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Am J Physiol Heart Circ Physiol 287: H419-H424, 2004. First published March 11, 2004; doi:10.1152/ajpheart.00699.2003
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Leukotriene B4 is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor

Magnus Bäck,1 Hong Qiu,2 Jesper Z. Haeggström,2 and Kiyoto Sakata1

1Division of Physiology, The National Institute of Environmental Medicine, Karolinska Institutet; and 2Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Submitted 23 July 2003 ; accepted in final form 4 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leukotriene B4 (LTB4) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB4 on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB4 for recording mechanical responses and measurements of mediator release, and LTB4 receptor (BLT1) expression was assessed by RT-PCR. Single concentrations of LTB4 induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB4 increased the release of histamine and thromboxane in the bath. The contractions induced by LTB4 were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT1 receptor antagonist U-75302. Pretreatment with all-trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB4, without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187 [GenBank] . Analysis by RT-PCR indicated the expression of a BLT1 receptor in the guinea pig aorta and that BLT1 receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB4 contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT1 receptor-mediated response can be upregulated by all-trans-retinoic acid.

histamine; retinoic acid; endothelium; thromboxane

LEUKOTRIENES (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism (15). Cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) are potent bronchoconstrictors and mediators of asthma (for a review, see Ref. 3), and leukotriene B4 (LTB4) is a potent chemoattractant for human leukocytes, including neutrophils, eosinophils, and T lymphocytes (10, 14, 18, 23, 33, 45). 5-LO and LTs have also been implicated in the development of atherosclerosis, because removal of only one 5-LO allele in mice lacking the LDL receptor confers a dramatic protection against atherosclerotic plaque formation (29). In addition, it was recently demonstrated that polymorphism in the 5-LO promoter is related to an increased carotid artery intima-media thickness (13) and that the gene encoding the 5-LO activating protein (FLAP) is associated with an increased risk for stroke and myocardial infarction (21). Moreover, expression of 5-LO and LT receptors has been demonstrated in atherosclerotic lesions of human vessels (42), but the exact mechanism of action of the LTs in atherogenesis remains to be established.

Previously, cysteinyl-leukotrienes have been reported to induce vasoconstriction as well as endothelium-dependent vascular responses (1–5, 43), whereas there are no previous reports that have associated LTB4 with systemic vasoconstriction. However, LTB4 displays contractile activity in the guinea pig lung parenchyma (11, 20, 38, 40), a response recently shown to contain a pulmonary vascular component (38). From these observations, the present study was initiated with the hypothesis that LTB4 could act as a vasoconstrictor, and the aim of the study was to characterize the responses induced by LTB4 in the guinea pig aorta.

The LTs exert their actions via membrane-bound G protein-coupled receptors consisting of two separate classes: CysLT receptors, activated by LTC4, LTD4, and LTE4 (2, 7), and BLT receptors, activated by LTB4 (7, 46, 47). There are two BLT receptor subtypes, BLT1 and BLT2, that can be pharmacologically recognized using receptor antagonists, where ONO-4057 has been reported to inhibit both BLT receptors, whereas U-75302 is a selective BLT1 receptor antagonist (47). In addition, the BLT1 receptor may be inducible because BLT1 receptor expression has been reported to be upregulated by all-trans-retinoic acid (31, 46), glucocorticoids (31, 44), interferon-{gamma}, and interleukin-5 (23). Therefore, the aim of the present study was also to examine the effects of all-trans-retinoic acid on the guinea pig aorta to determine whether this treatment upregulated BLT1 receptor expression in systemic vasculature and whether this could be correlated to an enhanced functional LTB4-induced response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Male Dunkin Hartley guinea pigs (300–450 g) were asphyxiated by CO2 and bled. The descending thoracic aorta was removed and cut into rings with a length of 2 to 3 mm. In some preparations, the endothelium was mechanically removed by gently rubbing the luminal surface with a metal forcep. Endothelium denudation was confirmed fuctionally (see below) and also histologically in sections of guinea pig aorta stained by hematoxylin and eosin at the end of the organ bath experiments.

All experiments were approved by the local animal ethics committee (Stockholms norra djurförsöksetiska nämnd, reference numbers N317/98 and N14/02).

Organ bath experiments. Preparations were placed in 5-ml organ baths containing Tyrode solution (composition in mM: 149.2 NaCl, 2.7 KCl, 11.9 NaHCO3, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, and 5.5 glucose) gassed with 6.5% CO2 in O2 at 37°C. Resting tensions were kept at 10 mN. Mechanical responses were recorded isometrically via Grass FT-03 force-displacement transducers connected to an EMKA data acquisition system.

After a 60-min equilibration period, with washes every 10 min, norepinephrine (10 µM) was added to the baths. After the plateau of the contraction was reached, cumulative concentrations of acetylcholine (0.1 nM-10 µM) were added, and the relaxant effect was used as an indication of an intact functional endothelium. Likewise, the lack of relaxation to acetylcholine was used to functionally confirm endothelial denudation. After an additional period of 60 min with washes every 10 min and readjustment of resting tension, the preparations were incubated for either 30 min or 4 h (all-trans-retinoic acid) in the absence (control) or presence of the different treatments before a single concentration of LTB4 was added. In some experiments the addition of LTB4 was repeated to examine tachyphylaxis, and in some experiments, norepinephrine, histamine, or calcium ionophore was added to the preparations after the incubation period.

Measurements of TXB2 and histamine. For measurements of mediators in the bath fluid, 120-µl aliquots were withdrawn just before and 2 and 10 min after administration of LTB4. Thromboxane (TX) B2 and histamine were measured using enzyme immunoassay kits from Cayman Chemical (Ann Arbor, MI) and Immunotech (Marseille, France), respectively. The TXB2 assay had a threshold corresponding to ~0.40 pM (15 pg/ml) bath concentration of TXB2, and the data derive from dilutions in the linear portion of the assay curve (between 0.5 and 3 pM). For this antibody, the cross-reactivity with 2,3-dinor-TXB2 is 8.2%, whereas all other tested prostanoids have cross-reactivities of <0.5%. The histamine measurements had a threshold of ~0.5 nM bath fluid concentration. The cross-reactivities with methyl-histamine, histidine, and serotonin were <0.05%.

Analysis of mRNA by RT-PCR. Guinea pig aortic tissue was incubated in either Tyrode solution or Tyrode solution containing retinoic acid (1 µM). The preparations were then frozen in liquid nitrogen after which the tissue was homogenized. Total RNA was prepared using TRIzol reagent (GIBCO Life Technologies), and the purity was assessed from the absorbance ratio A260/280.

RT-PCR was conducted using the GeneAmp/Perkin-Elmer RNA PCR kit as previously described (41). Briefly, total RNA in water was treated with DNase (Invitrogen, 1 U/µl) at room temperature for 15 min, after which EDTA (2.5 mM) was added, and the samples were heated (60°C, 10 min). The reverse transcriptase (RT) reaction was conducted at 42°C (20 min) in PCR buffer containing murine leukemia virus reverse transcriptase (2.5 U/µl), MgCl2 (5 mM), dNTPs (1 mM), RNase inhibitor (1 U/µl), deionized formamide (2.5%), and oligo d(T)16 primer (2.5 µM). Samples were amplified in PCR buffer containing Taq polymerase (0.05 U/µl), MgCl2 (5 mM), dNTPs (0.2 mM), deionized formamide (2.5%), and 1 µM of the respective primers, using a Perkin-Elmer/GeneAmp PCR system 2400. In nested PCR analysis, 30 and 15 cycles were used for the first (5'-TTCGAAAGGTGGTGAAGTTGAC-3'; 5-TGCTCTAATTTGCCCCACTTCT-3') and second (5'-CATCCCATTCCTTCCC CATGCC-3'; 5'-TGGTGGTGATCATCATCCTGGC-3') set of BLT1 receptor primers, respectively, based on the published sequence of the guinea pig BLT1 receptor (6, 28) designed to amplify a PCR product of 475 base pairs. One housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), was amplified as an internal standard using 20 cycles PCR with primers (5'-GCCAACATCAAGTGGGGTGATG-3'; 5'-GTCTTCTGGGTGGCAGTGATG-3') as previously described (12, 35), amplifying a PCR product of 310 base pairs (12).

The PCR products were separated by gel electrophoresis on 1% agarose stained with ethidium bromide and visualized and photographed under a UV transilluminator. For each sample, one negative control (without RT), which yielded no detectable bands, was used as an indication that the RT-PCR products did not come from genomic DNA. Nine guinea pigs were used for this part of the study, and detectable signals of both BLT1 and G3PDH were obtained in preparations from three of these animals, which were then used for quantification of PCR products.

Quantification of PCR products. Semiquantitative analysis of PCR products was performed using PicoGreen fluorescent dye (Molecular Probes; Eugene, OR), as previously described (36). One microliter of amplified DNA in 100 µl TE (10 mM Tris and 1 mM EDTA) buffer were mixed with an equal volume of PicoGreen reagent. Samples were incubated for 5 min at room temperature protected from light in a microtiter plate. The fluorescence was measured (excitation wavelength = 485 nm; emission wavelength = 538 nm) in a SpectraMax Gemini XS fluorometer. The fluorescence of the negative control was subtracted from each sample. The data derive from dilutions within the linear standard curve (between 0.002 and 2 µg/ml double-stranded DNA in the well).

Drugs. Norepinephrine, acetylcholine, histamine, indomethacin, mepyramine, and all-trans-retinoic acid were obtained from Sigma (St. Louis, MO). LTB4 was obtained from Cayman Chemicals (Ann Arbor, MI). Calcium ionophore A23187 [GenBank] was from Calbiochem (San Diego, CA). The following drugs were kindly provided by the respective pharmaceutical company: BAYu3405 (3R-3-[4-fluorophenylsulfonamido]-1,2,3,4-tetrahydro-9-carbazolepropanoic acid) from Bayer (Leverkusen, Germany), metiamide from SKB (Swedeland, PA), U-75302 (6-[6-{3-hydroxy-1E,5Z-undecadienyl}-2-pyridinyl]-1,5-hexanediol) from Pharmacia-Upjohn (Kalamazoo, MI), and ONO-4057 (5-[2-(2-carboxyethyl)-3-{6-(4-methoxyphenyl)-5E-hexenyl} oxyphenoxy] valeric acid) from ONO Pharmaceutical (Osaka, Japan). Oligonucleotides were purchased from CyberGene AB (Huddinge, Sweden).

Norepinephrine, acetylcholine, histamine, mepyramine, and metiamide were dissolved in Tyrode solution. ONO-4057 and A23187 [GenBank] were dissolved in dimethylsulfoxide. LTB4, BAYu3405, U-75302, and all-trans-retinoic acid were dissolved in ethanol. Indomethacin was dissolved in 10% ethanol and 10% 1 M Tris (pH 8.0) in distilled water. KCl was dissolved in distilled water. The final concentrations of ethanol or dimethylsulfoxide in the bath were always below 0.1%

The concentrations of the LTB4 were checked by UV spectrometry using the extinction coefficient 55,000 M–1·cm–1.

Data analysis. All results are expressed as means ± SE, and n indicates the number of animals. Contractions are expressed as a percentage of a final contraction to KCl (40 mM). Measurements of TXB2 and histamine are expressed as molar release per milligram tissue wet weight. Levels of BLT1 receptor mRNA are expressed relative to the housekeeping gene (G3PDH).

Statistically significant differences were determined by either a Student's t-test or one- or two-way ANOVA followed by Tukey test, as appropriate. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Single concentrations of LTB4 (30 nM-1 µM) induced concentration-dependent contractions of the guinea pig aorta (Fig. 1). A second application of LTB4 (300 nM) 1 h (with washes at 10-min intervals) after the first application of LTB4 (300 nM) did not cause any contraction of guinea pig aorta (first: 19.4 ± 2.6%, second: 1.5 ± 0.5%, n = 3, P < 0.05; Student's t-test). Endothelium denudation caused significant inhibition of the contraction induced by LTB4 (1 µM; intact: 37.5 ± 2.9%, denuded: 8.8 ± 3.7%, n = 5, P < 0.05; Student's t-test) but did not significantly alter the contractions induced by norepinephrine (10 µM; intact: 58.1 ± 2.0%, denuded: 63.3 ± 7.1%, n = 5).



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  		Fig. 1. Noncumulative administration of leukotriene B4 (LTB4) (n = 3–7) in endothelium-intact guinea pig aorta. Each point represents the mean ± SE.

 
The time course of the contractions induced by LTB4 is shown in Fig. 2. The response was rapid in onset with a time to maximal contraction of ~2 min, and the contraction had completely faded after 20 min. Treatment with either the cyclooxygenase inhibitor indomethacin or the thromboxane receptor antagonist BAYu3405, as well as treatment with the combination of the H1 receptor antagonist mepyramine (1 µM) with the H2 receptor antagonist metiamide (1 µM), caused a significant inhibition of the contractions induced by LTB4 (1 µM; Fig. 2). The combination of antihistamines with either BAYu3405 or indomethacin completely abolished the contraction (Fig. 2).



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  		Fig. 2. Time course of the contraction induced by LTB4 (1 µM) in endothelium-intact guinea pig aorta and the effects of indomethacin (10 µM, Indo, A), BAYu3405 (3 µM, BAY, B), and the combination of mepyramine (1 µM, Mep) with metiamide (1 µM, Met). Each point represents the mean ± SE of five experiments. *P < 0.05 vs. control; {dagger}P < 0.05 vs. BAY or Indo; $P < 0.05 vs. Mep + Met.

 
The concentrations of both histamine and TXB2, which is the stable metabolite of TXA2, were significantly increased 10 min after exposure to LTB4 (1 µM; Fig. 3).



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  		Fig. 3. Results of measurements of histamine (A) and thromboxane B2 (B) in the organ bath from endothelium-intact guinea pig aorta. Bath fluid was collected before and 2 and 10 min after LTB4 (1 µM) treatment. Each bar represents the mean ± SE of three to five experiments. *P < 0.05 vs. before treatment.

 
Both the unselective BLT1/BLT2 receptor antagonist ONO-4057 (10 µM) and the selective BLT1 receptor antagonist U-75302 (1 µM) significantly inhibited the contractions induced by LTB4 (1 µM; control: 36.1 ± 5.8%, U-75302: 0.2 ± 0.1%, n = 3, P < 0.05; control: 49.4 ± 8.7%, ONO-4057: 0.6 ± 0.1%, n = 4, P < 0.05; Student's t-test).

After 4-h incubation subsequent to the second equilibration period, the contractions induced by LTB4 (0.1 and 1 µM) were significantly greater in preparations treated with all-trans-retinoic acid (1 µM) compared with their parallel controls (Fig. 4). The contractions induced by LTB4 (1 µM) in the presense of all-trans-retinoic acid (1 µM) were inhibited by U-75302 (Fig. 4). All-trans-retinoic acid (1 µM) also significantly increased the release of histamine induced by LTB4 (Fig. 4). In contrast to the results obtained with LTB4, treatment with all-trans-retinoic acid (1 µM) did not alter either the contractions induced by histamine (10 nM-10 µM; Fig. 4) or the histamine release induced by calcium ionophore A23187 [GenBank] (100 nM; Fig. 4).



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  		Fig. 4. Effects of all-trans-retinoic acid on the contractions induced by LTB4 (A, n = 3–4) and histamine (B, n = 4) and on the histamine release induced by LTB4 (C, n = 5–7) and calcium ionophore A23187 [GenBank] (D, n = 6) in the organ bath containing guinea pig aorta. Bath fluid was collected 10 min after LTB4 or A23187 [GenBank] treatment. Each bar represents the mean ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. retinoic acid.

 
A typical example of RT-PCR products from guinea pig aorta is shown in Fig. 5. The fragments amplified with BLT1 receptor and G3PDH specific primers, respectively, had the expected sizes (Fig. 5). In addition, a more marked signal was detected in samples from preparations treated with retinoic acid (1 µM). Semiquantitative analysis of amplified PCR products by the fluorescent dye PicoGreen for the measurement of mRNA levels for the BLT1 receptor revealed a significantly higher fluorescence in preparations treated with retinoic acid compared with controls (Fig. 5).



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  		Fig. 5. RT-PCR comparing BLT1 receptor expression in the guinea pig aorta incubated in the absence (Contr) or presence of all-trans-retinoic acid (RA, 1 µM). A: ethidium bromide-stained gel of PCR products show positive signals corresponding to BLT1 receptor (BLT) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). B: results of semiquantitative analysis of the cDNA concentrations (n = 3) using PicoGreen fluorescence. Each bar represents the mean ± SE. *P < 0.05 vs. retinoic acid.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study provide the first evidence of a vasoconstrictive effect of LTB4 in a systemic vessel. LTB4 is synthesized in the circulation (39), and the expression of both 5-LO and LTA4 hydrolase, i.e., the enzymes involved in LTB4 biosynthesis, has been reported in systemic vessels (32, 42). In addition, the number of 5-LO-expressing cells is increased in advanced atherosclerotic lesions (42). Taken together, those studies suggest that high concentrations of LTB4 arise around the vessels and that the levels of LTB4 may be increased in vascular inflammatory conditions, such as atherosclerosis.

Interestingly, previous studies have failed to demonstrate an LTB4-induced systemic vasoconstriction. Although human internal mammary artery (1) and aorta from normal and spontaneously hypertensive rats (43) have been reported to be unresponsive to LTB4, there are experimental differences between those studies and the present. First, cumulative concentration-response curves were performed in the previous studies (1, 43), which may lead to receptor desensitization. The BLT1 receptor is rapidly desensitized by its ligand in isolated cells (7, 17), and tachyphylaxis of the contractile response to LTB4 has also been reported in the guinea pig lung (11, 38). Gaudreau et al. (17) recently showed that the desensitization of LTB4 responses involves the cytoplasmic tail of the BLT1 receptor and phosphorylation of threonine (Thr308). In the present study, a second administration of LTB4 did not induce contraction of the guinea pig aorta, indicating that a noncumulative dosing of LTB4 may be necessary to avoid tachyphylaxis. Another explanation as to the inability of previous studies to detect an LTB4-induced vasoconstriction may be related to the lower concentrations of LTB4 (100 nM) used in those studies (1, 43).

The vasoconstriction induced by LTB4 in the present study was indirect and mediated mainly via the release of histamine, as demonstrated by the inhibition with antihistamines and by the measurements of histamine release from the preparations after LTB4 challenge. In addition, LTB4 also stimulated the release of TXB2, and because either cyclooxygenase inhibition or TP receptor antagonism inhibited the LTB4-induced contractions, liberated TXA2 may also be involved in the contraction induced by LTB4. These mechanisms of contraction provide some key information. First, besides the observed vasoconstriction, the histamine released by LTB4 may increase microvascular permeability and, in addition to chemotaxis, further promote the inflammatory response induced by LTB4. Second, the vasoconstrictor TXA2 is in addition prothrombotic, supporting a role of LTB4 in the pathogenesis of atherosclerosis.

Endothelium denudation significantly inhibited the LTB4-induced contraction of the guinea pig aorta, suggesting that endothelial cells may be the vascular targets for LTB4. It has previously been reported that the endothelium releases histamine, TXA2, and prostaglandin H2 in response to stimulants (8, 19, 24), supporting that the endothelium indeed is able to produce the mediators released in response to LTB4 in the present study. Furthermore, LTB4 have been reported to increase intracellular calcium concentrations in endothelial cells (27, 34), and LTB4 stimulates endothelium-leukocyte adhesion by activation of endothelium (22, 30, 47). This is however in contrast to results in pulmonary arteries, where the effects of LTB4 recently were reported to be endothelium independent (38), and the reason for this apparent difference between systemic and pulmonary vessels remains to be established.

Both the unselective BLT receptor antagonist ONO-4057 and the selective BLT1 receptor antagonist U-75302 abolished the contraction induced by LTB4 in the guinea pig aorta, suggesting that this response was mediated only via BLT1 receptor activation. In support of these findings, the expression of a BLT1 receptor in the guinea pig aorta was confirmed by RT-PCR. In addition, treatment with all-trans-retinoic acid resulted in an upregulation of the BLT1 receptor expression and also enhanced the functional BLT1 receptor-mediated responses. This enhancement was not due to an increased sensitivity to released histamine, because all-trans-retinoic acid did not alter the contractions induced by histamine. In addition, the LTB4-induced release of histamine was augmented after treatment with all-trans-retinoic acid, whereas that induced by calcium ionophore was unaltered by this treatment. Taken together these results suggest that all-trans-retinoic acid induced a specific enhancement of the LTB4-induced response due to BLT1 receptor upregulation. Interestingly, in HL-60 cells, the BLT1 receptor upregulation by glucocorticoid has been reported to be correlated to enhanced LTB4-induced intracellular calcium mobilization and chemotaxis (31), further supporting the notion that BLT1 receptor upregulation may be one important regulator of LTB4-induced responses. Retinoic acid has also been reported to upregulate the expression of intercellular adhesion molecule-1 (9), transforming growth factor-{beta} (48), tissue-type plasminogen activator (25, 26), and fibroblast growth factor-2 (16) in endothelial cells, supporting that retinoic acid affects vascular functions. In addition to those effects, upregulation of the endothelial LTB4-induced effects suggested in the present study may also be a risk factor involved in the side effects that have been reported for retinoic acid treatment, including pulmonary distress symptoms and thrombotic complications (37).

In conclusion, the present study identifies LTB4 as an endothelium-dependent vasoconstrictor in the guinea pig aorta. The LTB4-induced response involved BLT1 receptor activation and release of histamine and TXA2. In addition to the vasoconstrictive effect, also other effects of the secondary released mediators, such as proinflammatory (histamine) and prothrombotic (TXA2) actions may be implicated in vascular responses to LTB4. In addition, the BLT1 receptor expression was upregulated by treatment with all-trans-retinoic acid, which was correlated to an increased functional response, suggesting that the vascular effects of LTB4 may be enhanced in pathological conditions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by grants to Professor Sven-Erik Dahlén from The Swedish Heart Lung Foundation, The Swedish Medical Research Council (projects 71X-9071 and O3X-10350), The Swedish Foundation for Health Care Sciences and Allergy Research, Ono Pharmaceuticals and Karolinska Institutet.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bäck, Cardiovascular Research Unit, Center for Molecular Medicine, Karolinska Hospital, CMM L8:03, SE-171 76 Stockholm, Sweden (E-mail: Magnus.Back{at}cmm.ki.se).

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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