O2 TMEM16A channels regulate endothelial cell vasodilator signalling pathways

Introduction Endothelial cell (EC) ion channels regulate blood vessel diameter via calcium-dependent nitric oxide production and plasma membrane hyperpolarization. While the role of calcium-permeable cation channels is well established, the physiological significance of EC anion channels is less understood. Recently, the calcium-activated chloride channel, TMEM16A (also known as anoctamin 1) has emerged as a potential regulator of vascular function via calcium signalling and mitochondrial function, though underlying mechanisms remain unclear. This study was designed to investigate the role of TMEM16A in regulating EC vasodilator signalling pathways.

Methods All experiments used blood vessels isolated from male Sprague-Dawley rats euthanized by Schedule 1 Procedures (Animals [Scientific Procedures] Act 1986, UK). Calcium activity was studied using flat-mounted mesenteric artery preparations in which ECs were loaded with the fluorescent calcium indicator, Cal-520/AM. Mitochondrial membrane potential was studied in freshly isolated aortic EC sheets, loaded with the mitochondrial membrane potential indicator, TMRE.

Results Blockade of calcium-activated chloride channels using the specific TMEM16A inhibitor, Ani9, significantly inhibited acetylcholine-evoked calcium activity in intact artery ECs (n=5). This effect was mimicked by three other TMEM16A channel blockers (MONNA, CaCHinh-A01 and Benzbromarone, n=5 each). Notably, the action of Ani9 was independent of mitochondrial function, as evidenced by the mitochondrial membrane potential in freshly isolated ECs, which remained unchanged following a ten-minute exposure to the drug. In contrast, MONNA, CaCHinh-A01 and Benzbromarone each caused rapid depolarization of the mitochondrial membrane potential.

Conclusion We show that TMEM16A channels are involved in EC calcium signalling pathways that regulate vascular tone. Notably, the effects of Ani9 were independent of mitochondrial function, whilst the other inhibitors caused mitochondrial depolarization. Mitochondrial depolarization likely depletes ATP levels, explaining their effects on calcium signalling. Our findings underscore the potential role of TMEM16A in EC blood flow control, and highlight potential off-target effects of certain inhibitors.