Glutathione monoethyl ester and inhibition of the oxyhemoglobin-induced increase in cytosolic calcium in cultured smooth-muscle cells

Object. The mechanism of arterial vasoconstriction caused by oxyhemoglobin production after subarachnoid hemorrhage was investigated. Methods. Using a fluorescent Ca++ indicator (fura-2 acetoxymethyl ester), t he change in the cytosolic intracellular Ca++ concentration, [Ca++](i), was measured in cultured rat vascular smooth-muscle cells exposed to oxyhemogl obin and other substances. Oxyhemoglobin induced transient elevation of smo oth-muscle cell [Ca++](i) in either the presence or absence of ethyleneglyc ol-bis (beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca++ r eleased by oxyhemoglobin was derived from [Ca++](i) stores. In contrast, me themoglobin had no effect on the smooth-muscle cells. Exposure of the cells to reactive oxygen species generated by xanthine plus xanthine oxidase yie lded the same results as with oxyhemoglobin, that is, transient elevation o f smooth-muscle cell [Ca++](i). Procaine (a Ca++ channel blocker) failed to inhibit the oxyhemoglobin-induced elevation of [Ca++](i). Ryanodine (a Ca+ channel opener) plus oxyhemoglobin caused markedly greater elevation of [ Ca++](i) than ryanodine alone, whereas thapsigargin Can adenosine triphosph ate [ATP]-dependent Ca++ pump inhibitor) plus oxyhemoglobin had no addition al effect when compared with thapsigargin alone. The oxyhemoglobin-induced elevation of [Ca++](i) could be blocked by an Fe++ chelator (ferene), but n ot by an Fe+++ chelator (deferoxamine mesylate). Treatment with either dith iothreitol or glutathione monoethyl ester markedly inhibited the oxyhemoglo bin-induced elevation of [Ca++](i). Conclusions. These results indicate that Fe++-catalyzed hydroxyl radicals g enerated from oxyhemoglobin-derived free radicals induce the elevation of [ Ca++](i) by inhibiting the ATP-dependent Ca++ pump rather than the Ca++ cha nnels in the sarcoplasmic reticulum and that thiols may prevent Ca++ pump i nactivation by inhibiting the oxidation of membrane sulfhydryl groups.