© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Hypoxia induces the release of a pulmonary-selective, Ca2+-sensitising, vasoconstrictor from the perfused rat lung
aDepartment of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, 30602, USA
bDepartment of Respiratory Medicine and Allergy, GKT School of Medicine, King's College London, Guy's Campus, London SE1 9RT, UK
* Corresponding author. Tel.: +1-706-542-3014; fax: +1-706-542-3015 troberts{at}vet.uga.edu
Received 18 July 2000; accepted 28 December 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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Objective: Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. Methods: The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. Results: Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca2+ channels, and was not associated with a rise in intracellular [Ca2+], suggesting Ca2+-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F2
. Conclusion: We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca2+ sensitisation, is independent of voltage-gated Ca2+ entry, and is potentiated by the presence of preconstriction.
KEYWORDS Arteries; Caclium (cellular); Hypoxia/anoxia; Pulmonary circulation; Vasoactive agents
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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Hypoxic pulmonary vasoconstriction is a unique homeostatic mechanism resident within the lung which maintains efficient ventilation-perfusion matching in the event of localised alveolar hypoxia, yet can lead to pulmonary hypertension when hypoxia is global. However, despite extensive research the exact mechanisms underlying HPV remain unresolved. In rat isolated intrapulmonary arteries (IPA) HPV typically takes the form of a biphasic response which is potentiated in the presence of a small degree of agonist-induced tone [1,2]. The first phase is transient in nature, reaching a peak within 5 min (phase 1), and is superimposed upon a more slowly developing, and sustained, constriction (phase 2). It has been proposed that the secondary rise in tension in this model of HPV may be the more physiologically relevant process since it is sustained [3], reflecting the profile of HPV in vivo, and because a similar transient phase 1 constriction can be observed in a variety of systemic arteries [2,4]. We have previously reported that the integrity of the endothelium is pivotal to the development of phase 2 [2]. This is in agreement with several other reports e.g. [5], although this is not a universal finding [6].
Dependence of phase 2 of HPV upon the endothelium would strongly suggest a role for an endothelium-derived constrictor factor (EDCF) in sustained HPV, and indeed hypoxia has been reported to induce the release of an EDCF, distinct from endothelin-1, from pulmonary endothelial cells of the pig [7]. In the present study we have utilised the salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat IPA functional bioassay in an attempt to determine whether hypoxia induces the release of a vasoconstrictor from the lung. We report that a vasoconstrictor(s) is indeed released during hypoxic perfusion of the rat lung, and that this constrictor activity shares some of the properties of phase 2 of HPV in the rat isolated IPA.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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Male Wistar rats (250–350 g) were anaesthetised with sodium pentobarbitone (55 mg/kg i.p.) and killed by cervical dislocation, as approved by the local Home Office Inspector. The heart, lungs and intestines were excised and placed in a physiological salt solution (PSS) containing (in mM): NaCl, 118; NaHCO3, 24; MgSO4, 1; NaH2PO4, 0.435; glucose, 5.56; Na-pyruvate, 5; CaCl2, 1.8; and KCl, 4.
2.1 Perfused lung preparation
The main pulmonary artery was cannulated and the lungs perfused with PSS, gassed at source with 95% air/5% CO2 (PO2 154±5 mmHg, pH 7.38±0.01, 37°C), at a rate of 15 ml/min which typically gave a perfusion pressure of between 10 and 14 mmHg. Hypoxia was induced after 1 h by gassing the PSS at source with 95% N2/5% CO2 (PO2 15±3 mmHg; pH 7.39±0.01; 37°C). The hypoxic challenge was maintained for 1 h, during which the perfusate was collected. Perfusion pressure was monitored continuously via a pressure transducer (Bell and Howell type 4-422) from a side port of the pulmonary cannula. In control experiments the lungs were perfused with normoxic PSS for 2 h, the perfusate from the final hour being collected.
2.2 Perfused mesenteric arterial bed
The mesenteric artery and vein were tied off near the caecum, and the remaining intestine was then dissected free from the arterial bed along the intestinal wall. The mesenteric arterial bed was then perfused as previously described in detail [8]. Briefly, the superior mesenteric artery was cannulated and then perfused with PSS (37°C, pH 7.40±0.01) at a rat of 5 ml/min, gassed at source with 95% air/5% CO2. Hypoxia was induced, and perfusate collected as per the protocol described for the perfused lung above.
2.3 Isolation of vasoactive substances from lung perfusate
Sep-Pak C18 cartridges (Sep-Pak C18 Environmental cartridge 23635, Millipore, Watford, UK) were prepared by perfusion with 30 ml of methanol, and subsequent washing with 30 ml of PSS. Perfusate from three lung or mesenteric bed preparations was then passed through the cartridge at a rate of 5 ml/min. The cartridges were washed with 10 ml of sterile water, and any substances retained by the cartridge eluted in three steps with 20 ml of 30% acetonitrile (ACN), followed by 20 ml of 50% ACN and finally 20 ml of 100% ACN, at a flow rate of 1 ml/min, into polypropylene tubes (50 ml Falcon tubes, Becton Dickinson, Oxford, UK). The fractions were then dried under nitrogen, re-dissolved in 1 ml of PSS and passed through a low protein binding 0.22 µm filter (Millex GV, Millipore, Watford). The fractions were then tested for constrictor activity on isolated rat IPA and mesenteric arteries (MA).
2.4 Isolation and mounting of small rat IPA and MA
IPA and MA (150–400 µm internal diameter, i.d.) were dissected free of connective tissue and mounted in a small vessel myograph (Cambustion AM10 myograph, Cambustion Ltd., Cambridge, UK), as previously described in detail [9,10], and gassed with 95% air/5% CO2 (PO2 140±7 mmHg; pH 7.39±0.01; 37°C). Arteries were subjected to a standard run-up procedure of four exposures to 80 mM KCl-PSS (KPSS, 4-min duration, isotonic replacement of NaCl by KCl) and the presence of a functioning endothelium was determined by acetylcholine challenge (1 µM) following PGF2 (10 µM) induced contraction as previously described [1,11].
2.5 Determination of basic physical characteristics
In an attempt to derive preliminary physical characteristics of the active fractions, these were passed through a microconcentrator (500 µl sample spun at 13 000 g for 90 min, Microcon 3 microconcentrator, Amicon Ltd., Stonehouse, Gloucestershire, UK) which allows only substances with molecular weights less than 3000 to pass through. Active fractions were also tested for heat stability by placing them in 1.5 ml Eppendorff tubes suspended in boiling water for 20 min. During the latter procedure the temperature of the fraction typically reached 97°C.
2.6 Effect of protein kinase C (PKC) inhibition and voltage-gated, calcium-channel inhibition upon the constrictor effect of the hypoxic extract
IPA were exposed to 150 µl of the 30–50% ACN fraction in 6 ml PSS for a period of 10 min, and then washed with PSS. After a further 30 min the arteries were then incubated with either the PKC-selective antagonist Ro 31-8220 (3 µM) or the voltage-gated calcium channel antagonist diltiazem (10 µM) for 20 min prior to re-exposure to 150 µl of the 30–50% ACN fraction for 10 min. Inhibition of PKC, and voltage-gated calcium channels was confirmed by exposure to either 0.3 µM 4b-phorbol dibutyrate (PdBu) or KPSS respectively after the second exposure to the 30–50% ACN fraction. In control experiments IPA were subjected to two exposures of 150 µl of the 30–50% ACN fractions, 50 min apart.
2.7 Estimation of intracellular [Ca2+] ([Ca2+]i)
IPA were loaded with the Ca2+-sensitive fluorophore fura PE-3, via incubation of the vessels with the acetoxymethyl ester of fura PE-3 (3 µM) for 2 h at room temperature, (Sigma–Aldrich Ltd., Poole, Dorset, UK). The vessels were then washed with PSS, the bath temperature raised to 37°C, and the myograph transferred to the stage of an inverted fluorescence microscope (Olympus IMT2, Olympus Ltd., London UK). Changes in [Ca2+]i were assessed by calculating the ratio of the light emitted through a wide band pass 500-nm emission filter when the vessel was illuminated at 340 and 380 nm respectively (Cairn spectrophotometer, Cairn Research Ltd., Newnham, Kent, UK) as previously described [11].
Tension is expressed as a percentage of the maximum constriction (Tk) observed to the final KCl-PSS exposure during the run up procedure. Results are expressed as mean±S.E.M., and Student's t test was used to compare the means. A difference was considered significant if P<0.05.
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3 Results and discussion |
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TopAbstract1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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Induction of hypoxia caused a small biphasic increase in perfusion pressure in the salt-perfused isolated rat lung preparation. This biphasic pressor response had a timecourse similar to HPV in the rat isolated IPA [9,11] and was rapidly reversed upon reoxygenation (Fig. 1).
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3.1 Effect of 0–30% ACN, 30–50% ACN, and 50–100% ACN fractions upon tension in isolated rat IPA
No constrictor activity was observed in either the 0–30% ACN, or the 50–100% ACN fractions (n = 3). However, the 30–50% ACN fraction was found to elicit a slowly developing and sustained constriction in rat IPA (Fig. 2, n = 6). This constrictor activity appeared to be associated with hypoxic perfusion as the equivalent 30–50% ACN fraction from normoxic perfused lungs produced significantly less vasoconstriction (150 µl 30–50% ACN fraction; hypoxic perfusate=29.9±1.0% Tk, n = 6; normoxic perfusate=6.3±1.6% Tk, n = 3, P<0.001). This constrictor activity was retained after passing the active samples through a microconcentrator (3000 molecular weight cut off, n = 3) and following heating to 97°C for 20 min (n = 3). No significant constrictor activity could be found in any of the fractions isolated from the mesenteric bed perfusate (n = 3).
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This suggests that vasoconstrictor release from the lung was stimulated specifically by hypoxia, rather than its production merely being an artefact of perfusion per se, and as such suggests that this fraction might contain a candidate for the EDCF released during HPV. In order to further examine this possibility it was important to establish whether the constriction to this fraction shared similar mechanistic features to those described for phase 2 of HPV in this preparation.
3.2 Selectivity of the active fraction for IPA
HPV, by its very definition, is a pressor response peculiar to the pulmonary circulation. When applied to rat MA the active fraction produced only a small transient increase in tension, compared to that produced in IPA (mesenteric maximum effect 3.8±0.9% Tk, n = 6, P<0.0001, Fig. 2). Dose response curves to the 30–50% ACN fraction were constructed by increasing the volume of the fraction added to the myograph chamber at 10-min intervals. Typically, a maximum effect was attained when 150 µl of this fraction was added to the myograph chamber (bath volume 6 ml, Fig. 3). Interestingly, as with HPV, the constrictor response observed to the 30–50% ACN fraction was rapidly reversed upon washing. The active fraction would, therefore, appear to be relatively selective for rat IPA over MA.
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3.3 Independence of the constrictor activity from voltage-gated Ca2+-entry
We have recently demonstrated that antagonists of voltage-gated Ca2+ entry have no effect upon phase 2 of HPV in this preparation [1]. Diltiazem (10 µM) was also without effect upon the contraction evoked by the active fraction (Fig. 4, n = 3), indicating that activation of voltage-gated calcium channels was not involved.
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3.4 Induction of PKC-independent Ca2+-sensitisation by the active fraction
We have previously reported that the development of phase 2 in rat IPA involves an apparent sensitisation of the contractile myofilaments to [Ca2+]i [11], which is, however, independent of PKC (a pathway which is known to cause Ca2+-sensitisation in this preparation [12]). Fig. 5 shows the effect of 50 µl of the active fraction upon force and [Ca2+]i in a 237-µm i.d. IPA. Upon addition to the myograph chamber the active fraction caused a slow-to-develop constriction without any concomitant rise in [Ca2+]i (n = 4, mean increase in tension 13.2±0.2% Tk, mean increase in fura ratio 0.1±0.1%). This apparent Ca2+-sensitisation appeared to be independent of PKC, since the PKC inhibitor Ro-31-8220 was without effect upon the contraction caused by the active fraction (3 µM, Fig. 4, n = 3). In contrast, 3 µM Ro-31-8220 completely abolished the constriction observed to 0.3 µM PdBu, confirming PKC inhibition at this concentration (n = 3, see also [11]).
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3.5 Potentiation of the active fraction constrictor response by agonist pre-stimulation
It is well established that HPV is potentiated in isolated arteries by a small degree of agonist induced stimulation e.g. [2]. Fig. 6 illustrates the effect of 25 µl of the active fraction added to the myograph chamber before and after 5 µM prostaglandin F2
. This volume of the active fraction produced only a small vasoconstriction in the absence of pre-stimulation, however when added in the presence of prostaglandin F2, it elicited a marked vasoconstriction (Fig. 6, 3.8±0.6% TK vs. 31±2.3% TK, n = 3, P<0.001).
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3.6 Evidence against endothelin-1 being the active constituent of the 30–50% ACN fraction
Endothelin-1 has attracted much attention as a potential mediator of HPV [6]. However, it is unlikely that the active component of the 30–50% ACN fraction is endothelin-1 since the constriction observed to this fraction was readily reversed upon washing, whereas it is well established that endothelin-1 induced vasoconstriction in this preparation is not [13]. This was confirmed in experiments using the ET-A and ET-B receptor antagonists BQ123 and BQ788 upon the active fraction. These antagonists (both 10 µM, used in combination, n = 3) had no effect upon the constriction to the active fraction (Fig. 4), but completely inhibited constriction to 100 nM endothelin-1 (n = 3).
Despite its first description over 50 years ago by Von Euler and Liljestrand [14], the exact mechanisms underlying sustained HPV remain unresolved. One area of great controversy in recent years has been the endothelial dependency of HPV. Although not a universal finding [6], several studies have observed that the endothelium is required for HPV, especially if it is sustained [2,5]. This would appear to provide evidence that the generation of sustained HPV involves the release of an EDCF which is specifically released by hypoxia, and is ‘confined’ to the pulmonary circulation by production and/or mechanism of action. Several reports have provided evidence that hypoxia does indeed induce the release of an EDCF from the pulmonary vasculature [7,15,16]. The most persuasive of these is the work by Gaine and co-workers [7] who described the release of an EDCF from pulmonary artery endothelial cells which was distinct from endothelin-1.
In the present study we have used a simple solid-phase extraction technique in an attempt to determine whether the perfused lung releases a vasoconstrictor(s) in response to hypoxia, and also to examine whether the resultant vasoconstriction possesses mechanistic characteristics which may indicate a possible role for the active component in the mediation/modulation of HPV. Although the present study is preliminary, several key properties of the active fraction conform closely to those predicted for a ‘unique chemical mediator’ in HPV [17]. The most important of these is its selective release during hypoxic, but not normoxic, perfusion and its relative selectivity for IPA over small MA.
One of the most interesting findings of the present study is the apparent Ca2+-sensitisation observed in response to the active fraction (Fig. 5). This again parallels the effects of the hypoxia upon this preparation [11] and is consistent with the potentiation of the constriction in the presence of an agonist (in this case PGF2) which raises intracellular Ca2+. The observation that this sensitisation is independent of PKC is also in accord with our previous findings regarding phase 2 [11]. The precise mechanisms by which the active fraction and hypoxia induce sensitisation require further study, although preliminary experiments have indicated a possible role for RhoA associated kinases (activation of which is known to induce myofilament Ca2+ sensitisation) in phase 2 [18]. If indeed the active fraction contains a Ca2+-sensitising agent, this would potentially be the first endogenous agonist (to our knowledge) which elicits constriction purely via Ca2+-sensitisation. However, this possibility remains speculative, since the active fraction may contain a number of vasoactive substances which may interact synergistically. The source of the active component also remains to be established, so to describe it as an EDCF would be premature.
In summary, we have presented a preliminary investigation suggesting that the lung releases a vasoactive agent(s) in specific response to hypoxia, which in turn preferentially constricts isolated IPA of the rat. The active component appeared to have a molecular weight of less than 3,000 and was relatively heat stable. This constriction appears to involve PKC-independent Ca2+-sensitisation, and is not due to endothelin-1. These results offer encouragement that the active component(s) of the fraction isolated may be involved in the underlying mechanism of HPV. Although speculative, we feel that the latter possibility is worthy of further investigation.
Time for primary review 22 days.
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Acknowledgments |
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TopAbstract1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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This work was supported by the Wellcome Trust (grant number 060565).
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References |
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TopAbstract1 Introduction2 Methods3 Results and discussionAcknowledgmentsReferences |
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