Introduction: Endothelial cell (EC) ion channels regulate the dynamic changes in blood vessel diameter through the release of various chemical mediators, for example, in the case of calcium-dependent release of nitric oxide (NO) to adjacent smooth muscle, as well as through hyperpolarisation of the plasma membrane. Mitochondria play a critical role in endothelium-dependent vascular function; loss of OXPHOS in ECs has been shown to inhibit calcium signalling 1. Some calcium-permeable cation channels, such as the calcium-sensitive channel TRPV4, have had their role well characterized2, yet the contributions of anion channels to control of blood vessel diameter is much less understood. The calcium-activated chloride channel, TMEM16A (otherwise known as anoctamin-1) has been suggested as a potential regulator of calcium signalling 3 as well as mitochondrial function, however, it’s mechanisms still remain unclear. This study was designed with the aim of investigating the role of TMEM16A in regulating EC vasodilator signalling pathways.
Methods: All experiments included used isolated blood vessels from healthy male Sprague-Dawley rats euthanized by Schedule 1 Procedures (Animals {Scientific Procedures] Act 1986, UK). Calcium activity was studied using mesenteric artery preparations, flat-mounted and loaded with the calcium-sensitive fluorophore, Cal520-AM. Freshly isolated aortic endothelial cell sheets were utilized to study mitochondrial membrane potential, loaded with the mitochondrial membrane potential sensitive fluorophore, TMRE.
Results: Antagonism of the calcium-activated chloride channel, TMEM16A with the specific blocker Ani9, did not inhibit the calcium response induced by acetylcholine in the intact arteries (n=5). However, the 3 other TMEM16A blockers (MONNA, CaCCinh-A01 and Benzbromarone, n=5 each) caused a significant decrease in calcium activity in the flat-mounted tissue. Similar activity was seen in the isolated aortic ECs with MONNA, CaCCinh-A01 and Benzbromarone all causing a rapid loss of the mitochondrial membrane potential (n=5). In contrast, the mitochondrial membrane potential was unaffected by a ten-minute treatment with Ani9 (n=5).
Conclusion: In this study, we show that TMEM16A channels are essential for the propagation of the calcium signalling pathways that underpin the control of vascular tone. Treatment with 3 of the well-studied TMEM16A blockers caused inhibition of calcium signalling and lead to the depolarisation of the mitochondrial membrane potential. With regards to Ani9, both EC calcium signalling and the mitochondrial membrane potential remained unaffected. Loss of the mitochondrial membrane potential will cause the reduced availability of mitochondrial ATP, accounting for the loss of EC calcium signalling. Our findings indicate a role for TMEM16A in the endothelium’s control of blood vessel diameter.