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Anesth Analg 2006;103:281-288
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000226094.94877.98

CARDIOVASCULAR ANESTHESIA

Extracellular Signal-Regulated Kinases Trigger Isoflurane Preconditioning Concomitant with Upregulation of Hypoxia-Inducible Factor-1{alpha} and Vascular Endothelial Growth Factor Expression in Rats

Chen Wang, MD, Dorothee Weihrauch, DVM, PhD, David A. Schwabe, BSEE, Martin Bienengraeber, PhD, David C. Warltier, MD, PhD, Judy R. Kersten, MD, Phillip F. Pratt, Jr, PhD, and Paul S. Pagel, MD, PhD

From the Departments of Anesthesiology, Medicine (Division of Cardiovascular Diseases), and Pharmacology and Toxicology, the Medical College of Wisconsin and the Clement J. Zablocki Veterans Affairs Medical Center; Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin.

Address correspondence and reprint requests to Paul S. Pagel MD PhD, Medical College of Wisconsin, MEB-M4280, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. Address e-mail to pspagel{at}mcw.edu.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTRODUCTION: Extracellular signal-related kinases 1 and 2 (Erk1/2) are mitogen-activated protein kinases that have been implicated in anesthetic preconditioning; but whether Erk1/2 triggers or mediates this beneficial effect and the mechanisms by which Erk1/2 produces cardioprotection are unknown. We tested the hypothesis that isoflurane preconditioning is triggered by Erk1/2 concomitant with upregulation of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and vascular endothelial growth factor (VEGF) expression in rats instrumented for hemodynamic measurement and subjected to a 30-min coronary artery occlusion and 2-h reperfusion.

METHODS: Rats randomly received IV 0.9% saline (control) or isoflurane (1.0 minimum alveolar concentration administered for 30 min and discontinued 15 min [memory period] before coronary occlusion) in the absence or presence of the selective Erk1/2 inhibitor PD 098059 (1 mg/kg in dimethylsulfoxide administered IV either 3 min before exposure to isoflurane [trigger] or 3 min after discontinuation of the drug [mediator]). Additional rabbits were pretreated with dimethylsulfoxide alone. Left ventricular tissue samples were obtained at selected intervals from additional groups of rats for Western immunoblot analysis of phospho-Erk1/2, HIF-1{alpha}, and VEGF protein expression.

RESULTS: Isoflurane significantly (P < 0.05) reduced infarct size (41% ± 8% of the left ventricular area at risk; triphenyltetrazolium chloride staining) as compared with control (59% ± 4%). PD 098059 administered before, but not after, isoflurane abolished this cardioprotection (61% ± 5% and 42% ± 9%, respectively). Isoflurane-induced increases in phospho-Erk1/2, HIF-1{alpha}, and VEGF expression were also inhibited by PD 098059 pretreatment.

CONCLUSIONS: The results indicate that Erk1/2 triggers isoflurane preconditioning concomitant with HIF-1{alpha} and VEGF upregulation in vivo.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Volatile anesthetics produce pharmacological preconditioning against ischemia-reperfusion injury, but the precise mechanisms responsible for these cardioprotective effects remain incompletely characterized (1). The extracellular signal-related kinases (Erk1/2) are mitogen-activated protein kinases (MAPK) that have been shown to play important roles in cell division, proliferation, and survival (2). Erk1/2 was previously implicated in ischemic and opioid-induced pharmacological preconditioning in rats (3). Reductions in myocardial infarct size and Erk1/2 phosphorylation produced by administration of desflurane before ischemia and reperfusion were abolished by a selective Erk1/2 inhibitor in vivo (4). These results provided evidence suggesting that Erk1/2 plays an important role in anesthetic-induced preconditioning, but whether Erk1/2 acts as trigger or mediator of this phenomenon is unknown. Thus, we tested the hypothesis that Erk1/2 triggers isoflurane preconditioning concomitant with Erk1/2 phosphorylation in vivo.

The protective effects of Erk1/2 may be related to activation of pro-survival signal transduction (e.g., 70-kDa ribosomal protein s6 kinase [p70s6K], endothelial nitric oxide synthase [eNOS]), phosphorylation and inactivation of the regulatory enzyme glycogen synthase kinase-ß, inhibition of the mitochondrial permeability transition pore, or attenuation of apoptotic cell death (2,5,6). Erk1/2-related signaling proteins also control the expression of several genes concerned with cell survival including hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), an important DNA-binding complex (7) whose activity is influenced by intracellular oxygen tension (8). Myocardial hypoxia is a potent inducer of HIF-1{alpha} protein expression (9), and the combination of reduced oxygen tension and activation of Erk1/2 signaling enhance HIF-1{alpha} expression and activity (10). Furthermore, HIF-1{alpha} upregulates transcription of vascular endothelial growth factor (VEGF) (11). This important angiogenic protein plays a central role in coronary collateral development in response to chronic myocardial ischemia (12), and enhanced expression of both HIF-1{alpha} and VEGF occurs during myocardial ischemia in rats (13). Erk1/2-dependent upregulation of HIF-1{alpha} and VEGF has also been shown during ischemic and hypoxic preconditioning (14). Collectively, these data indicate an alternative mechanism by which activation of Erk1/2 signaling contributes to cardioprotection against ischemia-reperfusion injury (15). Whether volatile anesthetics enhance HIF-1{alpha} and VEGF protein expression in an Erk1/2-dependent manner is unknown. Thus, we also tested the hypothesis that isoflurane upregulates HIF-1{alpha} and VEGF by activating Erk1/2.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. 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.

Surgical Instrumentation
Male Wistar rats weighing between 270 and 390 g were anesthetized with IP sodium thiobutabarbital (150 mg/kg) and received additional doses of the barbiturate (25 to 50 mg/kg) to ensure that pedal and palpebral reflexes were absent throughout the experimental protocol. Rats were instrumented for the measurement of systemic hemodynamics as previously described (16). Briefly, heparin-filled catheters were inserted into the right jugular vein and the right carotid artery for fluid or drug administration and measurement of arterial blood pressure, respectively. A tracheostomy was performed, the trachea was intubated with a cannula connected to a rodent ventilator, and the lungs were ventilated with an air-oxygen mixture (fractional inspired oxygen concentration = 0.33). A thoracotomy was performed in the left fifth intercostal space, and the pericardium was opened. A 6-0 Prolene ligature was placed around the proximal left descending coronary artery and vein in the area immediately below the left atrial appendage. The ends of the suture were threaded through a propylene tube to form a snare. Coronary artery occlusion was produced by clamping the snare onto the epicardial surface of the heart with a hemostat and was confirmed by the appearance of epicardial cyanosis. Reperfusion was achieved by loosening the snare and was verified by observing an epicardial hyperemic response. Hemodynamic data were continuously recorded on a polygraph and digitized using a computer interfaced with an analog-to-digital converter.

Experimental Protocol
The experimental design is illustrated in Figure 1. All rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Rats were randomly assigned to receive IV 0.9% saline (control) or the mitogen activated protein kinase-extracellular signal-regulated kinase (MEK-1) inhibitor PD 098059 (1 mg/kg; Erk1/2 is activated by phosphorylation via the upstream kinase MEK-1) (3) in the absence or presence of isoflurane (1.0 minimum alveolar concentration [MAC]). PD 098059 was dissolved in dimethylsulfoxide and administered IV 3 min before exposure to isoflurane (trigger) or 3 min after discontinuation of the volatile anesthetic (mediator). An additional group of rats were pretreated with DMSO alone before coronary artery occlusion and reperfusion. Isoflurane was administered for 30 min and discontinued 15 min (memory period) before coronary artery occlusion. End-tidal concentrations of isoflurane were measured using an infrared gas analyzer that was calibrated with known standards before and during experimentation. The MAC value of isoflurane used for rats in the current investigation was 1.4% (17). After discontinuation of the volatile anesthetic, end-tidal concentrations of isoflurane decreased to zero before coronary occlusion.


Figure 14
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  		Figure 1. Schematic illustration of the experimental protocols used in myocardial infarct size (panel A, top) and Western immunoblotting (panel B, bottom) experiments. CON = control; DMSO = dimethylsulfoxide; PD = PD 098059; ISO = isoflurane.

 

Determination of Myocardial Infarct Size
Myocardial infarct size was measured as previously described (18). Briefly, the coronary artery was reoccluded after the 2-h reperfusion period. Patent blue dye was administered IV to stain the normal region of the left ventricle, and the heart was rapidly excised. The left ventricle was cut into 5 or 6 cross-sectional pieces of 2-mm thickness. The blue-stained left ventricular normal zone was separated from the nonstained left ventricular area at risk and incubated at 37°C for 15 min in 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer adjusted to pH 7.4. Tissues were fixed overnight in 10% formaldehyde, and the infarcted tissue was carefully separated from the area at risk using a dissecting microscope. Infarct size was expressed as a percentage of the left ventricular area at risk.

Western Immunoblotting
Rats (n = 6 per group) were randomly assigned to receive 0.9% saline (control), 1.0 MAC isoflurane administered for 30 min, followed by memory periods of 15 or 165 min, or IV PD 098059 (1.0 mg/kg in dimethylsulfoxide) in the absence or presence of 1.0 MAC isoflurane administered for 30 min, followed by a memory period of 15 min. PD 098059 was administered IV 3 min before exposure to isoflurane or at a corresponding time point in rats that did not receive the volatile anesthetic. Coronary artery occlusion and reperfusion were not performed in this series of experiments. Left ventricular samples were collected from rats 45 min after interventions were initiated with the exception of rats exposed to isoflurane for 30 min followed by a 165 min memory period. Left ventricular samples were immediately frozen in liquid nitrogen and stored in a freezer at –70°C for subsequent analysis. Western immunoblotting was performed using previously described methods (19). Briefly, tissue was homogenized using a Polytron homogenizer in ice-cold lysis buffer containing a complete proteinase inhibitor cocktail (one tablet per 10 mL). The homogenate was further homogenized using a Sonifier Liquid Processor (Branson Ultrasonics Corp., Danbury, CT) and subsequently centrifuged at 18,000g for 30 min at 4°C to isolate total Erk 1/2 protein. The clarified supernatant was used to quantify Erk1/2 phosphorylation and determine total Erk1/2 and VEGF protein expression. Protein concentrations were determined using the Lowry method. Equivalent amounts (100 µg) of protein were mixed with Laemmeli buffer and heated at 95°C for 5 min before electrophoretic separation as described below.

The pellet obtained from the initial centrifugation was solubilized and resuspended in a lysis buffer (500 µL), vortexed, and used for immunoblot analysis of HIF-1{alpha} expression conducted after immunoprecipitation. Five-hundred micrograms of clarified supernatant in a volume of 500 µL of lysis buffer were gently agitated with protein A-Sepharose beads for 1 h to minimize nonspecific binding of cell proteins to the beads used to isolate the HIF-1{alpha} immunocomplex. These precleared supernatant fractions were then transferred to a separate 1.5 mL microcentrifuge tube and incubated for 24 h with goat polyclonal HIF-1{alpha} antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. The HIF-1{alpha}-antibody immunocomplex was isolated by incubation (2 h) with 50 µL of a 50% slurry of protein A-Sepharose beads. The beads were then washed 3 times with 500 µL of Tris-buffered saline (TBS), mixed with Laemmeli buffer (50 µL), and heated at 95°C for 5 min.

All samples were separated on a 4% to 20% polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. The membrane was stained with 0.1% Ponceau S (Sigma; St. Louis, MO) in 5% acetic acid for 5 min to verify equal loading of lanes using the method of Ping et al. (20). After blocking with 5% milk in TBS containing 0.1% Tween-20, nitrocellulose membranes were incubated overnight at 4°C in 5% milk and a 1:200 dilution of rabbit/goat polyclonal or mouse monoclonal antibodies [Phospho-p44/p42 MAPK (Thr (202)/Tyr (204), Cell Signaling Technology, Beverly, MA); Erk1 (C-16), VEGF (C-1), HIF-1{alpha} (C-19), Santa Cruz Biotechnology, Santa Cruz, CA]. Bands were visualized with horseradish peroxidase-conjugated secondary antibodies and chemiluminescent substrate. Quantitative analysis of the band densities was performed using UN-SCAN-IT gel Automated Digitizing System (Version 5.1, Silk Scientific Corporation, Orem, UT).

Statistical Analysis
Statistical analysis of data within and between groups was performed with multiple analysis of variance for repeated measures followed by Student-Newman-Keuls test. Changes were considered statistically significant when P < 0.05. All values are expressed as mean ± sd.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fifty-one rats were instrumented to obtain 45 successful myocardial infarct size experiments. Three rats were excluded because of technical difficulties during instrumentation. Three rats were excluded because intractable ventricular fibrillation occurred during coronary artery occlusion. There were no differences in baseline hemodynamics among groups (Table 1). Isoflurane significantly (P < 0.05) decreased heart rate, mean arterial blood pressure, and rate-pressure product in the presence or absence of PD 098059. Hemodynamics returned to baseline values 15 min after isoflurane had been discontinued (memory period) before the onset of coronary artery occlusion. DMSO and PD 098059 alone did not produce hemodynamic effects. Coronary occlusion and reperfusion resulted in similar alterations in mean arterial blood pressure and rate-pressure product in each experimental group. There were no differences in hemodynamics among groups during and after coronary occlusion. Body weight, left ventricular mass, left ventricular area at risk weight, and the ratio of area at risk to left ventricular mass were similar among groups (data not shown). Isoflurane significantly (P < 0.05) reduced infarct size (41% ± 8% of the left ventricular area at risk; Fig. 2) as compared with control (59% ± 4%). PD 098059 administered before but not after isoflurane abolished reductions in infarct size produced by the volatile anesthetic (61% ± 5% and 42% ± 9%, respectively). PD 98059 and DMSO did not affect infarct size when administered alone (60% ± 4% and 62% ± 9%, respectively).


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  		Table 1. Systemic Hemodynamics

 

Figure 24
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  		Figure 2. Myocardial infarct size expressed as a percentage of the left ventricular area at risk in rats receiving 0.9% saline (CON), PD 098059 (PD, 1.0 mg/kg) alone, the drug vehicle dimethylsulfoxide (DMSO), 1.0 minimum alveolar concentration (MAC) isoflurane (ISO), PD 098059 (1.0 mg/kg) administered before isoflurane (PD + ISO), and PD 098059 (1.0 mg/kg) administered after isoflurane (ISO + PD). Each point represents a single experiment. All data are mean ± sd. *Significantly (P < 0.05) different from CON.

 

Erk1/2 phosphorylation is illustrated in Figure 3. Total Erk1/2 expression was similar among groups. The densities of phosphorylated Erk1/2 were normalized against total Erk1/2 expression. Administration of 1.0 MAC isoflurane for 30 min followed by memory periods of 15 and 165 min significantly increased Erk1/2 phosphorylation (+55% ± 16% and +103% ± 8% change from control, respectively). PD 098059 alone did not alter Erk1/2 phosphorylation but abolished increases in Erk1/2 phosphorylation produced by administration of isoflurane for 30 min followed by a memory period of 15 min. Representative Western blots for HIF-1{alpha} and VEGF protein expression are illustrated in Figures 4 and 5, respectively. Administration of 1.0 MAC isoflurane for 30 min followed by a 15-min memory period significantly increased HIF-1{alpha} and VEGF expression (+61% ± 18% and +36% ± 17% change from control, respectively). Further increases in HIF-1{alpha} and VEGF expression were observed 165 min after exposure to isoflurane (+91% ± 33% and +63% ± 30% change from control, respectively). Pretreatment with PD 098059 abolished isoflurane-induced increases in HIF-1{alpha} and VEGF expression.


Figure 34
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  		Figure 3. Representative immunoblots of phospho-Erk (top lanes, top panel) and total Erk (bottom lanes, top panel) in rats receiving 0.9% saline (CON), PD 098059 (PD, 1.0 mg/kg), 1.0 minimum alveolar concentration (MAC) isoflurane (ISO) administered for 30 min, and PD 098059 (1.0 mg/kg) administered before isoflurane. Left ventricular tissue samples were obtained 15 or 165 min after administration of ISO or at a corresponding time point in rats that did not receive the volatile anesthetic. Histograms illustrating the relative density of phospho-Erk in each experimental group are depicted in the lower panel. All data are mean ± sd. *Significantly (P < 0.05) different from CON.

 

Figure 44
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  		Figure 4. Representative immunoblots (top panel) of HIF-1{alpha} in rats receiving 0.9% saline (CON), PD 098059 (PD, 1.0 mg/kg), 1.0 minimum alveolar concentration (MAC) isoflurane (ISO) administered for 30 min, and PD 098059 (1.0 mg/kg) administered before isoflurane. Left ventricular tissue samples were obtained 15 or 165 min after administration of ISO or at a corresponding time point in rats that did not receive the volatile anesthetic. Histograms illustrating HIF-1{alpha} expression in each experimental group are depicted in the lower panel. All data are mean ± sd. *Significantly (P < 0.05) different from CON.

 


Figure 54
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  		Figure 5. Representative immunoblots (top panel) of VEGF in rats receiving 0.9% saline (CON), PD 098059 (PD, 1.0 mg/kg), 1.0 minimum alveolar concentration (MAC) isoflurane (ISO) administered for 30 min, and PD 098059 (1.0 mg/kg) administered before isoflurane. Left ventricular tissue samples were obtained 15 or 165 min after administration of ISO or at a corresponding time point in rats that did not receive the volatile anesthetic. Histograms illustrating VEGF expression in each experimental group are depicted in the lower panel. All data are mean ± sd. *Significantly (P < 0.05) different from CON.

 

   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current results confirm the findings of many previous studies (1) demonstrating that administration and subsequent discontinuation of isoflurane before prolonged coronary occlusion and reperfusion reduces myocardial infarct size in rats. This isoflurane-induced cardioprotective effect was abolished by pretreatment with the selective MEK-1 inhibitor PD 098059. These data suggest that Erk1/2 plays a central role in isoflurane preconditioning and support previous findings indicating that activation of Erk1/2 is required for desflurane preconditioning in rats (4). To our knowledge, the current findings demonstrate for the first time that Erk1/2 acts as a trigger, but not a mediator, of isoflurane preconditioning because administration of PD 098059 before, but not after, exposure to isoflurane abolished decreases in infarct size produced by the volatile anesthetic. Furthermore, the current results demonstrate that isoflurane causes phosphorylation of Erk1/2 independent of subsequent ischemia and reperfusion. This isoflurane-induced increase in phospho-Erk1/2 expression was time-dependent and was also inhibited by PD 098059 pretreatment. The current results with isoflurane-induced Erk1/2 phosphorylation support previous findings in which the volatile anesthetic desflurane was examined (4). It appears highly likely that Erk1/2 phosphorylation by isoflurane activates several downstream pro-survival signaling molecules responsible for cardioprotection (2). We did not specifically identify other cardioprotective moieties located in this signaling cascade in the current investigation, but our laboratory recently demonstrated that at least two enzymes (eNOS and p70s6K) known to be influenced by Erk1/2 signaling mediate isoflurane-induced postconditioning in rabbits (21). Further investigation will be required to define Erk1/2-related signaling that may be responsible for isoflurane preconditioning.

The current results further demonstrate for the first time that isoflurane produces temporal upregulation of HIF-1{alpha} protein expression independent of ischemia and reperfusion. These increases in HIF-1{alpha} were abolished by PD 098059 pretreatment, providing evidence that activation of Erk1/2 by isoflurane stimulates the production of this protein. Previous studies have implicated MAPK-dependent signaling not only in HIF-1{alpha} transcription and translation but also in regulation of HIF-1{alpha} activity in the absence of reduced intracellular oxygen tension (22,23). p42/p44 MAPK (Erk1/2) was shown to directly phosphorylate and thereby enhance the HIF-1{alpha} transcriptional activity (24). PD 098059-induced inhibition of p42/p44 MAPK also abolished transcription of HIF-1{alpha} in PC12 cells in vitro (25). HIF-1{alpha} has been shown to translocate to the nucleus and bind to gene promoters, thereby enhancing transcription of glycolytic and angiogenic proteins (26). Intracellular hypoxia is a major stimulus for the production and enhanced activity of HIF-1{alpha}, but several other mediators (e.g., reactive oxygen species, nitric oxide, growth factors) have also been shown to be potent activators of HIF-1{alpha} under normoxic conditions (27). Our laboratory has demonstrated that isoflurane produces small quantities of superoxide anion independent of ischemia (28) and, furthermore, that these reactive oxygen intermediates play a key role in triggering isoflurane-induced preconditioning in vivo (29). Isoflurane was also recently shown to increase eNOS transcription and translation before prolonged coronary artery occlusion and reperfusion (30). Taken together, these data support the contention that isoflurane may enhance HIF-1{alpha} expression and activity by directly generating reactive oxygen species or producing nitric oxide. Our laboratory is currently conducting experiments to test this hypothesis. Notably, isoflurane-induced increases in HIF-1{alpha} protein mediated by Erk1/2 occurred within 15 min after discontinuation of the volatile anesthetic. These findings suggest that anesthetic preconditioning may occur by regulation of gene expression in addition to the previously demonstrated beneficial effects of volatile anesthetics on cardioprotective signal transduction.

HIF-1{alpha} has been shown to activate a variety of target genes including the angiogenic mitogen VEGF. HIF-1{alpha} upregulates VEGF transcription by binding to specific promotor sequences and preserves VEGF translation during hypoxic conditions by enhancing mRNA stability (31,32). The current results indicate isoflurane produces time-dependent increases in VEGF protein expression by activating an Erk1/2- and HIF-1{alpha}-mediated signaling pathway. These data indicate for the first time that exposure to a volatile anesthetic directly increases the production of a critical angiogenic factor. Brief, repetitive ischemic episodes upregulated VEGF mRNA before prolonged coronary artery occlusion in rats (33) and was associated with enhanced neovascularization in the ischemia region. The increase in VEGF transcription activation observed in these experiments was linked to activation of the {varepsilon} isoform of protein kinase C (PKC-{varepsilon}) (33). Our laboratory has shown that isoflurane preconditioning is mediated by activation of PKC-{varepsilon} and Src protein tyrosine kinase (PTK) in rats (34). Notably, VEGF protein has been shown to stimulate the kinase insert domain receptor tyrosine kinase that signals through Src PTK to activate PKC (35). Taken together, these data suggest the intriguing hypothesis that VEGF-mediated activation of PKC-{varepsilon} may play a role in isoflurane preconditioning. Furthermore, VEGF has also been shown to activate Erk1/2 through a reactive oxygen species-dependent mechanism, suggesting that isoflurane-induced increases in VEGF may combine with the simultaneous production of reactive oxygen species (28) to produce a "positive feedback" stimulation of Erk1/2-regulated cardioprotective signaling (36). These hypotheses are currently being investigated by our laboratory.

The current results must be interpreted within the constraints of several general (21,37) and specific potential limitations. PD 098059 has been shown to be a selective MEK-1-Erk1/2 inhibitor at the dose used in the current investigation (3). PD 098059 abolished isoflurane-induced reductions in infarct size and increases in phospho-Erk1/2, HIF-1{alpha}, and VEGF protein expression. Nevertheless, a dose-response relationship to PD 098059 was not performed, and the possibility that this drug may have inhibited other protein kinases that regulate cardioprotection or mediate HIF-1{alpha} or VEGF translation cannot be completely excluded from the analysis. Finally, we did not specifically measure HIF-1{alpha} and VEGF transcription in the current investigation, and therefore cannot distinguish whether isoflurane-induced increases in HIF-1{alpha} and VEGF protein expression occurred as a consequence of Erk1/2-mediated enhancement of mRNA transcription or stabilization of existing transcripts.

In summary, the current findings demonstrate that Erk1/2 triggers, but does not mediate, isoflurane preconditioning independent of alterations in the major determinants of myocardial oxygen consumption in vivo. The results further indicate that isoflurane produces time-dependent upregulation of phospho-Erk, HIF-1{alpha}, and VEGF protein expression in the absence of ischemia and reperfusion. These data suggest that isoflurane may exert cardioprotective effects by favorably affecting gene expression before coronary artery occlusion.


   Footnotes
 
Accepted for publication April 10, 2006.

Supported, in part, by American Heart Association Greater Midwest Affiliate grant AHA 0265259Z (to Dr. Weihrauch) and National Institutes of Health grants HL 054820 (to Dr. Warltier), GM 008377 (to Dr. Warltier), GM 066730 (to Dr. Warltier), and HL 063705 (to Dr. Kersten) from the United States Public Health Service (Bethesda, MD).


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press