TRPV4 channels contribute to ATP‐stimulated calcium influx in endothelial cells

Objective: We examined the role of TRPV4 channels in ATP‐stimulated Ca 2+ influx in endothelial cells (ECs) of the carotid artery (CA). Methods: Rat CAs were harvested, cut into segments, and organ cultured for three days with TRPV4 siRNA to selectively knockdown TRPV4 protein. Mock transfection and scrambled siRNA controls were performed in parallel. Knockdown was confirmed by real‐time PCR, Western, and immunofluorescence. Intracellular free Ca 2+ concentration was measured (fura‐2) in response to ATP in ECs isolated from organ cultured arteries. Results: TRPV4 mRNA and protein were significantly knocked down in whole CA in the TRPV4 siRNA group. EC‐specific expression of TRPV4 from organ cultured arteries was also significantly reduced in the siRNA group compared with control (immunofluorescence). Next, endothelial Ca 2+ responses to 4α‐PDD (a TRPV4 channel opener) and ATP were evaluated in mock and TRPV4 siRNA transfected arteries. The EC Ca 2+ increase to 4α‐PDD was significantly reduced (~50%) following TRPV4 knockdown, demonstrating significant functional loss of TRPV4 channels. Finally, stimulation with ATP revealed a significant reduction in the sustained Ca 2+ influx component whereas peak Ca 2+ responses were unchanged. Conclusion: These results demonstrate that ATP‐stimulated Ca 2+ influx in CA endothelial cells is partially dependent on TRPV4 channels. (AHA 0665100Y and 0230353N)