Release by hypoxia of a soluble vasoconstrictor from rabbit small pulmonary arteries

N. P. Talbot, P. A. Robbins and K. L. Dorrington*

University Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK

Corresponding author. E-mail: keith.dorrington@physiol.ox.ac.uk

Accepted for publication: May 29, 2003


    Abstract
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. Soluble pulmonary vasoconstrictors released in response to hypoxia have been reported in pig and rat preparations, but not in rabbit preparations.

Methods. We used myography to evaluate the contribution of a soluble factor to constriction in rabbit small pulmonary arteries (external diameter 300–475 µm) exposed to 45 min hypoxia (PO2=9 mm Hg).

Results. Hypoxia produced gradually intensifying constriction. Return to euoxia (PO2=145 mm Hg) for 30 min relaxed only approximately 30% of the constriction, whereas elution of the myograph bath yielded full relaxation. Reapplication of the eluent gradually restored the constriction to its pre-elution level over a 30-min period.

Conclusions. In this closed system, a soluble factor contributes substantially to hypoxic pulmonary vasoconstriction.

Br J Anaesth 2003; 91: 592–4

Keywords: lung, hypoxia; lung, pulmonary vasoconstriction


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Hypoxic pulmonary vasoconstriction (HPV) probably plays a substantial role in supporting oxygenation during anaesthesia, when matching of ventilation to perfusion can be impaired by atelectasis in dependent alveoli.1 Many studies have shown that a component of the constriction of pulmonary arteries exposed to hypoxia can be attributed to the presence of the endothelium.2 3 Studies are usually unable to distinguish between two modes of communication between endothelial and smooth muscle cells: that of a diffusible factor released from the endothelium and that of direct cellular connection between endothelial and smooth muscle cells.

Previous experiments have suggested a role in HPV for a soluble, diffusible factor that is capable of communicating over distances of several millimetres.4 5 However, the release of such a factor from isolated pulmonary arteries has yet to be demonstrated. We tested the hypothesis that a component of the vasoconstriction seen in small pulmonary arteries from the rabbit would be associated with a soluble factor, the effect of which could be removed from vessels and reapplied with their bathing solution.


    Methods and results
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
This study conformed to the United Kingdom Animals (Scientific Procedures) Act of 1986.

Male New Zealand White rabbits (mean (SEM) body weight 1.7 (0.1) kg, n=8) were anaesthetized with 1–2 ml i.v. of a mixture of ketamine 30 mg ml–1 (Fort Dodge Animal Health, Southampton, UK) and xylazine 7 mg ml–1 (Bayer, Leverkusen, Germany), anticoagulated with heparin sulphate 1000 U i.v. and then killed with a further 2–3 ml of the same anaesthetic mixture. The heart and lungs were excised and immersed in cold (<4°C) physiological salt solution A (PSS-A), containing (mM) NaCl 137, KCl 5.4, KH2PO4 0.4, CaCl2 1.3, MgSO4 0.8, Na2HPO4 0.3, glucose 5.6 and HEPES 14, and adjusted to pH 7.4 with NaOH. Under a dissecting microscope, segments of small pulmonary arteries (external diameter 382 (13) µm, length 1855 (34) µm, arising from the third division of the right pulmonary artery, n=16) were isolated, mounted in a dual myograph and bathed in PSS-B 15 ml at 38°C, containing (mM) NaCl 137, KCl 5.4, KH2PO4 0.4, CaCl2 1.3, MgSO4 0.8, Na2HPO4 0.3, glucose 5.6 and NaHCO3 21, pH 7.40 (0.01). After a period of equilibration, baseline wall tension was set by stepwise increases in internal circumference to be equal to that produced by a transmural pressure of 15 mm Hg in a cylindrical vessel of equal internal circumference. Contractile function was established at the start and the end of each experiment by brief exposure to a depolarizing solution of KCl 75 mM, produced by equimolar substitution of KCl for NaCl in PSS-B, which increased wall tension by 1.3 (0.2) mN mm–1, equivalent to a pressure increase of 132 (10) mm Hg.

During euoxia, the bathing solution was gassed continuously with 5% carbon dioxide, balance air 300 ml min–1, which produced an oxygen partial pressure (PO2) of 145 (2) mm Hg. Hypoxia was induced by switching to oxygen 1.3%, carbon dioxide 5%, balance nitrogen 300 ml min–1, which produced a PO2 of 8.5 (0.2) mm Hg. Bath PO2 was measured continuously using a Clark-type oxygen electrode.

Once contractile function had been confirmed and a stable baseline established, rings were exposed to hypoxia for 45 min. At the end of this period, euoxia was resumed for 30 min before the bath was eluted and the contents replaced with fresh, gassed PSS-B. During elution, bath contents were retained and transferred to a glass container within a water bath (38°C), where they were gassed with 5% carbon dioxide, balance air 300 ml min–1 for 20 min before being returned to the myograph bathing chamber for 30 min. At the end of this period, bath contents were discarded and replaced with fresh, gassed PSS-B.

Figure 1 illustrates the effect of hypoxia on pulmonary arterial rings. Changes in tension (T) are expressed as calculated changes in transmural pressure (P), using Laplace’s law in the form P=T/r, where r is the radius that a vessel of given internal circumference would have if it were cylindrical. In response to hypoxia (t=0 min), a constriction with a simple time-course was initiated within approximately 5 min, and developed gradually over 45 min (P<0.01, analysis of variance with repeated measures). Mean pressure change was +8.7 (2.1) mm Hg, approximately 7% of the response to KCl. On returning to euoxia (t=45 min), vessels continued to constrict for several minutes before undergoing partial relaxation to around 60% of the peak constriction. Elution of the bath (t=75 min) yielded full relaxation; there was no difference between wall tension at t=0 (14 (5) mm Hg) and t=90 min (13 (6) mm Hg; P>0.3, paired Student’s t-test). Reapplication of the eluent produced an initial rapid constriction followed by a more gradual constriction that reached the pre-elution level by 30 min. Again, elution of the bath yielded full relaxation.



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Fig 1 Effect of 45 min hypoxia (PO2=8.5 mm Hg) on rabbit isolated pulmonary artery rings. Arrows indicate elution of the bath and addition of fresh solution. Data are mean (SEM), n=16. Each point reflects the mean of data from previous 3 min.

 

    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
The main finding of this study is that hypoxia induces the release of a soluble factor that contributes substantially to HPV in rabbit isolated pulmonary arteries.

Similar findings have been reported recently in the rat and the pig. Using large (internal diameter (ID) 8–12 mm) preconstricted proximal porcine pulmonary artery rings mounted in a myograph, Gaine and colleagues4 showed that removal of the endothelium eliminated a contraction generated by hypoxia (PO2=8–12 mm Hg for 45 min), and that contraction could be restored by the close proximity of pulmonary valve leaflets, a rich source of endothelial cells. This experiment suggested a role in hypoxia-induced constriction for a soluble, diffusible factor that is capable of communicating over distances of several millimetres and that direct proximity between cells was not essential for them to communicate. Similarly, Robertson and colleagues5 collected perfusate leaving hypoxic rat lung (PO2=15 mm Hg), and were able to induce constriction of isolated small (ID 150–400 µm) preconstricted pulmonary arteries from the same species using a fraction of the perfusate with a molecular weight of less that 3000. Our findings confirm that release of a soluble factor occurs in the rabbit and demonstrate for the first time the release of such a factor from isolated pulmonary artery rings.

The identity of the soluble vasoconstrictor in HPV is unknown. The use of fluorescent intracellular Ca2+-sensitive dyes has shown that endothelium-dependent HPV in isolated rat pulmonary arteries occurs without an increase in intracellular Ca2+ concentration, suggesting that an endothelium-derived factor may sensitize the contractile apparatus to hypoxia.6 This was also a feature of the soluble factor released by hypoxia from rat lung.5 The vasoactive peptide endothelin-1 (ET-1) is released from the vascular endothelium in response to hypoxia, and has been shown to sensitize pulmonary vascular smooth muscle cells to an increase in intracellular Ca2+ concentration.7 However, the studies of both Gaine and colleagues4 and Robertson and colleagues5 used specific receptor antagonists to demonstrate that ET-1 did not account for the component of HPV that was dependent upon the release of a soluble factor.

In summary, a soluble vasoconstrictor is released from rabbit isolated small pulmonary arteries during hypoxia. This factor is stable at 38°C for more than 60 min and is responsible for a significant component of the constriction to 45 min hypoxia.


    Acknowledgements
 
This research was supported by the Dunhill Medical Trust. The authors would like to thank Mr David O’Connor for technical assistance.


    References
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
1 Hedenstierna G, Tokics L, Strandberg A, Lundquist H, Brismar B. Correlation of gas exchange impairment to development of atelectasis during anaesthesia and muscle paralysis. Acta Anaesthesiol Scand 1986; 30: 183–91[ISI][Medline]

2 Ward JP, Robertson TP. The role of the endothelium in hypoxic pulmonary vasoconstriction. Exp Physiol 1995; 80: 793–801[Abstract]

3 Liu Q, Sham JS, Shimoda LA, Sylvester JT. Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence. Am J Physiol 2001; 280: L856–65[ISI]

4 Gaine SP, Hales MA, Flavahan NA. Hypoxic pulmonary endothelial cells release a diffusible contractile factor distinct from endothelin. Am J Physiol 1998; 274: L657–64[ISI][Medline]

5 Robertson TP, Ward JP, Aaronson PI. Hypoxia induces the release of a pulmonary-selective, Ca2+-sensitising, vasocon strictor from the perfused rat lung. Cardiovasc Res 2001; 50: 145–50[CrossRef][ISI][Medline]

6 Robertson TP, Aaronson PI, Ward JP. Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization. Am J Physiol 1995; 268: H301–7[ISI][Medline]

7 Evans AM, Cobban HJ, Nixon GF. ET(A) receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway. Br J Pharmacol 1999; 127: 153–60[Abstract/Free Full Text]





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