Figure 7.

Direct interaction of TRPV4 with α2 integrin and Src tyrosine kinase. A, Proteins were extracted from DRG neurons isolated from alcohol-fed rats cultured for 2 d. TRPV4 was immunoprecipitated with anti-TRPV4 antibody (1:500) and immunoprecipitates were subjected to SDS gel electrophoresis. The membrane was then probed with anti-α2 integrin antibody (1:200) or with anti-Lyn antibody (1:300). IP, Immunoprecipitation; IB, immunoblot. B, Intradermal injection of a soup of inflammatory mediators in rat hindpaw induces a decrease in mechanical nociceptive thresholds in the rat. Spinal intrathecal injection of α2 integrin antisense (AS) ODN prevented the induced mechanical hyperalgesia (n = 10) and the effect of the antisense was reversible. Similarly, intradermal injection of anti-α2 integrin antibody (Ab; 100 ng/10 μl) before the intradermal injection of the inflammatory soup markedly reduced the mechanical hyperalgesia (n = 4). C, Small-diameter DRG neurons from TRPV4^+/+ and TRPV4^−/− mice were first challenged with a 30% hypotonic solution containing the inflammatory mediators PGE₂ and 5-HT (10 μm each) for 3 min and then challenged with a 30% hypotonic solution containing PGE₂, 5-HT, and the anti-α2 integrin antibody (5 μg/ml). The inflammatory mediator-induced increase in [Ca²⁺]_i is reversed in the presence of anti-α2 integrin antibody in TRPV4^+/+ mice (3.71 ± 1.03 before anti-α2 integrin antibody and 1.8 ± 0.3 after; n = 19; p < 0.0001, paired Student's t test), whereas it has no effect on [Ca²⁺]_i in TRPV4^−/− mice (1.8 ± 0.2 before and 1.7 ± 0.2 after anti-α2 integrin antibody; n = 30; p > 0.05, paired Student's t test). *p < 0.05.