Heat shock protein 90 modulates endothelial nitric oxide synthase activity and vascular reactivity in the newborn piglet pulmonary circulation.
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 (O(2)(*-)). We determined the effects of the Hsp90 antagonists geldanamycin (GA) and radicicol (RA) on basal and ACh-stimulated changes in vessel diameter, cGMP production, and Hsp90:eNOS coimmunoprecipitation 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 O(2)(*-) 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 coprecipitation. 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 O(2)(*-) generation.
Aschner
Judy L
JL
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA. judy.aschner@vanderbilt.edu
Foster
Susan L
SL
Kaplowitz
Mark
M
Zhang
Yongmei
Y
Zeng
Heng
H
Fike
Candice D
CD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D016227:Benzoquinones / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D003997:Dicarbethoxydihydrocollidine / Q000031:analogs & derivatives; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008833:Microcirculation / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D011652:Pulmonary Circulation / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D014655:Vascular Resistance / Q000502:physiology*
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018942:Macrolides; C514241:dihydroethidine; D013481:Superoxides; D009569:Nitric Oxide; D003997:Dicarbethoxydihydrocollidine; D000255:Adenosine Triphosphate; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; C035359:monorden; C001277:geldanamycin