. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol 285: H669-H678, 2003. First published April 3, 2003 10.1152/ajpheart.01025.2002 is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the ␣-or ␤-subunit of sGC show that the sGC ␤-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.smooth muscle cells; endothelium; vascular endothelial growth factor; bradykinin; cGMP accumulation SOLUBLE GUANYLATE CYCLASE (sGC), an ␣/␤-heterodimeric heme protein, catalyzes the conversion of GTP to cGMP in many cells including vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Activation of sGC is by direct binding of nitric oxide (NO) to the sGC heme prosthetic group. Formation of the nitrosyl heme adduct induces a conformational change in sGC that results in an increase in its enzymatic activity (21). The NO that activates sGC in various cells is the product of a reaction that is catalyzed by one of three distinct NO synthase (NOS) molecules, which catalyze the oxidation of L-arginine to produce L-citrulline and NO (1). In ECs, NO production is mediated by the constitutively expressed endothelial NOS (eNOS). Activation of eNOS is by Ca 2ϩ -calmodulin (CaM) after agonist-stimulated elevations in intracellular Ca 2ϩ concentrations. Two signaling pathways exist that involve eNOS and sGC. The first is an intercellular pathway whereby NO, which is produced by eNOS in ECs, diffuses to the underlying SMCs and promotes blood vessel relaxation (16). The second is an intracellular eNOS-sGC pathway that is essential for vascular endothelial growth factor (VEGF)-induced increases in EC permeability and proliferation (24,25,30).Initially, eNOS was thought to function as an isolated homodimer. It is now known, however, that eNOS exists in multiprotein complexes in which it interacts with other proteins. These pr...

Hsp90 inhibitors may offer a new pharmacological tool in the management of severe sepsis and severe sepsis-induced ALI.

Background:The activation of RhoA is a critical event in acute lung injury (ALI), but the role of nitration in this process is unresolved. Results: The nitration of RhoA at Tyr 34 produced GEF-like conformational changes that stimulate RhoA by decreasing GDP binding. Conclusion:We have identified a new mechanism of RhoA activation. Significance: Preventing RhoA nitration may be useful for the management of ALI.

ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60 Src , phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and ␤-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VEcadherin and ␤-catenin expression, and inhibited activation of pp60 Src and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of ␤-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsisassociated endothelial barrier dysfunction. endothelial permeability; radicicol; endotoxin; acute respiratory distress syndrome GRAM-NEGATIVE BACTERIAL SEPSIS affects more than 750,000 people annually in the United States alone (31); it is often associated with endothelial hyperpermeability leading to vascular leak syndromes, including acute respiratory distress syndrome (ARDS; Refs. 32, 44). Endotoxin or LPS, the bacterial cell envelope component responsible for most of the septic response, activates macrophages, neutrophils, and other cells to produce inflammatory mediators and free radicals that cause endothelial damage (25). Direct activation of endothelial cells by LPS is one of the earliest causes of endothelial dysfunction in sepsis (3,19).Heat shock protein 90 (hsp90), an abundant molecular chaperone, is involved with folding, maturation, and stabilization of numerous client proteins ranging from tyrosine kinases to several different types of transcription factors and proteins controlling growth and survival (49). Hsp90 inhibitors, such as radicicol, interact with the NH 2 -terminal ATP binding site of hsp90 and result in destabilization and degradation of client proteins (47). Hsp90 inhibitors have emerged as an attractive therapeutic modality for various types of cancer, causing combinatorial blockade of numerous growth-promoting and apoptosis-blocking pathways (45, 55).Regulation of endothelial permeability is a complex process involving signaling components that are associated with cytoskeletal and membrane-bound proteins that maintain cell-cell or c...