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 (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 +/- 1 nmol/mg protein vs. 18 +/- 1 nmol/mg protein in the control, n = 6 culture dishes, p < 0.05), an increase in [Ca2+](i) (278 +/- 24 nM vs. 71 +/- 2 nM 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 10 mM 2-DG led to a similar decrease in ATP content (14 +/- 2 nmol/mg vs. 18 +/- 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca2+](i) response of EC was biphasic with a peak after 1 min (183 +/- 6 nM vs. 71 +/- 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca2+](i). A 24-h pre-treatment with 10 mM 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. (C) 2013 Elsevier Inc. All rights reserved.