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Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide
Journal article   Peer reviewed

Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

Om Prakash Mishra, Qazi M. Ashraf and Maria Delivoria-Papadopoulos
Neurochemical research, v 35(1)
2010
PMID: 19590958

Abstract

Biochemistry Biomedical and Life Sciences Biomedicine Cell Biology General Neurochemistry Neurology Neurosciences Original Paper
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.

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Web of Science research areas
Biochemistry & Molecular Biology
Neurosciences
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