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Am J Physiol Lung Cell Mol Physiol 294: L733-L738, 2008. First published January 11, 2008; doi:10.1152/ajplung.00403.2007
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Up4A stimulates endothelium-independent contraction of isolated rat pulmonary artery

Yu Gui,1 Michael P. Walsh,1 Vera Jankowski,2 Joachim Jankowski,2 and Xi-Long Zheng1

1Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, the University of Calgary, Calgary, Alberta, Canada; and 2Charite-Universitatsmedizin Berlin, Berlin, Germany

Submitted 27 September 2007 ; accepted in final form 9 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate (Up4A) was reported as a novel endothelium-derived vasoconstrictive factor. Up4A contains both purine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of Up4A on contractility of isolated rat pulmonary artery. Up4A at 1–100 µM stimulated contraction in a concentration-dependent manner. Up4A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of Up4A was inhibited by suramin but not IP5I or desensitization of P2X receptors with {alpha},β-methylene-ATP ({alpha},β-Me-ATP). Up4A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by H1152. Furthermore, unlike ATP and UTP, Up4A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that Up4A is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. Up4A likely acts through a suramin-sensitive P2Y receptor. The contractile effect of Up4A involves the entry of extracellular Ca2+ and release of Ca2+ from intracellular stores but not Ca2+ sensitization via the RhoA/Rho kinase pathway. Up4A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone.

extracellular nucleotides; P2 receptors; smooth muscle contraction

INCREASING EVIDENCE SUGGESTS that extracellular nucleotides play important roles in the regulation of cardiovascular tone (22, 23). In blood vessels, nucleotides, such as ATP and UTP, can be released from adventitial nerves or from platelets and endothelial cells to induce either vasoconstriction or vasodilatation through cell surface purinergic (P2) receptors. P2 receptors are divided into two families: P2X (ligand-gated ion channels) and P2Y receptors (G protein-coupled receptors) (2). P2X receptors are mainly activated by ATP and its analogs, whereas P2Y receptors can be activated by ATP, UTP, and UDP, dependent on the subtypes of P2Y receptors involved (1). Both P2 receptors are expressed in smooth muscle and endothelial cells (33). Pharmacological studies coupled with expression of cloned receptors indicate that P2Y2 and P2Y4 in rat are selective for ATP and UTP, whereas P2Y6 favors UDP (2).

In the pulmonary vasculature, nucleotides stimulate P2 receptors either on endothelial cells to induce endothelium-dependent vasodilatation (21) or on smooth muscle cells to cause vasoconstriction (2, 26). In perfused rat lung, it has been reported that ATP is equipotent as UTP to induce suramin-insensitive vasoconstriction (26). However, in isolated rat intrapulmonary artery, UTP or UDP is a potent, whereas ATP is only a weak, vasoconstrictor (27). These results suggest that multiple P2 receptor subtypes exist in the pulmonary vasculature.

The mechanisms underlying smooth muscle contraction in response to nucleotides involve an increase in cytosolic Ca2+ concentration ([Ca2+]i; Refs. 9, 20, 28). The increase in [Ca2+]i in response to UTP can result from Ca2+ entry from the extracellular space (28) and release from intracellular stores (16, 20, 30). In addition, it has been reported that P2Y receptor-mediated aortic contraction involves the RhoA/Rho kinase pathway (13, 32). However, the molecular mechanisms involved in nucleotide-mediated constriction of the pulmonary artery are largely unknown.

Recently, uridine adenosine tetraphosphate (Up4A) was identified by us (15) as a novel endothelium-derived vasoconstrictive factor. This hypothesis is reinforced by our recent study (14) stressing increased plasma Up4A concentrations in hypertensives compared with normotensives. In isolated perfused kidney, Up4A stimulated vasoconstriction mainly through P2X receptors. Up4A is also the first dinucleotide found in living organisms that contains both purine and pyrimidine moieties, which are known to potentially activate both P2X and P2Y receptors. Since it is present at concentrations of 55.5 ± 15.2 nM in human plasma and can be released from endothelial cells by a variety of stimuli, such as ACh, endothelin, ATP, UTP, and mechanical stress (15), we hypothesized that Up4A may be involved in the regulation of vascular tone under physiological and pathological conditions.

The present study was designed to characterize the effects of Up4A on smooth muscle contractility in isolated rat pulmonary artery and to explore the signaling mechanisms whereby Up4A regulates smooth muscle contraction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drugs. Up4A was purchased from Axxora (San Diego, CA). ATP, ADP, UTP, UDP, 2-methylthio-ATP (2-MeSATP), {alpha},β-methylene-ATP ({alpha},β-Me-ATP), suramin, pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), diinosine pentaphosphate pentasodium salt hydrate (IP5I), EGTA, nitrendipine, H1152, phenylephrine (PE), and ACh chloride were purchased from Sigma-Aldrich Canada (Oakville, Ontario, Canada).

Tissue preparation. Male Sprague-Dawley rats (250–350 g) were cared for in accordance with the recommendations of the Canadian Council on Animal Care and killed by decapitation under halothane anesthesia. The protocol of animal study was approved by the Animal Care and Use Committee at the University of Calgary. The lung and heart were removed and maintained in Krebs solution (pH 7.4) containing 114 mM NaCl, 4.7 mM KCl, 0.8 mM KH2PO4, 1.2 mM MgCl2, 11 mM D-glucose, 25 mM NaHCO3, and 2.5 mM CaCl2. The left and right pulmonary arteries (diameter 1–1.5 mm) were dissected and cut into rings (~2 mm in length), each of which was mounted horizontally in a 5-ml myograph chamber (Danish Myo Technology A/S, Skejbyparken, Denmark) by two metal hooks. One of the hooks was connected to a force transducer, and the other to a micropositioner. In the case of endothelium-denuded preparations, the vessels were mechanically rubbed before mounting. The rings were consistently maintained in Krebs solution that was bubbled with a 95%-5% O2-CO2 gas mixture at 37°C. The tissues were allowed to equilibrate for 60 min at a resting tension of 1 g. Isometric tension was recorded using Myodaq/Myodata 2.1 software (Danish Myo Technology A/S). After equilibration, each ring was contracted with 0.1 µM PE followed by treatment with 1 µM ACh. Vessel segments displaying greater than 50% relaxation to ACh were considered to have intact endothelium. Preparations without endothelium did not relax to ACh.

Experimental procedures. Since there was no observed desensitization or cross desensitization, cumulative concentration responses to nucleotides were studied under basal tone conditions in the preparations with and without intact endothelium. Contraction by nucleotides was normalized relative to maximal tension elicited by 0.1 µM PE in the same tissue. In studies of the effects of antagonists and inhibitors (e.g., suramin, PPADS), reproducible contraction in response to Up4A was examined in the absence and presence of drugs (added 30 min before the application of Up4A). Vasodilator responses were evaluated in the endothelium-intact preparations that were precontracted with PE. When the contractile response to PE reached a stable plateau, cumulative concentrations of nucleotides were added. The interval between individual contractions was 1 h.

RT-PCR. Endothelium-intact pulmonary arteries were used for detection of P2Y2, P2Y4, and P2Y6 mRNA. Total RNA was extracted with the TRIzol reagent (Invitrogen, Burlington, Ontario, Canada) according to the recommendations of the manufacturer. One microgram of total RNA was reverse-transcribed with a first-strand cDNA synthesis kit using (N)6 primer (Pharmacia LKB Biotechnology, Uppsala, Sweden) at 37°C for 1 h. Two microliters of RT product were used to amplify the fragments of P2Y2, P2Y4, and P2Y6 using primers and conditions as previously described (8). The PCR products were separated using 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The expected products for P2Y2, P2Y4, and P2Y6 were 337, 377, and 450 bp, respectively. To confirm their identity, the PCR products amplified by P2Y2, P2Y4, and P2Y6 primers were purified with the Magic DNA Purification Kit (Pharmacia LKB Biotechnology) for DNA sequencing analysis (DNA Sequencing Facility, University of Calgary, Calgary, Alberta, Canada).

Data analysis. pD2 is the concentration of nucleotide that elicits 50% of the maximal observed response (Emax). Data represent the means ± SE (error bars on graphs in figures; n = number of separate experiments). Statistical comparisons of concentration-response parameters (pD2 and Emax) between two experimental groups were made using Student's t-test for paired data. Comparison of contractile responses to different nucleotides was made by one-way ANOVA followed by Newman-Keuls post hoc test (P < 0.05 was considered statistically significant).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The effect of Up4A on the isolated rat pulmonary artery under basal tone was first evaluated in endothelium-intact and -denuded preparations. Cumulative concentrations of Up4A (10 nM to 100 µM) were used. Up4A at concentrations of 1–100 µM induced concentration-dependent contraction in both preparations (Fig. 1, A and B). The Emax (% of the PE response) and pD2 of Up4A were 149% ± 21% and 10 ± 1.8 µM in the absence of endothelium and 138% ± 42% and 12 ± 2.3 µM in the presence of endothelium, respectively (n = 8; P > 0.05).


Figure 1
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  		Fig. 1. Uridine adenosine tetraphosphate (Up4A) induces endothelium-independent contraction of rat pulmonary artery. A and B: representative recordings of tension in response to increasing concentrations of Up4A (10 nM to 100 µM) in the endothelial-intact (A) and -denuded preparations (B). The lack of a relaxant response to ACh (1 µM) in phenylephrine (PE)-precontracted rings (B) confirmed effective removal of the endothelium. C and D: cumulative concentration-response curves for Up4A UTP, UDP, 2-methylthio-ATP (2-MeSATP), and ATP in endothelium-intact (C) and -denuded pulmonary arteries (D). The values (means ± SE, n = 6–8) are expressed as percentages of the maximal contraction elicited by PE (0.1 µM).

 
The constrictor effects of Up4A were then compared with other nucleotides. In the endothelium-intact pulmonary artery, ATP (up to 100 µM) did not cause contraction, whereas UTP and UDP induced minor contractions at 10–100 µM compared with the robust Up4A (1–100 µM)-induced constriction (Fig. 1C). In the endothelium-denuded preparation, the contraction induced by Up4A was similar to that in response to UTP or UDP (Fig. 1D). Emax values for UTP and UDP were 123% ± 21% and 125% ± 15% of the PE response, respectively. However, ATP and 2-MeSATP, a selective P2X agonist, elicited only weak contractions with Emax values of 29% ± 7% and 15% ± 3%, respectively (Fig. 1D). The rank order of potency was Up4A ≥ UTP ≥ UDP >> 2-MeSATP > ATP.

To explore whether P2X or P2Y receptors are involved in the Up4A-induced contraction, we examined the effects of IP5I (a P2X antagonist), suramin, and PPADS (nonselective P2Y receptor antagonists) on the contractile response to Up4A (10 µM). Pretreatment with suramin (50 µM), but not PPADS (10 µM) or IP5I (100 µM), significantly inhibited Up4A-induced contraction (Fig. 2A), suggesting that Up4A-induced contraction occurs via suramin-sensitive P2Y receptors but not P2X receptors. To further examine this possibility, {alpha},β-Me-ATP (a P2X agonist) was used to desensitize P2X receptors (19). After successive applications of {alpha},β-Me-ATP (10 µM), the arteries no longer responded to {alpha},β-Me-ATP but were still able to contract in response to Up4A (Fig. 2B). Up4A-induced tension after {alpha},β-Me-ATP treatment was 94% ± 6% of Up4A-evoked tension before {alpha},β-Me-ATP treatment (P ≥ 0.05; n = 5), supporting the conclusion that P2Y receptors are involved in Up4A-induced contraction.


Figure 2
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  		Fig. 2. Involvement of P2Y receptors in Up4A-induced contraction of rat pulmonary artery. A: the contractile responses to Up4A (10 µM) in the absence and presence of P2 receptor antagonists, suramin, pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), and diinosine pentaphosphate pentasodium salt hydrate (IP5I), respectively. Data are expressed as means ± SE (n = 6; *P < 0.01). N.S., not significant. B: representative recording of tension in response to Up4A before and after desensitization of P2X receptors achieved by successive applications of {alpha},β-methylene-ATP ({alpha},β-Me-ATP; 10 µM). The recording is representative of 5 experiments.

 
To determine the mechanisms underlying Up4A-induced contraction, we investigated the requirement for extracellular and intracellular sources of Ca2+ and whether the RhoA/Rho kinase pathway of Ca2+ sensitization is involved. The role of extracellular Ca2+ entry in the Up4A-induced response was first evaluated by sequestration of extracellular Ca2+ with EGTA for 10 min before addition of Up4A. EGTA (10 mM) pretreatment, which did not affect caffeine-induced contraction (data not shown), markedly inhibited Up4A-induced contraction. When EGTA was washed out, Up4A-induced contraction was restored (Fig. 3A). We next examined the effects of nitrendipine (a Ca2+ channel blocker) and thapsigargin (an inhibitor of Ca2+ release from the sarcoplasmic reticulum) on Up4A-induced constriction. Both nitrendipine (1 µM) and thapsigargin (1 µM) significantly inhibited the vasoconstrictor effect of Up4A (Fig. 3B; P < 0.01; n = 6). To determine whether Up4A-induced contraction involves the RhoA/Rho kinase pathway, we used H1152 (a highly selective Rho kinase inhibitor; Refs. 12, 31). H1152 (1 µM) had no effect on the contractile response to Up4A (97% ± 11% of Up4A-induced contraction, n = 6; P > 0.05).


Figure 3
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  		Fig. 3. Up4A-stimulated contraction involves extracellular Ca2+ entry and intracellular Ca2+ release. A: representative recording of the response to Up4A in the absence and presence of EGTA. EGTA (10 mM) was added 10 min before the application of Up4A (10 µM). The interval between each application of Up4A was 1 h. The recording is representative of 6 experiments. B: cumulative data showing the effects of nitrendipine (1 µM) and thapsigargin (1 µM) on Up4A-induced contraction. *P < 0.01 when compared with the control group (n = 6).

 
We also investigated whether Up4A has a relaxant effect on PE-precontracted tissue. In endothelium-intact pulmonary arteries, which relaxed in response to ACh, the addition of ATP or UTP, at concentrations of 100 nM to 10 µM, induced a concentration-dependent relaxation, and at 100 µM the relaxation was reversed, but the tension was still lower than that of PE-precontracted tissue (Fig. 4A). In contrast, Up4A only induced a small relaxant response at a concentration of 1 µM, which was reversed by 10 µM Up4A to a level exceeding the PE-preconstricted tension (Fig. 4A). UDP had a lower relaxant effect than UTP or ATP. In endothelium-denuded preparations, the addition of ATP, UTP, UDP, or Up4A did not cause relaxation (data not shown). Figure 4B summarizes the concentration-dependent responses of PE-precontracted tissues to the nucleotides. ATP (1–100 µM) and UTP (1–10 µM) induced significant reductions in tension in the PE-precontracted preparations, whereas Up4A did not cause significant relaxation. The rank of order of potency of nucleotides to induce endothelium-dependent relaxation was ATP ≥ UTP >> UDP > Up4A.


Figure 4
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  		Fig. 4. Up4A does not dilate the PE-precontracted rat pulmonary artery. A: representative recordings showing that both ATP (10 nM to 10 µM; bottom, right) and UTP (100 nM to 10 µM; top, right) induced relaxant responses in PE-precontracted preparations, whereas Up4A induced only a small relaxation at the concentration of 1 µM (top, left). UDP also caused weak relaxation (bottom, left). B: mean data of PE-elicited tension in the presence of cumulative concentrations of Up4A, UTP, UDP, or ATP. The relaxant responses in the presence of UTP or ATP (1–10 µM) were significant when compared with those in the absence of UTP or ATP (P < 0.05; n = 8).

 
The potency of nucleotides to induce relaxation of precontracted pulmonary arteries suggests that P2Y2/P2Y4 receptor subtypes are involved in the relaxation. However, since the rank of order of potency of nucleotides to evoke contraction was Up4A ≥ UTP ≥ UDP >> 2-MeSATP > ATP, it is likely that the P2Y6 subtype is involved in nucleotide-evoked contraction. In support of these conclusions, RT-PCR confirmed the expression of P2Y6, P2Y4, and P2Y2 mRNAs in rat pulmonary artery (Fig. 5).


Figure 5
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  		Fig. 5. Expression of P2Y receptors in the rat pulmonary artery. Expression of P2Y2, P2Y4, and P2Y6 mRNA was detected by RT-PCR as described in MATERIALS AND METHODS. The image is representative of 4 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The effects of different nucleotides on the rat pulmonary vasculature are diverse. It has been shown that UTP and UDP are equipotent constrictors, whereas ATP is a less effective constrictor in the large isolated intrapulmonary arteries (6, 27). Interestingly, in the large isolated intrapulmonary arteries, ATP is a potent, whereas UTP or UDP is only a weak, vasodilator (27). The vasoconstrictive activity of UTP and UDP can be inhibited (6, 11) or unaffected by suramin (26, 27). These results suggest complexity of the pulmonary vasculature in response to nucleotides, likely involving multiple P2 receptor subtypes.

Our data support the conclusion of Rubino et al. (27) that UTP and UDP have similar vasoconstrictor potencies. In contrast, 2-MeSATP and ATP are much less effective vasoconstrictors. Importantly, our study demonstrates that Up4A at concentrations of 1–100 µM induces concentration-dependent contraction of the isolated pulmonary arteries. Up4A is equipotent as UTP and UDP in the endothelium-denuded artery. However, Up4A is a much more effective vasoconstrictor than UTP, UDP, or ATP in endothelium-intact preparations. Unlike UTP and UDP, Up4A-stimulated contraction is independent of the endothelium. Although the concentrations of Up4A in human plasma are in the nanomolar range (15), which is close to that of ATP, the effective concentrations of Up4A observed in the present in vitro study are in the micromolar range. Given that Up4A can be released from endothelial cells by a variety of stimuli (15), it is reasonable to predict that Up4A could be as important as ATP and UTP in the regulation of vascular tone under pathophysiological conditions.

The present study demonstrated that the rank of order of potency of the nucleotides to induce contraction is Up4A ≥ UTP ≥ UDP >> 2-MeSATP ≥ ATP. Since P2X receptors are mainly selective for ATP and its analogs, and they are inactive to UDP and UTP (1, 2), the vasoconstriction activity of Up4A in the rat pulmonary artery likely involves P2Y but not P2X receptors. This conclusion was supported by the finding that the Up4A response was unaffected by IP5I and desensitization of P2X receptors. Among the subtypes of P2Y receptors, P2Y2, P2Y4, and P2Y6 are responsive to nucleotides with a pyrimidine moiety (2), ATP and UTP are equally effective on P2Y2 and rat P2Y4 receptors (3, 5, 24), and only P2Y6 is selective for UDP (7, 25). If Up4A, UTP, and UDP act through the same receptor, it is most likely that P2Y6 mediates the contraction. Expression of P2Y6 mRNA in the pulmonary artery was confirmed by RT-PCR (Fig. 5). However, the Up4A-induced contraction was inhibited, but not abolished, by suramin, which contrasts with the findings that P2Y6 is usually insensitive to suramin (4, 25). Thus Up4A mediates contraction probably via both suramin-sensitive and suramin-resistant P2Y receptors. In addition, Up4A-mediated contraction was not inhibited but rather slightly enhanced by PPADS. This could be due to inhibition of Up4A breakdown by PPADS, since it can inhibit the ectonucleotidases present in smooth muscle (17). This observation is consistent with the finding that PPADS potentiated, whereas suramin inhibited, UTP- and UDP-evoked contractions in isolated rat pulmonary arteries (6). Therefore, the native receptors mediating constriction of the pulmonary artery in response to Up4A cannot be identified with any of the currently known P2Y subtypes. We cannot rule out the possibility that Up4A acts through a novel suramin-sensitive, uridine-specific receptor to induce contraction.

It has been suggested that nucleotides induce vasodilatation, and the vasodilator effect of nucleotides is linked to P2Y receptors (10, 21). In the isolated rabbit pulmonary artery (21) and in the mouse aorta preparation (10), ATP, UTP, and UDP have been shown to induce vasodilatation. However, in the isolated rat intrapulmonary artery, it was reported that only ATP, not UTP, causes vasodilatation (27). The present study demonstrates that ATP and UTP (0.1–10 µM) induce marked endothelium-dependent relaxation of the PE-preconstricted pulmonary artery. In contrast, Up4A and UDP lack a vasodilator effect in the same preparation. Since P2Y2 and P2Y4 receptors are favorable for ATP and UTP, and expression of P2Y2 or P2Y4 mRNA was detected in the pulmonary artery (Fig. 5), it is likely that P2Y2/P2Y4 but not P2Y6 in the endothelial cells mediates the nucleotide-induced relaxation and that Up4A is not an agonist for the endothelial P2Y2/P2Y4 receptors.

It has been reported that external Ca2+ (16, 18, 29) and intracellular Ca2+ stores can contribute to the increase in [Ca2+]i in response to UTP (9, 18, 20, 30). In the present study, Up4A-induced vasoconstriction was inhibited by sequestration of extracellular Ca2+ with EGTA (Fig. 3A) and by blockage of Ca2+ influx with nitrendipine (Fig. 3B), suggesting the involvement of extracellular Ca2+ entry. In addition, Up4A-induced contraction involves the release of intracellular Ca2+, since thapsigargin inhibited the contractile response to Up4A (Fig. 3B). It will be important to investigate the mechanisms by which Up4A regulates extracellular Ca2+ entry and intracellular Ca2+ release in the future. Furthermore, the Up4A response was not affected by the Rho kinase inhibitor, H1152, indicating that the RhoA/Rho kinase pathway is not involved in Up4A-induced contraction.

Taken together, the present study demonstrates that Up4A is a potent vasoconstrictor, but not a vasodilator, of the isolated pulmonary arteries. Up4A-induced contraction may be mediated by a novel P2Y receptor and involves the entry of extracellular Ca2+ and release of Ca2+ from intracellular stores. Up4A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone via its vasoconstrictor action. Further study using other experimental models, such as an isolated perfused lung system with smaller diameter resistance vessels, will lead to better understanding of the pathophysiological roles of Up4A.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Heart and Stroke Foundation of Alberta to X.-L. Zheng, an equipment grant from the Canada Foundation for Innovation to M. P. Walsh, and a German Research Foundation (DFG) Grant (Ja-972/11-1) to J. Jankowski. X.-L. Zheng is the recipient of a New Investigator Award from the Heart and Stroke Foundation of Canada. M. P. Walsh is the recipient of a Canada Research Chair (Tier 1) and Alberta Heritage Foundation for Medical Research Scientist Award. Y. Gui is the recipient of a Postdoctoral Fellowship Award from the Alberta Heritage Foundation for Medical Research. V. Jankowski is a recipient of a Rahel-Hirsch-scholarship from the Charité (Germany).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. P. Walsh and X.-L. Zheng, Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1 (e-mail: walsh{at}ucalgary.ca and xlzheng{at}ucalgary.ca)

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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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