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

Abstract

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), the change in the cytosolic intracellular Ca++ concentration, [Ca++]i, was measured in cultured rat vascular smooth-muscle cells exposed to oxyhemoglobin and other substances. Oxyhemoglobin induced transient elevation of smooth-muscle cell [Ca++]i in either the presence or absence of ethyleneglycol-bis (β-aminoethylether)-N,N′-tetraacetic acid, indicating that Ca++ released by oxyhemoglobin was derived from [Ca++]i stores. In contrast, methemoglobin had no effect on the smooth-muscle cells. Exposure of the cells to reactive oxygen species generated by xanthine plus xanthine oxidase yielded the same results as with oxyhemoglobin, that is, transient elevation of 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 (an adenosine triphosphate [ATP]-dependent Ca++ pump inhibitor) plus oxyhemoglobin had no additional effect when compared with thapsigargin alone. The oxyhemoglobin-induced elevation of [Ca++]i could be blocked by an Fe++ chelator (ferene), but not by an Fe+++ chelator (deferoxamine mesylate). Treatment with either dithiothreitol or glutathione monoethyl ester markedly inhibited the oxyhemoglobin-induced elevation of [Ca++]i.Conclusions. These results indicate that Fe++-catalyzed hydroxyl radicals generated from oxyhemoglobin-derived free radicals induce the elevation of [Ca++]i by inhibiting the ATP-dependent Ca++ pump rather than the Ca++ channels in the sarcoplasmic reticulum and that thiols may prevent Ca++ pump inactivation by inhibiting the oxidation of membrane sulfhydryl groups.