Transient receptor potential vanilloid 4-dependent calcium influx, ATP release and expression of inflammatory factors in enteric glial cells

To investigate the role and physiological significance of transient receptor potential vanilloid receptor 4（TRPV4）in regulation of calcium influx in rat intestinal glial cells.

The expression of TRPV4 at mRNA and protein levels and its localization were detected by RT-qPCR, Western blotting and immunofluorescence assay in rat enteric glial cell line CRL-2690, rat intestinal epithelial cell line IEC6 and human embryonic kidney cell line HEK293T. The CRL-2690 cells were divided into GSK1016790A（GSK, TRPV4-specific agonist）group and GSK+HC067047（HC, TRPV4-specific inhibitor）group, hypotonic group and hypotonic+HC group, GdCl₃ group and GdCl₃+HC group, and CaCl₂ group and CaCl₂+HC group, while the activity of TRPV4 channel was assessed by Fura-2 fluorescent probe and single-cell fluorescent calcium ion assay. CRL-2690 cells were also divided into control group, GSK group and GSK+HC group, and the level of cellular ATP release was measured by luciferin-luciferase assay, and the mRNA levels of inflammatory genes GFAP, IL-1β, IL-6 and IL-10 were detected by RT-qPCR.

TRPV4 was expressed at mRNA and protein levels in rat intestinal glial cell line CRL-2690. GSK and low osmolarity stimulation significantly enhanced intracellular calcium fluorescence intensity in CRL-2690 cells, which was inhibited by HC（0.430 38±0.063 65 vs 0.004 23±0.005 78, P<0.000 1）. Calcium-sensing receptor（CaSR）activation-induced Ca²⁺ influx was also inhibited by HC（P<0.05）. Functionally, activation of TRPV4 promoted ATP release, expression of EGC reactive proliferation marker, GFAP, and inflammatory factors IL-1 β as well as IL-6, and inhibited the expression of anti-inflammatory factor IL-10 respectively. But all these effects were inhibited by HC treatment（P<0.05）.

Activation of TRPV4 induces extracellular Ca²⁺ influx and promotes ATP release and expression of inflammatory cytokines in enteric glial cell, which may be closely involved in the underlying mechanisms of bowel inflammation.