TRPV4 channel expression and function in blood‐brain barrier (BBB) microvessel endothelial cells

Abstract

RT-PCR analysis revealed TRPV4 mRNA expression in microvessels isolated from mouse brain, in rat brain microvessel endothelial cell cultures, and in an immortalized mouse BBB endothelial cell line, bEnd3. The analysis was extended with bEnd3 cells grown on coverslips. Both immunoblotting and immunohistochemistry verified expression of TRPV4 protein in the bEnd3 cells and immunofluorescent imaging showed TRPV4 within the cytoplasm and at the cell membrane. To evaluate function, intracellular calcium levels were measured in bEnd3 cells on coverslips using fura 2 fluorescence imaging (37 °C). Cells were activated by application of: 1) the PKC-inactive phorbol ester, 4α-phorbol 12,13-decanoate (4α-PDD, 100 nM), a selective activator of TRPV4; 2) the PKC-active phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM); and 3) mechanical stimulation via hypotonic swelling (225 mOsm/kg). All stimuli activated Ca influx and were partially inhibited by the general TRPV channel blocker, ruthenium red (RR, 1 μM). Similar responses were demonstrated in TRPV4-transfected HEK 293 cells, but not in non-transfected cells. It is concluded that TRPV4 is expressed in BBB endothelial cells and that it may function as a mechanosensitive channel similar to that previously demonstrated for TRPV4 function in TRPV4-transfected HEK 293 cells.