TRPV4-Mediated Relaxation of Pig Coronary Arteries is Dependent on KCa3.1 Channel Amplification of Endothelial Ca2+ Dynamics.

摘要

In coronary arteries of the heart, the endothelial cell (EC) layer contributes to arterial tone through a relaxing pathway known as endothelial-derived hyperpolarization (EDH). One form of EDH is the EC Ca2+-mediated activation of small (KCa2.3) and intermediate (KCa3.1) conductance Ca2+-activated K+ channels at the membrane to cause hyperpolarization of the underlying smooth muscle (SMC) and relaxation. Evidence from studies in mouse mesenteric arteries (MMAs) suggests that the vanilloid transient receptor potential channel 4 (TRPV4), a Ca2+ permeable channel that senses changes in blood flow, temperature, and pH, is involved in vasoregulation through Ca2+ regulation and interaction with KCa2.3 and KCa3.1. Work from our lab in MMAs also suggests that EC Ca2+ signals can be increased by KCa3.1-mediated positive feedback on TRPV4 channels. The specific relationship between complex Ca2+ signals, TRPV4, and KCa channels has not been fully described in human coronary arteries (HCAs). We have begun to characterize dynamic Ca2+ events in the pig coronary arteries (PCAs), a good model for HCAs, and our evidence suggests that increased EC Ca2+ signals predict substance P (SP)-mediated relaxation. We hypothesized that TRPV4 is expressed in PCAs, and that direct activation of TRPV4 both increases EC Ca2+ signals and elicits relaxation through the KCa2.3 and KCa3.1 channels. We used immunohistochemistry (IHC), Western Blot (WB), and qPCR to determine if TRPV4 was present in PCAs. IHC revealed specific labeling of TRPV4 protein along the base of the ECs, but not the SMCs. WB revealed a protein band at 120kDa in ECs that was not in the SMCs. qPCR revealed a distinct melting point for EC TRPV4 cDNA at 86.5°C. These results suggest that both TRPV4 protein and mRNA is present in the ECs of PCAs. Myography was used to measure changes in tone resulting from direct TRPV4 activation in the presence or absence of KCa2.3 and/or KCa3.1 channel inhibition. In PCAs precontracted with 10nM U46619, a thromboxane A2 analog, we found that treatment with the TRPV4-activator GSK1016790A elicited relaxation (27% of precontracted tone). In PCAs pretreated with the KCa2.3 inhibitor apamin, TRPV4-mediated relaxation was unaffected. However, pretreatment with the KCa3.1 inhibitor charybdotoxin abolished TRPV4-mediated relaxation. We then used high-speed confocal microscopy, open artery preparations, and the algorithm LC_Pro to determine if TRPV4 activation increases Ca2+ signals in the presence or absence of KCa3.1 channel inhibition. TRPV4 activation increased Ca2+ site recruitment by 50% and event recruitment by 54%. KCa3.1 inhibition abolished TRPV4-mediated Ca2+ increases. We found that direct activation of TRPV4 channels localized to the base of ECs elicits relaxation of PCAs and recruits sites of Ca2+ activity in the ECs. These responses were both found to be dependent on KCa3.1 channel-mediated signaling, but not KCa2.3. These findings may expose targets for therapeutic intervention in diseases such as coronary artery disease (CAD) where EC-mediated relaxation is compromised.