibitor (means±s.e.m.; n=8 per groups). Average fluorescence before the measurement is set to 1. (e) Effects of Cpd5J-4 on the CORT-induced release of ATP from astrocytes (means±s.e.m.; n=5 per group). Cells were treated with 100 ng ml⁻¹ CORT in the presence or absence of Cpd5J-4 (10 μM) for 4 h. **P<0.01; *P<0.05 significant difference between two groups (F_3,16=19.323, P<0.001, ANOVA with Tukey–Kramer's PHT). (f) Schematic diagram indicating the glucocorticoid-regulated diurnal oscillations in the threshold of mechanical allodynia in PSL mice. Temporal elevations in glucocorticoid levels induce the expression of SGK-1 in spinal astrocytes, thereby enhancing extracellular ATP release through pannexin-1 hemichannels. ATP released from astrocytes binds to P2Y₁₂ receptors on activated microglia. ATP-stimulated P2Y₁₂ receptors induce downstream events, which result in a decrease in the threshold of mechanical allodynia.