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(center dot-)). 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(center dot-) 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 AChmediated dilation by a mechanism that involves O-2(center dot-) generation.