TIME COURSE OF CHANGES IN CONCENTRATION OF INTRACELLULAR FREE CALCIUM IN CULTURED CEREBROVASCULAR SMOOTH-MUSCLE CELLS EXPOSED TO OXYHEMOGLOBIN

A culture of smooth muscle cells obtained from monkey middle cerebral arteries was developed to allow quantitative assessment of intracellular calcium and immunofluorescence analysis after various periods of exposure to oxyhemoglobin. Intracellular calcium concentration was examined for up to 7 days after a single exposure to oxyhemoglobin. Intracellular calcium concentrations were measured with the fluorescent dye fura-2 and were significantly elevated for 7 days after exposure to oxyhemoglobin (P < 0.01). Less than 2 minutes after application of oxyhemoglobin, there was marked elevation of intracellular calcium from the control value of 75 +/- 2 nmol/L to 240 +/- 28 nmol/L (P < 0.01 by analysis of variance). Intracellular calcium concentration of cells exposed for 24 hours to oxyhemoglobin and then grown in normal oxyhemoglobin-free medium fell close to normal levels on Days 3 and 7. On Day 3, the increase in intracellular calcium that followed repeated daily exposure to oxyhemoglobin was greater than that resulting from a single application of oxyhemoglobin (P < 0.01 by Student's t test), but by Day 7 the elevation produced by these different approaches was similar. Smooth muscle cells exposed to oxyhemoglobin showed a reduction in immunoreactivity to alpha-actin. These data support the hypothesis that disruption of intracellular calcium regulation and calcium overloading may be important in the process of cell injury, which results in vasoconstriction and sometimes cell death, after exposure to oxyhemoglobin.