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J Appl Physiol 98: 2370-2375, 2005. First published February 10, 2005; doi:10.1152/japplphysiol.00986.2004
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HIGHLIGHTED TOPICS
Biomechanics and Mechanotransduction in Cells and Tissues

Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. II. mtNOS activity

Tamara Zaobornyj,1 Laura B. Valdez,1 Pablo La Padula,2 Lidia E. Costa,2 and Alberto Boveris1

1Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, and 2Institute for Cardiological Research, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina

Submitted 7 September 2004 ; accepted in final form 3 February 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial nitric oxide (NO) production was assayed in rats submitted to hypobaric hypoxia and in normoxic controls (53.8 and 101.3 kPa air pressure, respectively). Heart mitochondria from young normoxic animals produced 0.62 and 0.37 nmol NO·min–1·mg protein–1 in metabolic states 4 and 3, respectively. This production accounts for a release to the cytosol of 29 nmol NO·min–1·g heart–1 and for 55% of the NO generation. The mitochondrial NO synthase (mtNOS) activity measured in submitochondrial membranes at pH 7.4 was 0.69 nmol NO·min–1·mg protein–1. Rats exposed to hypobaric hypoxia for 2–18 mo showed 20–60% increased left ventricle mtNOS activity compared with their normoxic siblings. Left ventricle NADH-cytochrome-c reductase and cytochrome oxidase activities decreased by 36 and 12%, respectively, from 2 to 18 mo of age, but they were not affected by hypoxia. mtNOS upregulation in hypoxia was associated with a retardation of the decline in the mechanical activity of papillary muscle upon aging and an improved recovery after anoxia-reoxygenation. The correlation of left ventricle mtNOS activity with papillary muscle contractility (determined as developed tension, maximal rates of contraction and relaxation) showed an optimal mtNOS activity (0.69 nmol·min–1·mg protein–1). Heart mtNOS activity is regulated by O2 in the inspired air and seems to play a role in NO-mediated signaling and myocardial contractility.

mitochondrial nitric oxide; heart contractility; acclimatization

NITRIC OXIDE (NO) IS AN INTERCELLULAR messenger and a regulator of respiration and cellular processes. This uncharged and diffusible molecule is produced in mammalian cells by the enzymatic cooxidation of L-arginine and NADPH by O2 to yield NADP, L-citrulline, and NO. Three main isoforms of NO synthase (NOS) with distinct genomic localization are known: the two constitutive enzymes, neuronal NOS (nNOS; 160 kDa) and endothelial NOS (eNOS; 140 kDa), and the inducible NOS (iNOS; 135 kDa). The Ca2+ dependence of NO synthesis distinguishes the NOS isoforms, with nNOS and eNOS having a much higher Ca2+ requirement than iNOS (1).

The presence of a mitochondrial NOS (mtNOS) was originally reported in inmunohistochemical studies (6) and followed by the determination of rat liver mitochondrial NO production and its biochemical characterization (22, 23). The observation of mitochondrial NO production was extended to other tissues, kidney (11), brain (31, 32, 41), thymus (15), diaphragm (7), and heart (7, 9, 19, 27, 48), weakening the idea of a cytosolic contamination of the mitochondrial preparations. Liver mtNOS was sequenced and identified as the {alpha}-isoform of nNOS, myristylated and phosphorylated at the COOH-terminal end (20). mtNOS activity has been found to be regulated by physiological effectors; it is downregulated by angiotensin II (9, 11) and thyroid hormones (16) and upregulated in cold acclimation (35). In addition, thymus mtNOS activity is markedly increased during early apoptosis (15).

An improvement in the resistance of the isolated heart to ischemia was recognized in the hearts of immature rabbits after normobaric hypoxia (4) and in the hearts of adult rats after intermittent hypobaric hypoxia (28). NO steady-state level is one of the factors that has been proposed to be determining in heart adaptation to low-oxygen pressure (5). Moreover, heart NO, regulated by the Ca2+ levels of the contraction and relaxation cycles, is essential for heart homeostasis and mechanical activity (36). Different NOS isoforms have been reported to participate in the adaptation to chronic hypoxia (5, 28), and liver mtNOS activity was found increased in rats subjected to acute hypoxia (29, 42). NO was identified as an effective regulator of heart mitochondrial functions inhibiting cytochrome oxidase activity (3, 14, 37, 39) and Ca2+ uptake (14), opening the ATP-sensitive K+ channel (26), and increasing superoxide anion (O2–) and hydrogen peroxide (H2O2) production rates (37). Moreover, both NO and H2O2 have been considered a pleiotropic signal that indicates high mitochondrial energy charge, with both molecules interacting with specific cytosolic target proteins involved in the modulation of the cellular cycle and the apoptosis pathway (9).

The aim of this work was to evaluate the effect of sustained hypobaric hypoxia and aging on heart mtNOS, along with other mitochondrial enzymes, and to correlate mtNOS activity with parameters of papillary muscle contractility and recovery after anoxia-reoxygenation. The cardioprotective effect of chronic hypoxia, observed as a retardation of the decline in the mechanical activity of papillary muscle upon aging and as an improved recovery after anoxia-reoxygenation, is reported in the accompanying paper (30).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals.   Biochemicals were purchased from Sigma Chemical (St. Louis, MO). Other reagents were of analytical grade.

Experimental design.   Seven-week-old Wistar male rats of the CHbbTHOM albino strain were chronically submitted to a simulated altitude of 5,000 m (53.8 kPa) in a hypobaric chamber, as described (30). Control sibling rats remained at sea-level atmospheric pressure (101.3 kPa). Both groups were maintained at 22°C on 12-h cycles of dark and light and had free access to food and water. Groups of five animals were killed at 2, 4, 8, 12, and 18 mo of age after 1, 10, 26, 45, and 74 wk, respectively, of exposure to hypobaric hypoxia or normoxia. The same animals were used in the study described in the accompanying paper (30). All procedures were in accordance with the 6344/96 regulation of Argentinian National Drug, Food and Medical Technology Administration (ANMAT) and the Guiding Principles for Research Involving Animals and Human Beings of the American Physiologycal Society.

Isolation of heart and left ventricle mitochondria.   Hearts and left ventricles deprived of the papillary muscles were weighed, chopped, and homogenized in an ice-cold homogenization medium (1:10) containing 0.23 M mannitol, 0.07 M sucrose, 1 mM EDTA, and 10 mM Tris·HCl, pH 7.4, for 30 s with a blade homogenizer (Kendro-Sorvall-Du Pont Institute, Asheville, NC) and by five strokes in a glass Teflon homogenizer. All of these operations were carried out at 2–4°C (10). The homogenates were centrifuged at 700 g for 10 min to discard nuclei and cell debris, and the supernatant was centrifuged at 7,000 g for 10 min. The mitochondrial pellet was washed and resuspended in the homogenization medium. The supernatant of the second centrifugation was used to determine the NOS activity in the postmitochondrial fraction.

Submitochondrial membranes.   Mitochondria were frozen and thawed three times and homogenized by passage through a 29-gauge hypodermic needle (10). Protein concentration was determined with the Folin reagent and bovine serum albumin as standard.

NO production and release.   NO production was measured in intact mitochondria, submitochondrial membrane (SMM), and postmitochondrial fraction by following spectrophotometrically at 577–591 nm [molar extinction coefficient ({epsilon}) = 11.2 mM–1·cm–1] (Beckman DU 7400 diode array spectrophotometer) the oxidation of oxyhemoglobin to methemoglobin, at 37°C (10). Control experiments adding 2 mM NG-monomethyl-L-arginine (L-NMMA) were performed, and L-NMMA-sensitive hemoglobin oxidation was considered due to NO formation that was expressed as nanomoles of NO per minute per milligram of protein. The reaction medium used to determine NO production by SMM and postmitochondrial fraction (0.5–0.8 mg protein/ml) consisted of 50 mM phosphate buffer (pH 7.0–7.4), 1 mM L-arginine, 1 mM CaCl2, 100 µM NADPH, 10 µM dithiotreitol, 4 µM CuZn-SOD, 0.1 µM catalase, and 20 µM oxyhemoglobin. The medium used to determine NO release from intact mitochondria (0.5 mg protein/ml) consisted of 0.23 M mannitol, 0.07 M sucrose, 1 mM EDTA, 5 mM phosphate buffer, 20 mM Tris·HCl, pH 7.4, 8 mM succinate, and 20 µM oxyhemoglobin, in the absence (state 4) or in the presence (state 3) of 0.5 mM ADP.

Mitochondrial electron transfer activities.   The membrane-bound activities of complexes I-III and IV were determined spectrophotometrically at 37°C with SMM suspended in 100 mM phosphate buffer, pH 7.4. For NADH-cytochrome c reductase (complexes I-III) activity, SMM (0.25 mg protein/ml) were added with 0.2 mM NADH, 25 µM cytochrome c3+, and 0.5 mM KCN, and the enzymatic activity was determined at 550 nm ({epsilon} = 19 mM–1·cm–1) (33). Cytochrome oxidase (complex IV) activity was determined in the same phosphate buffer added with 60 µM cytochrome c2+ (33). Reduced cytochrome c was prepared by reduction of cytochrome c3+ with sodium dithionite, followed by Sephadex G-25 chromatography. The rate of cytochrome-c oxidation was calculated as the pseudo-first-order reaction constant (k') per milligram protein.

Cytochrome content.   Cytochromes were determined at the indicated wavelength pairs and using the following extinction coefficients: cytochrome aa3, 605–630 nm ({epsilon} = 16 mM–1·cm–1) and cytochrome c, 550–540 nm ({epsilon} = 19 mM–1·cm–1). Cytochrome reduction was achieved by sodium dithionite addition (11).

Papillary muscle contractility.   Developed tension, maximal rate of contraction (+T), and maximal rate of relaxation (–T) were determined as reported in the accompanying paper (30).

Statistics.   Results are expressed as mean values ± SE. Two-way or one-way ANOVA was used, followed by the Student-Newman-Keuls test, to analyze differences between mean values. P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Heart mitochondria NO production.   The main biochemical requirements of heart mtNOS activity were determined in mitochondrial fragments (SMM) isolated from young adult male rats (2 mo old). The activity, measured at the optimal pH of 7.4, was 0.69 nmol NO·min–1·mg protein–1 (Table 1). Oxyhemoglobin oxidation depended on the presence of the substrates NADPH and L-arginine and the cofactor Ca2+ and was 92% inhibited by L-NMMA. This general pattern of biochemical activity makes mtNOS similar to eNOS and nNOS. Coupled respiring heart mitochondria, also isolated from young adult male rats, showed a NO release to the surrounding medium that was higher in resting state 4 (with substrate and without ADP; 0.62 nmol NO·min–1·mg protein–1) than in active state 3 (with substrate and ADP; 0.37 nmol NO·min–1·mg protein–1) (Table 2), as observed in liver and kidney mitochondria (49). The NO released from mitochondria is a fraction of the total mitochondrial NO production, because of the reaction of NO and O2– to form ONOO– in the mitochondrial matrix (47). The rate of NO release from mitochondria to cytosol is estimated as 29 nmol NO·min–1·g heart–1, considering that the perfused beating rat heart has a mitochondrial population that is 72% in state 4 and 28% in state 3 (8) [(0.72 x 0.62 nmol NO·min–1·mg protein–1 + 0.28 x 0.37 nmol NO·min–1·mg protein–1) x (53 mg protein/g heart)]. This value accounts for 55% of total cytosolic NO, taking as the other 45% the NO production by the postmitochondrial (cytosolic) fraction (24 nmol NO·min–1·g heart–1, Table 2).


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  		Table 1. Nitric oxide production in rat heart submitochondrial membranes as determined by the oxyhemoglobin assay at pH 7.4

 

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  		Table 2. Heart mitochondrial NO synthase activity and mitochondrial NO release

 
Effect of hypobaric hypoxia and aging.   Left ventricle mtNOS activity was significantly higher (20–60%) in rats submitted to hypobaric hypoxia than in their sibling controls kept at sea-level atmospheric pressure (Fig. 1). The quantitative effect depended on rat age, as mtNOS activity linearly decreased in control rats between 2 and 18 mo of age. The effect of hypobaric hypoxia was marked enough to counteract the age-associated decrease of mtNOS activity and to reach a higher activity than the one corresponding to young animals (2–4 mo old). In contrast, two other mitochondrial enzymatic activities, NADH-cytochrome c reductase and cytochrome oxidase, were not affected by hypobaric hypoxia, indicating a specificity for the effect of hypoxia on mtNOS activity.



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  		Fig. 1. Heart mitochondrial nitric oxide synthase (mtNOS) activity in rats submitted to hypobaric hypoxia (53.8 kPa; {bullet}) and in normoxia (101.3 kPa; {circ}) during aging. Values are means ± SE.

 
mtNOS, NADH-cytochrome c reductase, and cytochrome oxidase activities decreased by 20, 36, and 12%, respectively, upon aging from 2 to 18 mo (Figs. 1 and 2). There was a comparable age-dependent decline in the activity of these three mitochondrial enzymes, which are considered markers of aging (33). In agreement, cytochrome aa3 showed a 15% decrease on aging that was not observed for cytochrome c. The content of both cytochromes was similar in the left ventricles of hypoxic and normoxic animals (Fig. 3). Both groups of rats reached maximal cytochrome aa3 and cytochrome c contents at 4 mo of age, indicating that maturation from 2 to 4 mo is required to reach a maximal content of these mitochondrial components.



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  		Fig. 2. NADH-cytochrome c reductase (squares) and cytochrome oxidase (triangles) activities in heart mitochondria of rats submitted to hypobaric hypoxia (solid symbols) and in normoxia (open symbols). k', Pseudo-first-order reaction constant. Values are means ± SE.

 


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  		Fig. 3. Cytochrome aa3 (triangles) and cytochrome c (squares) contents in heart mitochondria for rats submitted to hypobaric hypoxia (solid symbols) and in normoxia (open symbols). Values are means ± SE.

 
Left ventricle mtNOS activity and papillary muscle mechanical activities.   Figure 4 shows a tridimensional plot of the dependence of both left ventricle mtNOS activity and papillary muscle developed tension on age in hypoxic and normoxic rats. The data of the normoxic rats are given by the five shaded bars on the left, and the data of the hypoxic rats are given by the five solid bars on the right. Maximal developed tension corresponds to an optimal mtNOS activity of 0.69 nmol NO·min–1·mg protein–1. Interestingly, the maximal developed tension was observed in young rats (2 mo old) kept at normoxia or submitted to 1 wk of hypobaric hypoxia. At 4, 8, and 12 mo of age, hypoxic rats showed an increase of 20, 35, and 45%, respectively, in mtNOS activity (solid bars) compared with their normoxic age-matched controls (shaded bars). That increase in mtNOS activity was related to higher developed tension (34, 64, and 62%), as can be seen by the height of the solid bars (hypoxia) compared with the height of the shaded bars (normoxia). At 18 mo, hypoxic rats showed a 60% increase in mtNOS activity but no significant difference in developed tension compared with their age-matched controls. The +T and –T of papillary muscle had a similar pattern to the one of developed tension (Fig. 2 in Ref. 30). Plots of papillary muscle +T and –T as a function of left ventricle mtNOS activity also identified an optimal mtNOS activity (0.69 nmol NO·min–1·mg protein–1) associated with the +T and –T (data not shown).



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  		Fig. 4. Tridimensional plot of papillary muscle developed tension, left ventricle mtNOS activity, and rat age. Solid bars correspond to rats submitted to hypobaric hypoxia, and shaded bars correspond to normoxic rats.

 
The developed tension of papillary muscle after an in vitro sequence of 60 min of anoxia-30 min of reoxygenation and left ventricle mtNOS activity in normoxic and hypoxic rats is plotted as a function of age in Fig. 5. Hypoxia was related to increased developed tension and mtNOS activity values at 8 mo of age: hypoxic rats showed an increase of 35% in mtNOS activity associated with a developed tension 100% higher than that of their age-matched controls. Once more, an optimal activity of the enzyme (0.69 nmol NO·min–1·mg protein–1) is observed for maximal developed tension. The +T and –T followed a similar pattern and showed a similar mtNOS activity (data not shown) (Figs. 5 and 6 in Ref. 30).



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  		Fig. 5. Tridimensional plot of papillary muscle developed tension after an in vitro sequence of 60-min anoxia and 30-min reoxygenation, left ventricle mtNOS activity, and rat age. Solid bars correspond to rats submitted to hypobaric hypoxia, and shaded bars correspond to normoxic rats.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Chronic hypobaric hypoxia (53.8 kPa, 2–16 mo) was an effective and specific upregulator of left ventricle mtNOS activity, with an effect that was marked enough to counteract the age-dependent decline in mtNOS activity. Simultaneously, we have observed that rat acclimatization to the Andes high altitude (Cerro de Pasco, 4,340 m, 61.3 kPa, 3 wk) also upregulated, up to 58%, whole heart mtNOS activity (24). It is likely that hypoxia-inducible factor-{alpha}, the oxygen-dependent gene regulator, mediates the upregulation of mtNOS expression by hypoxia (25, 43, 45). In this study, an increased left ventricle biochemical mtNOS activity was found associated with preserved papillary muscle contractile parameters and improved recovery after anoxia-reoxygenation in the hearts of aging rats. Decreased oxygen tension in the inspired air is now added to other physiological regulators of mtNOS activity. Heart, liver, and kidney mtNOS are downregulated by angiotensin II (10), and liver mtNOS is upregulated by cold acclimation (35) and downregulated by thyroid hormones (16).

Heart mtNOS activity has been determined in a series of previous studies (7, 9, 19, 27, 48). The relative contribution of the different subcellular NOS isoforms to the total cellular production and steady-state concentrations of NO is a subject that is starting to be understood. The distribution of NOS isoforms in the different cellular compartments and the NO diffusion between mitochondria and cytosol underlie the role of NO in the regulation of cellular homeostasis and in biochemical and genetic signaling. In the present study, the quantitative determination of NO release by heart mitochondria and the postmitochondrial fraction indicated that mitochondrial NO release accounts for 55% of the total NO in the heart cytosol of normoxic animals. Mitochondria, coupled with an intact permeability barrier, produced NO with intramitochondrial mtNOS substrates in a close to physiological situation. The postmitochondrial or cytosolic fraction, mainly carrying the eNOS of caveolae and plasma membrane from both cardiomyocytes and endothelial cells and in minor proportion the nNOS of sarcoplasmic reticulum and heart nerves (1, 5), fully supplemented with the NOS substrates and Ca2+, produced the remaining 45% of cytosolic NO, likely giving an overestimation of the real in vivo activity. Under chronic hypoxia, both mtNOS and the eNOS of caveolae and plasma membrane (5) appear to significantly contribute to an increased NO level in the cytosol as part of the mechanism of heart adaptation to hypoxia.

The biphasic relationship between left ventricle mtNOS activity and papillary muscle developed tension (Figs. 4 and 5) may indicate that heart NO levels have various regulatory roles that may exert either beneficial or detrimental effects (26, 44). In these considerations, it should be borne in mind that NO is a 30-Da molecule highly diffusible in aqueous and lipid phases that shows a rich biochemistry to support its role as the most versatile molecule of biological regulation and signaling. NO binds with high affinity to the iron chelated in iron-sulfur centers, as in guanylyl cyclase (1), and in heme rings (13, 17) and participates in the free radical-termination reaction with O2– to yield ONOO– (47). We observed an optimal mtNOS activity (0.69 nmol NO·min–1·mg protein–1) associated with the highest papillary muscle developed tension (Figs. 4 and 5) in the left ventricles of 2-mo-old rats that were either submitted to 1 wk of hypobaric hypoxia or kept at normoxia. Similar results were observed in papillary muscle developed tension assessed after in vitro anoxia-reoxygenation: the highest values were the ones corresponding to hypoxic and normoxic 2-mo-old animals. Using the measured optimal mtNOS activity and considering the NO-dependent inhibition of mitochondrial respiration (3, 9, 38, 47), we estimated a heart intramitochondrial NO steady-state concentration of 200 nM. This value is higher than the 50–100 nM NO measured in the cytosol of perfused rat heart (39), suggesting a physiological importance for NO diffusion from mitochondria to cytosol.

The upregulation of mtNOS activity in rats submitted to hypobaric hypoxia was related to a significant prevention of the age-associated decline in developed tension, +T, and –T. At 4, 8, and 12 mo of age, hypoxic rats showed an increase of ~20, 35, and 45%, respectively, in mtNOS activity. That increase was related to a slighter drop in developed tension as a function of age compared with normoxic rats. As a result, this parameter was very much preserved and significantly higher in hypoxic rats than in normoxic rats at 4 (34%), 8 (64%), and 12 mo (62%) of age. Similarly, mtNOS activity increase was related to a slower age-dependent decrease in papillary muscle developed tension after in vitro anoxia-reoxygenation. The well-established competitive NO inhibition of cytochrome oxidase (3, 13, 17) and the role of NO in signaling are alternative mechanisms by which mtNOS upregulation by hypoxia may be related to an improvement of heart contractile function.

The functional activity of mtNOS, i.e., the NO-mediated inhibition of mitochondrial respiration under physiological conditions, has been estimated as 16–25% for mammalian organs (2). Under physiological and pathophysiological conditions where heart perfusion and O2 levels become limiting for ATP production and contractility, the NO-inhibited respiration lowers the steepness of the O2 gradient in the normoxic-anoxic transition, allows O2 to diffuse further along its gradient, and extends the space of adequate tissue oxygenation away from the blood vessel (37, 46). The NO-supplemented condition will be associated with more areas with high enough ATP levels to sustain a homogeneous and synchronic contraction of the myofibrils.

The increased mtNOS activity and mitochondrial NO release in the hypoxic heart are a likely molecular mechanism of signaling, leading to better heart contractile function through maintained heart mass and avoiding apoptosis. Boyd and Cadenas (12) suggested that NO and H2O2 activate protein kinases by S-nitrosation in a process that is potentiated by glutathione depletion. Considering mitochondrial NO, interesting correlations between mtNOS activity and cellular cycle and proliferation have been observed (21) and termed the pleiotropic effect of mitochondrial NO release (10, 12). In heart, mtNOS activity relates to an optimal cardiac contractility, as reported here. Moreover, NO was reported to trigger mitochondrial biogenesis in cardiomyocytes and other cell types (34), and adaptation to chronic hypobaric hypoxia produced a moderate increase in the number of mitochondria per heart volume unit (18). The NO inhibition of electron transfer at ubiquinol-cytochrome c reductase (complex III) increases O2– and H2O2 production (39). Diffusion of both NO and H2O2 from mitochondria to cytosol would constitute a pleiotropic signal that indicates high mitochondrial energy charge to the cytosolic activation factors involved in cell cycle regulation.

However, at 18 mo of age, a difference of ~60% in mtNOS activity was associated with no significant difference in the contractile function of hypoxic and normoxic rats. Here, an excessive cytochrome oxidase inhibition and an increased ONOO– formation with the recognized mitochondrial toxicity of the latter species (40) may be the factors that explain the lack of effect of mtNOS upregulation associated with exhaustion of adaptation to hypobaric hypoxia.

In summary, heart mtNOS appears as a mitochondrial enzyme regulated by the O2 level in the inspired air. This upregulation appears to be associated with a preservation of contractile parameters for some time of exposure and may play a role in heart contractility and cellular NO signaling through mitochondrial NO release to the cytosol.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by research grants from the University of Buenos Aires (B075), Agencia Nacional de Promoción Científica y Tecnológica (PICT 00–8710), and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2271–00).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Zaobornyj, Fisicoquímica, Facultad de Farmacia y Bioquímica,, Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires, Argentina (E-mail: tamaraz{at}ffyb.uba.ar)

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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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