Journal of Applied Physiology AJP: Heart and Circulatory Physiology
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J Appl Physiol 98: 296-306, 2005. First published September 17, 2004; doi:10.1152/japplphysiol.00075.2004
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Metabolic activation of AMP kinase in vascular smooth muscle

L. J. Rubin,1,3,4,5 L. Magliola,2 X. Feng,1 A. W. Jones,2,3,4,5 and C. C. Hale1,3

Departments of 1Biomedical Sciences and 2Medical Pharmacology and Physiology, 3Dalton Cardiovascular Research Center, and Centers for 4Diabetes and Cardiovascular Health and 5Gender Physiology and Environmental Adaptations, University of Missouri, Columbia, Missouri

Submitted 22 January 2004 ; accepted in final form 10 September 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS AND PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N2 (N2-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was ~50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the {alpha}1- and {alpha}2-catalytic subunit followed by immunoblot analysis for [P]Thr172-AMPK indicates that {alpha}1-AMPK accounts for all activity. Little if any {alpha}2-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N2-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction

porcine; carotid artery; 2-deoxyglucose; smooth muscle; adenosine 5'-monophosphate protein kinase

AMP-ACTIVATED PROTEIN KINASE (AMPK) is a member of a family of serine-threonine kinases (ARKs, AMP-regulated kinases) that are traditionally activated by increases in the cellular AMP-to-ATP ratio. AMPK has been most highly studied in striated muscles where it is activated by such physiological stresses as exercise, exercise mimetics (electrical stimulation), and conditions that invoke hypoxia-ischemia (1, 24). Thus AMPK has been proposed to function as a metabolic master switch to coordinate cellular energy metabolism and ATP conservation and synthesis with physiological activity and metabolic demand (10). AMPK exists as a heterotrimeric enzyme consisting of a catalytic subunit ({alpha}) and two regulatory subunits ({beta} and {gamma}). Isoforms of each subunit exist ({alpha}1, {alpha}2, {beta}1, {beta}2, {gamma}1, {gamma}2, {gamma}3) with multiple combinations possible. AMP binds to the {gamma}-subunit of AMPK and facilitates phosphorylation of threonine 172 (Thr172) of the {alpha}-subunit by an upstream kinase, AMPK kinase (AMPKK), resulting in increased enzyme activity (34). Recent data suggest that the tumor suppressor protein LKB1 functions as an AMPKK in several cell types (15, 19, 32) and Ca2+/calmodulin-dependent protein kinase I can also phosphorylate Thr172 and activate AMPK (13).

In skeletal muscle, activation of AMPK is associated with increased translocation and activity of the GLUT-4 glucose transporter, phosphorylation, and inactivation of acetyl-CoA carboxylase, thus disinhibiting fatty acid oxidation, and activation of glycogen phosphorylase and thus stimulating glycolysis (6). In addition, AMPK phosphorylates and inhibits several key metabolic enzymes that regulate and coordinate ATP-consuming pathways, such as glycogen synthase and 3-hydroxy-3-methylglutaryl-CoA reductase (2). Thus AMPK is a key enzyme in regulation of cellular energy state and in striated muscle is a prime regulatory target for therapeutics aimed at metabolic disorders such as diabetes and obesity, particularly because AMPK is responsive to leptin and insulin-sensitizing drugs (21, 25).

Although considerable information exists regarding regulation of AMPK activation in a variety of cell types, particularly striated muscles, to date AMPK has not been demonstrated in vascular smooth muscle. Regulation of energy usage differs between striated and smooth muscle. In particular, vascular smooth muscle of large vessels such as the aorta or carotid develop and maintain contractile force with little or no measurable changes in cellular ATP or phosphocreatine, although the rate of O2 consumption increases (11, 18). Lower rates of phosphagen utilization during contraction (as compared with skeletal muscle) make it possible for intermediary metabolism to supply the energy needs of vascular smooth muscle (18). It is unknown whether AMPK plays a role in regulating metabolic pathways in smooth muscle during contractions.

Metabolic or hypoxic disturbances evoke a well-established, rapid vasodilation that is the hallmark of metabolic regulation of blood flow. Some of the mediators are intrinsic to the vascular tissues. For instance, blood vessels subjected to metabolic inhibition in vitro also respond with a rapid dilation. Endothelium-derived relaxing factors are suggested to play a significant role in metabolic relaxation. Hypoxia and metabolic inhibition also vasodilate blood vessels that are devoid of endothelium, however, and alter ion channel function of isolated smooth muscle cells. Thus vascular smooth muscle also possesses the intrinsic ability to respond by vasodilating to metabolic inhibition, although the mechanisms are not established. Under conditions of reduced substrate supply, i.e., replacement of glucose with 2-deoxyglucose (2DG), cellular phosphocreatine but not ATP has been shown to decrease in carotid artery with no change in tension induced by high potassium (12). Thus the correspondence between force and high-energy phosphate turnover in vascular smooth muscle is less coupled than in skeletal muscle. Recently, we have reported that coronary arteries from hyperlipidemic swine had reduced voltage-dependent potassium (KV) currents and were less able to dilate to the metabolic agonist adenosine under conditions of metabolic challenge (N2-2DG) (8). The present study provides an initial characterization of AMPK expression in porcine vascular smooth muscle. We asked whether AMPK activity was regulated by metabolic challenge during the associated changes in smooth-muscle contractile state.


   METHODS AND PROCEDURES
TOP
 ABSTRACT
 METHODS AND PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model and Tissue Collection

Sexually mature male (19) Yucatan miniature swine, 10–12 mo old (25–40 kg) were housed two per pen in 6 x 10-ft open-fence enclosures. Animals were fed 2.5% body wt with Purina Lab Mini Pig Chow (PMI, St. Louis, MO) once/day and kept on a normal 12:12-h light-dark cycle. Pigs were housed in the facility 3–4 mo before these studies. Pigs were anesthetized with ketamine (30 mg/kg) and pentobarbital sodium (35 mg/kg) and then injected with heparin (1,000 units/kg) before being euthanized by a left thoracotomy. Left and right carotid arteries were removed quickly from the animal, immediately placed in iced 0.5 mM CaCl2 Eagle's minimum essential medium (MEME, Sigma Chemical, St. Louis, MO), and cleaned of adherent connective tissue. For physiological measures of tension, carotids were sectioned into ~1.5-mm-length rings. For biochemical measures of AMPK activity, carotids were sectioned into ~5-mm length strips. All dissections were conducted in 0.5 mM Ca2+ MEME at 4°C. In addition, sections of porcine heart and skeletal muscle (right masseter and deltoid) were removed from the animal within 10 min of death and frozen by clamping the tissues between stainless steel blocks cooled to liquid N2 temperatures. Rat hindlimb muscles were obtained from male Sprague-Dawley rats anesthetized with isoflurane and euthanized by thoracotomy. Rat muscle was rapidly removed and frozen by clamping at liquid N2 temperatures. Samples were stored at –80°C until used. Animals were treated in accordance with institutional guidelines for humane animal care and use.

Functional Measures of Carotid Artery Rings

Carotid artery rings were prepared for measures of isometric tension. Ring axial length, as well as internal and external diameter, was measured with a calibrated Filar micrometer mounted on the dissection microscope. Rings were denuded of endothelium by gently rubbing the luminal surface over the edge of a scissors. Rings denuded of endothelium exhibited <5% relaxation to bradykinin (30 µM). Isometric tension was measured using two stainless steel wires that were passed through the lumen of each ring. One wire was connected to a force transducer (FT03, Grass Astro-Med), and the other connected to a micrometer microdrive (Stoelting), which allowed for stretching of each ring by known increments. Carotid rings were submerged in a 20-ml bath containing physiological saline solution (PSS: in mM, 138 NaCl, 5 KCl, 1.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 11.2 glucose, 10 HEPES) buffered to pH 7.4 and equilibrated at 37°C with 85% O2-15% N2 gas mixture. Rings were stretched and maintained at 3-g resting tension and equilibrated for 1 h. Each ring was then set to the individual optimum of its length-developed tension relationship (Lmax) as described previously (28). At optimal stretch, carotid rings were exposed to a maximal concentration of KCl (80 mM) to determine maximal relative reactivity. Rings were then contracted with increasing concentrations of endothelin-1 (ET-1) to achieve tensions 60–80% of the maximal KCl contraction. This concentration of ET-1 resulted in a stable background contractile response on which to assess the effects of relaxants and metabolic inhibition. After attainment of a stable contraction, individual rings were exposed to either 1) PSS with 2DG (10 mM) substituted for glucose and 100% N2 gas substituted for 85% O2-15% N2, 2) PSS with 2 mM 5-aminoimidazole-4-carboxamide ribofuranoside (AICAR, Sigma Chemical) in 85% O2-15% N2, or 3) PSS with 85% O2-15% N2 as time control. AICAR is a precursor to ZMP, which in some tissues mimics the ability of AMP to activate AMPK and is hydrolyzed more slowly than AMP (36). Rings were maintained in these solutions for 30 min and then exposed to a maximal concentration of sodium nitroprusside (SNP; 0.1 mM) to determine maximal relaxation for each ring. Data of isometric tension development were continuously recorded and collected by DATAQ computerized data-acquisition system for analysis by CODAS software.

AMPK Expression and Activity

AMPK expression was assessed by immunoblot analysis and kinase activity by immunoblot analysis and SAMS peptide using freshly obtained tissue from porcine carotids.

Tissue handling.   Sections of artery (5–10 mm length) were cleaned of adventitia, slit open longitudinally, and denuded of endothelium with the rough edge of a piece of filter paper. Samples were then weighed and suspended by a stainless steel wire in a 20-ml glass physiological chamber containing PSS bubbled with 85% O2-15% N2 at 37°C. At time 0, the solution was changed to PSS containing 2DG (10 mM) substituted for glucose, and the gas perfusion was switched to 100% N2. All solutions contained ET-1 (3 nM), added 10 min earlier to simulate conditions used for functional measures. ET-1 alone had no effect on AMPK, Akt, or Erk 1/2 phosphorylation. Vessels were incubated in either control or metabolic challenge solution for 30 min and then removed and rapidly frozen by compression between metal tongs precooled in liquid N2. Tissue samples were stored at –80°C until assayed, ~1–3 mo later. In some experiments (kinetics of activation and inactivation), the time samples were exposed to metabolic challenge, which was varied (1–30 min), or samples were returned to O2-PSS for predetermined times before freezing. Incubation in AICAR (2 mM) was 30 min. Experimental protocols included two control samples: one control sample was removed from O2-PSS at the start of the experiment (after preincubation) and frozen, and the second was frozen at the completion of the experimental protocol. There were no differences between these control samples, validating stability of the reagents and samples for the duration of these experiments.

In a control set, carotid rings were prepared 3–4 mm wide and mounted for tension measurements as described above. Adjacent sections of rings were suspended from wires with no applied tension in the same chamber. Both sets of rings were denuded of endothelium by gently rubbing the luminal surface over the edge of a scissors. Each set of rings then was exposed to a standard protocol for determination of Lmax and subsequent contraction by ET-1. After development of a stable contraction, duplicate sets of rings then were exposed to either vehicle control solution, metabolic challenge, AICAR (2 mM), metformin (2 mM), or phenformin (200 µM) for 30 min. Samples were then quickly removed from the apparatus by threading a wire through the lumen of the ring under tension and sliding it directly into liquid N2. Samples not under tension were also frozen and processed for immunoblots. These samples allowed for comparison of AMPK activation in the presence and absence of smooth muscle tension.

Immunoblot assessment of expression and activity.   Activity was measured by using the [P]Thr172 AMPK antibody that recognizes the amino acid sequence around Thr172 of AMPK that is stably phosphorylated by AMPKK. Heart and skeletal muscle control samples were obtained from freshly killed miniature swine (within 10 min) and rapidly frozen in liquid N2. Frozen samples of carotid artery, heart, and skeletal muscle were pulverized in liquid N2 and then homogenized in RIPA buffer (tissue weight in g x 9 ml buffer) using a Teflon-on-glass 20-ml Dounce homogenizer. RIPA buffer contained (in mM) 150 NaCl, 50 Tris·HCl (pH 7.4), 1 EGTA, 1 PMSF, 1 Na3VO4, 1 NaF, and 1% NP-40, and 1 µg/ml each of aprotinin and pepstatin. Homogenized samples were gently rocked for 20 min at 4°C and centrifuged (14,000 g, 10 min), and the supernatant was collected. Acid-insoluble protein was then precipitated by addition of trichloroacetic acid to attain a final concentration of 7%. The removed acid-soluble component consists primarily of connective tissue proteins, which are a major component in carotid samples. After sitting at room temperature for 10 min, samples were centrifuged (14,000 g, 10 min), and the protein pellet was collected. In a subset of samples, protein for immunoblots were obtained after partial purification of carotid artery protein as described below for SAMS assay. Pellets were resolubilized in neutralizing buffer (2:1 ratio of 250 mM Tris buffer: 0.1 N NaOH containing 0.1% SDS) and stored at 4°C overnight. Samples were centrifuged (14,000 g, 10 min), an aliquot of supernatant was removed for measurement of total protein, and the remaining protein was stored frozen at –80°C. An aliquot of known protein concentration was mixed with 2x PAGE sample buffer, heated to 95–100°C for 5 min, cooled on ice, and then centrifuged (11,000 g, 2 min). Samples then were loaded onto a 10% polyacrylamide gel (7 x 8 x 0.1 cm) prepared with a 4% stacking gel and proteins separated under denaturing conditions at room temperature.

Separated proteins were transferred to nitrocellulose membranes and probed with primary antibody diluted in PBS containing 5% nonfat dry milk overnight (4°C). Incubations with anti-[P]Thr172-AMPK also included 5% bovine serum albumin. Membranes were washed with double-distilled water and exposed where appropriate to either biotinylated goat anti-rabbit (Bio-Rad Laboratories, Hercules, CA; 1:5,000) or biotinylated rabbit anti-mouse (Sigma Chemical, 1:10,000) secondary antibody in PBS-5% milk for 1 h followed by horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA, 1:100,000) for 45 min at room temperature. Labeled bands were detected by chemiluminescence according to the manufacturer's instructions (Western Lightening Chemiluminescence Kit, Perkin-Elmer Life Sciences) followed by exposure to X-ray film (Kodak Hyperfilm ECL, Amersham Pharmacia Biotech). Where appropriate, after chemiluminescence detection, blots were stripped of reaction product in stripping buffer (62.5 mM Tris·HCl, 2% SDS, 100 mM 2-mercaptoethanol, 30 min at 50°C), washed in PBS (6 x 5 min), and reprobed with another primary antibody. For determination of activity, the antibodies against [P]Thr172 were applied first, stripped, and then reprobed with antibodies directed against total protein. Effective stripping of the first reaction product by this procedure was validated for each antibody set.

Immunoblot data analysis.   Kinase activity was quantified by densitometry. Films were scanned, and density of bands converted to arbitrary densitometry units via NIH ImageJ. Activity was calculated for each blot from the density ratio of phosphorylated protein to total protein for each kinase. Densitometry was performed either as a horizontal lane of eight samples for two independent gels (phosphorylated vs. total protein) or as a vertical lane of a composite image of both gels (merged using Photoshop) for each sample. There were no significant differences in outcome between these two methods; therefore, all analyses were performed using a horizontal lane of separate gels. The ratio was then normalized within blots to the ratio obtained for control samples (O2-PSS). At least two control samples were loaded on each gel, and the control value was considered the average of control ratios. Linearity of the analysis was predetermined for each antibody by assessing several primary antibody dilutions (bracketed around the manufacturers' recommendations) with increasing protein load in each lane (5–50 µg protein/lane). Optimal protein concentration for vascular smooth muscle was determined to be 20–30 µg/lane. In many cases, samples were loaded onto gels at two different protein loads to guarantee that both the phosphorylated and nonphosphorylated protein would fall within its own range of linearity.

Immunoprecipitation.   Immunoprecipitation of {alpha}1-AMPK or {alpha}2-AMPK was performed using Protein A Sepharose CL-4B beads (Amersham Bioscience, Piscataway, NJ). Beads were reconstituted into cell lysis buffer, and a 50% slurry was incubated with an antigen-antibody mixture for 3 h at 4°C. The antigen-antibody mixture was prepared by incubating 500 µl of cell lysate with primary antibody overnight at 4°C per manufacturer instructions. The antigen-antibody-bead complex was centrifuged and washed with lysate buffer (5x). The final pellet was resuspended in sample buffer and heated to 95°C for 5 min, and proteins were separated by immunoblot procedures as described above.

Antibodies.   Primary antibodies used for these studies include anti-{alpha}-pan AMPK (Upstate, Charlottesville, VA, 1:10,000), anti-{alpha}1-AMPK (Upstate, 1:1,000), anti-{alpha}2-AMPK (Upstate, 1:500; Zymed, San Francisco, CA, 1:500; Bethyl Laboratories, Montgomery, TX, 1:1,000), anti-[P]Thr172-AMPK (Cell Signaling Technologies, Beverly, MA, 1:1,000), anti-{beta}-AMPK (Upstate, 1:500), anti-Akt1/PKB{alpha} (Upstate, 1:5,000), anti-[P]Thr308-Akt1/PKB{alpha} (Upstate, 1:500), anti-[P]Ser473-Akt1/PKB{alpha} (Upstate, 1:500), anti-Erk 1/2 (Upstate, 1:20,000), anti-[P]Erk 1/2 mouse monoclonal (Upstate, 1:2,000), and anti-GAPDH mouse monoclonal (Chemicon International, 1:2,500).

SAMS assay.   The SAMS assay was used to measure vascular smooth-muscle kinase activity directed against a synthetic peptide, SAMS peptide (HMRSAMSGLHLVKRR), corresponding to the sequence around Ser79 of rat acetyl Co-A carboxylase, an endogenous target for AMP-dependent kinases (5). Unlike the native site in acetyl-CoA carboxylase, however, SAMS peptide contains alanine rather than serine at site 77 to eliminate phosphorylation by cyclic AMP kinase. Because vascular smooth muscle samples were not immunoprecipitated before SAMS assay, the values represent total kinase activity for all AMPK isoforms and possibly other, as-yet uncharacterized members of the AMPK family (33). SAMS peptide was synthesized by the Macromolecular Structure Analysis Facility at the University of Kentucky. The addition of two arginines at the COOH-terminal promotes binding to phosphocellulose paper, which permits separation of the peptide from unreacted ATP.

AMPK was partially purified by using a modification of the method described by Winder and Hardie (37) and was provided by Dr. Winder (personal communication). Briefly, frozen carotid artery samples were pulverized to a fine powder in a stainless steel mortar cooled in liquid N2. Frozen powder was homogenized (Omni International GLH) with 3 x 3-s bursts at setting no. 6 in ice-cold homogenization buffer (volume was 9 x tissue weight) and centrifuged (Ti50, 48,000 g, 30 min, 4°C). The homogenization buffer contained (in mM) 200 mannitol, 50 NaF, 10 Tris base, 1 EDTA, 10 2-mercaptoethanol, 0.5 mg/100 ml leupeptin, aprotinin, and {alpha}1-antitrypsin, buffered to pH 7.5 with NaOH. The supernatant was collected and the AMPK fraction was isolated by ammonium sulfate precipitation (144 mg/ml, 30 min, 4°C). The precipitate containing AMPK then was collected by centrifugation (48,000 g, 30 min, 4°C) and resuspended in 10% of the original homogenization buffer. The samples were centrifuged again (48,000 g, 30 min, 4°C) to remove denatured protein, and the supernatant was aliquoted and stored at –80°C until assayed.

AMPK activity was assayed from the supernatant fraction (5 µl) in a reaction mixture containing (in mM) 40 HEPES, 0.2 SAMS peptide, 0.2 AMP, 80 NaCl2, 0.8 EDTA, 0.8 dithiothreitol, 5 MgCl2, and 0.2 ATP spiked with 8% glycerol and 2 µCi [32P]ATP, at pH 7.0 in a final volume of 25 µl. After incubation (10 min at 37°C), a 15-µl aliquot was removed and spotted on Whatman P81 phosphocellulose filter paper. Unreacted [32P]ATP was removed from the filter by repeated washing with 1% phosphoric acid and one wash with acetone. The filters then were air dried, and radioactivity was quantified in 5 ml of scintillation cocktail (Ultima Gold XR).

AMPK activity was normalized to cell protein content. After ammonium sulfate precipitation, an aliquot of sample was removed from homogenization buffer, the protein was reprecipitated twice with trichloroacetic acid (final concentration 10%) pelleted by centrifugation, and the protein pellet was solubilized in 0.1 N NaOH. Base-soluble protein was determined by the Pierce bicinchoninic acid assay with BSA as a standard.

Data Analysis

Statistical significance for functional measures and kinase activity by either immunoblot or SAMS assay was determined by ANOVA with Tukey's post hoc test for multiple samples. All data met criteria for normality and equal variance. Immunoreactivity was quantified by using NIH ImageJ, and relative density was normalized within each lane by comparison 1) with GAPDH for relative expression of kinase protein or 2) between phosphorylated and total protein bands for kinase activity.


   RESULTS
TOP
 ABSTRACT
 METHODS AND PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional Responses

Functional responses of carotid rings were evaluated under conditions used to measure AMPK activity. Contraction and relaxation were assessed for endothelium-denuded rings under normal and metabolically challenged (N2-2DG) conditions. Under the conditions used here, ET-1 evoked stable contractions that exhibited no significant decline over 30–40 min (Fig. 1). Rings incubated in the AMPK activator AICAR (2 mM) relaxed ~12% (middle trace in Fig. 1 with averaged data shown in Fig. 1B). Metformin (2 mM) and phenformin (0.2 mM), which also are reported to activate AMPK in skeletal muscle, evoked a small and inconsistent increase in contractile force above that of ET-1 (data not shown). Rings exposed to metabolic challenge consistently exhibited rapid and pronounced relaxation that only transiently reached 100% (resting tension, bottom trace, Fig. 1A). Tension was partially recovered within 30 min to 20% of maximal force (80% relaxation). At the end of each experiment, maximal relaxation was determined by exposing each ring to a maximal concentration of SNP (0.1 mM). AICAR had no effect on maximal relaxations to SNP. Rings incubated in N2-2DG were unresponsive to SNP while maintained in N2-2DG. Therefore, subsequent addition of SNP did not cause any further relaxation, and the SNP induced relaxation in N2-2DG was significantly less than that in O2-PSS. Relaxation to SNP recovered fully on reexposure to normal O2-PSS (not shown), and there were no differences in maximal SNP relaxation for any treatment once the rings were returned to O2-PSS solution.



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  		Fig. 1. Physiological response of endothelium-denuded carotid artery rings to metabolic challenge. A: representative tracings of endothelin (ET)-1 constricted carotid artery ring responses to the vasodilator, sodium nitroprusside (SNP, 0.1 mM), the AMP-activated kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-ribofuranoside (AICAR), and metabolic challenge with 2-deoxyglucose plus N2 (N2-2DG). B: means ± SE (n = 5 animals) of relaxation responses expressed relative to initial resting tension (100%) at length-developed tension (Lmax). Data points for N2-2DG and AICAR were obtained after 30-min incubation and for N2-2DG + SNP and AICAR + SNP after an additional 10-min incubation in SNP. *P < 0.05 compared with SNP alone; #P < 0.05 compared with N2-2DG treatments.

 
AMPK Expression in Vascular Smooth Muscle

Because AMPK is a highly conserved protein, we predicted that it would be present in vascular smooth muscle. To validate AMPK {alpha}-subunit protein in vascular smooth muscle, we performed immunoblots on smooth muscle samples from endothelium denuded carotid arteries. As shown in Fig. 2A, lane a, {alpha}-pan AMPK antibody, which recognizes both {alpha}1- and {alpha}2-subunits of AMPK, consistently labeled a 63-kDa protein band. This same band was also labeled by an antibody directed against a region of AMPK that is phosphorylated and activated by AMPKK ([P]Thr172-AMPK, Fig. 2A, lane b). Thus two separate and distinct antibodies recognize the {alpha}-subunit of AMPK in vascular smooth muscle. Both antibodies also labeled a second band at ~70 kDa, which represents a nonspecific labeled protein. This band was observed after exposure to the secondary antibody alone (goat anti-rabbit IgG; Fig. 2A, lanes c and e) and also when blots were reacted with primary serum for mouse anti-human GAPDH and rabbit anti-mouse secondary antibody (Fig. 2A, lane d). A significant fraction of this nonspecific 70-kDa band was lost after the ammonium sulfate precipitation procedure used for partial purification of AMPK (Fig. 2B). To validate the presence and recovery of AMPK in partially purified samples used for subsequent SAMS assay (data described below), we subjected these protein samples to SDS-PAGE and immunoblotting using the {alpha}-pan antibody and [P]Thr172-AMPK antibodies. Partially purified extracts from both control (O2-PSS) and treated (N2-2DG) carotid artery expressed similar amounts of AMPK {alpha}-subunit ({alpha}-pan AMPK) (Fig. 2B, left). The same blots (stripped and reprobed), however, showed substantially more [P]Thr172-AMPK label in N2-2DG-treated samples compared with controls (Fig. 2B, right). Included on the blots is an aliquot of control (C) sample before partial purification showing both the AMPK band and the nonspecific 70-kDa band. All further immunoblots of AMPK expression and activity were performed on extractions of proteins not subjected to the ammonium sulfate precipitation purification.



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  		Fig. 2. Immunoblot demonstration of the catalytic {alpha}-subunit of AMPK in porcine carotid artery smooth muscle. A: lanes a–e were derived from the same protein sample, and a molecular weight marker set is shown to the left of each panel. a: *Band of {alpha}-subunit of AMPK (63 kDa) detected by rabbit anti-human {alpha}-pan AMPK antibody. b: Blot from a was stripped and reprobed with [P]Thr172 AMPK antibody to identify phosphorylated {alpha}-subunits of AMPK. c: Blot from b was stripped and reprobed with only the secondary antibody (goat anti-rabbit IgG) showing labeling of only a nonspecific ~70-kDa protein. d: Immunoblot demonstrating GAPDH with mouse anti-human GAPDH (36 kDa) that also demonstrates the nonspecific ~70-kDa protein. e: Virgin blot, never exposed to primary antiserum demonstrating nonspecific ~70-kDa protein with goat anti-rabbit secondary antiserum. B: immunoblot of partially purified protein used for SAMS assay from arteries under control [C, physiological saline solution plus O2 (O2-PSS) for 30 min] and metabolic challenge (MC, N2-2DG for 30 min) conditions demonstrating equal loading and similar recoveries of AMPK using {alpha}-pan AMPK antibody (left). Crude protein from another control sample (C) demonstrates the presence of AMPK and the 70-kDa nonspecific band. Note, partial purification reduced the 70-kDa band. The same blot was stripped and reprobed with anti-[P]Thr172-AMPK showing ~5-fold increase in signal of metabolic challenged tissues. MW, molecular weight markers.

 
The {alpha}1-subunit of AMPK appears to be the predominant form expressed in vascular smooth muscle (Fig. 3A). Fresh, nonstripped immunoblots containing duplicate sets of 50, 30, and 10 µg of carotid artery protein were prepared from the same gel and probed with either {alpha}1- or {alpha}2-AMPK antibody. Even at a relatively high load (50 µg), a band corresponding to {alpha}2-AMPK was barely detectable. Attempts to identify {alpha}2-AMPK employed antibodies from three separate commercial suppliers, and although distinct bands corresponding to AMPK were observed in rat skeletal muscle, as well as porcine heart and skeletal muscle with all antibodies, carotid artery failed to exhibit {alpha}2-AMPK labeling (Fig. 3B). Thus the lack of {alpha}2-AMPK signal in porcine carotid was not likely due to species selectivity of the antibodies. Vascular smooth muscle also was probed for the noncatalytic {beta}-subunit. Both the {beta}1- and {beta}2-subunits of AMPK were expressed in carotid smooth muscle with the {beta}1-isomer expressed at relatively higher levels (Fig. 3C). Direct comparison of porcine skeletal muscle (deltoid) and vascular smooth muscle indicates that {beta}-isoform expression patterns differ considerably between these muscle types. The {beta}2-isomer was more prominent in porcine skeletal muscle.



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  		Fig. 3. Immunoblot demonstrating expression patterns for {alpha}- and {beta}-subunits of AMPK in porcine tissues. A: representative blots showing relative expression of {alpha}1- and {alpha}2-subunit at 3 different protein loads (µg). Protein from the same sample was separated on individual gels rather than 1 blot, stripped, and reprobed to maximize signal to noise. Left lane in each blot demonstrates MW. B: validation of antibody detection of {alpha}2-AMPK in porcine tissues other than carotid. Representative blot shows {alpha}2-AMPK (63 kDa) in porcine skeletal muscle (PS, deltoid), porcine myocardium (PH), and rat hindlimb skeletal muscle (RS) but not porcine carotid (PC). Blots from tissues of 3 separate animals always indicated the strongest {alpha}2-signal in porcine heart with equal protein loading. C: immunoblot of porcine skeletal muscle and carotid smooth muscle for {beta}-subunit of AMPK.

 
Immunoprecipitation of {alpha}-Isoforms from Vascular Smooth Muscle

To determine which isoform of AMPK was activated by metabolic challenge, a carotid artery was divided into two segments: one treated as control, and the other exposed to N2-2DG. Protein samples from each treatment were separated into two fractions that were then immunoprecipitated with antibodies against either {alpha}1- or {alpha}2-AMPK. Blots were then probed with either {alpha}1- or {alpha}2-AMPK antibodies, stripped, and reprobed with [P]Thr172-AMPK antibody. Immunoprecipitation of vascular smooth muscle extracts with {alpha}1-AMPK antibody consistently yielded a 63-kDa band in the pellet (Fig. 4A). Reprobing these samples with anti-[P]Thr172-AMPK demonstrated a band in samples from metabolically challenged tissues but not control samples. In contrast, immunoprecipitation with {alpha}2-AMPK antibodies failed to demonstrate a band in the pellet that corresponded to AMPK when reprobed with anti-{alpha}2-AMPK or anti-[P]Thr172-AMPK. Antibodies from three suppliers failed to precipitate {alpha}2-AMPK from carotid smooth muscle. Figure 4B shows data from immunoprecipitation of porcine carotid smooth muscle and myocardial protein with {alpha}2-AMPK antibody. Although {alpha}2-AMPK antibodies failed to precipitate a protein from vascular smooth muscle, a 63-kDa band was apparent in the pellet from porcine heart (Fig. 4B, row a). Two bands were observed near 63 kDa in the crude protein sample from porcine heart using anti-{alpha}2-AMPK from Bethyl Laboratories (Fig. 4B) but not from other suppliers. The upper band, which was not present in porcine carotid smooth muscle, appeared to precipitate from the heart extract as demonstrated by its absence from the supernatant and presence in the immunoprecipitated pellet. Importantly, the precipitated {alpha}2-isoform showed no activity in either heart or smooth muscle samples when probed with the [P]Thr172-AMPK antibody (Fig. 4B, row b). These data indicate that the predominant catalytic isoform in vascular smooth muscle is {alpha}1-AMPK.



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  		Fig. 4. Immunoprecipitation (IP) of {alpha}1-AMPK and {alpha}2-AMPK. A: representative blot (n = 3) showing results of IP of either {alpha}1-AMPK or {alpha}2-AMPK from the same carotid artery protein samples (C, O2-PSS; MC, N2-2DG). Crude protein samples were loaded at dilutions used for IP. [P]Thr172-AMPK precipitates with {alpha}1-AMPK antibodies but not {alpha}2-AMPK antibodies. B: IP of extracts from PH and PC from C and MC samples with anti-{alpha}2-AMPK. Samples of crude protein and supernatant were loaded at equal volume. IP pellet represents an aliquot of the precipitated pellet. a: Blot was probed for {alpha}2-AMPK. PH shows 2 bands near 63 kDa in crude protein, but only 1 in the supernatant after IP. Carotid samples exhibit only 1 band at 63 kDa which is not immunoprecipitated and remains in the supernatant. b: The same blot was stripped and reprobed for [P]Thr172-AMPK. IP with {alpha}2-AMPK antibodies does not precipitate an active form of AMPK as detected by [P]Thr172-AMPK-antibodies in either heart or carotid samples. The prominent band at ~56 kDa represents reaction between the horseradish peroxidase-conjugated secondary antibody and the IgG heavy chains used in the IP. Representative blots of 3 experiments. MW is for 66 kDa.

 
SAMS Assay

The SAMS assay was used to measure the enzymatic activity of vascular smooth muscle. For comparison with other studies that used the SAMS assay, we also measured AMPK activity of porcine skeletal muscle (masseter muscle) and liver in normal glucose PSS. Liver contained the greatest basal AMPK activity (1.9 nmol phosphate incorporated·min–1·mg–1 protein), and skeletal muscle also contained considerable activity (0.3 nmol phosphate·min–1·mg–1). These values are essentially identical to those reported by Davies et al. (5) using the SAMS assay. Basal AMPK activity of carotid arteries in PSS was 0.04 ± 0.01 nmol phosphate·min–1·mg–1 (n = 7), which was considerably less than that of either liver or skeletal muscle. Treatment with either ET-1 or ET-1 plus AICAR had no effect on AMPK activity of carotid arteries (Fig. 5). However, AMPK activity increased more than twofold after 30-min incubation in ET-1 plus N2-2DG.



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  		Fig. 5. AMPK activity of carotid artery smooth muscle. AMPK activity was measured by the SAMS assay for samples in PSS, PSS with 10–8 M ET-1, PSS containing 10 mM 2-DG substituted for glucose and bubbled with 100% N2 (N2-2DG), and PSS containing 2 mM AICAR and bubbled with O2 (AICAR). Data represent means ± SE for n = 7 pigs with each pig contributing 1 sample per measurement. *Significantly different at P < 0.05.

 
Influence of Metabolic Challenge on Other Kinases

To determine whether phosphorylation of kinases other than AMPK was signaled by metabolic challenge, we probed for phosphorylation of Akt and Erk 1/Erk 2 kinases present in vascular smooth muscle and implicated in the regulation of contractile state. In contrast to the increased phosphorylation levels of AMPK, metabolic challenge consistently decreased phosphorylation of Akt and Erk 1/Erk 2 (Fig. 6) with no change in absolute levels of either kinase. Phosphorylation of Akt at Ser473 was present under basal conditions and unaffected by ET-1. Phosphorylation of Akt at Thr308 was not observed under any condition (data not shown). The decrease in phosphorylation of Ser473 of Akt was comparable to the decrease seen with the Akt inhibitor LY-294002 (10 µM, data not shown). The direct activators of AMPK, AICAR (2 mM), metformin (2 mM), and phenformin (0.2 mM), failed to consistently increase AMPK activity in carotid preparations. Lack of response was seen even in rings exposed to stretch identical to that used for functional measures (Fig. 7), although metabolic challenge significantly increased AMPK activity. The increase in activity due to metabolic challenge also was not altered by stretch.



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  		Fig. 6. Specificity of metabolic challenge for AMPK. Representative blots (right) demonstrating increased [P]Thr172 AMPK label in samples treated with N2-2DG, but not ET-1 alone. N2-2DG also caused a significant reduction in basal phosphorylation of Ser473-Akt and Erk 1 (left panel, means ± SE, n = 4 animals).

 


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  		Fig. 7. Effect of tension and AMPK agonists on AMPK activation. Duplicate carotid samples from each of 2 pigs were either stretched as for physiological measures of tension (Stretch) or suspended without tension (Susp) in the same tissue bath. Samples experienced repeated exposures to KCl, were constricted with ET-1, and then were treated with N2-2DG (MC), AICAR (2 mM), metformin (Met; 2 mM), or phenformin (Phen; 0.2 mM) for 30 min. Cont, control. There were no differences in response to metabolic challenge or agonist between stretched or suspended samples.

 
Kinetics of AMPK Phosphorylation

Using the [P]Thr172-AMPK antibody, we assessed phosphorylation and dephosphorylation kinetics of AMPK associated with metabolic challenge with N2-2DG. Segments of carotid artery were preincubated in O2-PSS for 30 min and then transferred to either fresh O2-PSS for 1 or 30 min or N2-2DG for variable time periods (0.5–30 min) before freeze-clamp. Increases in AMPK phosphorylation could be detected within 1 min of metabolic challenge and were significantly increased by 10 min (Fig. 8A). Levels continued to increase over the next 20 min to a level about threefold greater than controls. AMPK phosphorylation also was readily reversible in vascular smooth muscle (Fig. 8B). In another set of samples, paired vascular segments were exposed to metabolic challenge with N2-2DG for 10 min (~t, where t is time to half-maximal response). One sample from each set was then returned to O2-PSS for 1, 5, or 10 min, while the paired sample remained under metabolic challenge for the same time period. Within 1 min of return to O2-PSS, AMPK phosphorylation was reduced ~50% but did not reach statistical significance. AMPK activity returned to near baseline after a 5-min recovery period and was undetectable by 10 min of recovery, indicating that AMPK responds rapidly to changing metabolic state in vascular smooth muscle.



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  		Fig. 8. Time course of phosphorylation and dephosphorylation {alpha}-AMPK by N2-2DG in porcine carotid artery smooth muscle. A: time course of phosphorylation of AMPK by metabolic challenge. Top shows representative immunoblot. Numbers above each lane represent time (in min) of N2-2DG exposure. C, control samples in O2-PSS for 1 min (first lane) or 30 min (last lane). B: time course of dephosphorylation of AMPK. Top shows representative immunoblot. Numbers represent time (in min) samples were exposed to N2-2DG. Numbers preceded by "w" indicate wash-time that samples were exposed to O2-PSS after the 10 min of metabolic challenge. Data are means ± SE from duplicate samples taken from 4 animals. *Significantly different from corresponding nonwashout time point. {dagger}Significantly different from control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The Snf1/AMP-activated protein kinase family represents an important metabolic regulatory component that coordinates cellular catabolic and anabolic pathways to ensure adequate energy supply to meet cellular needs. Metabolic stress evokes a pronounced increase in blood flow mediated by relaxation of vascular smooth muscle to ensure delivery of adequate substrates to targeted tissues. The effect of metabolic stress on vascular smooth muscle, however, is largely overlooked because contraction is slow and cellular metabolic needs rarely exceed supply. However, data presented here demonstrate that metabolic challenge of porcine carotid smooth muscle rapidly activates AMPK. Increased activity was independent of the presence of the vasoconstrictor endothelin, the presence of endothelium, or the contractile state. Increased AMPK activity could be detected by both the SAMS assay and by increased stable phosphorylation of Thr172 of the catalytic {alpha}-subunit of AMPK. AMPK activity increased within 1 min of exposure to metabolic challenge and showed significant activity decline (t ~10 min) on return to metabolically supporting solutions. Agents reported to activate AMPK in skeletal muscle and liver (AICAR, metformin, and phenformin) had no effect on AMPK activity in carotid arteries. The predominant catalytic isomer expressed in porcine carotid smooth muscle was {alpha}1-AMPK with little, if any, {alpha}2-AMPK. Immunoprecipitation with either {alpha}1- or {alpha}2-AMPK antibodies revealed that only the {alpha}1-AMPK isoform was activated by metabolic challenge. These data are the first characterization of AMPK in vascular smooth muscle and demonstrate activation and inactivation kinetics controlled by metabolic challenge.

Unlike striated muscles in which AMPK activity is increased with contractile activity, contraction of vascular smooth muscle by the receptor-dependent agonist endothelin had no measurable effect on AMPK activity. Energetic regulation of smooth-muscle contraction has been extensively studied (see Ref. 14 for review), and contraction is known to increase O2 utilization with little or no change in either phosphocreatine or ATP concentrations (18, 39). Most likely, in this slowly contracting smooth muscle, energy demands for contraction are met by increased synthesis of ATP with little accumulation of AMP. Also, ET-1 regulates the contractile state via second-messenger systems that involve several stable phosphorylation steps that may not tax the energy state of the cell sufficient to activate AMPK. However, we cannot eliminate the possibility that the sensitivity for AMPK detection is not adequate or that AMPK activation occurred at time points other than those measured here.

AMPK activity of vascular smooth muscle also was unchanged by treatment with AICAR, metformin, or phenformin, agents shown to activate AMPK in striated muscle. AMPK of endothelial cells also was unaffected by metformin but was activated by AICAR (4, 23). It is unclear whether cell-dependent variations in response to AMPK agonists result from differences in subunit composition of the AMPK complex, differences in upstream regulatory signals, or an inability of the agonist to reach its target. Both vascular smooth-muscle and endothelial cells express primarily {alpha}1-AMPK. Although we show here that vascular smooth muscle also expresses predominantly {beta}1-AMPK, it is unclear from this work what the relative association is between {alpha}1-AMPK and either of the {beta}-subunits. We also have not evaluated expression patterns of {gamma}-subunits, which, via multiple CBS domains (31), are known to regulate the AMP sensitivity and energy-sensing ability of AMPK. The AMP dependence varies considerably with {gamma}-subunit expression, with {gamma}2 being more AMP dependent than {gamma}1, although most tissues other than brain express predominantly the {gamma}1-isoform. It is not clear whether {gamma}-subunits also participate in sensitivity to AICAR and its product ZMP. In endothelial cells, AICAR increased the activity of AMPK and PKB and stimulated eNOS phosphorylation. However, when endothelial cells were transfected with a dominant negative AMPK, AICAR's effects on AMPK and endothelial nitric oxide synthase phosphorylation were reduced, but PKB responses were unchanged, suggesting AICAR itself may have effects independent of AMPK activation under some conditions (23).

When subjected to a metabolic challenge (N2-2DG), however, AMPK activity increased significantly as measured by increased phosphorylation of Thr172 of the {alpha}-subunit and by incorporation of 32P into SAMS peptide. Although we did not immunoprecipitate {alpha}-subunit isoforms before the SAMS assay, the kinase activity measured by the SAMS assay in these samples is unlikely the result of {alpha}2-AMPK for reasons discussed above. Basal AMPK activity measured by the SAMS assay was 0.034 nmol·min–1·mg protein–1, within the range reported for {alpha}1-AMPK of rat striated muscles (0.05 nmol·min–1·mg–1; Ref. 7) but considerably less than that reported for cultured endothelial cells (~0.3 nmol·min–1·mg–1; Ref. 23), which also express primarily {alpha}1-AMPK. After metabolic challenge, AMPK activity increased threefold, similar to the changes reported for endothelial cells (23). Thus the basal and activated levels of vascular smooth-muscle AMPK are consistent with activity levels reported for other tissues and species. Although we attribute the kinase activity demonstrated in the SAMS assay to {alpha}1-AMPK, we cannot eliminate the possibility that some of this activity results from other, as-yet uncharacterized members of the AMPK family in vascular smooth muscle.

AMPK activity increased by metabolic challenge was associated with a rapid and highly significant decline in endothelin-evoked contractile force. The decline in force occurred within 60 s and was near maximal levels by 5 min of metabolic challenge. AMPK phosphorylation increased to measurable levels within 60 s of metabolic challenge and was near maximal by 30 min. Importantly, in these preparations, AMPK activation rapidly declined on return to normal O2-PSS. These changes occurred at time points when consistent changes in high-energy phosphates were not detected (11). This is surprising and may be due to small localized changes in AMP that could not be detected. However, in skeletal muscle, it was determined that the {alpha}1-AMPK isoform is considerably less sensitive to AMP than is the {alpha}2-AMPK isoform (30). Alternatively, rapid activation of AMPK may be regulated by upstream signaling events that are more sensitive to cellular metabolic state, as suggested for AMPKK and CaMKK (30). Thus rapid increases in cellular Ca2+ may play a role in AMPK activation, but upstream regulation was not examined in the present study. Recent reports suggest that LKB1 tumor suppresser protein kinase is an authentic AMPKK. LKB1 phosphorylates AMPK at Thr172 in yeast and mammalian fibroblasts, and LKB1-deficient cells fail to activate AMPK (15, 32). Whether LKB1 represents an obligatory step in AMPK activation, however, remains to be determined, as Sakamoto and colleagues (29) recently described activation of AMPK-{alpha}2 with no change in LKB1 activity in skeletal muscle after contraction, AICAR, and phenformin stimulation. The role of LKB1 in vascular smooth-muscle AMPK activity is unknown, although LKB1 deficiency is lethal in embryonic mice because of a failure in vascular development (38).

The time course for AMPK activation in carotid smooth muscle suggests that there may be both a rapid and a slow component of activation. It is unclear from the present data whether the rapid component reflects activation processes not directly dependent on AMP, whereas the slow process may be AMP dependent. Interestingly, recent studies of {alpha}2-AMPK activity in cultured vascular endothelial cells indicate that the rapid activation of AMPK by ONOO– occurs independent of changes in cellular high-energy phosphates, particularly AMP (40). Chronic activation of AMPK (>12 h) by hypoxia, however, was required to initiate angiogenic changes (26). The rapid decline of AMPK phosphorylation on return to PSS suggests that this enzyme also is tightly regulated by a protein phosphatase. Protein phosphatases 2A and 2C dephosphorylate and inactivate AMPK in vitro (30); however, the cellular form in vascular smooth muscle and its regulation is unknown.

Although AMPK is traditionally considered a major regulator of cellular anabolic and catabolic pathways that conserve and synthesize ATP, multiple nonmetabolic cellular targets have been identified, including endothelial nitric oxide synthase, cystic fibrosis transmembrane regulator, transcription factors, and components of second-messenger signaling pathways (3, 9). Associated with activation of AMPK by metabolic challenge in porcine carotid smooth muscle was a reduction in the steady-state phosphorylation of Akt and Erk 1/Erk 2. Activation of Erk/MAPK pathways has been controversial concerning its role in regulation of smooth-muscle contraction via thin-filament regulation (22). Also, recent evidence implicates phosphatidylinositol 3 (PI3)-kinase and its substrate Akt in both calcium-dependent and calcium-independent regulation of smooth-muscle force (16, 35). In the study by Su and colleagues (35), inhibition of the PI3-kinase pathway, measured as decreased phosphorylation of Akt, also inhibited agonist and depolarization-induced force development of porcine carotid media. Interestingly, CaMKK is considered a weak or alternate AMPKK to phosphorylate AMPK (13), and AMPK in turn phosphorylates myosin light-chain kinase in vitro (20). Thus considerable indirect data suggest that AMPK may be involved in smooth-muscle contractile regulation. It is unclear from the present data, however, whether AMPK is upstream or downstream of Akt or ERK 1/2. Cross talk between AMPK and Akt has been demonstrated for several cell systems, including endothelial cells (27) and the myocardium (17). In endothelial cells, it appears AMPK and Akt both are activated, whereas, in the myocardium, Akt acts as a negative regulator of AMPK activity. Thus it is possible that AMPK of vascular smooth muscle is freed from inhibition by Akt during metabolic challenge. Clearly, additional studies are necessary to determine the role of Akt in metabolic challenge and vascular smooth-muscle relaxation, possibly employing knockout models of either AMPK or Akt. Data from the present study, however, demonstrate that AMPK is activated over the time course of metabolic relaxation that activity results primarily from {alpha}1-AMPK and that metabolic challenge is associated with inhibition of unique signaling pathways (PI3-kinase/Akt) that may regulate smooth-muscle contraction. Further studies to elucidate the mechanism of AMPK activation in smooth muscle and its integration with other signaling pathways will be critical in understanding the complex kinase pathways known to regulate smooth-muscle contraction and relaxation processes.


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 ABSTRACT
 METHODS AND PROCEDURES
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REFERENCES
 
This work was supported by a grant from National Aeronautics and Space Administration and by National Heart Lung and Blood Institute Grants HL-52490 and HL-15852.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. Rubin, Dept. of Biomedical Sciences, Univ. of Missouri, Columbia, MO 65211 (RubinL{at}missouri.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.


   REFERENCES
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 ABSTRACT
 METHODS AND PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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