Heat shock protein 90-eNOS interactions mature with postnatal age in the pulmonary circulation of the piglet

• Published in: American journal of physiology. Lung cellular and molecular physiology 2009-03, Vol.296 (3), p.L555-L564
• Main Authors: Aschner, Judy L, Zeng, Heng, Kaplowitz, Mark R, Zhang, Yongmei, Slaughter, James C, Fike, Candice D
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Age Factors; Animals; Animals, Newborn; Cells; Gene expression; Heat shock proteins; Hogs; HSP90 Heat-Shock Proteins - metabolism; In Vitro Techniques; Lungs; NG-Nitroarginine Methyl Ester - pharmacology; Nitric oxide; Nitric Oxide Synthase Type III - metabolism; Proto-Oncogene Proteins c-akt - metabolism; Pulmonary arteries; Pulmonary Artery - drug effects; Pulmonary Artery - growth & development; Pulmonary Artery - metabolism; S-Nitroso-N-Acetylpenicillamine - pharmacology; Swine; Vascular Resistance
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.90456.2008

1 Department of Pediatrics, 2 The Vanderbilt Kennedy Center, 3 The Vanderbilt Center for Molecular Toxicology, and 4 Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee Submitted 22 August 2008 ; accepted in final form 4 January 2009 Binding of endothelial nitric oxide synthase (eNOS) to the chaperone protein, Hsp90, promotes coupled eNOS synthetic activity. Using resistance level pulmonary arteries (PRA) from 2-day-, 5- to 7-day-, and 12-day-old piglets, we tested the hypothesis that Hsp90-eNOS interactions are developmentally regulated in the early neonatal period. PRA were isolated for coimmunoprecipitation and immunoblot analyses or cannulated for continuous diameter measurements using the pressurized myography technique. NOS inhibition caused less constriction in PRA from 2-day- compared with 5- to 7-day- and 12-day-old piglets. No age-related differences were found in dilation responses to an NO donor or in protein expression of Hsp90, phospho-eNOS (Ser 1177 ), Akt, phospho-Akt, or caveolin-1. Compared with the older animals, PRA from 2-day-old piglets had higher total eNOS expression but displayed less binding of eNOS to Hsp90 and Akt. Hsp90 antagonism with radicicol induced greatest constriction in PRA from 12-day-old piglets. ACh stimulation caused dilation in PRA from 5- to 7-day- and 12-day-old but not 2-day-old animals, despite rapid and equivalent ACh-mediated eNOS phosphorylation (Ser 1177 ) in all three age groups. Hsp90 inhibition abolished ACh-mediated dilation in PRA from the older piglets. ACh failed to stimulate Hsp90-eNOS binding in 2-day-old but induced a significant increase in Hsp90-eNOS coimmunoprecipitation in PRA from the older age groups, which was blocked by Hsp90 antagonism. We conclude that physical interactions between Hsp90 and eNOS mature over the first weeks of life, likely contributing to the postnatal fall in pulmonary vascular resistance and changes in agonist-induced pulmonary vascular responses characteristic of the early neonatal period. Akt; development; nitric oxide Address for reprint requests and other correspondence: J. L. Aschner, The Monroe Carell Jr. Children's Hospital at Vanderbilt, Division of Neonatology, 11111 Doctor's Office Tower, 2200 Children's Way, Nashville, TN 37232-9544 (e-mail: judy.aschner{at}vanderbilt.edu )