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Am J Physiol Heart Circ Physiol 286: H561-H569, 2004. First published October 9, 2003; doi:10.1152/ajpheart.00736.2003
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Heat shock protein 90 and tyrosine kinase regulate eNOS NO· generation but not NO· bioactivity

Jingsong Ou,1,3 Jason T. Fontana,5 Zhijun Ou,1,3 Deron W. Jones,1 Allan W. Ackerman,1 Keith T. Oldham,1,3,4 Jun Yu,5 William C. Sessa,5,* and Kirkwood A. Pritchard, Jr.1,2,3,4,*

Division of 1Pediatric Surgery, Departments of Surgery and 3Pharmacology and 2Toxicology, and the 3Cardiovascular Center and 4Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 5Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536

Submitted 7 August 2003 ; accepted in final form 5 October 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 () 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 production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated 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.

endothelial nitric oxide synthase; geldanamycin; radiciol

NUMEROUS STUDIES demonstrate that heat shock protein 90 (HSP90) plays an important role in modulating endothelial nitric oxide (NO) synthase (eNOS) function in ways that directly influence endothelial and vascular physiology (14, 16, 19, 36, 40, 44, 46). For example, vascular endothelial growth factor (VEGF), estrogen, histamine, and shear stress have all been shown to enhance the association of HSP90 with eNOS to increase NO· generation, which, in turn, increases vascular permeability and promotes vasodilation (6, 14). In addition, overexpression of HSP90 increases NOS activity (14), phosphorylation (4, 12), and NO· release (4, 12, 14). Moreover, geldanamycin (GA), an inhibitor of the ATPase function of HSP90, blocks acetylcholine-induced endothelium-derived NO· production but not the vasodilatory effects of NO· donor drugs such as sodium nitroprusside and nitroglycerin (14, 40, 43). However, recent reports raised concerns about this protein-protein interaction based on the fact that GA can redox cycle to generate (3, 9), which could impair vascular function by scavenging NO· rather than by inhibiting HSP90. In addition, before it was realized that GA and radicicol (RAD) were inhibitors of HSP90 ATPase function, these ansamycin antibiotics were considered inhibitors of tyrosine kinases based in part on the fact that HSP90 function is required for the conformational maturation of tyrosine kinases such as c-src and raf and the observation that treatment with GA caused ubiquitin-dependent degradation of these proteins (25, 37).

In this report we examine the effects of GA and RAD on several aspects of the eNOS-dependent function in vivo and in vitro, including vascular permeability, NO· production, vasodilation, and stimulated generation. In addition, we compare the effects of inhibiting tyrosine kinase signaling with genistein (GE) with the effects of inhibiting HSP90 with RAD to determine the extent to which broad-based blockade of tyrosine kinases is similar to inhibiting the ATPase function of HSP90 in regulating eNOS function. Our findings suggest that GA and RAD are equally efficacious in inhibiting eNOS, and although GA may redox cycle to generate , the amount generated is not physiologically important with respect to eNOS-dependent function (i.e., vascular permeability and vasodilation). Moreover, GE inhibition of tyrosine kinases is not equivalent to RAD inhibition of HSP90 in regulating eNOS function. On the contrary, GE + RAD pretreatments led to a super-induced state of uncoupled eNOS activity when the cultures were subsequently stimulated.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. For vascular permeability and vasodilation studies, male Swiss mice (25–30 g) and male C57BL6 mice (20–25 g), respectively, from Jackson Laboratories (Bar Harbor, ME) were used. For the hydroethidine studies, male Sprague-Dawley rats from Harlan (Indianapolis, IN) were used. The animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals by the National Research Council.

Measurements of Evans blue leakage: VEGF-stimulated vascular permeability. Male Swiss mice (25–30 g) were pretreated for 45 min with N{omega}-nitro-L-arginine methyl ester (L-NAME, 15 mg/kg), GA (0.1 and 1 mg/kg), or RAD (3 mg/kg) via intraperitoneal injection. Plasma leakage in mouse skin was studied by using human VEGF165 as an agonist (300 ng). Mice (25–30 g) were anesthetized with ketaminexylazine, and a catheter was introduced in the left jugular vein for administration of Evans blue (30 mg/kg; Sigma, St. Louis, MO). VEGF was applied 1 min (intradermal injection) after the administration of Evans blue onto the dorsal and ventral surfaces of the right ear, and saline was injected into the other ear as a control. After 30 min, ears were removed, blotted dry, and weighed. The anesthetized mice were then euthanized by exsanguination. Evans blue was extracted from the ears with 500 µl of formamide for 24 h at 55°C and measured spectrophotometrically at 610 nm as previously described (5).

Measurements of acetylcholine-induced vasodilation. Arterioles (180–250 µm) were isolated from branches of the mandibular carotid artery from anesthetized C57BL6 mice after euthansia by exsanguination. The isolated arterioles were transferred to MOPS, buffered to pH 7.4 (in mM: 3 MOPS, 2 CaCl2·2H2O, 0.02 EDTA, 5 glucose, 4.7 KCl, 1.17 MgSO4·7H2O, 145 NaCl, 1.2 NaH2PO4·H2O, and 2 pyruvic acid), gently cleaned of excess tissue, ligated to micropipettes, and pressurized to 60 mmHg. Isolated, pressurized microvessels were preconstricted with U44617 (10–8–10–7 M) to no more than 50% of the maximum vessel diameter (Dmax), which was determined after the pressurized vessel was warmed to to 37°C. Dose-response curves to sequential additions of ACh (10–7–10–5 M) were recorded by videomicroscopy. Pressurized arterioles were pretreated with GA (20 µM) or RAD (20 µM) for 30 min, and MOPS buffer was removed and replaced with fresh MOPS buffer for 10 min. This wash with MOPS buffer was replaced, the pressurized arteriole was preconstricted with U46619 [GenBank] , and vascular responses to ACh (10–7–10–5 M) were recorded as described above. The MOPS buffer was then replaced with fresh MOPS buffer, the dilated arteriole was preconstricted with U46619 [GenBank] , and vascular responses to Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate, 10–7–10–5 M) were recorded as above. At the end of this protocol, endothelium-independent vasodilation was determined with papaverine (10–4 M). To determine eNOS-dependent vasodilation, the pressurized carotid arterioles were incubated with L-NAME (100 µM, 20 min), and the arteriole was then preconstricted with U46619 [GenBank] . Vasodilation in response to ACh or DETA-NONOate was determined by videomicroscopy as described above. In all cases, papaverine dilated L-NAME-, GA-, or RAD-treated arterioles to 85–95% of Dmax.

Endothelial cell culture. Bovine coronary or aortic endothelial cells were expanded and maintained in RPMI-1640 media containing 20% FBS, antibiotics, and mycotics. The endothelial cell cultures were passaged with trypsin-EDTA and used for experiments between passage 5 and 7.

Measurements of VEGF-stimulated NO· generation. For measurement of NO·, the release of nitrite, the stable breakdown product of NO· in aqueous medium, was determined as previously described (39). In brief, endothelial cell cultures were equilibrated for 30 min at 37°C in serum-free Dulbecco's modified Eagle's medium and pretreated with GA or RAD for 30 min. To stimulate NO· release, VEGF (50 ng/ml) was added for 30 min, and the supernatant was collected for analysis by NO· chemiluminescence. Samples (10 µl) were refluxed in glacial acetic acid containing sodium iodide and nitrite quantified in a Seivers NO chemiluminescence analyzer as previously described (39). Nitrite concentrations were calculated after subtraction of background levels.

Hydroethidine measurements of production in native endothelial cells on isolated rat aortas. To determine the effects of GA and RAD on vascular endothelial cell generation, isolated rat aortic segments were treated at time 0 ± L-NAME (500 µM). The aortic segments were pretreated with RAD and GA at time 30 min for 30 min and washed, and at time 60 min, they were incubated in Hank's balanced salts solution (HBSS) containing hydroethidine (10 µM) for 30 min ± L-NAME as previously described (45). Fluorescent confocal microscopy was used to capture fluorescent intensity images as described (45).

Measurements of stimulated production. The protocol for stimulated measurements was as follows. At time 0, L-NAME (1 mM) was added to endothelial cell cultures in six-well plates. At 30 min, vehicle (V = dimethylsulfoxide) or RAD (20 µM) was added to the untreated cultures and L-NAME-treated cultures. At 60 min, the four test groups were washed three times with HBSS. After the final HBSS wash, the test groups were incubated with 1 ml HBSS containing ferricytochrome c (50 µM) and A-23187 (5 µM) without and with L-NAME (1 mM) for 30 min. Rates of release of were calculated from SOD-inhibitable ferricytochrome c absorbance at 550 nm using the molar extinction coefficient ({epsilon} = 21,000 M–1·cm–1). Data were compared with rates of release from independent wells incubated with HBSS containing ferricytochrome c and superoxide dismutase (1,000 U/ml). To determine the role of tyrosine kinases on stimulated generation, we pretreated the cultures with GE (100 µM, 45 min), a broad-spectrum tyrosine kinase inhibitor (28), with and without RAD (20 µM, 30 min) and measured the changes in SOD-inhibitable ferricytochrome c reduction ± L-NAME. Each experiment was performed in triplicate, and the cell protein for each well was analyzed in duplicate. Results are expressed as means ± SE (in nmol·min–1·mg cell protein–1).

Statistics. Statistical analysis was by the Student's t-test for experiments with two groups and ANOVA with a Newman-Keuls test as a post hoc test for experiments with more than two groups for determining levels of significance. Minimum levels of significance were set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF-driven changes in vascular permeability are dependent on NO· derived from eNOS, because this response is largely eliminated in eNOS–/– mice and in mice treated with L-NAME (13). To determine whether GA and RAD inhibit VEGF-induced changes in permeability, we quantified VEGF-driven extravasation of Evans blue dye into the interstitium of the ears of mice pretreated with the inhibitors. As seen in Fig. 1A, intradermally administered VEGF markedly increased vascular permeability, an effect abrogated by L-NAME (intraperitoneal administration 45 min before VEGF challenge). GA pretreatments dose dependently (0.1–1 mg/kg) reduced leakage of Evans blue, which is opposite of what would be expected of a compound that generates (10, 22). Because the generated via GA redox cycling may scavenge NO· (2, 3, 9), we repeated the study with RAD, a specific inhibitor of HSP90 that does not redox cycle but is less potent and more hydrophillic (3). As seen in Fig. 1B, RAD (3 mg/kg) also decreased VEGF-induced vascular leakage by >66%. These data show that GA and RAD share similar effects on NO·-dependent vascular leakage in response to VEGF.



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  		Fig. 1. Inhibition of heat shock protein 90 (HSP90) blocks vascular endotelial growth factor (VEGF)-induced increases in vascular permeability. A: VEGF increases leakage of Evans blue dye into the ears of mice, which is ablated by N{omega}-nitro-L-arginine methyl ester (L-NAME, 15 mg/kg) and geldanamycin (GA, 0.1 and 1 mg/kg). C, control. B: VEGF increases leakage of Evans blue dye into the ears of mice, which is significantly reduced by radicicol (RAD, 3 mg/kg). C: representative photographs of Evans blue leakage induced by VEGF and reduced by GA into the ears of mice. CT, control. *P < 0.05, n = 5 mice per group.

 

To determine the cellular mechanisms by which the ansamycin antibiotics decreased vascular permeability, we next examined their effects on VEGF-stimulated NO· production by endothelial cell cultures. As seen in Fig. 2, VEGF induced marked increases in nitrite production that were clearly decreased by pretreatment with GA or RAD. It is important to note that both inhibitors reduced stimulated nitrite production. If nitrite production was not inhibited by these compounds, then it could be argued that they reduced NO·-dependent increases in permeability by inactivating NO· with generated via redox cycling. These data indicate that if GA redox cycles to generate in vivo, then the amount produced may not be physiologically important.



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  		Fig. 2. GA and RAD inhibit VEGF-induced increases in endothelial cell nitrite production. Bar graph shows that VEGF (50 ng/ml) increases nitrite () production by endothelial cell cultures. Pretreatment of endothelial cell cultures with either GA (20 µM) or RAD (20 µM) for 30 min results in a significant decrease in VEGF-stimulated nitrite production. *P < 0.05, n = 8.

 

To appreciate the relative levels of generated in native endothelial cells when eNOS activity is blocked either directly with L-NAME or indirectly via targeting HSP90, we incubated isolated aortic segments from rats with L-NAME or pretreated them with GA or RAD (20 µM, 30 min) and then performed the hydroethidine assay on washed vessels in the presence and absence of L-NAME as described (45). As seen in Fig. 3, L-NAME increased ethidine staining in the endothelium of control aortic segments, confirming that under normal conditions eNOS generates NO·, which scavenged before it had a chance to react with hydroethidine (30, 31). In contrast, GA markedly increased ethidine staining, an effect that was partially reduced by L-NAME. RAD increased ethidine staining, but not to the extent that GA did, and L-NAME decreased ethidine staining to the levels observed in controls treated with L-NAME. On the basis that GA redox cycles (2) and RAD does not (3), these data suggest that GA increased generation in native endothelium by uncoupling eNOS activity and redox cycling, whereas RAD increased by uncoupling eNOS activity. These data begin to explain why only a portion of the increase in stimulated generation was significantly reduced by L-NAME in endothelial cell cultures pretreated with GA (30).



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  		Fig. 3. Effects of GA and RAD on superoxide anion () generation in native endothelial cells on aortic segments. These pseudocolorized images show the fluorescent intensity of ethidine staining in the endothelium on aortic segments in the presence and absence of L-NAME (500 µM). Left, baseline ethidine staining in untreated and L-NAME-treated control segments. L-NAME treatment slightly increases ethidine staining confirming the role of nitric oxide (NO·) in scavenging before it has a chance to react with hydroethidine. Middle, ethidine staining in aortic segments that were pretreated with GA and treated with L-NAME demonstrates that GA markedly increases ethidine staining, which is reduced by L-NAME. Right, ethidine staining in aortic segments that were pretreated with RAD and treated with L-NAME. Pretreatment with RAD markedly increases ethidine staining compared with controls (left) but not as high as with pretreatment with GA. L-NAME markedly reduces ethidine staining in aortic segments, pretreated with RAD to control levels (left) below ethidine staining seen in GA- and L-NAME-treated segments. Images are representative of 3 independent experiments.

 

Although few differences were observed between GA and RAD in their ability to inhibit VEGF-driven permeability in vivo or stimulated NO· production in vitro, the possibility that from GA could inhibit NO·-dependent vasodilation still remained. To test this possibility, we pretreated pressurized arterioles with GA and RAD and then determined the effects of this pretreatment on ACh- and DETA-NONOate-induced vasodilation of washed, pressurized arterioles. Arterioles were incubated also with L-NAME, and the effects of inhibiting eNOS on ACh- and DETA-NONOate-induced vasodilation were determined. L-NAME, GA, and RAD ablated ACh-induced vasodilation (Fig. 3, A and B). DETA-NONOate, a NO· donor that releases NO· without cellular metabolism (15), increased vasodilation of arterioles incubated with GA, RAD, and L-NAME to essentially the same extent except at the highest concentration used (10–5 M), where the vasodilation of arterioles in the presence of L-NAME was greater than the vasodilation of arterioles pretreated with the ansamycins (Fig. 3C). These data are consistent with previous reports using GA on isolated vessels (6, 14, 19, 40) and extends the published findings with direct comparisons to RAD, which does not redox cycle (3), and to L-NAME, which blocks eNOS as a substrate-analog competitive inhibitor (18). Although GA may redox cycle to generate , these data reveal that the amount of from GA, under the experimental conditions employed here, in fact, does not significantly alter NO·-dependent vasodilation, in contrast to previous conjecture (9). Because neither agent inhibited papaverine-induced vasodilation (data not shown), an endothelium-independent event (21), we can conclude with confidence that GA and RAD inhibited vasodilation by an endothelium- and eNOS-dependent mechanism, not redox cycling.

We then measured generation in cultured endothelial cells. Under control conditions, untreated cultures generated low levels of when stimulated with the receptor-independent agonist A23187 [GenBank] (Fig. 5A). L-NAME increased the release of from stimulated control cultures confirming that eNOS generated NO·, which scavenges before it had a chance to escape and react with ferricytochrome c (20, 27, 30, 31, 45). Pretreatment of cultures with RAD increased stimulated generation compared with the levels produced by stimulated control cultures, similar to findings from a previous report using GA (30). When RAD-pretreated cultures were stimulated in the presence of L-NAME, marked decreases in stimulated production were observed. Such reciprocal changes in stimulated generation in the absence or presence of L-NAME confirm that RAD inhibited NO· by uncoupling eNOS activity, not by redox cycling with eNOS, as occurs with other redox cycling agents (49, 51). Data here obtained from confluent cultures are consistent with findings from previous studies where proliferating endothelial cell cultures were used (26) and with studies where measurements of cGMP were used to assess the effects of geldanamycin on basal and bradykinin-stimulated NO· activity (52). Taken together these reports suggest that HSP90 plays an important role in NO· generation and cGMP accumulation under basal and stimulated conditions.Go



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  		Fig. 5. Effects of RAD, GE, and GE + RAD on stimulated generation from endothelial cell cultures. A: L-NAME (LN, 1 mM) increases A23187 [GenBank] (5 µM)-stimulated generation in control cultures (C + LN) compared with stimulated generation from control cultures (C) in the absence of LN. Pretreatment with RAD (20 µM, 30 min) also increases stimulated generation, which L-NAME (RAD + LN) reduces to control levels. These data show that pretreatment with RAD results in uncoupled eNOS activity when the cultures are subsequently stimulated. *P < 0.05, n = 5. B: LN (1 mM) increases A23187 [GenBank] (5 µM)-stimulated generation compared with stimulated generation from controls (C). Pretreatment with GE (100 µM, 40 min) slightly increases stimulated generation, which is unaltered by L-NAME (GE + LN). These data show that pretreatment with GE, a broad-spectrum tyrosine kinase inhibitor, does not uncouple eNOS activity when the cultures are subsequently stimulated (**P < 0.01, n = 8). C: LN (1 mM) increases A23187 [GenBank] (5 µM)-stimulated generation compared with stimulated generation from controls (C). Pretreatment with GE (100 µM, 40 min) + RAD (20 µM, 30 min) markedly increases stimulated generation, which is ablated by L-NAME (GE + RAD + LN). These data show that pretreating cultures with an inhibitor of tyrosine kinase activity and HSP90 ATPase function results in a state of superinduced uncoupled eNOS activity when the cultures are subsequently stimulated (**P < 0.01, n = 6–8).

 


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  		Fig. 4. Effects of GA and RAD on endothelium-dependent vasodilation. Graphs show the vasodilatory responses of isolated, pressurized arterioles (180–250 µm, 60 mmHg) to ACh, inhibition by pretreatment with GA and RAD, and vasodilatory responses to DETA-NONOate after inhibition with GA, RAD, and L-NAME. A: pretreatment with L-NAME (100 µM, 20 min) inhibits ACh-induced vasodilation (10–7-10–5 M) (n: C = 7; L-NAME = 5). B: pretreatment with GA or RAD (20 µM, 30 min) inhibits ACh-induced vasodilation (10–7-10–5 M) (n: GA = 6; RAD = 5). C: DETA-NONOate (10–7-10–5 M) increases vasodilation of arterioles in the presence of L-NAME or pretreated with GA or RAD to essentially the same levels except at the highest concentration where L-NAME arterioles vasodilated to a greater extent than arterioles pretreated with GA or RAD (**P < 0.01, n: GA = 8; RAD = 8; and L-NAME = 13).

 

As GA and RAD inhibit the ATPase function of HSP90, which is required for conformational maturation of tyrosine kinases (25, 29, 37, 42), these agents may uncouple eNOS via inhibiting tyrosine kinase activity. To test this hypothesis we repeated the studies using GE, a broad-spectrum tyrosine kinase inhibitor that has been shown to inhibit eNOS-dependent NO· generation (8, 28). Pretreatment of the cultures with GE increased stimulated production by a mechanism that was not inhibited by L-NAME (Fig. 5B). In contrast, pretreatment with GE + RAD super-induced stimulated generation by a mechanism that was nearly ablated by L-NAME (Fig. 5C). The rate of generated in cultures pretreated with GE + RAD was significantly increased compared with cultures pretreated with RAD alone [P < 0.023; RAD = 0.054 ± 0.012 (n = 5) vs. GE + RAD = 0.138 ± 0.033 nmol·min–1·mg cell protein–1 (n = 6)]. These data suggest that both the tyrosine kinase pathway and HSP90-dependent signaling act in concert to preserve "coupled" eNOS activity. Blockade of tyrosine kinase signaling, per se, has been shown to reduce NO· release (28) but, as can be seen here, does not result in uncoupled eNOS activity, whereas blockade of HSP90 ATPase function reduces NO· release and promotes uncoupled eNOS activity upon stimulation. These data are consistent with the notion that HSP90 interactions with eNOS play a critical role in mediating eNOS function and suggest that tyrosine kinase activity, either directly or indirectly, possibly by influencing HSP90 function, work synergistically with HSP90 to suppress uncoupled eNOS activity.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The findings reported here support the concept that HSP90 plays an essential role in modulating eNOS-regulated vascular permeability and vasodilation and suggest that tyrosine kinase- and HSP90-dependent signaling pathways act in concert to prevent uncoupled eNOS activity. Moreover, direct side-by-side comparisons of two inhibitors of the ATPase function of HSP90, GA and RAD, reveal that both drugs antagonize HSP90-eNOS signaling. These comparisons also revealed subtle differences in mechanisms by which they increase generation in vascular endothelium. Although GA redox cycles to generate (2, 9) and RAD does not (3, 26), the low levels of from GA are likely of little physiological consequence. This conclusion is based on physiologically relevant data showing few differences in the ability of GA and RAD to inhibit eNOS-dependent vascular responses in vivo or ex vivo. When HSP90- and tyrosine kinase-dependent signaling is intact, eNOS generates NO· upon activation. Inhibition of HSP90 with RAD increased stimulated generation by an L-NAME-inhibitable mechanism, whereas blockade of tyrosine kinases with GE, a broad-spectrum inhibitor, did not. Inhibiting both pathways at the same time, however, superinduced eNOS-dependent generation in stimulated cultures. This suggests that tyrosine kinase blockade, per se, reduces NO· release, perhaps through an effect on signaling cascades upstream of eNOS [i.e., calcium mobilization (41, 47)], but is unlikely mediated via dissociating HSP90-dependent signaling from eNOS. The importance of tyrosine kinase signaling to HSP90-dependent changes in eNOS function is manifested only when tyrosine kinase-dependent and HSP90-dependent pathways are both antagonized. Thus the effects of GA and RAD on inhibiting NO· release cannot be explained through generalized inhibition of tyrosine kinases.

Although the binding affinities of GA and RAD for HSP90 are high (17, 38), the chemical nature of GA in promoting oxidative stress (2) and the central role of HSP90 in protein folding (25, 37) may confound interpretation of findings using GA or RAD as pharmacological tools, if not put into perspective. Previous reports raised concerns about the utility of GA to study vessel function because, as a benzoquinone-containing compound, this antineoplastic agent can redox cycle to generate (3, 9). It was argued that from GA could scavenge NO· to inhibit NO·-dependent changes in vascular function rather than by inhibiting HSP90-dependent signaling (3, 9). However, previous data have shown that GA does not block the actions of two NO· donors sodium nitroprusside and nitroglycerin (14, 40, 43), and data here show that GA and RAD both block ACh-induced vasodilation completely and only marginally reduce the action of DETA-NONOate at the highest concentration used.

Findings here are in conflict with previous reports by Dikalov et al. (9) whose experimental protocols biased results in favor of redox cycling rather than inhibition of HSP90-dependent signaling. However, our observations are consistent with a previous report (26) showing that pretreatment of proliferating endothelial cells with RAD results in uncoupled eNOS activity upon stimulation. The effect of GA and RAD on NO· responses at high concentrations may also reflect a newly described interaction of HSP90 with soluble guanylyl cyclase (52). Furthermore, examination of our data shows that if were derived from GA alone, then L-NAME would not have inhibited stimulated generation in cultures pretreated with ansamycins, which it does (30). In addition, if the amount of generated by GA redox cycling was significant, then GA should have impaired DETA-NONOate-induced vasodilation to a greater extent than RAD, which it did not.

We find that RAD is as effective as GA in preventing VEGF-induced increases in permeability and NO· release from endothelial cells and ACh-induced increases in vasodilation. If this were not the case, then RAD would not have inhibited VEGF-induced NO· production or ACh-induced vasodilation, which it does. Careful examination of all the GA and RAD data generated in vivo, in isolated vessels and in cultured cells, reveals many conspicuous similarities that would not have transpired if from GA redox cycling were significant. This abundance of physiologically relevant and biochemical data showing that GA and RAD are similar underscores the importance of appreciating the experimental context in which inhibitors are used to avoid erroneous conclusions that the generated by stimulated endothelial cell cultures pretreated with GA in our previous report (30) could be completely accounted for by redox cycling (9, 11). The data reported here clearly show that the amount of generated via GA redox cycling did not account for all of the generated by uncoupled eNOS activity, which begins to explain why the physiological effects of GA and RAD are similar.

Early on, the ansamycin antibiotics were classified as inhibitors of tyrosine kinases, such as c-src and raf, because these enzymes required chaperones for proper maturation (25, 37). Because the ansamycin antibiotics may alter eNOS via the tyrosine kinase pathway, we determined the effects of GE, a broad-spectrum tyrosine kinase inhibitor, on stimulated generation. We observed that GE pretreatments increased stimulated production, which was further increased by L-NAME, suggesting that GE partially inhibited eNOS activity. Such findings are in contrast to the effects of GA and RAD, which uncoupled eNOS activity (26, 30). The observation that L-NAME did not inhibit from cultures pretreated with GE is important for two reasons. First, the increase in stimulated generation by GE-treated cultures is important because it demonstrates that not all agents that inhibit NO· release uncouple enzyme activity. Second, after stimulation, cultures pretreated with GE + RAD were observed to generate at rates that were greater than two and three times the rates of generation induced by pretreating the cultures with either RAD or GE, respectively. When these observations are interpreted by the fact that GA, RAD, and GE all inhibit eNOS (26, 28, 30) but only GA and RAD uncouple eNOS activity in stimulated cultures, it suggests HSP90-dependent and tyrosine kinase-dependent signaling act together to promote coupled eNOS activity. Our observation that L-NAME further increased stimulated production in cultures pretreated with GE is consistent with the idea that generated from non-eNOS-dependent oxidative enzymes [i.e., xanthine oxidase, NADPH oxidoreductase or mitochondria (24, 32, 34)] can be detected only when NO· generation is blocked, thereby allowing to escape the endothelium and react with ferricytochrome c (31). Our data demonstrate that both signaling pathways must be blocked to super induce uncoupled eNOS activity. Had we not used both inhibitors for pretreatment, this superinduced state of uncoupled eNOS activity would not have been observed. We interpret such changes in eNOS function to mean that the tyrosine kinase and HSP90 signaling pathways work in concert to preserve coupled eNOS activity. Although a recent review (35) cites numerous articles showing that a wide variety of tyrosine kinases are substrates for HSP90, to our knowledge, findings here appear to be the first to suggest that these two pathways may act together, either directly or indirectly, to maintain coupled eNOS activity.

Findings here support the concept that eNOS function, as defined by the ability of the enzyme to generate NO· or upon demand, is modulated, in part, by HSP90-dependent signaling (30). Because many phosphorylation and dephosphorylation events are sensitive to oxidative stress (1, 7) and eNOS production of NO· is increased by a p38 map kinase pathway in isolated hearts from neonatal rabbits raised in hypoxic environments (33), it is interesting to speculate that what controls the ability of eNOS to generate NO· and is tightly controlled cell signaling.

The notion that cell signaling controls eNOS function is very different from uncoupling eNOS via oxidation of tetrahydrobiopterin (48, 50). Yet, site-directed mutagenesis studies provide proof of concept for this new mechanism where transfection of a dephospho/phospho-eNOS T497A/S1179D construct results in the expression of a highly active but uncoupled eNOS mutant (23). From these observations, the T497 phosphorylation site, in conjunction with HSP90, may actually be an intrinsic molecular switch that regulates the balance of NO· and generation from eNOS (23). In contrast when tetrahydrobiopterin is lost due to oxidation, eNOS activity will remain uncoupled until the synthesis of and/or dietary supplementation of the cofactor occurs at rates that exceed consumption (50). Because GA redox cycles to generate , we attempted to address this concern by supplementing endothelial cell cultures with sepiapterin, an immediate precursor to tetrahydrobiopterin in the salvage pathway (53), before incubation with GA and A23187 [GenBank] stimulation (30). Supplementation with sepiapterin decreased control levels of stimulated superoxide generation but had no effect on stimulated generation from eNOS in cultures pretreated with GA (30). These findings were interpreted to mean that GA uncoupled eNOS activity by altering HSP90 interactions and not by oxidation of tetrahydrobiopterin. The fact that RAD essentially does the same thing as GA without redox cycling provides further support for the idea that cell signaling modulates coupled eNOS activity. When these two mechanisms are put into perspective, a new concept begins to emerge where vascular function is controlled by a continuum of interrelated and dependent mechanisms. Under conditions of low oxidative stress, phosphorylation/dephosphorylation and HSP90-dependent signaling directs coupled and uncoupled eNOS activity in vascular endothelium to modulate vasodilation to meet demand. Under conditions of high oxidative stress, oxidation of tetrahydrobiopterin results in prolonged states of uncoupled eNOS activity, which impairs vasodilation via loss of an essential cofactor (50). The relative importance of these two pathways in controlling coupled eNOS activity relative to vascular function and vascular disease remains to be established.

In conclusion, HSP90-dependent signaling modulates eNOS function in ways that directly influence vascular endothelial cell function with respect to permeability and vasodilation. Although GA and RAD are highly specific for inhibiting the ATPase function of HSP90, side reactions that increase and dependence of tyrosine kinase on chaparones for maturation demanded that the mechanisms by which the ansamycins impair vascular function be revisited. Our data show that although GA may redox cycle to generate , the dominant mechanism by which it impairs vascular endothelial cell function is by inhibiting HSP90-dependent signaling. Broad-based blockade of tyrosine kinase and inhibition of HSP90-dependent signaling suggest that the tyrosine kinase and HSP90 signaling pathways act in concert to inhibit eNOS-dependent generation. Future studies aimed at understanding the exact mechanisms and the enzyme involved by which these two pathways modulate eNOS may lead to a better understanding in the cellular events driving HSP90 association with eNOS and the signaling mechanisms governing the balance of NO· and production from eNOS.


   ACKNOWLEDGMENTS
 
GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-61417, HL-71214, P01-HL-68769 (to K. A. Pritchard) and RO1-HL-57665, HL61371, and HL-64793 (W. C. Sessa) and also by the Marie Z. Uihlein endowed chair award (to K. T. Oldham) from the Children's Hospital Foundation (Milwaukee, WI).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Pritchard, Jr., Dept. of Surgery, Div. of Pediatric Surgery, Cardiovascular Center, M4060, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: KPRITCH{at}mcw.edu).

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.

* W. C. Sessa and K. A. Pritchard contributed equally to the experimental design as senior authors. Back


   REFERENCES
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