title: 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.
abstract: 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.
authors: Department of Pediatrics, Drexel University College of Medicine and St. Christopher's Hospital for Children, Philadelphia, PA 19102, USA. omprakash.mishra@drexelmed.edu
authors: Om Prakash
authors: OP
authors: Mishra
authors: Qazi M
authors: QM
authors: Ashraf
authors: Maria
authors: M
authors: Delivoria-Papadopoulos
mesh_terms: 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*
chemical_list: D009569:Nitric Oxide; D014443:Tyrosine; D052248:Nitric Oxide Synthase Type I