Objective-We explored the role of endoplasmic reticulum (ER)-mitochondria Ca 2+ cross talk involving voltage-dependent anion channel-1 (VDAC1)/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 in endothelial cells during hypoxia/reoxygenation (H/R), and investigated the protective effects of acetylcholine. Approach and Results-Acetylcholine treatment during reoxygenation prevented intracellular and mitochondrial Ca 2+ increases and alleviated ER Ca 2+ depletion during H/R in human umbilical vein endothelial cells. Consequently, acetylcholine enhanced mitochondrial membrane potential and inhibited proapoptotic cascades, thereby reducing cell death and preserving endothelial ultrastructure. This effect was likely mediated by the type-3 muscarinic acetylcholine receptor and the phosphatidylinositol 3-kinase/Akt pathway. In addition, interactions among members of the VDAC1/ glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex were increased after H/R and were associated with mitochondrial Ca 2+ overload and cell death. Inhibition of the partner of the Ca 2+ channeling complex (VDAC1 siRNA) or a reduction in ER-mitochondria tethering (mitofusin 2 siRNA) prevented the increased protein interaction within the complex and reduced mitochondrial Ca 2+ accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER-mitochondria Ca 2+ cross talk by inhibiting the VDAC1/glucoseregulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 expression. Phosphatidylinositol 3-kinase siRNA diminished acetylcholine-mediated inhibition of mitochondrial Ca 2+ overload and VDAC1/glucoseregulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex formation induced by H/R. Conclusions-Our data suggest that ER-mitochondria interplay plays an important role in reperfusion injury in the endothelium and may be a novel molecular target for endothelial protection. Acetylcholine attenuates both intracellular and mitochondrial Ca 2+ overload and protects endothelial cells from H/R injury, presumably by disrupting the ER-mitochondria interaction.

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Background and Purpose The activation of M3 cholinoceptors (M3 receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)‐induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca2+/calmodulin‐dependent protein kinase II (CaMKII) regulation. Experimental Approach Rats were given choline (10 mg·kg−1, i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M3 receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), was injected (0.12 μg·kg−1, i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca2+‐cycling proteins were also assessed. Key Results Choline treatment attenuated I/R‐induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up‐regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca2+‐cycling proteins, including Na+/Ca2+ exchanger, inositol 1,4,5‐trisphosphate receptor, sarcoplasmic reticulum Ca2+‐ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4‐DAMP. Conclusions and Implications Our data suggest that inhibition of the ROS‐mediated CaMKII pathway and modulation of Ca2+‐cycling proteins may be novel mechanisms underlying choline‐induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M3 receptors in the vasculature, providing a new therapeutic strategy for I/R‐induced vascular injury. Linked Articles This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

The Ca(2+)-sensing receptor (CaSR) plays an important role in regulating vascular tone. In the present study, we investigated the positive effects of the vagal neurotransmitter acetylcholine by suppressing CaSR activation in mesenteric arteries exposed to hypoxia/reoxygenation (H/R). The artery rings were exposed to a modified 'ischemia mimetic' solution and an anaerobic environment to simulate an H/R model. Our results showed that acetylcholine (10(-6) mol/L) significantly reduced the contractions induced by KCl and phenylephrine and enhanced the endothelium-dependent relaxation induced by acetylcholine. Additionally, acetylcholine reduced CaSR mRNA expression and activity when the rings were subjected to 4 h of hypoxia and 12 h of reoxygenation. Notably, the CaSR antagonist NPS2143 significantly reduced the contractions but did not improve the endothelium-dependent relaxation. When a contractile response was achieved with extracellular Ca(2+), both acetylcholine and NPS2143 reversed the H/R-induced abnormal vascular vasoconstriction, and acetylcholine reversed the calcimimetic R568-induced abnormal vascular vasoconstriction in the artery rings. In conclusion, this study suggests that acetylcholine ameliorates the dysfunctional vasoconstriction of the arteries after H/R, most likely by decreasing CaSR expression and activity, thereby inhibiting the increase in intracellular calcium concentration. Our findings may be indicative of a novel mechanism underlying ACh-induced vascular protection.