Figure 5. TNFα activates sphingosine kinase 1 to enhance spiral modiolar artery Ca²⁺ sensitivity.

(A) Under resting conditions, GFP-Sk1 was homogenously distributed throughout the cytosol. (B) TNFα (1ng/mL; 2hrs) stimulated a redistribution of GFP-Sk1 to plasma membrane. (C) Expression of a dominant inactive Sk1 mutant dramatically reduced the resting SMA Ca²⁺ sensitivity (i.e., a rightward shift in the Ca²⁺/tone relationship), compared to controls. TNFα augmented Ca²⁺ sensitivity in control SMAs, but not those expressing the dominant-negative Sk1 mutant Sk1^G82D (dia_max Control= 90±7µm, n=7; dia_max Sk1^G82D=92±7µm, n=7). (D) The effects of the chemical inhibition of sphingosine kinase (dimethyl-sphingosine; DMS; 3µmol/L, 30min) were similar to that of Sk1^G82D expression: it reduced resting Ca²⁺ sensitivity and prevented the Ca²⁺ sensitivity increase following subsequent application of TNFα (dia_max: 87±9µm, n=7; paired observations). (E) DMS also reversed the TNFα-stimulated enhancement of SMA Ca²⁺ sensitivity (dia_max: 86±4µm, n=5; paired observations). (F) TNFα failed to increase Ca²⁺ sensitivity in SMAs expressing the non-phosphorylatable, but catalytically active Sk1 mutant Sk1^S225A. * denotes a significant difference (p<0.05) in the dose-response relationships.