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

Abstract

Objective 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. Methods 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. Results 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). Conclusion 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.

Keywords

• enteric glial cells
• transient receptor potential vanilloid 4
• ca2+ signal
• adenosine triphosphate
• inflammation