Regulation of cerebellar nitric oxide production in response to prolonged in vivo hypoxia.
The aim of this study was to assess the influence of prolonged in vivo hypoxia on cerebellar nitric oxide (NO) production. Conscious rats were exposed to 10% O2 (balanced N2) for 12 or 48 hr (arterial PO2 between 35 and 39 mmHg). The animals were then killed, and the cerebella were quickly frozen. NO production was measured in vitro by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline. Protein and mRNA expressions of the neuronal (nNOS) and endothelial (ecNOS) isoforms of nitric oxide synthases were assessed by using immunoblotting and semiquantitative reverse transcription-polymerase chain reaction, respectively. We also measured mRNA expression of GTP cyclohydrolase I, the rate-limiting enzyme in the synthesis of NOS cofactor, tetrahydrobiopterin, and mRNA and protein expressions of argininosuccinate synthase and argininosuccinate lyase, essential enzymes for the recycling of L-citrulline to L-arginine. Prolonged in vivo hypoxia resulted in a time-dependent increase in cerebellar nitric oxide synthase activity and a significant rise in mRNA and protein expressions of nNOS isoform; however, ecNOS protein expression declined significantly. There was also a rise in mRNA expression of GT cyclohydrolase I; however, neither mRNA nor protein expression of argininosuccinate synthase and argininosuccinate lyase changed significantly in hypoxic animals. These results suggest that prolonged hypoxia increases cerebellar NO formation as a result of upregulation of cerebellar nNOS expression, whereas ecNOS expression declines. We propose that cofactor availability for NO production may also increase during hypoxia because of upregulation of GTP cyclohydrolase I expression. Recycling of L-citrulline to L-arginine, however, remains unchanged.
Guo
Y
Y
Critical Care and Respiratory Divisions, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Ward
M E
ME
Beasjours
S
S
Mori
M
M
Hussain
S N
SN
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000235:genetics / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D002531:Cerebellum / Q000098:blood supply / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D004790:Enzyme Induction; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism*; D002534:Hypoxia, Brain / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D001708:Biopterins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase; D001123:Argininosuccinate Lyase; C003402:sapropterin