Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways.


Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 microm).


APC decreased myocardial infarct size compared with control experiments (26 +/- 6% vs. 46 +/- 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 +/- 4%). Sepiapterin alone had no effect on infarct size (46 +/- 3%) but restored APC during hyperglycemia (21 +/- 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (47 +/- 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 +/- 3%). Isoflurane increased nitric oxide production to 177 +/- 13% of baseline, and this action was attenuated by high glucose concentrations (125 +/- 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 +/- 12% and 175 +/- 9%; 10 and 100 microm, respectively).


The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central role in cardioprotection that is favorably modulated by volatile anesthetics and dysregulated by hyperglycemia. Enhancing the production of BH4 may represent a potential therapeutic strategy.

  • ❖ Myocardial protection through anesthetic preconditioning relies on nitric oxide production and is disrupted by hyperglycemia

  • ❖ Tetrahydrobiopterin (BH4) and heat shock protein (Hsp) 90 also regulate nitric oxide production and may interact with anesthetic preconditioning

  • ❖ In rabbits, hyperglycemia decreased, whereas cardiac anesthetic preconditioning increased BH4and Hsp 90 association with nitric oxide synthase. A BH4precursor rescued nitric oxide synthase activity and nitric oxide production during hyperglycemia

  • ❖ Enhancing BH4production may enhance anesthetic preconditioning during hyperglycemia

NITRIC oxide derived from endothelial nitric oxide synthase (eNOS) is a critical mediator of anesthetic preconditioning (APC) against myocardial infarction,1–3and this molecule protects against ischemia and reperfusion injury through activation of intracellular signaling pathways and direct effects on mitochondria.4The volatile anesthetic isoflurane activates eNOS, as indicated by phosphorylation of Serine 1177,1and nitric oxide production is directly related to the extent of eNOS phosphorylation.5Conversely, hyperglycemia decreases the availability of nitric oxide6and is an independent predictor of increased cardiovascular morbidity and mortality.7APC is abolished by diabetes and acute hyperglycemia,2,8but interestingly, cardioprotection is restored by treatment with an 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor through a nitric oxide-mediated mechanism.2 

There are several important regulators of eNOS function that are potentially modifiable by hyperglycemia and volatile anesthetics. For example, heat shock protein (Hsp) 90 is a physiologic binding partner of eNOS that regulates eNOS phosphorylation and modulates subsequent nitric oxide production.9–11We recently demonstrated that Hsp90 impacts APC through protein–protein interactions, thereby, enhancing nitric oxide production in endothelial cells and decreasing myocardial ischemia and reperfusion injury.1In contrast, diabetes and hyperglycemia have been shown to impair Hsp90–eNOS interactions.12It is unknown whether attenuated associations between Hsp90 and eNOS might account for the deleterious effects of hyperglycemia on APC signal transduction.

eNOS is also regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4), a reduced unconjugated protein and essential cofactor for the normal function of this enzyme. Decreases in BH4contribute to endothelial dysfunction in diabetes,13whereas BH4supplementation decreases reactive oxygen species production and restores endothelial function induced by acute hyperglycemia,14diabetes,15–17and hypercholesterolemia.18,19The actions of volatile anesthetics to alter BH4concentrations have not been investigated previously.

Thus, the aim of this study was to test the hypothesis that hyperglycemia abolishes APC through Hsp90- and BH4-dependent mechanisms. Experiments in vivo  were conducted to determine whether the metabolic precursor of BH4, sepiapterin, restores APC during hyperglycemia. In vitro  experiments were conducted to define the mechanisms whereby hyperglycemia disrupts isoflurane-enhanced eNOS chaperone (Hsp90) and cofactor (BH4) function using ozone chemiluminescence to measure nitric oxide, high performance liquid chromatography to measure BH4, and immunoblotting and confocal microscopy to measure Hsp90–eNOS interactions.

All the experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin (Milwaukee, WI). Furthermore, all conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals.

In Vivo  Myocardial Infarction Model

Male, New Zealand white rabbits were anesthetized with intravenous sodium pentobarbital (30 mg/kg) and instrumented as previously described.1,20Briefly, a tracheotomy was performed, and rabbits were ventilated with positive pressure using an air–oxygen mixture (30% fractional inspired oxygen concentration). Arterial blood gas tensions and acid–base status were maintained within a normal physiologic range by adjusting the respiratory rate or tidal volume throughout the experiment. Heparin-filled catheters were positioned in the right carotid artery and the left jugular vein for continuous measurement of arterial blood pressure and fluid and drug administration (0.9% saline; 15 ml · kg−1· h−1), respectively. After thoracotomy, a silk ligature was placed around the left anterior descending coronary artery approximately halfway between the base and the apex for the production of coronary artery occlusion and reperfusion. Coronary artery occlusion was verified by the presence of epicardial cyanosis and regional dyskinesia in the ischemic zone, and reperfusion was confirmed by observing an epicardial hyperemic response.

The experimental protocol is illustrated in figure 1. All rabbits underwent a 30-min coronary artery occlusion followed by 3 h of reperfusion. Rabbits were randomly assigned to preconditioning with 30 min of isoflurane (2.1%, 1 minimum alveolar concentration; APC) followed by a 15-min washout. In separate experimental groups, rabbits were randomly assigned to receive 0.9% saline or 15% dextrose in water to increase blood glucose concentrations (glucometer) to approximately 270 mg/dl in the presence or absence of APC,2with and without pretreatment with intravenous sepiapterin (2 mg/kg),21which is converted to BH4intracellularly, the NOS inhibitor N  (G)-nitro-l-arginine methyl ester (10 mg/kg), or the sepiapterin reductase antagonist N -acetylserotonin (15 mg/kg).22 

Myocardial infarct size was measured as previously described.23Briefly, the left ventricular area at risk for infarction was separated from the normal area, and the two regions were incubated at 37°C for 20 to 30 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 m phosphate buffer adjusted to pH 7.4. After overnight storage in 10% formaldehyde, infarcted and noninfarcted myocardial areas within the area at risk were carefully separated and weighed. Myocardial infarct size was expressed as a percentage of the area at risk. Rabbits that developed intractable ventricular fibrillation and those with an area at risk less than 15% of total left ventricular mass were excluded from subsequent analysis.

Human Coronary Artery Endothelial Cells

Human coronary artery endothelial cells isolated from healthy coronary arteries (Cells Applications, San Diego, CA) were cultured without cryopreservation, propagated to fifth passage in growth medium (Cells Applications) and used for experiments between the fourth and the fifth passage. Cells were used for experiments when approximately 70 to 80% confluent. Human coronary artery endothelial cells were seeded on cell culture dishes (100 mm) and maintained at 37°C in growth medium (Cell Applications). Twenty-four hours before experimentation, the growth medium was removed and replaced with normal (d-glucose 5.5 mm, mannitol 14.5 mm, NaCl 81 mm, KCl 4.0 mm, CaCl21.6 mm, pH 7.4) or high (d-glucose 20.0 mm, NaCl 81 mm, KCl 4.0 mm, CaCl21.6 mm, pH 7.4) glucose media having the same osmolarity (290 mosmol/L). Additional cells were pretreated with sepiapterin (10 or 100 μm) for 1 h before exposure to isoflurane or air (control). Isoflurane was administered for 60 min, and the anesthetic concentrations (0.42 mm; the equivalent of 1 minimum alveolar concentration) were continuously monitored by a gas analyzer (POET IQ; Critcare System, Waukesha, Wisconsin), at continuous air flow (0.7 l/min) in a specific incubator chamber (Billups-Rothenberg, Del-Mar, CA) maintained at 37°C.1Because gas flow can induce shear-stress-dependent nitric oxide release,24the control group was exposed to air alone at the same flow rate.

Ozone Chemiluminescence

Nitrite concentration corresponding to the stable breakdown product of nitric oxide in aqueous solution was quantified by ozone chemiluminescence. Previous evidence suggests that nitric oxide release in response to stimuli in coronary endothelial cells peaks at 60 min.1,10Therefore, nitric oxide measurements were performed 60 min after isoflurane exposure. Samples (20 μl) were refluxed in glacial acetic acid containing potassium iodide and nitrite quantified in a nitric oxide chemiluminescence analyzer (Sievers Instruments, Boulder, CO) as described previously.25Nitrite concentrations were calculated after subtraction of background levels and normalized to protein content (Bradford method).

High Performance Liquid Chromatography

BH4and 7,8-dihydrobiopterin (BH2) were quantified by high performance liquid chromatography with an electrochemical detector (ESA Biosciences CoulArray® system Model 542, Chelmsford, MA) as described previously.26Endothelial cell pellets were immediately lysed in 300 μl of 50 mm phosphate buffer (pH 2.6) containing 0.2 mm diethylenetriaminepentaacetic acid and 1 mm dithioerythritol (freshly added) by shearing cells with a 28-gauge tuberculin syringe. Samples were centrifuged (12000g , 10 min, 4°C), and supernatants were filtered through a 10-kD cutoff column (Millipore, Billerica, MA). One hundred eighty microliters of the flow through was analyzed by using a Synergi Polar-RP column (Phenomex, Torrance, CA) eluted with argon-saturated 50 mm phosphate buffer (pH 2.6). Multichannel colorimetric detection was set between 0 and 600 mV. One channel was set at −250 mV to verify the reversibility of BH4oxidative peak detection. Calibration curves were constructed by summation of peak areas collected at 0 and 150 mV for BH4and 280 and 365 mV for BH2. Intracellular concentrations were calculated using authentic BH4and BH2as standards. Cellular BH4and BH2levels were then normalized to cell protein concentration and expressed as the ratio of BH4to BH2.27 


Human coronary artery endothelial cells were lysed in 500 μl of lysis buffer [20.0 mm 3-(N-morpholino)propanesulfonic acid, 2.0 mm EGTA, 5.0 mm EDTA, protease inhibitor cocktail (1:100; Sigma-Aldrich, St. Louis, MO), phosphatase inhibitors cocktail (1:100; Calbiochem, San Diego, CA), 0.5% detergent (Nonidet P-40 detergent pH 7.4, Sigma-Aldrich)] 20 min after the beginning of isoflurane exposure, because this time period corresponds to the peak of eNOS phosphorylation in coronary endothelial cells in response to various stimuli.1,10,11Fifteen to 25 μg of protein was loaded onto precast 7.5% tris-HCl gels (Criterion, BioRad, Hercules, CA) and transferred to polyvinylidene fluoride membranes. After blocking the membranes in 5% milk in tris-buffered saline, immunoblots were performed with rabbit monoclonal antiphospho-eNOS (Ser 1177;1:1,000; Cell Signaling Technology, Danvers, MA) and rabbit polyclonal anti-eNOS (1:5,000; Santa Cruz Biotechnologies, Delaware, CA) and were incubated overnight at 4°C. Membranes were washed and incubated with secondary antibodies horseradish peroxidase-conjugated donkey antirabbit IgG for eNOS (1:10,000; Santa Cruz Biotechnologies) and goat antirabbit for phospho-eNOS (1:10,000; Bio-Rad Laboratories, Hercules, CA). Membranes were developed using the ECLplus Western blot chemiluminescence detection reagent (Bio-Rad Laboratories), and densitometric analysis was carried out by using image acquisition and analysis software (Scion Image, Frederick, Maryland).

Confocal Microscopy

Human coronary artery endothelial cells were cultured on gelatin-coated slides as described previously 1to visualize colocalization of Hsp90 and eNOS. After 60 min of isoflurane exposure, cells were fixed in 1% paraformaldehyde, permeabilized in 0.5% TritonX-100 (Sigma-Aldrich), and incubated for 30 min at 37°C with primary monoclonal antibody anti-eNOS (1:100; Biomol International, Plymouth, PA) in phosphate-buffered saline. Incubations with corresponding biotinylated secondary antibodies Alexa 488 conjugated (1:1,000; Invitrogen, Eugene, OR) were conducted for 30 min at 37°C. After washing with phosphate-buffered saline, cells were incubated for 30 min at 37°C with monoclonal antibody anti-Hsp90 (1:50; Santa Cruz Biotechnologies). Incubations with corresponding biotinylated secondary antibodies Alexa 546 conjugated (1:1,000; Invitrogen) were conducted for 30 min at 37°C followed by 1:1000 TO-PRO-3 (nuclear stain; Molecular Probes, Eugene, OR) for 5 min at room temperature and washed again with phosphate-buffered saline. Images were visualized using confocal microscopy (Nikon Eclipse TE 200-U microscope with EZ C1 laser scanning software, Melville, NY) at excitation wavelengths of 488/546/633 nm and emission wavelengths of greater than 520/578/661 nm for eNOS, Hsp90, and TO-PRO-3 respectively. The number of double-stained cells indicating colocalization of Hsp90 and eNOS were counted and expressed as a percentage of the total cell count.


Data were expressed as mean ± SD. Comparison of two means was performed using the Student's-t  test. Comparison of several means was performed using one-way (one factor tested) or two-way (systemic hemodynamics: two factors tested) analysis of variance, when appropriate, and the post hoc  test Newman-Keuls test. Hemodynamic data were analyzed with repeated measures analysis of variance. All P  values were two-tailed, and a P  value less than 0.05 was considered significant. Statistical analysis was performed using NCSS 2007 software (Statistical Solutions Ltd., Cork, Ireland).

Influence of Hyperglycemia on APC: Modulation of BH4and eNOS In Vivo 

Eighty rabbits were instrumented to obtain 76 successful experiments in which infarct size was measured. Four rabbits were excluded because intractable ventricular fibrillation occurred during coronary artery occlusion (two in the control group, one in the hyperglycemia alone group, and one in the hyperglycemia with APC group). Arterial blood gas tensions were maintained within the physiologic range in each group (data not shown). Systemic hemodynamics were similar at baseline among groups (table 1). Intravenous dextrose similarly increased (P < 0.05) blood glucose concentrations during coronary artery occlusion compared with baseline values in the presence (257 ± 33 mg/dl vs.  124 ± 11 mg/dl) or absence of APC (281 ± 46 mg/dl vs.  110 ± 33 mg/dl), during APC with sepiapterin (260 ± 62 mg/dl vs.  123 ± 9 mg/dl), and with N  (G)-nitro-l-arginine methyl ester (295 ± 42 mg/dl) or N -acetylserotonin (288 ± 17 mg/dl). Left ventricular mass, area at risk mass, and the ratio of area at risk to left ventricular mass were similar between groups (table 2). APC decreased myocardial infarct size compared with control experiments (26 ± 6% vs.  46 ± 3% of the left ventricular area at risk, respectively; P < 0.05: fig. 2). Hyperglycemia alone had no effect on infarct size but abolished the protective effects of APC (44 ± 2% vs.  43 ± 4% of the left ventricular area at risk, respectively). Sepiapterin did not influence infarct size compared with control experiments (46 ± 3% vs.  46 ± 3%), but it restored the cardioprotective effect of APC during hyperglycemia (46 ± 3% vs.  21 ± 3%, respectively; P < 0.05). The beneficial actions of sepiapterin to restore APC during hyperglycemia were blocked by the NOS inhibitior N  (G)-nitro-l-arginine methyl ester (47 ± 2%) and the BH4synthesis inhibitor N -acetylserotonin (46 ± 3%). Sepiapterin had no effect on infarct size during hyperglycemia (n = 4; 39 ± 3%). N  (G)-nitro-l-arginine methyl ester3or N -acetylserotonin alone (n = 4; 46 ± 2%) did not alter the extent of myocardial necrosis.

Effects of Glucose on Isoflurane-dependent Nitric Oxide Production and BH4in Human Coronary Artery Endothelial Cells

Isoflurane significantly increased nitric oxide production in human coronary artery endothelial cells compared with control experiments (612 ± 63 nmol/mg vs.  344 ± 11 nmol/mg of protein, respectively; P < 0.05: fig. 3A), and this action was significantly attenuated by high (20 mm) glucose conditions (379 ± 32 nmol/mg of protein; fig. 3B). Sepiapterin alone (100 μm) slightly increased (418 ± 37 nmol/mg of protein) nitric oxide production compared with control experiments and enhanced isoflurane-dependent nitric oxide production (757 ± 99 nmol/mg of protein) in the absence of high glucose. Hyperglycemic conditions had a deleterious effect on nitric oxide production by isoflurane (125 ± 6% vs.  177 ± 13% of control values, respectively; fig. 4), whereas sepiapterin dose-dependently restored (149 ± 12 and 175 ± 9%, 10 and 100 um, respectively) isoflurane-enhanced nitric oxide concentrations in the presence of elevated glucose.

BH4/BH2was increased by isoflurane when compared with control experiments (0.11 ± 0.03 vs.  0.06 ± 0.01, respectively; P < 0.05: fig. 5A). Increases in glucose concentration had no effect on BH4/BH2(0.07 ± 0.01) but abolished increases in the ratio of reduced to oxidized biopterin produced by isoflurane (0.05 ± 0.01). Sepiapterin, in both low and high concentrations, profoundly increased BH4/BH2(fig. 5B) in the presence of increased glucose. During hyperglycemic conditions and low dose sepiapterin, isoflurane did not further augment BH4/BH2levels. However, this ratio was further increased when isoflurane was combined with high dose sepiapterin.

eNOS Activation and Hsp90–eNOS Interactions during APC and Increased Glucose

Isoflurane increased the ratio of phospho-eNOS to total eNOS during normoglycemic but not hyperglycemic conditions (135 ± 17% vs.  85 ± 19% of baseline values, respectively; P < 0.05: fig. 6). Sepiapterin significantly enhanced (132 ± 26% of control values) isoflurane-induced eNOS activation (phospho-eNOS) during hyperglycemia (fig. 6). Similarly, isoflurane increased colocalization of Hsp90 with eNOS (fig. 7) in cells cultured in normal but not high glucose media.

The loss of nitric oxide bioavailability because of reduced synthesis or scavenging by oxidative species is the sine qua non of endothelial dysfunction and an independent predictor of adverse cardiovascular events.28Dysregulation of eNOS plays a critical role in the pathogenesis of cardiovascular disease during hyperglycemia and diabetes mellitus,29hypercholesterolemia,18hypertension,30aging,31and chronic smoking.32The current results extend previous findings demonstrating that diabetes and hyperglycemia may increase cardiovascular risk by impairing ischemic and pharmacologic preconditioning2,33,34and further demonstrate that this action is mediated by glucose-induced modulation of eNOS chaperone and cofactor function during APC.

Hsp90 is a highly abundant molecular chaperone protein involved in protein folding and maturation, and it is a physiologic binding partner and regulator of eNOS.9Impairment of eNOS–Hsp90 interaction disrupts nitric oxide-dependent signaling and increases the production of superoxide anion.1,9,10We have previously demonstrated that APC increases the association between Hsp90 and eNOS.1Hsp90 antagonists, geldanamycin and radicicol, abolished infarct size reduction afforded by either anesthetic or ischemic preconditioning, and inhibition of Hsp90 prevented isoflurane-induced production of nitric oxide.1The current results similarly indicate that hyperglycemia attenuates APC, in part, by disrupting Hsp90 association with eNOS and decreasing nitric oxide production. This finding confirms previous work showing that hyperglycemia and diabetes induce translocation of Hsp90 to the outside of endothelial cells, which decreases Hsp90-eNOS association and subsequent nitric oxide production.12 

BH4is an essential cofactor that also regulates nitric oxide synthesis.18,35eNOS enzyme consists of a reductase domain that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to the flavoproteins, flavin adenine dinucleotide, and flavin mononucleotide; and a heme-containing oxygenase domain that binds BH4, molecular oxygen, and substrate l-arginine.36Electrons transferred from the reductase domain to the oxygenase domain enable ferric heme to bind oxygen, forming a ferrous-dioxy complex. A second electron may be preferentially transferred from BH4to activate oxygen and catalyze l-arginine hydroxylation. Thus, in the presence of adequate substrate and BH4, heme and oxygen reduction are coupled to the synthesis of nitric oxide. However, in the presence of low concentrations of intracellular BH4, electron transfer within the active site of eNOS becomes uncoupled from l-arginine oxidation, and molecular oxygen is reduced to superoxide anion.37,38 

The intracellular regulation of BH4is dependent on the cellular redox state.36Therefore, it is likely that hyperglycemia and accompanying increases in oxidative stress adversely modify eNOS regulation and cardioprotection during APC by modulating biopterin concentrations. For example, the oxidant species peroxynitrite that is increased by hyperglycemia has been shown to oxidize BH4to the catalytically incompetent pterin species BH2. This action leads to uncoupling of eNOS and enhanced production of superoxide anion by the uncoupled enzyme.39,40Hyperglycemia may overwhelm the natural antioxidant defense mechanisms that maintain BH4in its reduced form.27Experimental findings demonstrate that high concentrations of glucose decrease intracellular BH4/BH2levels in parallel with decreases in nitric oxide production and overproduction of superoxide anion.15,27,41In fact, superoxide anion is the sole product of recombinant eNOS either in the absence of BH4or in the presence of excess BH2.27Conversely, increases in BH4/BH2favor an increased production of nitric oxide production, a decrease in superoxide anion,18,35and maintenance of eNOS in its active phosphorylated state.42 

BH4is synthesized in the cell cytoplasm by either de novo  or salvage pathways.43The enzyme guanosine 5′-triphosphate cyclohydrolase I is the first and rate-limiting step in the biosynthesis of BH4through the de novo  pathway. BH4is also synthesized from BH2through the activity of two salvage pathway reduced nicotinamide adenine dinucleotide phosphate-dependent enzymes, sepiapterin reductase, and dihydrofolate reductase. Although sepiapterin is not an endogenous precursor of BH4, it can serve as an effective substrate for BH4synthesis through the salvage pathway.

The current results demonstrate that isoflurane increases BH4/BH2levels concomitantly with enhanced eNOS activation (increased phospho-eNOS to total eNOS) and nitric oxide production in human coronary artery endothelial cells. In contrast, isoflurane failed to increase BH4/BH2, phosphorylated eNOS and nitric oxide production during hyperglycemia, confirming that hyperglycemia adversely impacts isoflurane-induced nitric oxide signaling. In contrast, sepiapterin dose-dependently restored eNOS function during hyperglycemia. Although low concentrations (10 μm) of sepiapterin profoundly increased BH4/BH2levels, 10-fold higher concentrations (100 μm) of sepiapterin were required to restore APC-induced nitric oxide production during hyperglycemia. This result could indicate that excess concentrations of BH4might be required during hyperglycemia to compensate for sustained eNOS dysfunction due to impaired Hsp90–eNOS association.

Hyperglycemia blocked the cardioprotective effects of APC; however, isoflurane-induced reductions of myocardial infarct size were restored by sepiapterin during increases in blood glucose concentration. Thus, our findings indicate that BH4is an important cofactor that affects outcome after ischemia and reperfusion injury in the setting of hyperglycemia. This contention is supported by other evidence that myocardial ischemia and reperfusion decreased intracellular BH4concentrations in parallel with overproduction of superoxide anion.44Conversely, BH4supplementation enhanced left ventricular developed pressure and decreased left ventricular end-diastolic pressure after ischemia and reperfusion injury in the isolated perfused rat heart.45Supplementation with BH4enhanced nitric oxide production and decreased superoxide anion release during hypercholesterolemia,18chronic smoking,32diabetes,16,46and acute hyperglycemia.14,27,47Interestingly, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors have been shown to stimulate the synthesis of BH4by increasing the expression of guanosine 5′-triphosphate cyclohydrolase I.47We previously demonstrated that simvastatin restored ischemic preconditioning during hyperglycemia through a nitric oxide-dependent mechanism.2Taken together, these results suggest that pharmacologic strategies that target BH4either directly (sepiapterin) or indirectly (statins) may enhance eNOS signaling and cardioprotection during diabetes and hyperglycemia.

Hyperglycemia also impairs APC by increasing concentrations of deleterious reactive oxygen species8and by attenuating activation of ATP-regulated potassium channels.34ATP-regulated potassium channels are known downstream targets of nitric oxide, and superoxide anion has previously been shown to decrease nitric oxide concentrations by reacting with and inactivating this cardioprotective molecule. Thus, hyperglycemia produces a wide range of adverse effects on multiple components of the APC signaling pathway, including alterations in the regulation of eNOS, and on downstream effectors such as ATP-regulated potassium channels.

The current results should be interpreted within the constraints of several potential limitations. The role of BH4, Hsp90, and eNOS during APC was investigated only with isoflurane. Although a recent study performed in humans confirmed a protective effect of other halogenated anesthetics such as sevoflurane on endothelial function,48we cannot substantiate that the current results indicate a class effect of volatile anesthetic agents on eNOS regulation. The actions of isoflurane on Hsp90 interactions with eNOS, BH4, and nitric oxide were measured in human coronary artery endothelial cells in vitro . Results may be different in cardiomyocytes or myocardium in vivo . The role of paracrine interactions between endothelial cells that produce nitric oxide and effects on neighboring cardiomyocytes during cardioprotection in vivo  are unknown and is an important focus for future investigations. There is remarkable consistency in the identified signaling pathways demonstrated by experiments conducted in multiple animal and human models. Although the current results obtained in human coronary artery endothelial cells confirmed those obtained in rabbits in vivo , altered anesthetic signaling mechanisms that are dependent on species or cell lineage cannot be totally excluded from the analysis. Different severity of hyperglycemia was used during in vivo  (moderate; approximately 14 mm), when compared with in vitro  (severe; 20 mm), experiments. Moderate hyperglycemia was used during myocardial infarction experiments because severe hyperglycemia alone increases infarct size. In contrast, endothelial cells in culture are relatively resistant to moderate hyperglycemia.

In conclusion, the current results demonstrate that hyperglycemia adversely modulates APC through Hsp90 chaperone and BH4cofactor function. Although isoflurane enhances eNOS function by increasing Hsp90–eNOS interactions and the ratio of reduced to oxidized biopterin, these actions are attenuated by hyperglycemia. Conversely, sepiapterin, a precursor of BH4, restores APC-induced cardioprotection in the presence of hyperglycemia.

The authors thank David Schwabe, B.S., Research Technician, for technical assistance and Shelly Logsdon, Administrative Assistant, for manuscript preparation (both Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin).

Amour J, Brzezinska AK, Weihrauch D, Billstrom AR, Zielonka J, Krolikowski JG, Bienengraeber MW, Warltier DC, Pratt PF Jr, Kersten JR: Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning. Anesthesiology 2009; 110:317–25
Gu W, Kehl F, Krolikowski JG, Pagel PS, Warltier DC, Kersten JR: Simvastatin restores ischemic preconditioning in the presence of hyperglycemia through a nitric oxide-mediated mechanism. Anesthesiology 2008; 108:634–42
Chiari PC, Bienengraeber MW, Weihrauch D, Krolikowski JG, Kersten JR, Warltier DC, Pagel PS: Role of endothelial nitric oxide synthase as a trigger and mediator of isoflurane-induced delayed preconditioning in rabbit myocardium. Anesthesiology 2005; 103:74–83
Burwell LS, Brookes PS: Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia-reperfusion injury. Antioxid Redox Signal 2008; 10:579–99
McCabe TJ, Fulton D, Roman LJ, Sessa WC: Enhanced electron flux and reduced calmodulin dissociation may explain “calcium-independent” eNOS activation by phosphorylation. J Biol Chem 2000; 275:6123–8
Giugliano D, Marfella R, Coppola L, Verrazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, D'Onofrio F: Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 1997; 95:1783–90
Grundy SM, Benjamin IJ, Burke GL, Chait A, Eckel RH, Howard BV, Mitch W, Smith SC Jr, Sowers JR: Diabetes and cardiovascular disease: A statement for healthcare professionals from the American Heart Association. Circulation 1999; 100:1134–46
Kehl F, Krolikowski JG, Weihrauch D, Pagel PS, Warltier DC, Kersten JR: N-acetylcysteine restores isoflurane-induced preconditioning against myocardial infarction during hyperglycemia. Anesthesiology 2003; 98:1384–90
Pritchard KA Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, Sessa WC: Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem 2001; 276:17621–4
Chen JX, Lawrence ML, Cunningham G, Christman BW, Meyrick B: HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium. J Appl Physiol 2004; 96:612–20
Chen JX, Meyrick B: Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium. Lab Invest 2004; 84:182–90
Lei H, Venkatakrishnan A, Yu S, Kazlauskas A: Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes. J Biol Chem 2007; 282:9364–71
Satoh M, Fujimoto S, Haruna Y, Arakawa S, Horike H, Komai N, Sasaki T, Tsujioka K, Makino H, Kashihara N: NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy. Am J Physiol Renal Physiol 2005; 288:F1144–52
Ihlemann N, Rask-Madsen C, Perner A, Dominguez H, Hermann T, Kober L, Torp-Pedersen C: Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects. Am J Physiol Heart Circ Physiol 2003; 285:H875–82
Bagi Z, Toth E, Koller A, Kaley G: Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH(4). Am J Physiol Heart Circ Physiol 2004; 287:H626–33
Heitzer T, Krohn K, Albers S, Meinertz T: Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia 2000; 43:1435–8
Pannirselvam M, Simon V, Verma S, Anderson T, Triggle CR: Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br J Pharmacol 2003; 140:701–6
Cosentino F, Hurlimann D, Delli Gatti C, Chenevard R, Blau N, Alp NJ, Channon KM, Eto M, Lerch P, Enseleit F, Ruschitzka F, Volpe M, Luscher TF, Noll G: Chronic treatment with tetrahydrobiopterin reverses endothelial dysfunction and oxidative stress in hypercholesterolaemia. Heart 2008; 94:487–92
Fukuda Y, Teragawa H, Matsuda K, Yamagata T, Matsuura H, Chayama K: Tetrahydrobiopterin restores endothelial function of coronary arteries in patients with hypercholesterolaemia. Heart 2002; 87:264–9
Chiari PC, Pagel PS, Tanaka K, Krolikowski JG, Ludwig LM, Trillo RA Jr, Puri N, Kersten JR, Warltier DC: Intravenous emulsified halogenated anesthetics produce acute and delayed preconditioning against myocardial infarction in rabbits. Anesthesiology 2004; 101:1160–6
Qu XW, Thaete LG, Rozenfeld RA, Zhu Y, De Plaen IG, Caplan MS, Hsueh W: Tetrahydrobiopterin prevents platelet-activating factor-induced intestinal hypoperfusion and necrosis: Role of neuronal nitric oxide synthase. Crit Care Med 2005; 33:1050–6
Tang XL, Takano H, Xuan YT, Sato H, Kodani E, Dawn B, Zhu Y, Shirk G, Wu WJ, Bolli R: Hypercholesterolemia abrogates late preconditioning via a tetrahydrobiopterin-dependent mechanism in conscious rabbits. Circulation 2005; 112:2149–56
Tanaka K, Weihrauch D, Kehl F, Ludwig LM, LaDisa JF Jr, Kersten JR, Pagel PS, Warltier DC: Mechanism of preconditioning by isoflurane in rabbits: A direct role for reactive oxygen species. Anesthesiology 2002; 97:1485–90
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM: Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399:601–5
Sessa WC, Garcia-Cardena G, Liu J, Keh A, Pollock JS, Bradley J, Thiru S, Braverman IM, Desai KM: The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. J Biol Chem 1995; 270:17641–4
Whitsett J, Picklo MJS, Vasquez-Vivar J: 4-Hydroxy-2-nonenal increases superoxide anion radical in endothelial cells via stimulated GTP cyclohydrolase proteasomal degradation. Arterioscler Thromb Vasc Biol 2007; 27:2340–7
Crabtree MJ, Smith CL, Lam G, Goligorsky MS, Gross SS: Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS. Am J Physiol Heart Circ Physiol 2008; 294:H1530–40
Lerman A, Zeiher AM: Endothelial function: Cardiac events. Circulation 2005; 111:363–8
Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM: Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest 2003; 112:725–35
Schulz E, Jansen T, Wenzel P, Daiber A, Munzel T: Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension. Antioxid Redox Signal 2008; 10:1115–26
Blackwell KA, Sorenson JP, Richardson DM, Smith LA, Suda O, Nath K, Katusic ZS: Mechanisms of aging-induced impairment of endothelium-dependent relaxation: Role of tetrahydrobiopterin. Am J Physiol Heart Circ Physiol 2004; 287:H2448–53
Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, Munzel T: Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: Evidence for a dysfunctional nitric oxide synthase. Circ Res 2000; 86:E36–41
Kehl F, Krolikowski JG, Mraovic B, Pagel PS, Warltier DC, Kersten JR: Hyperglycemia prevents isoflurane-induced preconditioning against myocardial infarction. Anesthesiology 2002; 96:183–8
Kersten JR, Montgomery MW, Ghassemi T, Gross ER, Toller WG, Pagel PS, Warltier DC: Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels. Am J Physiol Heart Circ Physiol 2001; 280:H1744–50
Bevers LM, Braam B, Post JA, van Zonneveld AJ, Rabelink TJ, Koomans HA, Verhaar MC, Joles JA: Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase. Hypertension 2006; 47:87–94
Forstermann U: Janus-faced role of endothelial NO synthase in vascular disease: Uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal. Biol Chem 2006; 387:1521–33
Whitsett J, Martasek P, Zhao H, Schauer DW, Hatakeyama K, Kalyanaraman B, Vasquez-Vivar J: Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution. Free Radic Biol Med 2006; 40:2056–68
Wever RM, Luscher TF, Cosentino F, Rabelink TJ: Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation 1998; 97:108–12
Milstien S, Katusic Z: Oxidation of tetrahydrobiopterin by peroxynitrite: Implications for vascular endothelial function. Biochem Biophys Res Commun 1999; 263:681–4
Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG: Endothelial regulation of vasomotion in apoE-deficient mice: Implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 2001; 103:1282–8
Bagi Z, Koller A: Lack of nitric oxide mediation of flow-dependent arteriolar dilation in type I diabetes is restored by sepiapterin. J Vasc Res 2003; 40:47–57
Cai S, Khoo J, Channon KM: Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells. Cardiovasc Res 2005; 65:823–31
Shi W, Meininger CJ, Haynes TE, Hatakeyama K, Wu G: Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells. Cell Biochem Biophys 2004; 41:415–34
Dumitrescu C, Biondi R, Xia Y, Cardounel AJ, Druhan LJ, Ambrosio G, Zweier JL: Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc Natl Acad Sci U S A 2007; 104:15081–6
Verma S, Maitland A, Weisel RD, Fedak PW, Pomroy NC, Li SH, Mickle DA, Li RK, Rao V: Novel cardioprotective effects of tetrahydrobiopterin after anoxia and reoxygenation: Identifying cellular targets for pharmacologic manipulation. J Thorac Cardiovasc Surg 2002; 123:1074–83
Sasaki N, Yamashita T, Takaya T, Shinohara M, Shiraki R, Takeda M, Emoto N, Fukatsu A, Hayashi T, Ikemoto K, Nomura T, Yokoyama M, Hirata K, Kawashima S: Augmentation of vascular remodeling by uncoupled endothelial nitric oxide synthase in a mouse model of diabetes mellitus. Arterioscler Thromb Vasc Biol 2008; 28:1068–76
Ding QF, Hayashi T, Packiasamy AR, Miyazaki A, Fukatsu A, Shiraishi H, Nomura T, Iguchi A: The effect of high glucose on NO and O2 through endothelial GTPCH1 and NADPH oxidase. Life Sci 2004; 75:3185–94
Lucchinetti E, Ambrosio S, Aguirre J, Herrmann P, Harter L, Keel M, Meier T, Zaugg M: Sevoflurane inhalation at sedative concentrations provides endothelial protection against ischemia-reperfusion injury in humans. Anesthesiology 2007; 106:262–8