Submitted on July 5, 2006
Accepted on February 26, 2007
Heat shock protein 90 modulates endothelial nitric oxide synthase activity and vascular reactivity in the newborn piglet pulmonary circulation
Judy L. Aschner1*, Susan L. Foster2, Mark R. Kaplowitz3, Yongmei Zhang3, Heng Zeng4, and Candice D. Fike3
1 Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States; The Vanderbilt Kennedy Center, Nashville, Tennessee, United States; Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States
2 Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States
3 Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States; Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States
4 Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States
* To whom correspondence should be addressed. E-mail: judy.aschner{at}vanderbilt.edu.
Heat Shock Protein 90 (Hsp90) binding to endothelial nitric oxide synthase (eNOS) is an important step in eNOS activation. The conformational state of bound Hsp90 determines whether eNOS produces nitric oxide (NO) or superoxide (·O2-). We determined the effects of the Hsp90 antagonists, geldanamycin (GA) and radicicol (RA), on basal and acetylcholine (ACh)-stimulated changes in vessel diameter, cGMP production and Hsp90:eNOS co-immunoprecipitation in piglet resistance-level pulmonary arteries (PRA). In perfused piglet lungs we evaluated the effects of GA and RA on ACh-stimulated changes in pulmonary arterial pressure (Ppa) and perfusate accumulation of stable NO metabolites (NOx-). The effects of GA and RA on ACh-stimulated ·O2- generation was investigated in cultured pulmonary microvascular endothelial cells (PMVEC) by dihydroethidine (DHE) oxidation and confocal microscopy. Hsp90 inhibition with GA or RA reduced ACh-mediated dilation, abolished the ACh-stimulated increase in cGMP and reduced eNOS:Hsp90 co-precipitation. GA and RA also inhibited the ACh-mediated changes in Ppa and NOx- accumulation rates in perfused lungs. ACh increased the rate of DHE oxidation in PMVEC pretreated with GA and RA but not in untreated cells. The cell-permeable superoxide dismutase mimetic, M40401, reversed GA-mediated inhibition of ACh-induced dilation in PRA. We conclude that Hsp90 is a modulator of eNOS activity and vascular reactivity in the newborn piglet pulmonary circulation. Uncoupling of eNOS with GA or RA inhibits ACh-mediated dilation by a mechanism that involves
·O2-.
