Role of the endothelial caveolae microdomain in shear stress–mediated coronary vasorelaxation

Tong Lu,Xiao-Li Wang,Qiang Chai,Xiaojing Sun,Garry C Sieck,Zvonimir S Katusic,Hon-Chi Lee

doi:10.1074/jbc.m117.786152

Tong Lu, Xiao-Li Wang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m117.786152

Copy DOI

Abstract

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress-mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI2) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca2+ ([Ca2+] i ) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae-TRPV4-SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.

Highlights

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress–mediated vasodilation (SSD)
We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies
Further evaluation to confirm the purity of the BCEC preparations was performed by incubating the cells with FITC-conjugated bovine–specific monoclonal anti-CD31 antibodies (1:10 clone CO.3E1D4, Thermo Fisher Scientific) or with control nonimmune mouse IgG conjugated with FITC

Summary

To whom correspondence should be addressed

Activation of endothelial ion channels by shear stress mouse mesenteric arteries through TRPV4 activation [23]. A major impediment to progress in our understanding of the regulation of ion channel function in vascular endothelial cells is the change of phenotype of these cells under culture conditions We have overcome this barrier by studying freshly isolated bovine coronary endothelial cells. We were able to directly measure ionic currents in freshly isolated single ECs from coronary arteries in response to SS, demonstrating the regulation of TRPV4 and SK3 function by SS. These novel findings provide mechanistic insights into the molecular and ionic signaling mediating SSD in the coronary vasculature and have significant scientific and clinical implications

Results

Discussion

Experimental procedures