Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels.

We tested the hypothesis that the ubiquitous store-operated Ca(2+) entry (SOCE) pathway contributes to histone-induced endothelial Ca(2+) events. We also considered an alternate hypothesis: cationic electrostatic interactions between histones and negatively charged phospholipids deform endothelial membranes and thereby allow extracellular Ca(2+) entry. A role for SOCE in histone responses was ruled out by genetic ablation of the ORAI1/2/3 channel trio; yet, histone effects were blocked by application of the multivalent cation gadolinium Gd(3+). Using live cell video microscopy of endothelial cells labeled with membrane dye FM1-43, we recorded plasma membrane movements including vesiculation, blebbing, and ruffling of lamellipodia over 60 minutes following histone exposure. These cell membrane theatrics were markedly different from the uniform pattern of exocytosis and subsequent blebbing produced by calcium overload with ionomycin. The membrane permeabilization produced by histones, and not ionomycin, was transient and a subset of cells recovered membrane integrity within 1 hour. Removal of extracellular Ca(2+) prevented histone-induced intracellular Ca(2+) overload while surprisingly exacerbating plasma membrane deformation. Conversely, decreasing the density of the negative charge surface by adding calcium or or increasing extracellular Ca(2+) levels effectively screened common membrane phospholipids from interactions with labeled histones and prevented endothelial damage in cells exposed to histones. Collectively these results indicate that low extracellular Ca(2+) levels enhance interactions between histones and endothelial cell membrane phospholipids to increase cytotoxicity. Importantly, this supports the concept of aggressive Ca(2+) repletion during resuscitation to prevent hypocalcemia, stabilize endothelial cell membranes and improve cardiovascular recovery from shock.