Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Abstract

. 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...

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

“…Caveolae are therefore regulatory platforms for the cascade of events resulting in NO production. Although the natural receptor for NO, sGC has been detected in association with the plasma membrane (Zabel et al, 2002;Venema et al, 2003), and sGC is translocated to caveolar domains to be sensitized by NO (Zabel et al, 2002); the interaction with caveolin-1 and the role of this association with the plasma membrane in blood vessel relaxation are unknown. We observed that the relaxation induced by the sGC activator YC-1 is impaired in the presence of methyl-␤-cyclodextrin in rat aorta.…”

Structural and Functional Characterization of the Dimerization Region of Soluble Guanylyl Cyclase

“…Binding of ␤ 1 [204 -408] appears to have a greater impact on the stability of ␣ 1 , as indicated by our findings that ␣ 1 protein was more readily down-regulated than ␤ 1 protein and that ␤ 1 [204 -408] was readily detected in ␤ 1 but not in ␣ 1 coimmunoprecipitates. These phenomena might relate to the fact that only ␤ 1 but not ␣ 1 interacts with and is stabilized by heat shock protein hsp90 (6). It is tempting to speculate that expression of alternatively spliced variants or isoforms of the ␤ subunit exposing modified N-or C-terminal portions but retaining the dimerization region may contribute to the fine tuning of cellular cyclase activity through targeted degradation of the resultant complexes.…”

New insights into the pathophysiology of cyclosporine nephrotoxicity: a role of aldosterone

“…Mechanisms implicated were suppression of osteopontin, TGF-␤, and intrarenal C-reactive protein expression together with higher eNOS expression (70). However, the beneficial effect of pravastatin in very high doses (20 mg/kg) was not observed in rats that received lower doses (5 mg/kg).…”