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Am J Physiol Heart Circ Physiol 293: H402-H408, 2007. First published March 16, 2007; doi:10.1152/ajpheart.00854.2006
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Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta

Kevin B. Atkins,1 Antoine Prezkop,1 James L. Park,1 Jharna Saha,1 Damon Duquaine,1 Maureen J. Charron,3 Ann Louise Olson,4 and Frank C. Brosius, 3rd1,2

Departments of 1Internal Medicine and 2Physiology, University of Michigan Medical School, Ann Arbor, Michigan; 4Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and 3Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York

Submitted 9 August 2006 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.

hypertension; myosin phosphatase

THE INSULIN-RESPONSIVE GLUCOSE transporter GLUT4 is expressed in vascular smooth muscle (VSM) (3, 4, 7, 24). We previously showed (1, 31) that in arterial VSM GLUT4 participates in constitutive, non-insulin-dependent glucose uptake. This unusual property distinguishes VSM from other tissues that express GLUT4 since in those tissues GLUT4 largely resides in intracellular vesicles until translocated to the plasma membrane in response to insulin or other physiological stimuli (8). In addition, we have reported that GLUT4 expression is decreased in large arteries of hypertensive rats and mice (1, 2, 31) and that arterial reactivity in arteries from GLUT4 knockout (KO) mice is increased compared with vessels from wild-type animals (31), similar to arteries from hypertensive animals. Since hypertensive vessels exhibit increased sensitivity to agonist stimulation, it is important to ascertain what effect chronic changes in GLUT4 expression exert on the vascular abnormalities associated with hypertension.

Agonist stimulation of smooth muscle leads to a rise in intracellular Ca2+ with a resultant binding of Ca2+ to calmodulin. This Ca2+-calmodulin complex activates myosin light chain kinase (MLCK), which in turn phosphorylates myosin light chain (MLC). Phosphorylation of MLC enables interaction of myosin with actin, initiating the cross-bridge cycle of contraction. It has been observed, however, that intracellular free Ca2+ concentration is not always a predictor of MLC phosphorylation or force of contraction induced by agonist stimulation. The increased phosphorylation of MLC and force of contraction at a constant Ca2+ concentration is termed Ca2+ sensitization (10, 17–19, 23). Conversely, a decreased level of MLC phosphorylation and force of contraction at a given Ca2+ concentration is termed desensitization. The major mechanism of sensitization and desensitization involves regulation of myosin light chain phosphatase (MLCP). This phosphatase is a heterotrimer composed of a 38-kDa catalytic subunit, a 20-kDa subunit of unestablished function, and a 110- to 130-kDa regulatory subunit, MYPT1 (15, 20). In general, the activity of this phosphatase can be modulated either via direct inhibition of the phosphatase by CPI-17 or by changes in the degree of phosphorylation of MYPT1 (18, 19), which is regulated by a variety of kinase pathways including the Rho A/Rho kinase pathway (19).

Smooth muscle myosin heavy chain 2 (SM2) and h-caldesmon, markers of mature smooth muscle phenotype, are reduced in aortas of hypertensive rats (2). In addition, the expression of phosphorylated (p)Akt is reduced in hypertensive aortas, and our previous data (2) and those of others (16, 22, 27) suggest that Akt may mediate the expression of the mature smooth muscle phenotype.

Given the effect of chronic reduction in GLUT4 expression on vascular sensitivity, we tested the hypothesis that maintenance of GLUT4 expression would reduce hypertension-induced increased vascular reactivity and possibly prevent the development of hypertension itself. We tested this hypothesis in wild-type and GLUT4 transgenic (TG) mice in the deoxycorticosterone acetate (DOCA)-salt model of hypertension. In addition, we examined whether pAkt and smooth muscle marker expression is reduced in hypertensive aortas of mice and what effect GLUT4 expression might exert.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. GLUT4 TG mice were generated as previously described (30) by insertion of an 11.5-kb KpnII/EcoRI fragment of the human GLUT4 gene into the pronuclei of fertilized mouse embryos. GLUT4 TG animals were identified by PCR amplification of tail DNA using primers 5'-GAGTATTTAGGGCCAGATGAGAAC-3' and 5'-GGTTACAAATAAAGCAATAGCATCAC-3', which amplify a 590-bp region unique to the human GLUT4–11.5 construct. These mice have been backcrossed onto a C57BL/6J background for >10 generations. GLUT4 KO mice have been described previously (21, 31) and have also been backcrossed onto a C57BL/6J background for >10 generations. These mice were compared without further manipulation for the experiments reported in Fig. 4B.


Figure 4
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  		Fig. 4. Phosphorylated (p)MYPT1 levels in aortas of sham-treated and DOCA-salt WT and GLUT4 TG mice (A) and in normotensive GLUT4 TG, WT, and GLUT4 TG knockout (KO) mice (B). pMYPT1 immunoblots of aortic lysates (20 µg) from mice after 8 wk are representative of 3 separate trials (n ≥ 8; A) or from untreated, non-uninephrectomized mice (n = 4; B).

 
For all other experiments, adult male mice (25–30 g) were anesthetized with Ketaset and xylazine (ip), and their left kidneys were removed. A DOCA-impregnated Silastic implant (2,000 mg/kg) was placed subcutaneously behind the skull in 50% of the mice. After surgery, DOCA-salt mice were maintained on salt water (1% NaCl plus 0.2% KCl), and sham-treated mice received tap water. Systolic blood pressure in conscious mice was measured by the tail cuff method (6 Channel Blood Pressure System, IITC, Woodland Hills, CA). After 8 wk mice were anesthetized with pentobarbital (50 mg/kg ip), and both diaphragms were cut. The aortas from aortic arch to just above the renal arteries were removed, carefully cleaned, and either used for reactivity or snap frozen in liquid nitrogen and stored at –80°C. The procedures used in our study were approved by the University of Michigan Committee on the Use and Care of Animals. The University of Michigan Unit for Laboratory Animal Medicine provided veterinary care. The University of Michigan is accredited by the American Association for Accreditation of Laboratory Animal Care. The animal care and use program conformed to the standards in the Guide for Care and Use of Laboratory Animals (DHHS Pub. No. 86-23).

Immunoblotting. Frozen vascular samples were ground in a chilled pestle, suspended in the presence of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer (125 mmol/l Tris·HCl, pH 6.8, 4% SDS, 20% glycerol, 100 mmol/l PMSF, 10 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A), and sonicated, as previously reported (1, 2, 31). Lysates were run on 10% SDS-PAGE and immunoblotted with antibodies for GLUT4, GLUT1, caldesmon, smooth muscle (SM) {alpha}-actin, SM2, pAkt (Ser473) and Akt, as previously described (2), and pMYPT [Thr696; Santa Cruz; phosphorylation at this site has been widely demonstrated to occur in hypertensive vessels (20, 23)] and MYPT (BD Biosciences). All blots were within the linear range and, with the exception of those for pAkt (Akt) and pMYPT (MYPT), were normalized to SM {alpha}-actin, the expression of which we have found is unaffected by hypertension (2). Immunoblots for GLUT1 and GLUT4 exhibit multiple bands due to the highly glycosylated nature of these proteins (1, 2, 7, 21, 31).

Vascular reactivity experiments. These experiments were performed as previously reported (31). Briefly, mouse aortas were cleaned and cut into 2-mm-length rings. Endothelium was removed by gently rubbing the lumen of the aortic ring with 4-0 silk suture. The denuded aortic rings were mounted in a myograph system (Danish Myo Technology, Aarhus, Denmark). Vessels were bathed with warmed (37°C), aerated (95% O2-5% CO2) physiological salt solution (PSS; mmol/l: 130 NaCl, 4.7 KCl, 1.18 KHPO4, 1.17 MgSO4, 1.6 CaCl2, 14.9 NaHCO3, 5.5 dextrose, 0.03 CaNa2 EDTA). Rings were set at 700 mg of passive tension and equilibrated for 1 h, with washing every 20 min. Before performance of concentration-response curves, vessels were contracted with isotonic PSS containing 120 mmol/l KCl in which an equimolar quantity of KCl was substituted for NaCl (KPSS). After the KPSS was washed out, the vessels were contracted with serotonin (5-HT; 1 µmol/l) and subsequently treated with acetylcholine (10 µmol/l) to test for absence of endothelium. All arteries were then contracted with KPSS, allowed to plateau, and washed. Cumulative concentrations of 5-HT were added to the bath to establish a concentration-response curve. Contractions to 5-HT were expressed as a percentage of the 120 mmol/l KPSS contraction.

Statistical evaluation. Data are expressed as means ± SE. Unpaired two-tailed Student's t-test was used to compare results from two populations. For the reactivity experiments, agonist EC50 values were calculated with a nonlinear regression analysis with the algorithm [effect = maximum response/1 + (EC50/agonist concentration)] in the computer program GraphPad Prism (San Diego, CA). For all other comparisons, including vascular reactivity experiments, ANOVA with Scheffé's post hoc analysis was performed. Differences were considered to be statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Systolic blood pressure increased significantly after 8 wk of DOCA-salt treatment in both wild-type and GLUT4 TG mice compared with their respective sham-treated controls. There was no difference between the two DOCA-salt treated groups in final blood pressure (Fig. 1). GLUT4 KO mice had basal systolic blood pressure similar to that of wild-type C57BL/6J mice, as previously determined (31).


Figure 1
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  		Fig. 1. Effect of deoxycorticosterone acetate (DOCA)-salt treatment on systolic blood pressure (BPsys) in wild-type (WT) and GLUT4 transgenic (TG) (G4) mice. Blood pressure was taken by tail cuff at the end of 8 wk. These data were derived from 3 separate trials with a final n ≥ 8 mice/group. Sham, sham-treated mice.

 
As seen before in several rodent models of hypertension (1, 2, 31), immunoblots demonstrated that GLUT4 expression was markedly decreased in aortas of wild-type DOCA-salt animals (Fig. 2A). GLUT4 levels were significantly higher in the GLUT4 TG sham-treated mice than in wild-type sham-treated mice and were not decreased in GLUT4 TG DOCA-salt mice compared with either GLUT4 TG or control sham-treated mice. GLUT1 expression was unchanged in aortas of wild-type DOCA-salt animals compared with sham-treated controls (Fig. 2B). Interestingly, GLUT1 expression was lower in GLUT4 TG sham-treated animals compared with wild-type sham-treated animals. GLUT1 expression increased in aortas of GLUT4 TG DOCA-salt-treated mice compared with GLUT4 TG sham-treated mice, to levels quite similar to those of wild-type DOCA-salt- and sham-treated animals.


Figure 2
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  		Fig. 2. GLUT4 (A) and GLUT1 (B) expression in aortas of sham-treated and DOCA-salt WT or GLUT4 TG mice. The representative GLUT4 and GLUT1 immunoblots of aortic lysates (20 µg) were performed after 8 wk of treatment, and combined data were derived from 3 separate trials (n ≥ 8).

 
Measurement of vascular reactivity indicated that sensitivity and total force generation in response to 5-HT were increased in aortas of wild-type DOCA-salt mice compared with wild-type sham-treated mice (Fig. 3A), as found previously in DOCA-salt hypertensive rats (14). When compared with wild-type hypertensive vessels, the enhanced sensitivity to 5-HT was significantly reduced in aortas from GLUT4 TG DOCA mice, although not to the levels of the sham-treated mice (Fig. 3, A and B). Most strikingly, maximal force generation in the aortas from GLUT4 TG DOCA-salt mice was virtually identical to that of the sham-treated groups (Fig. 3A). Total force generation in response to high concentrations of extracellular potassium was not different among the four groups (Fig. 3C), indicating that enhanced agonist-induced reactivity was specifically affected in DOCA-salt hypertension in these models.


Figure 3
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  		Fig. 3. Reactivity to serotonin (5-HT) by endothelium-denuded aortic rings of sham-treated and DOCA-salt WT and GLUT4 TG mice. A: concentration-response curves are expressed as % of the contraction elicited by 120 mM physiological salt solution with KCl substituted for NaCl (KPSS). B: EC50 values for each set of animals. C: total force generation in response to 120 mM KPSS in endothelium-denuded aortic rings of sham-treated and DOCA-salt WT and GLUT4 TG mice.

 
DOCA-salt hypertension increased MYPT1 phosphorylation on Thr696 in aortas of wild-type mice compared with wild-type sham-treated mice. There was no comparable increase in MYPT1 phosphorylation in aortas of DOCA-salt treated GLUT4 TG mice compared with respective sham-treated mice (Fig. 4A). A comparison of constitutive MYPT1 phosphorylation in aortas among naive (i.e., non-uninephrectomized) wild-type, GLUT4 TG, and GLUT4 KO mice indicated that basal MYPT1 phosphorylation was greatest in GLUT4 KO aortas and least in GLUT4 TG aortas, suggesting an inverse correlation of MYPT1 phosphorylation and GLUT4 expression (Fig. 4B). However, this apparent association was not seen in the sham-uninephrectomized animals shown in Fig. 4A, in which MYPT1 phosphorylation was apparently (but not statistically significantly) greater in the TG than in the wild-type animals.

Previously, we showed (2) that DOCA-salt hypertension in rats is associated with decreased expression of markers of the mature smooth muscle phenotype. We therefore determined whether changes in such markers occur in DOCA-salt hypertension in mice and, if so, whether such changes are prevented by preservation of normal GLUT4 expression. The expression of h-caldesmon and SM2 was not different between wild-type and GLUT4 TG sham-treated animals (Fig. 5). Moreover, the expression of both h-caldesmon and SM2 decreased similarly in both wild-type DOCA-salt and GLUT4 TG DOCA-salt mice (Fig. 5). In aortas of DOCA-salt hypertensive rats we have observed (2) that Akt phosphorylation is decreased. There was a similar decrease in Akt phosphorylation (Ser473) in aortas of wild-type DOCA-salt mice compared with wild-type controls (Fig. 6). Interestingly, Akt phosphorylation in GLUT4 TG sham-treated mice was significantly decreased compared with wild-type sham-treated mice, and there was no further change due to DOCA-salt treatment (Fig. 6).


Figure 5
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  		Fig. 5. h-Caldesmon (A) and smooth muscle myosin heavy chain 2 (SM2; B) expression in aortas of sham-treated and DOCA-salt, WT, and GLUT4 TG mice. h-Caldesmon and SM2 immunoblots of lysates (20 µg) of aortas from mice were performed after 8 wk of treatment, and combined data were derived from 3 separate trials (n ≥ 8).

 

Figure 6
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  		Fig. 6. Phosphorylated (p)Akt levels in aortas of sham-treated and DOCA-salt WT and GLUT4 TG mice. pAkt immunoblots of lysates (20 µg) of aortas from mice after 8 wk of treatment are shown. Data are representative of 3 separate trials (n ≥ 8).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously found (1, 31) that several rat and murine models of hypertension are associated with a marked decrease in GLUT4 expression in arterial smooth muscle. This reduction in GLUT4 levels corresponds to enhanced arterial sensitivity to agonists such as 5-HT and ergonovine. Since abrogation of GLUT4 expression by genetic knockout is associated with both increased total force generation as well as enhanced sensitivity to agonist stimulation (31), we hypothesized that preservation of GLUT4 expression during hypertension would prevent the increased vascular reactivity associated with hypertension. For the present studies we utilized the DOCA-salt hypertension model in both rats and mice, since this commonly studied model is associated with substantial changes in both GLUT4 and arterial reactivity. Indeed, while preservation of GLUT4 expression did not affect blood pressure, it did prevent the increases in total force generated in response to 5-HT stimulation and also partly prevented the increased sensitization demonstrated in the wild-type DOCA-salt vessels. Given our previous findings of enhanced reactivity to both 5-HT and norepinephrine in GLUT4 KO animals (31), our present data confirm that reduction in GLUT4 is associated with enhanced arterial reactivity in hypertension and that preservation of GLUT4 expression is associated with prevention of these reactivity changes.

These findings suggest that the effects of GLUT4 on arterial reactivity are independent of blood pressure and represent a direct VSM phenomenon. However, because the GLUT4 TG and GLUT4 KO animals manifest altered GLUT4 expression in all tissues that normally express GLUT4, it is conceivable that the effects on arterial reactivity are not directly related to changes in VSM GLUT4 expression but due to systemic effects of altered GLUT4 expression in other tissues. Nonetheless, given the findings that GLUT4 is reduced only in VSM and not in other tissues in rodent models of hypertension (1, 31), that GLUT4 KO arteries have contractile abnormalities very similar to those from hypertensive animals (31), and that GLUT4 TG animals preserve GLUT4 levels in hypertensive VSM and do not develop hypertensive vascular abnormalities, it seems very likely that the reactivity changes directly result from the VSM GLUT4 changes. Final proof of this concept will await development and testing of VSM-specific GLUT4 TG and GLUT4 KO animals.

Potentially pertinent to the effect of GLUT4 expression on arterial reactivity is the finding that phosphorylation of the regulatory subunit of myosin phosphatase, MYPT1, did not increase in aortas of GLUT4 TG hypertensive mice as it did in aortas of wild-type hypertensive mice. Although there was a statistical difference in MYPT1 phosphorylation between naive GLUT4 TG and wild-type mice (Fig. 4B), this was not the case in uninephrectomized animals (Fig. 4A). While we have no explanation at present for this discrepancy other than that nephrectomy may have affected basal MYPT1 phosphorylation, it is nonetheless important to emphasize that prevention of hypertension-associated reduction in GLUT4 expression clearly and statistically was correlated with prevention of hypertension-induced increases in MYPT1 phosphorylation (Fig. 4A). Furthermore, knockout of GLUT4 resulted in increased MYPT1 phosphorylation in normotensive mice. MYPT1 phosphorylation leads to reduced myosin phosphatase activity (18, 19). The increased reactivity of aortas from hypertensive wild-type mice can be explained, at least in part, by increased Ca2+ sensitization resulting from inhibition of dephosphorylation of MLC by myosin phosphatase (18, 19, 23). Reciprocally, the lack of increased responsiveness to contractile agonists in the aortas of hypertensive GLUT4 TG mice may be due to a desensitization, or at least inhibition of sensitization, from preserved MLCP activity and decreased basal MLC phosphorylation. One of the major protein kinases that phosphorylates MYPT1 is Rho kinase (12), which in turn is activated by the activation of RhoA. Activation of the RhoA/Rho kinase pathway comprises at least part of the mechanism of increased arterial reactivity in the DOCA-salt model of hypertension (25, 32, 35). The resultant prolonged increase in Rho/Rho kinase activity and in resultant arterial reactivity could potentiate long-term vascular changes typical of hypertension such as enhanced stiffness, medial hypertrophy, and vascular remodeling (26).

The mechanisms by which GLUT4 affects MYPT phosphorylation and possibly RhoA/Rho kinase activity have not yet been elucidated. However, there is the potential for interactions of GLUT4 with serotonergic signaling pathways. 5-HT2A and 5-HT2B receptors and other G protein-coupled receptors activate heterotrimeric G proteins G12/13 (13, 34) or Gq/11 (6, 9) to signal to RhoA in VSM cells (VSMC). Given the apparent localization of both GLUT4 and 5-HT2 receptors in or near lipid rafts/caveolae (5, 11, 31), GLUT4 may interact with these receptors and thereby affect signaling through G12/13 or Gq/11 to RhoA/Rho kinase. Recent studies have found that the downstream glycolytic enzyme phosphofructokinase is also closely associated with caveolae and recruited to caveolae by caveolin-1 in VSMC (33), strongly suggesting that the entire glucose uptake and glycolytic machinery is localized to these structures. These findings support the concept that changes in GLUT4 expression could alter expression or activity of downstream signaling molecules directly or indirectly, by regulating the expression or function of 5-HT2 receptors or other G protein-coupled receptors. In addition, since caveolin-1 has been shown to modulate 5-HT2A signaling by facilitating the interaction of 5-HT2A receptors with Gq (5), altered GLUT4 expression could indirectly alter G protein-coupled receptor signaling via effects on caveolin-1.

In agreement with what we previously showed in aortas from DOCA-salt hypertensive rats (2), markers of the mature VSM phenotype were reduced in aortas of DOCA-salt hypertensive mice. However, the decline in expression of h-caldesmon and SM2 was similar in aortas from wild-type DOCA-salt and GLUT4 TG DOCA-salt mice, suggesting that the expression of GLUT4 had no effect on the expression of these markers. There was a reduction in Akt phosphorylation in aortas of wild-type DOCA-salt mice, as others have observed in aortas of DOCA-salt hypertensive rats (28). Interestingly, pAkt levels were reduced in normotensive GLUT4 TG mice, with no further decrease observed with hypertension. Multiple studies have found that Akt phosphorylation plays an important role in maintaining marker expression (2, 16, 22, 29), and we have demonstrated (2) that restoration of marker expression in hypertensive vessels is associated with increased pAkt expression. There was not, however, any decrease in caldesmon or SM2 in aortas of normotensive GLUT4 TG mice. It is possible that under normotensive conditions reduction in pAkt expression is not sufficient to induce dedifferentiation.

The findings that preserved GLUT4 expression can prevent enhanced VSMC contractility, and a biochemical hallmark of Ca2+ sensitization in hypertension underline the potential importance of GLUT4 reduction in the altered contractile responses found in hypertensive arteries. It will be important to dissect the mechanisms by which GLUT4 expression and function lead to these seemingly protective responses.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-65667 and HL-60156 to F. C. Brosius and American Heart Association Scientist Development Grant 0430045N to J. L. Park.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. B. Atkins and F. C. Brosius 3rd, Univ. of Michigan, 1560 MSRB2, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0676 (e-mail: katkins{at}umich.edu, fbrosius{at}umich.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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