Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia.
We studied the effect of focal cerebral ischemia on inducible (iNOS) and constitutive (cNOS) nitric oxide synthase enzymatic activities in the affected brain. The middle cerebral artery (MCA) was occluded in spontaneously hypertensive rats. Animals were killed 1, 2, 4, and 7 days later. cNOS and iNOS enzymatic activities were determined in the infarcted cortex using the assay of Bredt and Snyder. cNOS was assayed in the presence of calcium, whereas iNOS was assayed in the absence of calcium and in the presence of tetrahydrobiopterin. The validity of the iNOS assay was verified in rats treated with bacterial lipopolysaccharide. In these animals, the magnitude of the induction of iNOS enzymatic activity in lung, spleen, and brain paralleled the expression of iNOS mRNA, assessed by reverse-transcription polymerase chain reaction. After MCA occlusion, calcium-dependent (cNOS) activity was markedly reduced only in lesioned cerebral cortex at days 1-7 (p < 0.001; analysis of variance and Tukey's test). In contrast to cNOS, calcium-independent (iNOS) activity was induced substantially in the infarct (p < 0.005) but not in the contralateral intact cortex (p > 0.05). iNOS activity peaked at day 2 and was not different from baseline at day 7 (p > 0.05). No NADPH diaphorase-positive neurons were observed in the area of the lesion at days 1-7. Macrophages appeared at day 2 and invaded the infarcted tissue by day 7. At this time, numerous glial fibrillary acidic protein-positive astrocytes were observed within the lesion. The results suggest that the decline in calcium-dependent (cNOS) activity reflects loss of NOS neurons within the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
Iadecola
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Laboratory of Cerebrovascular Biology and Stroke, University of Minnesota Medical School, Minneapolis.
Xu
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Zhang
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el-Fakahany
E E
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Ross
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ME
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000473:pathology; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000201:enzymology; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D002546:Ischemic Attack, Transient / Q000201:enzymology* / Q000473:pathology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D008297:Male; D009252:NADPH Dehydrogenase / Q000032:analysis; D009474:Neurons / Q000201:enzymology / Q000473:pathology; D019001:Nitric Oxide Synthase; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR; D013154:Spleen / Q000201:enzymology
D005904:Glial Fibrillary Acidic Protein; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D001708:Biopterins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D009252:NADPH Dehydrogenase; C003402:sapropterin; D002118:Calcium