Objective: The aim was to examine the relationship between cellular metabolism and intracellular [Ca2+] in vascular endothelial cells, focusing on the timing, mechanism, and reversibility of intracellular [Ca2+] changes resulting from ATP depletion.
Methods: Cultured rat aortic endothelial monolayers were loaded with indo-1 and exposed for 30 min to: (1) glucose-free buffer, (2) 10 mM deoxyglucose or iodoacetic acid (0.1 or 2.5 mM) to inhibit glycolysis, or (3) 2 mM NaCN to inhibit oxidative phosphorylation with or without glucose. In other experiments, the pH sensitive fluorescent indicator SNARF-1 was used to assess the relationship between observed changes in [Ca2+] and pH.
Results: While glucose deprivation resulted in a minor increase in [Ca2+], glycolytic inhibition resulted in a larger, slowly developing, sustained increase in [Ca2+]. Endothelial [Ca2+] was not affected by inhibition of oxidative phosphorylation alone, whereas a rapid, sustained, and largely reversible increase (approximately 102 nM) occurred when NaCN exposure was combined with glucose deprivation. The increase in [Ca2+] during glucose-free NaCN exposure was not altered when calcium influx was prevented by removal of extracellular calcium, but was abolished following depletion of an intracellular calcium store by the endoplasmic reticular Ca(2+)-ATPase inhibitor thapsigargin. In SNARF-1 loaded monolayers, inhibition of glycolysis with iodoacetic acid decreased intracellular pH by 0.33(SEM 0.10) units whereas inhibition of oxidative phosphorylation in the absence of glucose increased intracellular pH by 0.17(0.05) units. While these divergent pH changes were noted, [Ca2+] increased in both groups.
Conclusions: A metabolically sensitive endoplasmic reticular calcium store is rapidly and reversibly released in vascular endothelial cells. Endothelial [Ca2+] is shown to be dependent on glycolytic energy production. In the endothelial cell, brief periods of inhibition of oxidative phosphorylation in the absence of glucose rapidly affect intracellular calcium pools rather than leading to calcium influx due to non-specific cellular damage. Effects on intracellular pH alone cannot account for the changes in [Ca2+].