Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion.

Ischemia and Reperfusion (I/R) injuries are associated with coronary artery hypercontracture. They are mainly originated by an exacerbated response to agonists released by endothelium such as Endothelin (ET-1), involving the alteration in intracellular calcium handling. Recent evidences have highlighted the implication of Store-Operated Calcium Channels (SOCC) in intracellular calcium homeostasis in coronary artery. However, little is known about the role of SOCC in the regulation of coronary vascular tone under I/R. The aim of this study was to evaluate the role of SOCC and l-type Ca2+ channels (LTCC) in coronary artery vasoconstriction originated by ET-1 in I/R. We used Left Anterior Descendent coronary artery (LAD) rings, isolated from Wistar rats, to study the contractility and intracellular Ca2+ concentration ([Ca2+]i) under a simulated I/R protocol. We observed that responses to high-KCL induced depolarization and caffeine-induced Ca2+ release are attenuated in coronary artery under I/R. Furthermore, ET-1 addition in ischemia promotes transient and small rise of [Ca2+]i and coronary vascular tone. Meanwhile, these effects are significantly potentiated during reperfusion. The resulting ET-1-induced vasoconstrictions and [Ca2+]i increase were abolished by; GSK-7975A and gadolinium, inhibitors of SOCC; and nifedipine a widely used inhibitor of LTCC. Interestingly, using in situ Proximity Ligation Assay (PLA) in isolated coronary smooth muscle cells we found significant colocalization of LTCC CaV1.2 isoform with Orai1, the pore forming subunit of SOCC, and TRPC1 under I/R. Our data suggest that hypercontraction of coronary artery induced by ET-1 after I/R involves the co-activation of LTCC and SOCC, which colocalize significantly in the sarcolemma of coronary smooth muscle cells.