OBJECTIVE: To identify the subtype of transient receptor potential (TRPs) channel involved in stretch-induced injury of human brain microvascular endothelial cells (HBMEC) and to explore the mechanism responsible for eNOS expression. METHODS: TRPs expression was examined by Western blot and immunocytofluoresence in the cultured HBMEC. Mechanical stretch was performed by mini-type multi-functional bio-impact machine. The levels of free calcium ion in cells were examined by the flow cytometry. The eNOS expression was detected by Western blot. RESULTS: The mRNA and protein expression of TRPV4 was detected in HBMEC by qRT-PCR, Western blot and immunocytofluoresence. The levels of free calcium ion in the stretch-treated HBMEC was significantly decreased in the presence of TRPV4 specific inhibitor (P<0.001), but there was no difference in calcium levels between the stretch and the control or unspecific inhibitor group (P=0.072 or 0.308). The levels of eNOS protein in the stretch-treated HBMEC were reduced in the presence of TRPV4 specific inhibitor or NOS inhibitor (P<0.05), but it was not changed compared with that in the control group (P>0.05). CONCLUSION: The eNOS expression is up-regulated under the condition of mechanic stretch, which is related to the activation of TRPV4, resulting in the influx of calcium.
OBJECTIVE: To identify the subtype of transient receptor potential (TRPs) channel involved in stretch-induced injury of human brain microvascular endothelial cells (HBMEC) and to explore the mechanism responsible for eNOS expression. METHODS: TRPs expression was examined by Western blot and immunocytofluoresence in the cultured HBMEC. Mechanical stretch was performed by mini-type multi-functional bio-impact machine. The levels of free calcium ion in cells were examined by the flow cytometry. The eNOS expression was detected by Western blot. RESULTS: The mRNA and protein expression of TRPV4 was detected in HBMEC by qRT-PCR, Western blot and immunocytofluoresence. The levels of free calcium ion in the stretch-treated HBMEC was significantly decreased in the presence of TRPV4 specific inhibitor (P<0.001), but there was no difference in calcium levels between the stretch and the control or unspecific inhibitor group (P=0.072 or 0.308). The levels of eNOS protein in the stretch-treated HBMEC were reduced in the presence of TRPV4 specific inhibitor or NOS inhibitor (P<0.05), but it was not changed compared with that in the control group (P>0.05). CONCLUSION: The eNOS expression is up-regulated under the condition of mechanic stretch, which is related to the activation of TRPV4, resulting in the influx of calcium.