Cardiovascular Research

Cardiovascular Research 1997 33(1):223-229; doi:10.1016/S0008-6363(96)00171-X
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

-Toxin-permeabilised rabbit fetal ductus arteriosus is more sensitive to Ca²⁺ than aorta or main pulmonary artery

Catherine A. Crichton^a,*, Gordon C.S. Smith^b and Godfrey L. Smith^a

^aInstitute of Biological and Life Sciences, West Medical Building, Glasgow University, Glasgow, G12 8QQ, UK
^bDept of Obstetrics and Gynaecology, Glasgow Royal Infirmary, Glasgow, UK

Received 31 December 1995; accepted 14 July 1996

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objectives: The Ca²⁺ sensitivity of contractile protein-generated tension production was measured in the smooth muscle of the rabbit ductus arteriosus and compared with two neighbouring fetal blood vessels (main pulmonary artery and aorta). The effect of prostaglandin E₂ (PGE₂), 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), cyclic adenosine 3',5'-monophosphate (CAMP) and forskolin (an activator of adenylate cyclase) on Ca²⁺-activated force generated by preparations from ductus arteriosus was also examined. Methods: Strips of smooth muscle from the three vessels were permeabilised using crude -toxin from Staphylococcus aureus. The relationship between [Ca²⁺] and force production was then measured in the three tissues and the effect of PGE₂, cAMP, IBMX and forskolin was examined on submaximal Ca²⁺-activated force (0.3 µM Ca²⁺) in preparations from rabbit ductus arteriosus. Results: Permeabilised smooth muscle from fetal rabbit ductus arteriosus was significantly more sensitive to Ca²⁺ (EC₅₀, 0.20 µM) than its two neighbouring blood vessels aorta (EC₅₀, 0.52 µM) and main pulmonary artery (EC₅₀, 0.72 µM). Submaximal Ca²⁺-activated force (0.3 µM Ca²⁺) was depressed by PGE₂ (1 nM) in the presence of IBMX (10 µM), by cAMP (10 and 100 µM) and by forskolin alone (0.1 µM and 1 µM). Conclusion: PGE₂-mediated depression of Ca²⁺-activated force in the smooth muscle of the ductus arteriosus may play a role in the maintenance of a patent ductus arteriosus in the fetus. The intrinsically high Ca²⁺ sensitivity of smooth muscle contractile proteins may aid the sustained vasoconstriction of the ductus when the PGE₂ levels fall after birth.

KEYWORDS -Toxin; Calcium sensitivity – Ductus arteriosus – Prostaglandin – cAMP – Rabbit, vascular myocytes – Rabbit arteries

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The ductus arteriosus is a fetal shunt blood vessel which extends between the main pulmonary artery and the aorta[1, 2]. In fetal life, it diverts deoxygenated blood away from the pulmonary circulation to the descending aorta and ultimately to the placenta. At delivery of the fetus, the smooth muscle in the wall of the ductus arteriosus contracts, closing the vessel [2]. Later the lumen closes permanently by necrosis of the vessel wall and the eventual formation of the ligamentum arteriosus.

Prostaglandins, principally prostaglandin E₂ (PGE₂), are implicated in maintaining patency of the ductus arteriosus in the fetus and are produced within the smooth muscle of this vessel [3, 4]. Circulating concentrations of PGE₂ (1–2 nM) [5] are in the range which dilate the ductus and are thought to maintain the ductus in the patent state in utero. Circulating concentrations of PGE₂ fall to 10% of fetal levels within hours of birth [5] and increasing oxygen tension decreases the sensitivity of the ductus to the dilator effect of PGE₂ [6–8]. Prostaglandins E₁ and E₂ are administered to the neonate in conditions where it is desirable to maintain ductus patency and indomethacin, a cyclo-oxygenase inhibitor which blocks synthesis of prostaglandins, is the main medical therapy for patent ductus arteriosus in the neonate [9]. It is thought that PGE₂ dilates the ductus through activation of adenylate cyclase, as PGE₂ increases intracellular concentrations of cyclic adenosine 3',5'-monophosphate (cAMP [10]). Furthermore, the receptor mediating the effects of PGE₂ on the ductus has been identified as the EP₄ sub-type [11]. The receptor has been cloned and when transfected into cultured cells is coupled to adenylate cyclase [12–14].

Closure of the ductus is in part due to elimination of the effects of PGE₂. However, it is the immediate rise of PO₂ at birth that is thought to mediate principally the ductus closure. One possible mechanism is the inactivation of K_ATP channels and subsequent depolarisation of the smooth muscle cells [15, 16].

The aim of this study was to examine the Ca²⁺ sensitivity of tension production by the contractile proteins of the smooth muscle from rabbit ductus arteriosus and to compare this with the Ca²⁺ sensitivity of the smooth muscle from two adjacent blood vessels: aorta and main pulmonary artery. Permeabilisation of the smooth muscle by -toxin from Staphylococcus aureus renders the surface membrane permeable to molecules below a molecular weight of 1000 Da while retaining functional membrane bound receptors and their associated G-proteins and enzyme systems [17–19]. This technique allows examination of the intracellular effects of receptor activation. This study has also examined the effect of PGE₂, cAMP and forskolin on submaximal Ca²⁺-activated force.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1. Tissue
At day 28 of their 31-day gestation, pregnant New Zealand White rabbits were killed with an intravenous dose of sodium pentobarbital (Euthatal) followed by exsanguination. The fetuses were rapidly removed by Caesarean section and decapitated before onset of respiration, and the ductus arteriosus, aorta and the main pulmonary artery were quickly dissected. Small strips of circular muscle were dissected from the three vessels (approximately 100 µm in diameter and 2–3 mm in length) and attached to a force transducer (Akers, AE17625; SensoNor a.s., Norway) and a fixed point with snares in a small tissue bath (0.96 ml). Whilst superfused with Tyrodes' solution (Solution C; Table 1), a small amount of resting tension (0.15 mN) was applied. All diagrams show active isometric tension generated by the preparation. Isolation of the smooth muscle layer from the wall of the vessel allows the smooth muscle responses to be studied in the absence of endothelium-derived factors. The investigation was performed in accordance with the Home Office Guidance on the operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London.

View this table: [in this window] [in a new window]   Table 1 Solution composition (in mmol.1–1 except where stated) The pH of Solutions A and B was 7.2; free Mg2+ was 2.3 mM; Solution C had a pH of 7.4. Experiments were carried out at room temperature (20–22°C). Pi = inorganic phosphate.

 
2.2. -Toxin permeabilisation procedure
The smooth muscle preparation was permeabilised by superfusing with a mock intracellular solution (Solution A, Table 1; [Ca²⁺] 100 µM) containing crude -toxin from Staphylococcus aureus (final concentration 2 mg/ml) [19]. Tension rose slowly over a 10–15 min period as the Ca²⁺ gained access to the myofilaments. When tension had plateaued, the -toxin was removed and the [Ca²⁺] lowered to 1 nM (Solution B, Table 1). Lowering the [Ca²⁺] caused the muscle to relax. Experiments were carried out at room temperature to limit deterioration of maximum Ca²⁺-activated force and at ambient oxygen tension.

2.3. Solution composition
Solution composition is given in Table 1. The Ca²⁺ buffer ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used to control [Ca²⁺]. The solutions contained adenosine triphosphate (ATP) and phosphocreatine (PCr) to support contraction of the permeabilised muscle. A range of [Ca²⁺] was obtained by mixing Solutions A and B in differing proportions. The equilibrium concentrations of metal ions were calculated using a computer programme with affinity constants for H⁺, Ca²⁺, and Mg²⁺ binding to EGTA taken from Smith and Miller [20]. The affinity constants used for ATP and PCr are those quoted by Fabiato and Fabiato [21]. Corrections for ionic strength, EGTA purity and the principles of the calculation are detailed elsewhere [20]. All chemicals were purchased from Sigma, UK, except -toxin (Glasgow University) and PGE₂ (Upjohn, Kalamazoo, MI, USA). The stock solution of PGE₂ [(5Z,11,13E,15S)-11,15-dihydroxy-9-oxoprosta-5,13-dien-1-oic acid)] was dissolved in ethanol. All experiments were performed in the presence of 1 µM indomethacin to inhibit intrinsic prostaglandin production by the smooth muscle.

2.4. Statistics
Experiments were carried out on tissue from at least 4 separate animals unless otherwise stated. The fitted curves drawn through the data in Fig. GR2 and Fig. GR3 were the best fit curves using the Hill equation (see legend, Fig. GR2) using the computer programme Fig-P (Biosoft, UK). The values for the calculated variables within the Hill equation (T_max, K_m and N) are quoted with the 95% confidence interval (CI) in the legends of Fig. GR2 and Fig. GR3. Statistically significant differences between the curves in Fig. GR2 and Fig. GR3 were calculated by comparing the 95% confidence limits on the variables. All EC₅₀ values quoted are the reciprocal of the calculated K_m in the Hill equation. Statistically significant differences in the data shown in Fig. GR4 and Fig. GR5 were tested using the appropriate Student t-test and the data are expressed as mean values ± standard error of the mean (s.e.m.).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR2 Cumulated data examining the relationship between Ca2+ and tension production by the contractile proteins in -toxin-permeabilised rabbit ductus arteriosus, rabbit aorta and rabbit main pulmonary artery. Data plotted are the steady-state tensions (mean ± s.e.m.) from 4 experiments from different blood vessels. Continuous line represents the best-fit curve of the following form of the Hill equation: where T/Tmax is a fraction of maximal Ca2+-activated force (Tmax) and Km is the apparent affinity constant of the myofilaments for Ca2+. Values quoted are the means ± 95% CI. Tissue Tmax Km (x 106 M) EC50 (x 10–7 M) N Ductus arteriosus 0.99±0.03 5.01±0.46 2.00±0.18 1.81±0.25 Aorta 0.97±0.07 1.93±0.53 5.18±1.38 1.27±0.39 Pulmonary artery 0.97±0.05 1.39±0.25 7.19±1.54 1.44±0.34

 

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR3 The relationship between Ca2+ and tension in the presence and absence of 1 µM indomethacin in -toxin-permeabilised rabbit ductus arteriosus. Data plotted are the steady-state tensions (mean ± s.e.m.) from 4 experiments from different blood vessels. Continuous line represents the best fit curve of the Hill equation given in Fig. GR2. Values quoted are the means ± 95% CI. Tmax Km (x 106 M) EC50 (x 10–7 M) N Indomethacin 1 µM 0.99±0.03 5.01±0.46 2.00±0.18 1.81±0.25 Indomethacin 0 0.98±0.05 4.03±0.69 2.48±0.44 1.54±0.35

 

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR4 The effect of IBMX (1 and 10 µM) and PGE2 (1 nM) on submaximal Ca2+ activated force (0.3µM Ca2+) in -toxin permeabilised rabbit ductus arteriosus. IBMX and PGE2 were added to the bathing medium as indicated above the trace. Bathing [Ca2+] was changed as indicated below the tension trace.

 

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR5 Mean effect of IBMX (10 µM) and PGE2 (1 nM) on submaximal Ca2+-activated force (0.3 µM Ca2+) in -toxin-permeabilised rabbit ductus arteriosus. Tension has been expressed as a percentage of the Ca2+-activated force measured as 0.3 µM Ca2+. Columns represent the averaged data and bars the s.e.m. from 4 experiments from different vessels (P < 0.01).

 

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1. Comparison of the Ca²⁺ sensitivity of force production in -toxin-permeabilised ductus arteriosus, aorta and main pulmonary artery
The Ca²⁺ sensitivity of force production was measured by cumulatively raising the [Ca²⁺] from 1 nM to 100 µM for 10 min at each [Ca²⁺] in -toxin-permeabilised smooth muscle from ductus arteriosus, aorta and main pulmonary artery. Typical examples of normalised Ca²⁺-activated force are shown for each of the tissues in Fig. GR1. Preparations derived from ductus arteriosus achieved on average a maximum force of 0.4 ± 0.05 mN, n = 25; aorta, 0.1 ± 0.02 mN, n = 4; main pulmonary artery, 0.3 ± 0.02 mN, n = 4 (mean ± s.e.m.). The threshold [Ca²⁺] for activated force production was 0.1 µM in the three permeabilised muscles. However, maximum Ca²⁺-activated force was achieved at 10 µM for smooth muscle from ductus arteriosus, but not until 100 µM in aorta or pulmonary artery.Fig. GR1 suggests that the rate of development of tension development is more rapid in the preparations from ductus arteriosus than from aorta and main pulmonary artery; however, this was not a consistent observation and there appeared to be no significant difference in the rate of rise of tension at any of the [Ca²⁺]'s used in the study.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR1 Relationship between Ca2+ and tension in -toxin-permeabilised rabbit ductus arteriosus (panel A), rabbit aorta (panel B) and rabbit main pulmonary artery (panel C). [Ca] was raised as indicated by the line below the tension trace.

 
The relationship between Ca²⁺ and tension for the three permeabilised smooth muscles is shown in more detail inFig. GR2. The curves drawn through the data (see legend, Fig. GR2) are expressed as a percentage of the response at 100 µM Ca²⁺. Rabbit ductus arteriosus (RDA) is significantly more sensitive to Ca²⁺ over the range of [Ca²⁺] examined (1 nM-100 µM) than either aorta (RA) or main pulmonary artery (RPA) [RDA, EC₅₀ 2.00 ± 0.18 x 10^–7 M; RA, EC₅₀ 5.18 ± 1.38 x 10^–7 M; RMPA, EC₅₀ 7.19 ± 1.54 x 10^–7 M (mean ± 95% CI, n = 4), P < 0.05], whereas there is no significant difference between the curves drawn though the data for aorta and main pulmonary artery [RA, EC₅₀ 5.18 ± 1.38 x 10^–7 M; RMPA, EC₅₀ 7.19 ± 1.54 x 10^–7 M (mean ± 95% CI, n = 4), P > 0.05].

3.2. The effect of 1 µM indomethacin on Ca²⁺ sensitivity of permeabilised ductus arteriosus
Fig. GR3 shows the relationship between Ca²⁺ and tension production by the contractile proteins in -toxin-permeabilised ductus arteriosus in the presence and absence of 1 µM indomethacin. Indomethacin was present in all solutions to inhibit intrinsic PGE₂ production by the smooth muscle [6, 2]. The results suggest that indomethacin (1 µM) caused a small increase in the Ca²⁺ sensitivity of the contractile proteins of -toxin-permeabilised ductus arteriosus, however, the effect was not statistically significant [1 µM indomethacin, EC₅₀ 2.00 ± 0.18 x 10^–7 M; 0 indomethacin, EC₅₀ 2.48 ± 0.44 x 10^–7 M (mean ± 95% CI, n = 4), P > 0.05].

3.3. The effect of PGE₂ on submaximal Ca²⁺-activated force in permeabilised ductus arteriosus
Increasing [Ca²⁺] from 1 nM to 0.3 µM produced a sustained increase in force on average 67 ± 4% (mean ± s.e.m., n = 4) of maximum Ca²⁺-activated force. Fig. GR4 shows the effect of 1 nM PGE₂ on submaximal Ca²⁺-activated force (0.3 µM). PGE₂ was added to the bathing medium, while maintaining the [Ca²⁺] at 0.3 µM in either the absence or presence of 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). IBMX (1 and 10 µM) and PGE₂ (1 nM) alone had no significant effect on submaximal Ca²⁺-activated force (0.3 µM). However, in the presence of IBMX (10 µM), PGE₂ (1 nM) significantly depressed submaximal Ca²⁺-activated force. These data are summarised in Fig. GR5. IBMX (10 µM) and PGE₂ (1 nM) depressed submaximal Ca²⁺-activated force to 96 ± 4% (mean ± s.e.m, n = 4, P > 0.05) and 83.5% (± 9%, mean ± s.e.m., n = 4, P > 0.05), respectively, of the force achieved at 0.3 µM Ca²⁺ alone. In combination, IBMX and PGE₂ depressed submaximal Ca²⁺-activated force to 60 ± 1% (mean ± s.e.m., n = 4, P < 0.01) of the force achieved at 0.3 µM Ca²⁺ alone.

3.4. The effect of cAMP on submaximal and maximal Ca²⁺-activated force in permeabilised ductus arteriosus
cAMP concentrations within the physiologically relevant range of 1 nM to 1 µM had little effect on maximal Ca²⁺-activated force. However, 10 µM cAMP caused a reduction (to 78 and 81% of control values in two experiments) while 100 µM depressed force further (to 30 and 35% of control values). As observed by previous workers[22, 23], cAMP was more effective at submaximal [Ca²⁺] (1 µM Ca²⁺). Under these conditions, 100 µM cAMP inhibited force to approximately 7% of the control value (8 and 6%). As mentioned in the Discussion, these results are similar to the sensitivities observed in other smooth muscle types when exogenous cAMP is added in the absence of phosphodiesterase inhibitors [22, 23].

3.5. The effect of forskolin on submaximal Ca²⁺-activated force in permeabilised ductus arteriosus
Increasing concentrations of forskolin (0.1–10 µM), an activator of adenylate cyclase, depressed submaximal Ca²⁺-activated force (0.3 µM Ca²⁺) in a dose-dependent manner in permeabilised ductus arteriosus. A typical example of these effects is shown in Fig. GR6. On average, submaximal Ca²⁺-activated force was depressed to 98.7 ± 1.3% (mean ± s.e.m., n = 4, P > 0.05) of its original value by 0.1 µM forskolin; 60.6 ± 11.6% (mean ± s.e.m., n = 4, P > 0.05) by 1 µM forskolin and 25.0 ± 6.4% (mean ± s.e.m., n = 4, P < 0.01) by 10 µM forskolin. These effects of forskolin were partially reversible.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR6 The effect of forskolin (0.1, 1 and 10 µM) on submaximal Ca2+-activated force (0.3 µM Ca2+) in -toxin-permeabilised rabbit ductus arteriosus. Forskolin and Ca2+ concentrations in the bathing medium were changed as indicated above and below the tension trace.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The results presented in this paper show that tension production by contractile proteins of -toxin-permeabilised smooth muscle from rabbit ductus arteriosus has a higher Ca²⁺ sensitivity than that observed in smooth muscle from its two neighbouring vessels, namely the aorta and main pulmonary artery. We have also shown that PGE₂ (1 nM) depresses submaximal Ca²⁺-activated force and further results suggests that PGE₂ acts via the stimulation of adenylate cyclase and the production of cAMP.

4.1. Comparing the Ca²⁺ sensitivity of rabbit ductus arteriosus, aorta and main pulmonary artery
One other study has examined the Ca²⁺ sensitivity of the contractile proteins of ductus arteriosus smooth muscle[24] isolated from fetal lambs. The Ca²⁺ sensitivity reported was significantly lower than that observed in this study. The reason for this disparity is unknown—certainly comparing results across species may not be valid, or the difference may arise from the use of saponin to permeabilise the smooth muscle cells [24] rather than -toxin from Staphylococcus aureus (present study). It is known that essential contractile proteins are lost from saponin-permeabilised smooth muscle [25, 26], yet the selective permeabilisation produced by -toxin will prevent the loss of cytosolic proteins.

Tension production by the smooth muscle from rabbit ductus arteriosus was significantly more sensitive to Ca²⁺ than the muscle from two neighbouring vessels (i.e., aorta and main pulmonary artery). Indeed, of the smooth muscles investigated in this laboratory rabbit ductus arteriosus is the most sensitive to Ca²⁺. The [Ca²⁺] required to achieve half the maximal Ca²⁺-activated force in a variety of smooth muscle preparations is as follows (µM): 0.20, rabbit ductus arteriosus; 0.25, rat myometrium; 0.33, rat portal vein; 0.37, rat anococcygeus; 0.43, human umbilical artery; 0.52 rabbit aorta (this study); 0.72 rabbit main pulmonary artery (this study); and 1.04, rabbit portal vein. One point to note is that the experiments described in this study were carried out at ambient oxygen tension, but that much lower oxygen tensions are normally experienced by the ductus arteriosus in utero [1, 2]. It is conceivable that oxygen tensions may affect the Ca²⁺ sensitivity of the contractile proteins and further investigations are required to examine this point.

4.2. Effect of IBMX, PGE₂, cAMP and forskolin on submaximal Ca²⁺-activated force
Previous studies have shown that the intact ductus arteriosus relaxes in response to PGE₂ when precontracted by oxygen or an agonist such as noradrenaline [7, 27]. These studies also demonstrated that indomethacin, a cyclo-oxygenase inhibitor, will contract the isolated ductus arteriosus, suggesting that the intact vessel synthesises and releases prostaglandins that act on the smooth muscle to produce a chronic vasodilation. In this study indomethacin had no effect on the developed tension of permeabilised smooth muscle from rabbit ductus arteriosus, indicating that there was no significant endogenous prostaglandin production by the smooth muscle under these experimental conditions.

Reports in the literature suggest that cAMP mediates the intracellular effects of PGE₂ [10, 28]. Increased intracellular [cAMP] is known to lower intracellular [Ca²⁺] (via the stimulation of the sarcoplasmic reticulum Ca²⁺ pump) and reduce the responsiveness of the contractile proteins to Ca²⁺ and in these two ways reduce tone in a range of smooth muscle types [29]; however, work on the smooth muscle from the ductus arteriosus is rare.

This study reports that addition of cAMP to permeabilised smooth muscle from rabbit ductus arteriosus markedly reduces Ca²⁺-activated force. As with previous studies [22, 23], the effective concentrations of cAMP were much higher than the expected intracellular levels due to the activity of phosphodiesterase within the preparation; much higher sensitivities are observed in the presence of phosphodiesterase inhibitors [30]. Further evidence for the role of cAMP in this tissue is the observation that forskolin, a specific stimulator of the enzyme adenylate cyclase, markedly reduces Ca²⁺-activated force in permeabilised smooth muscle from ductus arteriosus. The concentrations of forskolin needed to produce effects in the permeabilised preparation are greater than in the intact muscle [27]. Possible explanations for this are: (i) diffusion of endogenously produced cAMP out of a permeabilised preparation may prevent the development of high localised concentrations; (ii) the further relaxation caused by the cAMP-mediated decrease of intracellular [Ca²⁺] in intact cells will not apply in permeabilised preparations since the [Ca²⁺] next to the contractile proteins is fixed by the external bathing solution.

Ca²⁺-activated force was depressed in the permeabilised smooth muscle from the ductus arteriosus by PGE₂, but only in the presence of a phosphodiesterase inhibitor (IBMX). These results suggest that cAMP mediates the effects of PGE₂ on the contractile proteins. Comparable levels of PGE₂ markedly decrease tone in intact ductus arteriosus without the addition of IBMX [7]. This disparity between intact and permeabilised preparations may reflect the absence of a barrier to the loss of cAMP generated by the stimulation of adenylate cyclase from permeabilised preparations.

In summary, the experimental work presented in this paper suggests that circulating PGE₂ can maintain ductus arteriosus patency in the fetus in part by depressing the Ca²⁺ responsiveness of contractile proteins of the smooth muscle within the wall of the ductus. This effect appears to be mediated via cAMP. The higher Ca²⁺ sensitivity of the contractile proteins of the ductus arteriosus over the neighbouring vessels may be beneficial to the function of the vessel in utero. Normal cytosolic [Ca²⁺] (100–200 nM) within the smooth muscle of the ductus arteriosus will generate a greater tone than in other vascular smooth muscle; thus the properties of the contractile proteins may contribute to the closure of the ductus when the PGE₂ levels fall after birth.

	Acknowledgements

 
We are grateful to the Wellcome Trust for supporting this research. G.C.S.S. was supported by a Wellcome Trust Clinical Research Fellowship.

	Notes

 
^* Corresponding author. Tel. +44 141 330-5963; Fax +44 141 330-4612.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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