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1Vascular Biology Center, 2Department of Pediatrics, and 3Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2500; and 4Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611-3008
Submitted 2 December 2002 ; accepted in final form 1 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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- 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
/-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 Ca2+-calmodulin (CaM) after
agonist-stimulated elevations in intracellular Ca2+
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 proteins include Akt (10), caveolin-1 (13, 19, 23), heat shock protein 90 (HSP90; Refs. 12, 26), certain G protein-coupled receptors (18, 22), eNOS-interacting protein (NOSIP; Ref. 7), and dynamin-2 (6). Interactions of eNOS with Akt, caveolin-1, HSP90, and G protein-coupled receptors are regulated in an agonist-dependent manner (9, 12, 18, 22, 26). Currently, no protein partners for sGC are known, and the enzyme is thus believed to exist in the cytoplasm as a free protein that does not interact with other proteins. However, because sGC is a downstream intracellular effector of NO, in the present study we have examined whether sGC forms a complex with eNOS in bovine aortic ECs (BAECs). We have also defined the role of HSP90 in mediating the sGC-eNOS interaction and identified the subunit of sGC that is responsible for complex formation. In addition, we investigated whether complex formation is regulated in an agonist-dependent manner and whether the capacity of sGC to be activated by NO is dependent on HSP90 activity.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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1- and
1-subunits were a generous gift from Dr. Andreas
Papapetropoulos, University of Athens, Athens, Greece. Cell culture. In-house harvested BAECs were passaged from primary cultures and used in experiments during passages 2–5. Cultures were maintained in M199 supplemented with 10% fetal bovine serum, 5% iron-supplemented calf serum, 20 µg/ml L-glutamine, 1x MEM amino acid and vitamin solutions, 0.6 µg/ml thymidine, 500 IU/ml penicillin, and 500 µg/ml streptomycin. In-house harvested rat aortic SMCs (RASMCs) were also passaged from primary cultures and used in experiments during passages 2–5. Cultures were maintained in DMEM that contained 10% fetal bovine serum, 500 IU/ml penicillin, and 500 µg/ml streptomycin.
Preparation of soluble and particulate fractions of BAECs and RASMCs. Cells were lysed in hypotonic buffer that contained 50 mM Tris · HCl, pH 7.4, 5 mM EGTA, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1% PMSF. Samples were homogenized by 30 up-and-down strokes in a tight-fitting Dounce homogenizer followed by sonication. Lysates were then centrifuged at 100,000 g for 30 min to obtain particulate (pellet) and soluble (supernatant) fractions. After resuspension of the pellet to the original volume of the homogenate, equal volumes of supernatant and pellet fractions were analyzed via immunoblot with anti-sGC antibody and densitometry of the immunoblots.
Immunoprecipitation and immunoblotting. BAECs and RASMCs were lysed in ice-cold buffer that contained 20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 1% Triton X-100, 1% PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 5 µg/ml aprotinin. Lysates were centrifuged at 10,000 g for 25 min to remove insoluble material. Anti-sGC, -eNOS, and -HSP90 antibodies were then added for 3 h at 4°C. Protein A/G agarose was added, and samples were incubated for an additional 3 h at 4°C. Immunoprecipitated proteins bound to the agarose beads were then washed twice, and proteins were eluted from the beads by boiling the samples in SDS sample buffer. Agarose beads were pelleted by centrifugation, and protein supernatants were analyzed on SDS polyacrylamide gels. Proteins were transferred to nitrocellulose membranes by electroblot. Membranes were then probed with anti-eNOS, -sGC, or -HSP90 antibodies or nonimmuno-Ig isotypes to confirm specificity of interaction. Bound proteins were visualized using the Pierce SuperSignal chemiluminescence substrate.
Expression and purification of eNOS in a baculovirus system. Bovine eNOS was expressed in a baculovirus-Sf9 insect cell system and purified to >95% homogeneity as described previously (29).
In vitro binding assays. Plasmid constructs that encode GST-sGC
1- and 1-subunits
(GST-sGC-1 and -1, respectively) were
created by subcloning the full-length cDNAs for human sGC-1
and sGC-1 into the GST fusion protein-cloning vector
pGEX-4T-1. Fusion proteins and a GST nonfusion protein were expressed in
Escherichia coli and purified by affinity binding to
glutathione-Sepharose as described by Frangioni and Neel
(11). GST or GST-sGC fusion
proteins (100 pmol each, quantified by Bio-Rad protein assay) prebound to
glutathione-Sepharose beads were incubated overnight (with shaking at 4°C)
with 100 pmol purified HSP90, purified eNOS, or a combination of both purified
proteins in a buffer that contained 50 mM Tris · HCl, 20% glycerol, and
1% PMSF. Beads were washed six times in buffer that contained 50 mM HEPES, pH
7.5, 1.0 M NaCl, 1 mM EDTA, 0.5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 1% PMSF
when binding assays were performed with purified HSP90 only. The same wash
buffer was used for assays with purified eNOS or the combination of eNOS and
HSP90 except for a change in salt concentration to 0.2 M. Bound proteins were
eluted with 100 mM reduced glutathione, 150 mM NaCl, 3 mM EDTA, and 7.5 mM
Tris·HCl, pH 8.0. Eluted proteins were then immunoblotted with
anti-HSP90 or anti-eNOS antibodies either separately or in combination.
Colocalization studies. BAECs and RASMCs grown on glass coverslips were fixed in 100% methanol at –20°C for 20 min, washed in PBS, blocked in 5% goat serum, and incubated with rabbit polyclonal anti-sGC and mouse monoclonal anti-HSP90 antibodies. After being washed and blocked as described, specimens were exposed to secondary rhodamine-tagged anti-rabbit and fluorescein-tagged anti-mouse antibodies and to Hoechst 33258. Preparations were examined using an Olympus IMT-2 microscope equipped for epifluorescence digital imaging. With the use of separate FITC, rhodamine, and 4',6-diamidino-2-phenylindole filter sets, image stacks were collected with 0.2-µm spacing between images at each wavelength and were processed for digital deconvolution optical sectioning using Microtome software (Vaytek; Fairfield, IA). Corresponding optical sections at the three separate wavelengths were pseudocolored and overlaid using IPLab Spectrum software (Scanalytics; Fairfax, VA).
cGMP accumulation. Estimation of cGMP accumulation was carried out by a modification of the procedure of Ishii et al. (17) that was originally designed to measure NO release by determining sGC activity in RFL-6 rat lung fibroblast reporter cells. BAEC or RASMC culture medium was removed and replaced by Locke's solution that contained 154 mM NaCl, 5.6 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5.6 mM glucose, 10 mM HEPES, pH 7.4, 20 U/ml superoxide dismutase (SOD), and 0.3 mM isobutyl methyl xanthine. Cells were treated for 15 min with various concentrations of SNP following either no pretreatment or pretreatment with geldanamycin (1 µg/ml for 1 h) and then were lysed in ice-cold 20 mM sodium acetate, pH 4.0. Lysates were frozen at –20°C until assay for cGMP concentration using an enzyme immunoassay kit.
Determination of extracellular release of superoxide anion. BAECs or RASMCs grown in 24-well multiwell plates were washed with Earle's balanced salt solution (EBSS) and were incubated for 60 min with EBSS that contained 1 or 10 µg/ml geldanamycin and 40 µM ferricytochrome c with or without 350 U/ml Cu/Zn SOD. SOD-inhibitable reduction of ferricytochrome c to ferrocytochrome c was assessed spectrophotometrically at 550 nm, and superoxide production was estimated based on the extinction coefficient of 21.1 mM–1 · cm–1.
Whole animal studies. Experiments were approved by the
Institutional Committee for Animal Use in Research and Education and adhered
to the American Physiological Society standards of humane animal
experimentation. Sprague-Dawley rats (250 g) were anesthetized,
tracheostomized, and artificially ventilated, and catheters were introduced
into the left carotid artery and right jugular vein. Systemic arterial
pressure was continuously recorded on a Gould recorder. Bolus injections of
SNP in saline (0.4–0.7 ml iv, 1–3 µg/ml) were given before and
60 min after administration of 3 mg/ml iv geldanamycin. Each animal received
two injections of equal volume and concentration before and after geldanamycin
administration, and the maximal fall in mean arterial pressure (MAP) values
was averaged. The volume and concentration of SNP before geldanamycin
administration were adjusted to produce an 
30-mmHg maximal decrease in
MAP.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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1 in
BAECs and 16 ± 1.9% (n = 6) of sGC-1 in
RASMCs exist in the particulate fraction of these cells
(Fig. 1). These numbers
remained essentially the same following treatment with geldanamycin (1
µg/ml for 1 h, n = 3 for each cell type). These data suggest that
at least in the case of BAECs, the term "soluble guanylate
cyclase" may be somewhat of a misnomer.
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Successive enzymes in intracellular signaling pathways are often physically associated in multiprotein complexes. Moreover, it was shown previously (12) that eNOS forms a complex with HSP90. Therefore, we examined whether sGC, eNOS, and HSP90 exist in a trimeric complex in BAECs. BAECs (passages 2–5) were grown in serum-containing medium and lysed, and lysates were immunoprecipitated with anti-eNOS, -HSP90, or -sGC antibodies. Immunoprecipitated proteins were then immunoblotted with an anti-eNOS antibody. As shown in Fig. 2A, anti-HSP90 and -sGC antibodies coimmunoprecipitated eNOS, which suggests that the three proteins do indeed form a complex in BAECs. To further confirm this conclusion, lysates were immunoprecipitated using anti-eNOS, -HSP90, or -sGC antibodies, and immunoprecipitates were immunoblotted with an anti-HSP90 antibody. Anti-eNOS and -sGC antibodies coimmunoprecipitated HSP90 in these experiments (Fig. 2B), thereby providing further evidence for the existence of an sGC/eNOS/HSP90 complex in BAECs. In an additional set of experiments, BAEC lysates were immunoprecipitated with anti-eNOS, -HSP90 or -sGC antibodies, and immunoprecipitated proteins were immunoblotted with an anti-sGC antibody. In these experiments, the anti-eNOS and -HSP90 antibodies coimmunoprecipitated sGC (Fig. 2C), which again confirms the existence of an sGC/eNOS/HSP90 complex in these cells.
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We also examined the potential interactions of sGC and HSP90 in cultured RASMCs. Coimmunoprecipitation experiments showed that anti-sGC antibodies coprecipitated HSP90 (Fig. 3A) and that anti-HSP90 coprecipitated sGC (Fig. 3B). These data suggest that an sGC/HSP90 complex exists in RASMCs that is similar to the sGC/eNOS/HSP90 complex found in BAECs.
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The amount of eNOS associated with HSP90 is increased following stimulation of ECs with the eNOS-activating agonists VEGF, histamine, and estrogen (12, 26). We therefore examined whether sGC interactions with eNOS and HSP90 in BAECs might increase in response to BK or VEGF stimulation. BAECs were serum starved overnight and then treated with BK (1 µM) or VEGF (20 ng/ml) for 0, 1, 5, or 10 min. Cells were lysed, and lysates were immunoprecipitated with an anti-sGC antibody. Immunoprecipitated proteins were then immunoblotted with either anti-eNOS or anti-HSP90 antibody. As shown in Fig. 4A, BK stimulated an increased association of sGC with both eNOS and HSP90 that was maximal at 1 min and declined thereafter. Likewise, VEGF stimulated an increased association of sGC with eNOS and HSP90 that was maximal at 5 min (Fig. 4B). These results suggest that sGC interactions with eNOS and HSP90 are regulated in an agonist-dependent manner.
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Complex formation between HSP90 and HSP90-interacting proteins generally requires the ATP-dependent conversion of HSP90 from an inactive to an active conformation. For example, formation of a trimeric complex of HSP90, the aryl hydrocarbon receptor (AhR), and AhR-interacting protein (AIP) is dependent on HSP90 activity (4). To determine whether the interactions of sGC with eNOS or HSP90 are dependent on HSP90 activity, we used a specific inhibitor of HSP90 activity, geldanamycin (14). BAECs were serum starved overnight and then treated with BK (1 µM) for 0, 1, and 5 min following either no pretreatment or pretreatment with geldanamycin (1 µg/ml for 1 h). Cells were lysed, and lysates were immunoprecipitated with an anti-sGC antibody. Immunoprecipitates were immunoblotted with anti-eNOS and -HSP90 antibodies. As shown in Fig. 5, A and B, geldanamycin inhibited BK-stimulated complex formation of sGC with both eNOS and HSP90, which suggests that complex formation requires that HSP90 be in an active conformation. Interestingly, geldanamycin inhibited sGC-eNOS association by 20–40%, whereas it inhibited sGC-HSP90 association by 45–91%. The significance of these differences is not apparent.
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To determine whether HSP90 or eNOS interacts directly with sGC and, if so,
with which subunit, we prepared GST fusion proteins that contained the
full-length human sGC-1 and -1 subunits.
The fusion proteins and a GST nonfusion protein were expressed in E.
coli and purified by affinity chromatography on glutathione-Sepharose.
The GST fusion proteins or GST only were then used in in vitro binding assays
with purified human HSP90, purified bovine eNOS, or a combination of the two
purified proteins. GST or GST fusion proteins prebound to
glutathione-Sepharose beads were incubated with HSP90, eNOS, or both proteins
overnight at 4°C, washed six times in buffer that contained either 0.2 M
or 1.0 M NaCl, and eluted with reduced glutathione. The wash buffers used with
purified eNOS or purified eNOS plus purified HSP90 contained only 0.2 M NaCl
to maximize our chances of detecting binding, because it was reported
previously that the eNOS-HSP90 complex disassembles in a highionic strength
buffer that contains 0.5 M NaCl
(12). Eluted proteins were
immunoblotted with anti-HSP90, -eNOS antibody, or a combination of both
antibodies. As shown in Fig. 6,
left, HSP90 bound to GST-sGC-1 but not to GST only
or GST-sGC-1 subunit. Furthermore, it appears that HSP90
binds to sGC-1 with very high affinity, because the binding
interaction is not disrupted by very high-stringency washes in buffer that
contains 1.0 M NaCl. However, eNOS did not interact with either of the sGC
subunits in the same experiment (Fig.
6, middle), which suggests that eNOS does not interact
directly with either subunit of sGC. However, when eNOS was incubated with the
GST fusion proteins in the presence of HSP90, eNOS binding to
GST-sGC-1 was detected
(Fig. 6, right). In
addition, when eNOS was incubated together with HSP90 and
GST-sGC-1 in the presence of geldanamycin (1 µg/ml),
binding of both HSP90 and eNOS to GST-sGC-1 was completely
blocked. These data suggest that the eNOS-sGC interaction is indirect, and
that it occurs due to binding of both eNOS and sGC to HSP90 with HSP90
functioning as an adaptor protein for the eNOS-sGC interaction.
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To confirm the in vivo subcellular colocalization of sGC and HSP90, BAECs and RASMCs were fixed, stained with either anti-HSP90 (fluorescein green) antibody, anti-HSP90 (rhodamine red) antibody, or both, and examined using fluorescence deconvolution optical sectioning microscopy. As shown in Fig. 7, wide extranuclear cytoplasmic distribution of sGC and HSP90 was observed in both cell types. Colocalization of the two proteins was confirmed by the overlay of pseudo-colored red and green images, which resulted in a yellow signal at sites of colocalization throughout most of the extranuclear domain in BAECs and RASMCs stained with both anti-sGC and -HSP90 antibodies. These data make it appear highly likely that sGC and HSP90 coexist in common subcellular compartments.
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One potential role for HSP90 in the sGC/eNOS/HSP90 complex is to facilitate sGC-eNOS interactions in a manner similar to that described recently for the HSP90/AhR/AIP complex (4). Another possible role is that HSP90 affects sGC either directly or indirectly to make the enzyme more susceptible to activation by NO. To test this possibility, BAECs were either not pretreated or were pretreated with geldanamycin (1 µg/ml for 1 h) and then exposed for 15 min to 0, 1, 10, or 100 µM of the NO donor SNP. The amount of cGMP produced in the absence and presence of geldanamycin, basally, or in response to increasing concentrations of SNP was then quantified by enzyme immunoassay. Geldanamycin had little or no effect on basal cGMP production in BAECs. However, geldanamycin significantly attenuated SNP stimulation of cGMP synthesis. Exposure to 100 µM SNP for 15 min produced a 4.3 ± 0.2-fold increase (mean ± SE, n = 3 wells, each in duplicate) in cGMP accumulation in BAECs that was reduced to a 1.9 ± 0.3-fold increase (n = 3 wells, each in duplicate) following pretreatment with geldanamycin (Fig. 8A). The effect of geldanamycin (1 µg/ml for 1 h) on SNP-induced cGMP accumulation in RASMCs was also investigated. At 1 µM concentration, SNP produced a 17.1 ± 1.4-fold (n = 3 wells, each in duplicate) increase in cGMP content of RASMCs in 15 min, which is consistent with the much higher levels of sGC activity that are known to exist in RASMCs compared with ECs. Pretreatment with geldanamycin reduced the SNP-induced increase in cGMP concentration to 8.0 ± 0.7-fold (Fig. 8B). Recent reports suggest that geldanamycin stimulates the production of superoxide anion (8). Superoxide could interact with NO to form peroxynitrite and reduce the amounts of NO that are available to interact with sGC independent of the actions of geldanamycin on HSP90. To prevent this possibility, all cGMP-accumulation experiments were performed in the presence of SOD. In addition, the ability of geldanamycin to produce superoxide was investigated in both BAECs and RASMCs following a 60-min incubation in the absence of SOD. As shown in Fig. 9, at 10 µg/ml, geldanamycin elicited significant increases in superoxide levels, but at 1 µg/ml (the concentration used in all our studies), there was no significant increase from basal superoxide levels.
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The effects of in vivo delivery of geldanamycin to anesthetized rats (n = 5) on SNP-induced decreases in arterial blood pressure were also examined. Bolus injections of SNP in saline (0.4–0.7 ml iv, 1–3 µg/ml) were given before and 60 min after administration of 3 mg/ml geldanamycin. Baseline MAP was 114 ± 7 mmHg. SNP produced an immediate and transient decrease in systolic pressure (SAP) values, diastolic pressure (DAP) values, and MAP values that averaged –27 ± 7, –30 ± 5, and –35 ± 3 mmHg, for SAP, DAP, and MAP, respectively. After geldanamycin treatment, the same doses and volumes of SNP elicited reduced responses (–11 ± 3, –6 ± 1, and –8 ± 1 mmHg, respectively; Fig. 10). When expressed as a percent change from baseline, before geldanamycin administration, SNP produced 25 ± 2, 25 ± 6, and 25 ± 4% decreases in SAP, DAP, and MAP, respectively; these values were reduced to 10 ± 2, 18 ± 5, and 13 ± 2% decreases from baseline 1 h after geldanamycin administration. After geldanamycin administration, MAP increased maximally by 10 ± 2 mmHg or 10 ± 3% from baseline.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Garcia-Cardeña et al. (12) reported previously that geldanamycin (10 µg/ml) does not influence the vasorelaxant properties of the NO donor nitroglycerin on rat aortic rings, nor does it (at a 1 µg/ml concentration) influence the cGMP accumulation in response to 100 µM SNP in human umbilical vein ECs. Shah et al. (27) also reported that there is no effect of geldanamycin (3 µg/ml) on SNP-incluced vasodilation of perfused rat mesenteric vessels. In contrast, in the present study, we found that geldanamycin (1 µg/ml) inhibits SNP-induced cGMP accumulation in early-passage BAECs and RASMCs as well as SNP-induced vasodilation in anesthetized rats. The reason for this discrepancy is not clear but may relate to the differences in the model systems [BAECs and RASMCs vs. human umbilical vein ECs (HUVECs); in vivo vs. perfused mesenteric bed and aortic rings]. In experiments most comparable to the ones reported here, Garcia-Cardeña et al. (12) measured cGMP accumulation in response to SNP in HUVECs; unfortunately, they did not mention the degree of control SNP-induced sGC activation (i.e., fold increase in cGMP levels by SNP compared to vehicle); they only cite SNP-stimulated cGMP levels in the presence or absence of geldanamycin. Obviously, the degree of inhibition by geldanamycin depends on the degree of stimulation of sGC by SNP; thus if SNP only poorly stimulates sGC (as for example in late-passage cells that may express significantly less sGC), the effect of geldanamycin may be very small or even masked. We harvest and grow our own cells and use them in passage 2 or 5; they express robust amounts of sGC.
An additional point that should be made is that whereas in most cases the effects of geldanamycin on HSP90 are to reduce its activity by blocking interaction of the protein with ATP, in other cases, the compound exerts its effects by targeting HSP90-interacting proteins for degradation (28). Thus it is possible that the effects of geldanamycin on sGC in the present study may be due at least in part to sGC degradation, which suggests that additionally or alternatively, HSP90 may serve to stabilize sGC as is the case for another protein of the sGC/eNOS/HSP90 complex, Akt (2).
It appears likely that the sGC/eNOS/HSP90 complex involves several additional proteins. Already, Akt, caveolin-1, select G protein-coupled receptors, NOSIP, and dynamin-2 have been shown to associate with HSP90 directly or indirectly (6, 7, 10, 13, 22); others are currently under investigation (Fig. 11). It is therefore reasonable to speculate that for optimum cellular function, several multimeric dynamic complexes exist with components that vary according to the subcellular localization of the complex and the magnitude of physiological or pathological stimulation of the cell.
The functional significance of sGC-HSP90 interaction remains to be fully determined. Endothelial sGC plays a critical role in VEGF-induced increased cell proliferation and permeability (24, 25, 30), angiogenic processes implicated in solid tumor growth, myocardial revascularization, and diabetic retinopathies. Smooth-muscle sGC is a major regulator of vascular tone and remodeling, which are processes implicated in heart disease, hypertension, stroke, shock, and erectile dys-function. One likely reason for the existence of these complexes is that they serve to target intracellular NO produced by eNOS and iNOS directly to sGC and thus prevent inactivation of NO by reaction with superoxide anion (15) and prevent production of the highly toxic molecule peroxynitrite (see Fig. 11). Peroxynitrite formation is injurious to cells because it modifies and inactivates tyrosine residues in proteins, a process that is implicated in the pathology of human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis (3). Additionally, the sGC/eNOS/HSP90 interaction may be important in facilitating the actions of NO on leukocyte or platelet vessel-wall interactions including expression of adhesion proteins on the endothelium and platelet aggregation. Our in vivo data also raise the possibility of a previously unrecognized role of the HSP90-sGC complex in the optimum mechanism of action of NO donors such as SNP and nitroglycerin. More studies are needed to discern whether the observed functional effects of HSP90-sGC association are due to optimization of sGC activity, sGC stabilization, or a yet-unknown mechanism.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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