Objectives: To examine erythrocyte intracellular calcium dynamics in clinical sepsis and experimental endotoxemia.
Design: Prospective, multiexperimental study utilizing in vitro manipulation and evaluation of human erythrocytes.
Setting: University research laboratory.
Patients: Healthy, elective surgical patients, "septic" surgical patients, and normal volunteers.
Interventions: For all experimental studies, whole blood specimens were incubated with 2 micrograms/mL of Escherichia coli endotoxin (experimental) or an equivalent volume of phosphate buffered saline (control). Incubations were performed in specimens pretreated with 0.4 mM of verapamil and/or 50 mM of dantrolene. Incubations were performed in the presence and absence of extracellular calcium. Incubations were also performed utilizing pre- and posttreatment with 1 mM of adenosine 5'-triphosphate (ATP) and/or 30 mM of adenosine.
Measurements and main results: Free cytosolic calcium concentration was determined by fluorescent spectroscopy, utilizing the calcium chelator, FURA-2AM. Sepsis was associated with a significant increase in erythrocyte intracellular calcium concentration as compared with nonseptic controls (96.26 vs. 45.38 nM; p < .001). Similar changes could be induced by endotoxin incubation of whole blood (84.52 vs. 40.45 nM; p < .001). This endotoxin-induced increase was independent of extracellular calcium concentration and was only partially ameliorated by calcium-channel blockade. Inhibition of intracellular calcium release was ineffective in altering the endotoxin-induced increase in the erythrocyte intracellular calcium value. In contrast, pretreatment with either adenosine or ATP minimized these increases. Posttreatment with ATP, but not adenosine, allowed partial reversal of this endotoxin-induced increase in intracellular calcium.
Conclusions: Sepsis induces alterations of erythrocyte intracellular calcium homeostasis. A significant increase in free cytosolic concentrations of intracellular calcium is characteristic of this altered homeostasis. These changes are reproducible by the incubation of whole blood with endotoxin. This increase in cytosolic calcium concentration appears to be independent of extracellular calcium concentration, transmembrane calcium channels, and/or intracellular calcium stores. It can, however, be modulated through provision of high-energy phosphates and/or their precursors to the cell itself.