Nitric oxide preconditioning regulates endothelial monolayer integrity via the heat shock protein 90-soluble guanylate cyclase pathway.
ABSTRACT Large (pathological) amounts of nitric oxide (NO) induce cell injury, whereas low (physiological) NO concentrations often ameliorate cell injury. We tested the hypotheses that pretreatment of endothelial cells with low concentrations of NO (preconditioning) would prevent injury induced by high NO concentrations. Apoptosis, induced in bovine aortic endothelial cells (BAECs) by exposing them to either 4 mM sodium nitroprusside (SNP) or 0.5 mM N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) for 8 h, was abolished by 24-h pretreatment with either 100 microM SNP, 10 microM spermine NONOate, or 100 microM 8-bromo-cGMP (8-Br-cGMP). Repair of BAECs following wounding, measured as the recovery rate of transendothelial electrical resistance, was delayed by 8-h exposure to 4 mM SNP, and this delay was significantly attenuated by 24-h pretreatment with 100 microM SNP. NO preconditioning produced increased association and expression of soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90). The protective effect of NO preconditioning, but not the injurious effect of 4 mM SNP, was abolished by either a sGC activity inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or a HSP90 binding inhibitor (radicicol) and was mimicked by 8-Br-cGMP. We conclude that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the HSP90/sGC pathway. HSP90/sGC may thus play a role in the protective effects of NO-generating drugs from injurious stimuli.
Article: Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.[show abstract] [hide abstract]
ABSTRACT: The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.AJP Heart and Circulatory Physiology 12/2007; 293(5):H3080-7. · 3.71 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Nitric oxide (NO) is capable of promoting either cell death or cell survival depending on cell type and experimental conditions. In this study, the possible effects of NO on the viability of lens epithelial cells were investigated in an explant model used previously to identify cellular changes associated with posterior capsule opacification following cataract surgery. Rat lens epithelial explants prepared from weanling rats were cultured in a serum-free medium for five days with or without the addition of the nitric oxide synthase inhibitor, L-N(omega)-nitro-L-arginine methyl ester (L-NAME), using the inactive enantiomer D-NAME as a control. Alternatively, explants were cultured for nine days with or without the NO donor, sodium nitroprusside. Explants were assessed morphologically and immunohistochemically or by determining DNA content. In the presence of L-NAME but not in controls, progressive rounding up and detachment of cells from the lens capsule occurred, leading to extensive cell loss. Affected cells showed apoptosis-like cell-surface blebbing and nuclear fragmentation. Conversely, inclusion of sodium nitroprusside suppressed the morphological changes and spontaneous cell loss that occurred when sparsely covered explants were cultured for nine days, increased cell coverage fourfold during that period, and prevented the expression of the transdifferentiation markers alpha-smooth muscle actin and fibronectin. In addition, whereas L-NAME exacerbated cell loss induced by culturing with 50 pg/ml transforming growth factor-beta2, sodium nitroprusside offered protection. This study points to a previously unidentified role for NO as an endogenously produced survival factor for lens epithelial cells, raising the possibility of using NO deprivation as a means of removing residual lens cells following cataract surgery and thereby preventing posterior capsule opacification.Molecular vision 01/2008; 14:983-91. · 2.20 Impact Factor