Mechanism of increased tyrosine (Tyr(99)) phosphorylation of calmodulin during hypoxia in the cerebral cortex of newborn piglets: the role of nNOS-derived nitric oxide.
The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr(99) phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr(99) and total) of calmodulin determined by Western blot using anti-phospho-(pTyr(99))-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr(99) calmodulin (ODxmm(2)) was 78.55 +/- 10.76 in Nx, 165.05 +/- 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 +/- 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 +/- 13.69 in Nx, 156.17 +/- 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 +/- 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr(99) phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr(99) phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr(99) phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.
Mishra
Om Prakash
OP
Department of Pediatrics, Drexel University College of Medicine and St. Christopher's Hospital for Children, Philadelphia, PA 19102, USA. omprakash.mishra@drexelmed.edu
Ashraf
Qazi M
QM
Delivoria-Papadopoulos
Maria
M
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000201:enzymology / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010766:Phosphorylation; D013552:Swine; D014443:Tyrosine / Q000378:metabolism*
D009569:Nitric Oxide; D014443:Tyrosine; D052248:Nitric Oxide Synthase Type I