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Am J Physiol Heart Circ Physiol 287: H2606-H2611, 2004. First published August 26, 2004; doi:10.1152/ajpheart.00600.2004
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Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS

Thomas Krieg,1 Qining Qin,1 Sebastian Philipp,1 Mikhail F. Alexeyev,2 Michael V. Cohen,1,3 and James M. Downey1

Departments of 1Physiology, 2Pharmacology, and 3Medicine, University of South Alabama, College of Medicine, Mobile, Alabama 36688

Submitted 16 June 2004 ; accepted in final form 5 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the rabbit heart, bradykinin and ACh trigger preconditioning by a mechanism involving ATP-sensitive potassium channel-dependent production of reactive oxygen species (ROS). Recent evidence indicates that the pathway by which bradykinin causes ROS generation includes nitric oxide synthase (NOS) and protein kinase G (PKG). On the other hand, Akt was shown to be essential for ACh to generate ROS. This study determines whether these two G-coupled receptor agonists indeed have similar signaling targets, i.e., whether Akt is involved in bradykinin's pathway and whether NOS is involved in ACh's pathway. Isolated adult rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 µM) caused a 51.4 ± 14.8% and 39.8 ± 11.7% increase, respectively, in ROS production (P < 0.005). Coincubation of either agonist with Akt inhibitor (20 µM) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP, 1 µM) also increased the ROS signal by 40.8 ± 15.7%, but this increase was unaffected by Akt inhibitor (39.0 ± 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors N{omega}-monomethyl-L-arginine monoacetate (100 µM) and L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO, 5 µM). L-NIO also blocked the anti-infarct effect of ACh (550 µM) in isolated rabbit hearts exposed to 30 min of regional ischemia. We conclude that both bradykinin and ACh trigger ROS generation by sequentially activating Akt and NOS.

nitric oxide; N{omega}-monomethyl-L-arginine monoacetate; L-N5-(1-iminoethyl)ornithine; S-nitroso-N-acetyl penicillamine

THE CARDIOPROTECTIVE EFFECTS of ischemic preconditioning in cardiomyocytes are triggered by the binding of several Gi-coupled surface receptors including adenosine A1/A3, bradykinin (BK) B2, and opioid {delta}-receptors by their respective agonists (26). The latter two receptors are thought to trigger the preconditioned state through a complex pathway that includes activation of phosphatidylinositol 3-kinase (PI3-kinase) (17, 21, 23), opening of mitochondrial ATP-sensitive potassium channels (7), and production of reactive oxygen species (ROS) (2, 7, 24). Much of our work on this pathway has examined effects of the well-characterized muscarinic receptor. Although its agonist acetylcholine (ACh) is not released during ischemic preconditioning, the muscarinic receptor typifies Gi-coupled receptors and its activation mimics ischemic preconditioning. Protection is dependent on all of the above elements (7) including PI3-kinase (21). PI3-kinase activates multiple downstream pathways through phospholipid-dependent kinases (PDK)1 and PDK2. Among those downstream targets is Akt, also known as PKB.

ACh clearly leads to phosphorylation of Akt in the rabbit heart (14). The only available inhibitor of Akt, sold by Calbiochem (La Jolla, CA) as "Akt inhibitor," unfortunately can also inhibit PI3-kinase in a nonspecific manner (10). This makes Akt inhibitor less than ideal for testing whether Akt actually carries the signal for preconditioning or whether its phosphorylation is simply an epiphenomenon in response to the augmented PI3-kinase activity. To overcome this problem we (13) used genetic manipulation to attenuate Akt's activity in a cell model in which ROS production in response to ACh treatment was monitored. However, because we were using a lipid transfection system, we had to limit our experiments to A7r5 rat vascular smooth muscle cells as transfection efficiency was found to be poor in adult rabbit cardiomyocytes. Expression of the dominant-negative (dn)Akt construct blocked ACh-stimulated ROS production in the transfected cells and provided evidence for Akt's involvement. The obvious shortcoming of that study, however, was the failure to observe these changes in cardiomyocytes. In the present study we circumvented this limitation by designing an adenovirus vector that could efficiently infect cardiomyocytes. Thus we are now finally able to test whether Akt is a required link in the pathway by which an agonist, either ACh or BK, triggers ROS formation in adult rabbit cardiomyocytes.

Three isoforms of Akt have been identified, Akt1, -2, and -3 (PKB-{alpha}, -{beta}, and -{gamma}), and it is still not fully known which isoforms are activated under various physiological conditions (16). In the present study, we targeted Akt1, which is known to be expressed at high levels in the heart and is activated through the hierarchical phosphorylation of both Thr308 and Ser473. In cardiomyocytes infected with a viral genome primed to produce copies of inactive Akt, any authentic Akt would be so diluted that a signal attempting to phosphorylate and activate Akt would be effectively extinguished, thus blocking further signal transduction.

Additionally, we probed whether activation of nitric oxide (NO) synthase (NOS) is unique to BK's signaling pathway leading to protection or whether NOS is more ubiquitously involved in the protection of other Gi-coupled agonists. We had previously found (18) that a NOS blocker, N{omega}-nitro-L-arginine methyl ester (L-NAME), could block BK's protective effect, thus implicating NO production as a critical link in this pathway. Because endothelial NOS (eNOS) is a known downstream target of Akt, it seemed reasonable to speculate that PI3-kinase activated Akt, which would in turn stimulate eNOS. If that were true, then protection from ACh should also be blocked with a NOS antagonist. Unfortunately, L-NAME also has muscarinic receptor-blocking effects, thus making it an inappropriate tool for this study. In the present investigation we tested whether L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO) or N{omega}-monomethyl-L-arginine monoacetate (L-NMMA), potent NOS antagonists that do not affect muscarinic receptors, could block ACh's protective signal.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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This study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996).

Adult rabbit myocytes. Rabbit ventricular myocytes were isolated as described previously in detail (1). Briefly, hearts of New Zealand White rabbits were excised and retrogradely perfused with calcium-free Krebs-Henseleit-HEPES buffer containing collagenase (type 2, 200 U/ml; Worthington, Lakewood, NJ) at 37°C. Viable myocytes were separated by repetitive slow-speed centrifugation and made calcium tolerant by stepwise restoration of calcium in the medium to 1.25 mM. Usually, 30–35 million viable, calcium-tolerant cells were extracted per heart.

Immediately after the isolation and separation procedure, cells were plated on laminin-coated 24-well plates (Becton Dickinson, Bedford, MA) with creatine (5 mM)-, L-carnitine (2 mM)-, and taurine (5 mM)-supplemented medium 199 (CCT-medium 199) as described by Piper et al. (20) and Mitcheson et al. (15). Penicillin (100 U/ml) and streptomycin (100 µg/ml) were added as antibiotics. Cells were stored in incubators at 37°C in air enriched with 5% CO2. Cells were left undisturbed and allowed to equilibrate for at least 18 h.

Experimental design. Each experiment started with a change of medium for 10 min. The medium was then removed and replaced with medium containing the drug or the drug plus blocker (if required) and reduced MitoTracker red (1 µM) as a dye. The reduced form of the probe is nonfluorescent. When the probe is oxidized by ROS, it then becomes fluorescent. The oxidized product is bound to thiol groups and proteins within the mitochondria. After incubation with MitoTracker for 15 min, cells were washed twice with fresh MitoTracker-free CCT-medium 199. The wash serves to remove the unbound and thus voltage-dependent pool of dye held in the cells. After cells are washed, fluorescence becomes stable for at least 30 min.

Experiments were performed in which the effect of the blocker 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor, 20 µM), L-NMMA (100 µM), or L-NIO (5 µM) on ROS production in response to ACh (250 µM), S-nitroso-N-acetyl-penicillamine (SNAP, 1 µM), or BK (400 nM) was measured. The blocker was present in the medium during the 10-min period before the addition of MitoTracker and the agonist. Previous investigations documented that the described protocol of a timed incubation followed by washing permits reliable measurement of ROS generation and minimizes any possible influence of changing mitochondrial transmembrane potential (12, 19).

Virus infection. An adenovirus encoding hemagglutinin (HA)-tagged dnAkt and coexpressing enhanced green fluorescent protein (EGFP) was used. The dnAkt plasmid was described previously (13). Briefly, in this NH2-terminal HA-tagged mutant of Akt1, the two major regulatory phosphorylation sites (Thr308 and Ser473) and the phosphate transfer residue in the catalytic site (Lys179) were replaced by alanine residues rendering the protein inactive. Recombinant adenoviruses were generated according to established protocols using a set of commercially available plasmids (AdMax; Microbix, Toronto, Ontario, Canada). The shuttle plasmid pDC512 was modified to incorporate a cytomegalovirus promoter, followed by dnAkt, an internal ribosome entry site of the encephalomyocarditis virus, and then the EGFP gene. Recombinant adenovirus was generated by cotransfecting HEK-293 cells (Microbix) with shuttle plasmid and pBHGfrt(del)E1,3FLP genomic plasmid and purified. Virus infection was performed by incubating cultured cardiomyocytes with 1 x 107 plaque-forming units/ml for 24 h. There was a >95% infection rate, as documented by fluorescent green, rod-shaped myocytes expressing EGFP. To further prove successful infection with the HA-tagged dnAkt, a Western blot analysis after electrophoresis on an SDS gel was performed with an anti-HA primary antibody (Sigma, St. Louis, MO). Appropriate control experiments were conducted. The effect of infection by the dnAkt mutant on ROS production by unstimulated cardiomyocytes was evaluated. We also studied the influence of infection with an "empty vector" containing the EGFP gene without the dnAkt plasmid on unstimulated cardiomyocytes and on those exposed to ACh.

Measurement of ROS production. Experiments were designed such that four different conditions were always simultaneously evaluated. Mitochondrial ROS generation was analyzed by measuring the fluorescence of at least 40 individual rod-shaped cells that were randomly selected within each well. The average fluorescence for the selected cells in each well was computed and compared with the average single-cell fluorescence in the respective control well in the same chamber. Thus the treated cells were only compared with untreated cells of the same age and isolation and stained with the same MitoTracker lot. Single-cell fluorescence was quantified as described previously (12). Each set of experiments was repeated five to eight times on different days with cells of different ages. Approximately 200–400 typical rod-shaped cells contributed data for each experiment. In experiments with infected cells, measurements were performed only in fluorescent green cells expressing EGFP.

Isolated heart model. As previously described (7), a 2-0 silk suture was passed around a branch of the left coronary artery of New Zealand White rabbits to form a snare by passing the ends of the thread through a small vinyl tube. The heart was rapidly excised, mounted on a Langendorff apparatus by the aortic root, and perfused with oxygenated, warmed Krebs buffer. Perfusion pressure was set at 75 mmHg by adjusting the height of the reservoir. A fluid-filled latex balloon was inserted into the left ventricle and inflated to set an end-diastolic pressure of 5 mmHg. All hearts were allowed to equilibrate for 30 min before the protocols were started.

For the infarct studies, four groups of hearts were studied. All hearts were subjected to 30 min of regional ischemia and then reperfused for 2 h. Control hearts received no treatment. A second group of hearts was treated with ACh (0.55 mM) for 5 min, followed by 10 min of washout before the long ischemia. A third group was also treated with ACh, and, in addition, L-NIO (5 µM) was added to the perfusate for 15 min starting 5 min before and ending 5 min after ACh treatment. This protocol allowed 5 min of washout before coronary occlusion. Although we used L-NMMA to inhibit NOS in the cardiomyocytes, we found it too expensive to use in the isolated heart model. L-NIO is much less expensive and also does not affect muscarinic receptors. A fourth group was treated with L-NIO alone. Hearts treated with ACh were electrically paced until the onset of ischemia to prevent slowing of the heart.

Infarct size measurement. As previously detailed (7), the risk zone was delineated with 2- to 9-µm-diameter green fluorescent microspheres (Duke Scientific, Palo Alto, CA) and infarction with triphenyltetrazolium chloride staining. The areas of infarct and risk zone were determined by planimetry of each slice, and volumes were calculated by multiplying each area by slice thickness and summing them for each heart. Infarct size is expressed as a percentage of the risk zone.

Chemicals. All drugs required for cell isolation and culture were purchased from Sigma. Reduced MitoTracker red was purchased from Molecular Probes (Eugene, OR). Akt inhibitor was from Calbiochem, and ACh, BK, and anti-HA antibody were from Sigma. L-NIO and L-NMMA were obtained from Tocris Cookson (Ellisville, Mo). Horseradish peroxidase-linked anti-rabbit IgG antibody, cell lysis buffer, and Lumi GLO were purchased from Cell Signaling Technology (Beverly, MA). Either distilled water or DMSO was used to dissolve the drugs and to prepare stock solutions. The final DMSO concentration was kept below 1%.

Data analysis. Fluorescence measurements provide data in arbitrary units. To remove the variability caused by different MitoTracker lots, cell age, and environmental conditions, average cell fluorescence was calculated and compared with that of simultaneously studied control cells as described above. Therefore, fluorescence data are provided as a percentage of the respective control (means ± SE). To further minimize the possible influence of these variables on the data, ANOVA for repeated measures with Tukey's post hoc test was used to evaluate differences in mean fluorescence of the groups within the same experiment. Baseline hemodynamic variables and risk zone and infarct size data among groups were compared by one-way ANOVA with Tukey's post hoc test. A value of P < 0.05 was considered significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Infection with dnAkt. Infection of the myocytes with adenovirus containing dnAkt and EGFP resulted in an infection rate of >95% as evaluated by counting the fluorescent green rod-shaped cells expressing EGFP. Furthermore, Fig. 1 presents a Western blot probed with anti-HA antibody. A prominent band at ~60 kDa corresponding to the HA-tagged dnAkt's expected molecular mass was seen only in the infected cells.



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  		Fig. 1. Representative immunoblot for anti-hemagglutinin (HA) antibody in untreated cells (WT) and cells infected with adenovirus (inf) containing dominant-negative (dn) HA-tagged Akt plasmid. Note the appearance of a prominent band at the expected molecular mass of 60 kDa only in the infected cells.

 
Inhibition of Akt. Exposing cardiomyocytes to ACh or BK led to a robust and highly significant increase in fluorescence and thus ROS production in normal cells [39.8 ± 11.7% (P = 0.002) and 51.4 ± 14.8% (P < 0.001), respectively], whereas the responses to these receptor agonists were totally abolished in virus-infected cells expressing dnAkt. The infection process with the dnAkt virus itself had no effect on ROS generation (Fig. 2). In addition, infection with an empty virus containing only the EGFP gene without the dnAkt construct had no effect on unstimulated cardiomyocytes and did not block ACh's effect. A pharmacological inhibitor of Akt also blocked the ACh-induced increase of ROS (Fig. 3). Although this pharmacological inhibitor is a potent antagonist of the Akt-PI3-kinase pathway, the use of a molecular approach to block Akt was necessary because the Akt inhibitor we used is known to also have nonspecific inhibitory effects on PI3-kinase that would thus preclude precise determination of the site at which the antagonist was interfering with signaling.



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  		Fig. 2. Reactive oxygen species (ROS) production as measured by cell fluorescence (means ± SE) expressed as % of that of simultaneously studied untreated control myocytes. Treatment of myocytes with both ACh and bradykinin (BK) increased ROS generation in normal, uninfected cells compared with the baseline fluorescence seen in untreated cells. Adenovirus infection (inf) with dnAkt totally abolished ACh- and BK-induced ROS generation (ACh-inf and BK-inf; P = not significant vs. control). Fluorescence was not affected in unstimulated, infected cells. Adenovirus infection with an empty virus containing only the enhanced green fluorescent protein gene and not the dnAkt plasmid did not affect the stimulatory effect of ACh on ROS production. Furthermore, this empty vector had no effect in unstimulated cardiomyocytes.

 


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  		Fig. 3. ACh leads to a significant increase in ROS generation in adult rabbit ventricular myocytes. Cotreatment with Akt inhibitor completely abolished this increase, whereas the inhibitor alone had no effect. Data are presented as change in cell fluorescence (means ± SE) expressed as % of that of simultaneously studied untreated control myocytes.

 
NOS blockade. It is known that Akt phosphorylates and therefore activates eNOS. We tested the involvement of NOS with the selective inhibitors L-NMMA and L-NIO in isolated myocytes, using ROS production as an end point. Coincubation with either L-NMMA or L-NIO prevented ACh-induced ROS generation, whereas the blockers themselves had no effect (Fig. 4). The NO donor SNAP (1 µM) increased the ROS signal by 40.8 ± 15.7% (P = 0.019), but this increase was unaffected by Akt inhibitor (39.0 ± 6.4%; P = 0.014), indicating that activation of Akt occurs upstream of NOS activation (Fig. 5).



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  		Fig. 4. Both of the nitric oxide (NO) synthase (NOS) inhibitors N{omega}-monomethyl-L-arginine monoacetate (L-NMMA) and L-N5-(1-iminoethyl)ornithine dihydrochloride (L-NIO) abolished the increase in ROS generation stimulated by ACh in adult rabbit cardiomyocytes. Neither antagonist alone had any effect. Data are presented as change in cell fluorescence (means ± SE) expressed as % of that of simultaneously studied untreated control myocytes.

 


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  		Fig. 5. Exposure of adult rabbit cardiomyocytes to the NO donor S-nitroso-N-acetyl penicillamine (SNAP) increased ROS generation, whereas coincubation with Akt inhibitor did not affect this increase, supporting a role for Akt activation upstream of NOS. Akt inhibitor alone had no influence. Data are presented as change in cell fluorescence (means ± SE) expressed as % of that of simultaneously studied untreated control myocytes.

 
To confirm the above observation in the whole heart model, ACh was administered in the presence of the selective NOS blocker L-NIO (22). There were no differences in baseline heart rate, left ventricular developed pressure, or coronary flow among the groups (Table 1). Infusion of L-NIO did not change hemodynamics. Average risk zone volumes were comparable in all groups (Table 2). Infarct size as a percentage of the region at risk was 30.5 ± 2.9% in the control group. As expected, the brief infusion of ACh before ischemia significantly reduced infarction to 9.3 ± 3.5% (P < 0.001 vs. control; Fig. 6). Coadministration of the NOS blocker L-NIO blocked ACh's cardioprotection (29.6 ± 3.3% infarction), whereas L-NIO alone had no effect. Risk zone volume was plotted against infarct size for all groups to ensure that risk zone volume was not having an independent effect on infarct size (Fig. 7). The regression line for the group exposed to ACh was shifted downwards, and this line was significantly different from the others by analysis of covariance, suggesting that cardioprotection was indeed the cause of the smaller infarcts.


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  		Table 1. Hemodynamic data

 

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  		Table 2. Infarct size data

 


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  		Fig. 6. Effect of ACh and the NOS inhibitor L-NIO on infarct size expressed as % of the risk zone. {circ}, Individual experimental points; {bullet}, means ± SE. Pretreatment with 5 min of ACh before the long ischemia was protective. However, bracketing the infusion of ACh with L-NIO blocked protection. L-NIO alone had no effect on infarct size. *P < 0.002 vs. other groups.

 


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  		Fig. 7. Plot of infarct size against risk zone volume for all 4 infarct groups to ensure that risk zone volume was not having an independent effect on infarct size. The regression line for the group exposed to ACh was shifted downward, and this line was significantly different from the regression lines for the control group, hearts treated with ACh in the presence of the NOS inhibitor L-NIO, and hearts exposed to L-NIO alone, suggesting that cardioprotection was indeed the cause of the smaller infarcts.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we found that expression of a dominant-negative form of Akt1 in rabbit cardiomyocytes blocks the ability of either ACh or BK to trigger ROS formation. These data provide the strongest evidence yet that activation of Akt is a necessary event in the signaling by which ACh or BK triggers the preconditioned state. We have also shown that the NOS blocker L-NIO selectively blocks ACh's protection in isolated rabbit hearts. Because a pharmacological blocker of Akt did not attenuate the increased ROS generation induced by a direct NO donor, Akt must reside upstream from NOS. Akt has a number of phosphorylation targets, including eNOS. It seems likely that Akt directly phosphorylates eNOS in this pathway.

Yao and Gross (25) tested for the involvement of NOS in the anti-infarct effect of ACh in the canine heart. In that study, the protection from ACh was blocked by L-NAME but not L-NMMA. They concluded that NOS was not involved in ACh's protective mechanism and that L-NAME had merely acted as a muscarinic blocker. There has been considerable controversy as to whether L-NAME blocks muscarinic receptors. In 1993, Buxton et al. (4) reported that L-NAME displayed atropine-like activity in several tests including ACh-mediated contraction of rabbit colon smooth muscle with an IC50 value of ~10 µM. Binding studies revealed an affinity of muscarinic receptors for L-NAME (9). However, functional studies failed to detect an atropine-like effect (3, 6, 9). Koss (11) was unable to inhibit nonendothelial cholinergic responses with L-NAME in vascular smooth muscle. Also, L-NAME failed to displace scopolamine from spinal cord membranes (8). On the other hand, in vivo experiments on the microcirculation of the rat diaphragm support a possible antimuscarinic effect of L-NAME (5). Thus it remains unresolved whether L-NAME actually has muscarinic blocking properties.

Nonetheless, the studies by Yao and Gross (25) are in obvious conflict with the present findings. We found that both L-NMMA and L-NIO blocked ROS generation from ACh in cardiomyocytes. Furthermore, L-NIO, which has not been implicated as a muscarinic blocker, antagonized ACh's anti-infarct effect in isolated hearts. There is strong evidence for involvement of NOS in BK-triggered preconditioning. We found that BK protection could be blocked not only by L-NAME in isolated hearts but also by the guanylyl cyclase blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (18), indicating that NO produced by NOS acts to generate cGMP. Both ACh and BK seem to trigger the preconditioned state through a similar pathway. BK's ability to trigger ROS generation in cardiomyocytes is dependent on PI3-kinase (18), as is the case for ACh (19). The present study reveals that Akt activation is also required for both ACh and BK to trigger ROS production. Thus it seems reasonable that ACh would utilize NOS in its pathway as does BK. Our data now confirm that both agonists also are dependent on NOS activation to produce protection. We do not know why L-NMMA failed to block ACh's protection in the canine heart (25). Possible explanations include underdosing or a species difference.

It is reassuring that ACh and BK use many of the same signaling elements because they both bind Gi-coupled receptors. Signaling by opioid agonists has not yet been extensively studied, but because the {delta}-receptor is also Gi coupled we would expect the signaling pathway to be similar. Curiously, adenosine, a fourth Gi-coupled receptor, deviates from this general scheme and bypasses both opening of ATP-sensitive potassium channels and ROS production in its triggering pathway (7). It is not understood how this latter Gi-coupled receptor can trigger a different pathway, but during evolution the redundancy of triggering pathways probably has produced a survival benefit.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-20468 and HL-50688.


   ACKNOWLEDGMENTS
 
Present address of T. Krieg: Klinik für Innere Medizin B, Ernst-Moritz-Arndt Universität, 17487 Greifswald, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Downey, Dept. of Physiology, MSB 3074, Univ. of South Alabama, College of Medicine, Mobile, AL 36688 (E-mail: jdowney{at}usouthal.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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 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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