title: Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity.
abstract: An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.
authors.affiliation: Division of Pediatric Surgery, Department of Surgery, Cardiovascular Center, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: Jingsong
authors.initials: J
authors.lastname: Ou
authors.forename: Jason T
authors.initials: JT
authors.lastname: Fontana
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Ou
authors.forename: Deron W
authors.initials: DW
authors.lastname: Jones
authors.forename: Allan W
authors.initials: AW
authors.lastname: Ackerman
authors.forename: Keith T
authors.initials: KT
authors.lastname: Oldham
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.forename: William C
authors.initials: WC
authors.lastname: Sessa
authors.forename: Kirkwood A
authors.initials: KA
authors.lastname: Pritchard
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001160:Arterioles / Q000187:drug effects* / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects* / Q000502:physiology; D002339:Carotid Arteries / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology / Q000502:physiology; D014664:Vasodilation / Q000187:drug effects* / Q000502:physiology
chemical_list: D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D007783:Lactones; D018942:Macrolides; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D019833:Genistein; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D011505:Protein-Tyrosine Kinases; C035359:monorden; D000109:Acetylcholine; D019331:NG-Nitroarginine Methyl Ester