Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

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Cytosolic free calcium concentration ([Ca2+]i) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca2+]i overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca2+]i overload can be prevented by lithium treatment. [Ca2+]i and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-d-glucose (5mM; 2-DG) plus sodium cyanide (5mM; NaCN) caused a significant decrease in cellular ATP content (14±1nmol/mg protein vs. 18±1nmol/mg protein in the control, n=6 culture dishes, P<0.05), an increase in [Ca2+]i (278±24nM vs. 71±2nM in the control, n=60 cells, P<0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10mM 2-DG led to a similar decrease in ATP content (14±2nmol/mg vs. 18±1nmol/mg in the control, P<0.05) with a delay of 5min. The [Ca2+]i response of EC was biphasic with a peak after 1min (183±6nM vs. 71±1nM, n=60 cells, P<0.05) followed by a sustained increase in [Ca2+]i. A 24-h pre-treatment with 10mM of lithium chloride before the inhibition of ATP synthesis abolished both phases of the 2-DG-induced [Ca2+]i increase. This effect was not observed when lithium chloride was added simultaneously with 2-DG.We conclude that lithium chloride abolishes the injurious [Ca2+]i overload in EC and that this most likely occurs by preventing inositol 3-phosphate-sensitive Ca2+-release from the endoplasmic reticulum. Though further research is needed, these findings provide a novel option for therapeutic strategies to protect the endothelium against imminent barrier failure.