INCREASE IN RAT AORTIC ENDOTHELIAL FREE CALCIUM-MEDIATED BY METABOLICALLY SENSITIVE CALCIUM-RELEASE FROM ENDOPLASMIC-RETICULUM

Objective: The aim was to examine the relationship between cellular me tabolism and intracellular [Ca2+] in vascular endothelial cells, focus ing on the timing, mechanism. and reversibility of intracellular [Ca2] changes resulting from ATP depletion. Methods: Cultured rat aortic e ndothelial monolayers were loaded with indo-1 and exposed for 30 min t o: (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 oxi dative 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+], glycolyti c inhibition resulted in a larger, slowly developing, sustained increa se in [Ca2+]. Endothelial [Ca2+] was not affected by inhibition of oxi dative phosphorylation alone, whereas a rapid, sustained, and largely reversible increase (similar to 102 nM) occurred when NaCN exposure wa s combined with glucose deprivation. The increase in [Ca2+] during glu cose-free NaCN exposure was not altered when calcium influx was preven ted by removal of extracellular calcium, but was abolished following d epletion of an intracellular calcium store by the endoplasmic reticula r Ca2+-ATPase inhibitor thapsigargin. In SNARF-1 loaded monolayers, in hibition of glycolysis with iodoacetic acid decreased intracellular pH by 0.33(SEM 0.10) units whereas inhibition of oxidative phosphorylati on in the absence of glucose increased intracellular pH by 0.17(0.05) units. While these divergent pH changes were noted, [Ca2+] increased i n both groups. Conclusions: A metabolically sensitive endoplasmic reti cular calcium store is rapidly and reversibly released in vascular end othelial cells. Endothelial [Ca2+] is shown to be dependent on glycoly tic energy production. In the endothelial cell, brief periods of inhib ition of oxidative phosphorylation in the absence of glucose rapidly a ffect intracellular calcium pools rather than leading to calcium influ x due to non-specific cellular damage. Effects on intracellular pH alo ne cannot account for the changes in [Ca2+].