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Cardiovascular Research 2001 51(1):140-150; doi:10.1016/S0008-6363(01)00275-9
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Copyright © 2000, European Society of Cardiology

Perinatal development influences mechanisms of bradykinin-induced relaxations in pulmonary resistance and conduit arteries differently

P.J Boelsa,*, J Deutscha, B Gaob and S.G Hawortha

aVascular Biology & Pharmacology Unit Institute of Child Health, University College London, 3-Guilford Street, London WC1N 1EH, UK
bPharmacology Royal Free Hospital School of Medicine University College London Rowland Hill Street, London NW3 2PF, UK

* Corresponding author. Tel.: +44-20-7905-2348; fax: +44-20-7905-2370 pboels{at}ich.ucl.ac.uk

Received 10 August 2000; accepted 19 February 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Note added in proof
 References
 
Objective: As bradykinin (BYK) relaxes conduit (EPA) and resistance (RPA) pulmonary arteries from both perinatal and adult lungs, we investigated whether this vasodilator's relaxation-mechanisms were altered during perinatal development, differed between EPA and RPA and differed with other endothelium-dependent vasodilators, acetyicholine (ACH) and substance P (SP). Methods: Arteries from mature foetal (5 days), neonatal ({approx}5 min), newborn (60–84 h) and adult pigs (≥6 months) were isolated, mounted for in vitro isometric force recording, activated with PGF2{alpha} (30 µmol/l) and relaxed with BYK (10 pmol/l–1 µmol/l), SP (10 pmol/l–0.1 µmol/l) or ACH (1 nmol/l–1 mmol/l). Results: (i) BYK: L-NAME (100 µmol/l) attenuated relaxations in foetal EPA ({approx}55%) but nearly abolished them in the adult ({approx}80%). In RPA, L-NAME nearly abolished ({approx}90%) relaxations in the foetus and this effect diminished progressively with age to {approx}20% in the adult. Indomethacin (IND, µmol/l) attenuated relaxations in neonatal ({approx}25%), new-born and adult EPA (both {approx}45%). Together, L-NAME and IND abolished relaxations in all EPA and in neonatal RPA but not in older RPA. SKF525a (100 µmol/l) attenuated relaxations in foetal RPA ({approx}4%), diminishing in the adult RPA to {approx}10%. Together, SKF52Sa and L-NAME largely abolished relaxations in postnatal RPA ({approx}80%). Activation with K+=125 mmol/l attenuated relaxations in adult EPA ({approx}80%), foetal RPA ({approx}45%) and neonatal RPA ({approx}75%) and abolished relaxations in RPA from older ages. (ii) ACH: L-NAME abolished relaxations in new-born EPA and RPA. In adult EPA, combined L-NAME and IND moderately attenuated relaxations. (iii) SP: Combined application of L-NAME and IND attenuated relaxations to a similar degree in new-born and adult EPA and RPA. Conclusions: In postnatal EPA, BYK-relaxations depend completely on prostaglandin- and NO-synthesis whereas those to SP (at all ages) and ACH (in the adult) do not. In RPA, BYK-relaxations develop from being completely dependant on the sole release of NO (foetus) to being almost completely independent of it (adult), a situation mimicked partially by SP but not by ACH, which, in new-born RPA is completely dependent on NO. BYK-relaxations in postnatal RPA depend on the release of a hyperpolarising factor generated through an SKF525a-sensitive pathway in conjunction with NO. The mechanisms of endothelium-dependent BYK-relaxations in the pulmonary vascular bed undergo diverging alterations, depending on the stage of development and arterial size/function. These changes are specific for BYK as they differ from those obtained from ACH or SP.

KEYWORDS ACH, acetylcholine; AD, adult (≥6 months old); BYK, bradykinin; CCRR, cumulative concentration response-relation; COX, cyclo-oxygenase; EDHF, endothelium-derived hyperpolarising factor; EDNO, endothelium-derived nitric oxide; EDPG, endothelium-derived prostaglandins; EPA, elastic conduit-type large intrapulmonary arteries; FT, mature foetal (5 days to term); NB, newborn, adapted to extrauterine life (60–84 h old); NE, neonatal with partially inflated lungs (≤5 min old); NO, nitric oxide; NOS, nitric oxide synthase; PG, prostaglandins; RPA, muscular resistance-type small intrapulmonary arteries; SP, substance P

This article is referred to in the Editorial by R.D. Higgins and M. Keszler (pages 7–8) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 Note added in proof
 References
 
There is consensus in the literature that bradykinin (BYK) can dilate pulmonary arteries of the foetus [1–3] and the neonate [1,4,5] with the possible exception of large, elastic, conduit-type pulmonary arteries [1,6], in which this response emerges only after the first rapid phase of extra-uterine pulmonary adaptation has been completed [7]. These relaxations appear to be endothelium-dependent [1] (see however Ref. [8]) and involve two endothelium-derived relaxing factors, namely nitric oxide (EDNO) and prostaglandins (EDPG), the latter to a varying extent [2,5,9–12]. It remains unclear from these studies whether the relative contributions of EDNO and EDPG to BYK-induced vasodilatation were similar in the foetus, the neonate and the adult. Several lines of evidence, either direct or indirect, suggest otherwise. Firstly, the relaxant effects of BYK in adult porcine pulmonary arteries in vitro have been shown to be independent of EDPG-synthesis and to be partly dependent on both EDNO and an endothelium-derived hyperpolarising factor (EDHF) [13]. These observations contrast with those obtained in perfused piglet lungs where L-NAME or indomethacin each could almost completely abrogate the vasodilator response to BYK [5]. However, extrapolations from intact lung studies to experiments on isolated arteries are difficult to make: arterial size may profoundly influence the type of endothelium-derived relaxing factor released with smaller, resistance-type arteries, utilising less EDNO and more of the as yet uncharacterised EDHF [14,15]. More specifically for large and small pulmonary arteries, numerous other differences have been documented already [16–20]. Secondly, BYK relaxations in conduit-type elastic perinatal arteries appear to be much more prone to desensitisation than in similar adult arteries [1]. Thirdly, the amounts and activities of nitric oxide synthase (NOS) and cyclo-oxygenase (COX) in pulmonary arteries and lungs change considerably within the perinatal period and between the perinatal period and adult life [21–26]. Fourthly, BYK releases increasing amounts of prostacyclin from ovine pulmonary arteries as determined between the mature foetal state and 4 weeks postnatally [26]. Fifthly, inhibition of prostaglandin-synthesis in ovine arteries has varying effects on the BYK-relaxations, depending on postnatal age [10].

From this literature survey, it is unclear whether the relaxations induced by BYK, which were shown to be similar in foetal, neonatal, new-born and adult resistance pulmonary arteries [1], were mediated by identical underlying mechanisms. It is also unclear whether these mechanisms would be uniquely activated by BYK, or also by other endothelium-dependent vasodilators and whether there would be differences between conduit and resistance pulmonary arteries. It was the objective of the present paper to investigate, through pharmacological in vitro manipulation, the mechanisms through which BYK elicits relaxations in pulmonary arteries of the perinatal period and to compare these mechanisms with those operating in mature, adult arteries. The foetal ovine pulmonary circulation has been extensively studied [2,3,8,10–12,21,25,26]. However, with increasing age, BYK loses its vasodilator capacity in ovine pulmonary arteries [2,10,11,16]. Therefore, porcine pulmonary arteries were chosen, taking advantage of the fact that BYK, unlike other endothelium-dependent vasodilators (acetylcholine, ACH; substance P, SP), elicits relaxation in both large conduit-type and small resistance-type arteries at every age [1]. However, wherever possible, comparisons were made with the mechanisms by which ACH- or SP-induced relaxations in arteries of comparable size and age.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 Note added in proof
 References
 
2.1 Animals
Large elastic conduit-type (EPA) and small muscular resistance-type (RPA) pulmonary arteries were obtained from mature foetal (5 days before term), neonatal ({approx}5 min old, lungs partially inflated), new-born (60–84 h old) and adult (≥6 months old) mixed breed pigs (Landrace/Large White) and mounted for isometric force recording as described previously [1]. Perinatal piglets weighed between 0.5 and 2.5 kg. All animal experimentation was carried out as described in the license issued by the Home Office in accordance with the Animals (Scientific Procedures) Act 1986.

2.2 Experimental protocols and calculations
After mounting, all arteries were stretched transversally for maximal isometric force to K+=125 mmol/l (dimensions and amplitude of the contraction in Table 1). All experimental protocols were as described previously [1]. In a first series of experiments, cumulative concentration–response relations (CCRR) to BYK (acetate salt, Sigma, 10 pmol/l to 1 µmol/l, log unit steps, except foetal and neonatal EPA, single application of 1 µmol/l) were obtained from PGF2{alpha} (30 µmol/l, tromethamine salt, Cayman Chemical) or K+=125 mmol/l contracted vessels, quantified against the maximal relaxation induced by papaverine (100 µmol/l, Sigma) and fitted to a sigmoidal relation yielding Rmax (maximal relaxation) and pD2 (concentration for half-maximal relaxation) [1]. A protocol employing a single rather than cumulative concentrations of BYK was applied in the EPA from foetal and neonatal vessels as the former procedure resulted in transient relaxations whereas the latter did not result in any relaxations, probably due to ongoing desensitisation processes [1]. In a second series of experiments, ACH (1 nmol/l to 1 mmol/l, log unit steps) was added to PGF2{alpha} contracted new-born EPA and RPA and adult EPA. ACH does not relax adult RPA in a concentration-dependent manner [1] and this observation was confirmed in the present study: in eight adult RPA a single application of ACH (1 µmol/l) yielded a small and unreproducible relaxation of 13±8% but all these vessels relaxed to the subsequent application of 0.1 µmol/l BYK (58±3%). ACH-induced relaxations were not fitted to a sigmoidal relation as the profile of the effects was distinctly biphasic: relaxations at the lower followed by contractions at the higher (≥10 µmol/l) concentrations. In a third series of experiments, SP (10 pmol/l to 0.1 µmol/l, log unit steps) was added to PGF2{alpha}-contracted adult EPA and RPA. The relaxations were fitted to a sigmoidal relation as described above. In EPA and RPA from new-born animals, SP was added at a single concentration (0.1 µmol/l) as this improved the amplitude of relaxation in the small vessels significantly (Student's t-test, unpaired, 35±4%, 18) when compared to the amplitudes obtained after concentration-dependent addition (6±3%, 6).


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  		Table 1 Basic characteristics of the pulmonary arteriesa

 
Agents interfering with the endothelial-dependent vasodilator pathways (L-NAME, NOS-inhibitor, 100 µmol/l, HCl-salt, Cayman Chemical), indomethacin (unspecific inhibitor of COX-I and COX-II, 10 µmol/l, Sigma) or SKF525a (proadifen, unspecific inhibitor of cytochrome P450 enzymes, thought to participate in EDHF-synthesis [27,28], 100 µmol/l, HCl-salt, Calbiochem) were added 15–20 min before addition of PGF2{alpha} with paired control tissues (same animal) receiving vehicle.

The level of spontaneous tone and the amplitude of the PGF2{alpha}-induced contractions were expressed as percentage of the amplitude of K+=125 mmol/l induced contractions obtained in the same tissues at the beginning and end of the experiment. Amplitudes of contractions and spontaneous tone were calculated from a baseline as obtained from papaverine [1].

The effect of drugs on Rmax and pD2 was evaluated by subtracting the values obtained in presence of the drugs from corresponding values of the paired control tissues (Table 2).


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  		Table 2 Effects of indomethacin, L-NAME and SKF525a on bradykinin-relaxations in pulmonary arteriesa

 
In addition, an overall estimate of the contribution of an endothelium-derived factor to the relaxant effects of BYK was obtained from calculating the area above the CCRR (averages, Sigmaplot 3.0.2.) [29]. For BYK-induced relaxations obtained under control conditions for each age and type of artery, the area above CCRR was labelled BYK(TOTAL). The difference between the areas above the curve for relaxations obtained under control conditions and during L-NAME (normalised to BYK(TOTAL)) was attributed to EDNO (BYK(NO)). A similar calculation was performed for indomethacin (BYK(PG)), (Fig. 7), for the combination of indomethacin and L-NAME (BYK(NO&PG)) and for SKF525a.


Figure 7
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  		Fig. 7 Age-dependence of contribution of EDNO and EDPG to BYK-induced relaxations (solid lines, RPA; dotted lines, EPA). Relative contribution of EDNO (bullet,{blacksquare}, based on inhibitory effects of L-NAME, Table 2, Fig. 2, Section 2.2) or EDPG ({circ},{square}, based on inhibitory effects of indomethacin, Table 2 and Fig. 1) to the total relaxant effect of BYK (set at the value of 1 for each age and type of artery).

 
2.3 Statistics
All values are reported as mean±S.E.M. with N or number in brackets the number of different animals. A difference of P<0.05 was considered to indicate statistical significance. Statistical tests used were (as indicated): (i) paired Student's t-test; (ii) unpaired Student's t-test; (iii) simple ANOVA, separately for EPA and RPA, with each of the four ages as the controlled variable, Rmax, pD2, the amplitudes of contraction (K+=125 mmol/l (mN/mm2) or PGF2{alpha} (% of K+=125 mmol/l)), circumference (mm) or length (mm) as experimental variables and 5 a priori contrasts (foetus–adult, neonatal–adult, new-born–adult, foetus–neonatal, neonatal–new-born) and (iv) General Linear Model Repeated Measures ANOVA (for ACH-relaxations).


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 Note added in proof
 References
 
3.1 Initial observations
The circumference of EPA and RPA at the various ages, as well as the amplitude of K+=125 mmol/l and PGF2{alpha}-induced contractions were comparable to similar data described previously (Table 1) [1]. The circumference from adult EPA was 4-fold larger than corresponding perinatal values. A similar, but much less pronounced (1.3-fold) pattern was observed in RPA. K+=125 mmol/l induced contractions were larger (2-fold) in adult RPA and EPA than in comparable perinatal vessels. The relative (% of K+=125 mmol/l) amplitude of PGF2{alpha}-induced contractions was higher in perinatal EPA (2-fold) and RPA (1.4-fold) than in comparable adult arteries. Thus the transition from foetal to postnatal life had little effect on the contractile parameters of the vessels studied and only maturation to adult life was accompanied by an increased contractile response to high K+.

3.2 Bradykinin
The concentration-dependent relaxations by BYK (except for foetal and neonatal EPA) were adequately described by a sigmoidal concentration–response relation (Figs. 1 and 2Go) characterised by the parameters Rmax (%) and pD2 (–logED50, mol/l). Thus, in control EPA (no drugs added), Rmax-values were 30±5 (16, single application), 21±7 (6, single application), 43±6 (12, CCRC) and 71±7 (20, CCRC) respectively for foetal, neonatal, new-born and adult. The values of all perinatal EPA differed significantly from corresponding values in the adult (a priori contrast, simple ANOVA). The pD2-values for new-born and adult were 8.2±0.2 and 8.0±0.2 respectively. In RPA, of all ages (no drugs added), Rmax-values were respectively 71±3 (17), 82±7 (7), 71±4 (13) and 90±2 (17) with pD2-values 8.7±0.1, 9.0±02, 9.1±0.1, 9.1±02. In this series of data, both the foetal and the new-born values were significantly different from corresponding values in the adult (a priori contrast, simple ANOVA).


Figure 1
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  		Fig. 1 Effects of indomethacin on BYK-relaxations in PGF2{alpha}-contracted RPA or EPA from foetal, neonatal (5 min), new-born (60–84 h) and adult lungs. Comparison of indomethacin (10 µmol/l, bullet{blacksquare}) with paired controls ({circ}{square}). For N, effects of indomethacin on foetal and neonatal EPA and statistical analysis of differences, see Table 2.

 

Figure 2
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  		Fig. 2 Effects of L-NAME (100 µmol/l) on BYK-relaxations in PGF2{alpha}-contracted EPA and RPA from foetal, neonatal, new-born and adult lungs. For N, effects of L-NAME on foetal and neonatal EPA and statistical analysis of differences, see Table 2. L-NAME increased spontaneous tone significantly in foetal EPA (from 24±3 to 32±4%), neonatal EPA (from 29±7 to 46±10%), new-born EPA (from 50±10 to 82±7%), neonatal RPA (from 35±9 to 40±9%), new-born RPA (from 16±2 to 26±5%) and adult RPA (from 19±4 to 26±4%). L-NAME increased the amplitude of the PGF2{alpha}-induced contraction significantly in new-born EPA (from 99±7 to 107±7%) and in RPA from all ages (FT, from 109±10% to 138±12%; NE, from 102±13% to 148±11%; NB, from 111±8% to 140±10% and AD, from 80±7% to 93±7%). All tests are paired Student's t-test. Symbols as in Fig. 1.

 
3.2.1 The attenuating effects of indomethacin increase postnatally (Fig. 1)
Application of indomethacin had no significant effects on spontaneous tone except in neonatal and new-born EPA. Here, spontaneous tone was reduced from 39±5% to 31±4% (8, NE) and from 43±3% to 33±4% (17, NB, paired Student's t-test). Application of indomethacin increased the amplitude of the PGF2{alpha}-induced contraction in new-born EPA (from 103±6% to 119±5%, 17, paired Student's t-test) and attenuated it in foetal RPA (from 112±8% to 89±8%, 7).

In foetal and neonatal EPA, indomethacin did not significantly alter the amplitude of the relaxation induced by a single application of BYK (Table 2). In EPA from older ages, indomethacin significantly attenuated BYK-induced relaxations (Fig. 1, Table 2). BYK(PG) was 0.07 in foetal and 0.09 in neonatal EPA and increased to 0.46 in new-born and adult EPA (Fig. 7). Indomethacin significantly attenuated the BYK-induced relaxations by reducing Rmax in neonatal RPA and by shifting pD2 by {approx}0.5units in adult RPA (Fig. 1 and Table 2). The attenuating effects of indomethacin on Rmax depended on age with significant differences between foetal and neonatal and between neonatal and adult stages (Table 1). BYK(PG) was –0.09 in foetal RPA and was 0.20, 0.12 and 0.19 postnatally (neonatal, new-born and adult, respectively, Fig. 7).

3.2.2 The age-dependency of the attenuating effects of L-NAME increases in EPA but decreases in RPA (Fig. 2)
Application of L-NAME (100 µmol/l) increased spontaneous tone significantly (paired Student's t-test) in all perinatal EPA and postnatal and adult RPA (see legend Fig. 2). The amplitude of the PGF2{alpha}-induced contraction was significantly (paired Student's t-test) increased in RPA from all ages but only in new-born EPA (see legend Fig. 2).

L-NAME significantly attenuated BYK-induced relaxations in EPA by reducing Rmax at all ages (Table 2 and Fig. 2). The attenuating effects of L-NAME on Rmax in EPA increased with age with significant differences between the foetal and the neonatal and between the foetal and adult stages (Table 2). In the new-born, no reliable fit of the CCRR could be obtained. BYK(NO) showed a biphasic age-dependency being 0.56 in foetal EPA, 1.0 in neonatal EPA, and {approx}0.80 in EPA from older ages (Fig. 7).

In foetal and neonatal RPA, L-NAME respectively abolished and significantly attenuated BYK-induced relaxations by reducing Rmax and shifting pD2 by respectively {approx}0.4 units and {approx}0.8 units (Table 2 and Fig. 2). No statistically significant effects were noted in the adult. The attenuating effects of L-NAME on Rmax in RPA decreased with age with significant differences between the foetal and the neonatal and between the foetal and adult stages (Table 2). BYK(NO) decreased from 0.90 in foetal RPA to 0.20 in adult RPA (Fig. 7).

3.2.3 Combined application of L-NAME and indomethacin abrogates relaxations in EPA but not in postnatal RPA (Fig. 3)
Combined application of L-NAME and indoemthacin to new-born and adult EPA completely inhibited the BYK-induced relaxations (Fig. 3). This protocol also inhibited relaxations after a single application of 1 µmol/l BYK in foetal EPA (paired Student's t-test, from 33±9% to –1.9±0.4%, 4). As L-NAME already completely inhibited relaxations in neonatal EPA, this protocol was not executed in these preparations. In new-born and adult RPA, combined application of L-NAME and indomethacin only attenuated (compare Fig. 3 and Fig. 1) BYK-induced relaxations. In neonatal RPA, where the attenuating effect of L-NAME was intermediate, the combined application of L-NAME and indomethacin completely attenuated the relaxation (Fig. 3). As L-NAME already completely abolished relaxations in foetal RPA, this protocol was not performed in these vessels. BYK(NO&PG) decreased from 0.95 in neonatal RPA to 0.22 in new-born and 0.42 in adult RPA.


Figure 3
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  		Fig. 3 Effect of combined application of L-NAME (100 µmol/l) and indomethacin (10 gmol/l) on BYK-relaxations in PGF2{alpha}-contracted EPA and RPA from neonatal, new-born and adult lungs. {square}, neonatal (RPA, 4); {bigtriangleup}, new-born, (EPA, 5, RPA, 6); {diamond}, adult, (EPA, 5; RPA, 6).

 
Complete abrogation of the relaxant response to BYK by combined application of L-NAME and indomethacin occurred while the sum of the contributions of EDNO and EDPG were larger (postnatal EPA, synergy) or smaller than unity (foetal EPA and neonatal RPA, amplification). In the latter case, the effect of combined NOS- and COX-inhibition was larger than could be predicted from the effects of separately inhibiting NOS or COX. The persistence of endothelium-dependent relaxant effects after combined application of both NOS- and COX-inhibitors suggested the participation of a non-NO, non-PG vasodilator (possibly EDHF [14,15,30]).

3.2.4 K+=125 mmol/l inhibits relaxations in postnatal RPA and adult EPA (Fig. 4)
To establish whether the vasodilatation by BYK after combined NOS- and COX-inhibition could be mediated by a hyperpolarising factor, we activated arteries by K+=125 mmol/l (high K+), which, by virtue of its extracellularly imposed depolarisation, would prevent a putative EDHF from exerting its effects while at the same time maintaining the degree of contraction obtained by applying PGF2{alpha}. BYK-induced relaxations in high K+-contracted EPA were not significantly different from those of PGF2{alpha}-contracted EPA in foetal (Fig. 4, legend) and new-born (compare Fig. 4 and Fig. 1) EPA but there was a significant reduction of Rmax but not of pD2 in the adult.


Figure 4
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  		Fig. 4 BYK-relaxations in K+=125 mmol/l contracted EPA and RPA. {circ}, foetus, (6); {square}, neonate (4); {bigtriangleup}, new-born, (EPA, 5, RPA, 4); {diamond}, adult, (EPA, 6; RPA, 8). Relaxation to BYK=1 µmol/l in foetal EPA was 29±7%, (5) during high K+ and 27±7% during PGF2{alpha} (no difference, paired Student's t-test). In adult EPA, Rmax was significantly (unpaired Student's t-test) reduced from 71±4% (20) in PGF2{alpha}-contracted EPA to 39±4% (6) in high K+-contracted EPA. pD2-values of foetal and neonatal K+-contracted RPA were not significantly different from those of PGF2{alpha}-contracted arteries. Rmax-values of foetal and neonatal RPA were significantly smaller (unpaired Student t-test) than those of PGF2{alpha}-contracted arteries (respectively 43±5 and 28±3% in high K+-contracted RPA and 71±3 (17) and 82±7% (7) in PGF2{alpha}-contracted RPA).

 
BYK relaxed high K+-contracted, foetal and neonatal RPA with significantly smaller Rmax but similar sensitivities (PD2) when compared with PGF2{alpha}-contracted preparations (Fig. 4, legend). There were no significant relaxations any more in new-born and adult RPA. Thus the effects of high K+, whilst establishing a major contribution of hyperpolarisation for the relaxant effects of BYK in RPA, also revealed a further age-dependency of the mechanisms of BYK-relaxations in both EPA and RPA.

3.2.5 SKF525a inhibits BYK-relaxations in postnatal RPA only in the presence of L-NAME (Figs. 5 and 6Go)
Having established that hyperpolarisation could play a role in BYK-relaxations, we tested whether a factor, synthesised through the cytochrome P450-pathway [27], was involved. The concentration of SKF525a used has been described to attenuate BYK-induced relaxations in porcine coronary arteries [28]. SKF525a did not influence spontaneous tone in any of the arteries. Contractions induced by PGF2{alpha}(30 µmol/l) were reduced significantly (paired Student's t-test) in foetal RPA (from 141±9% to 87±20%, 6) and adult RPA (from 131±9% to 57±9%, 6).


Figure 5
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  		Fig. 5 Effects of SKFS25a (100 µmol/l, bullet,{blacksquare}) on BYK-relaxations in PGF2{alpha}-contracted EPA and RPA compared with paired controls ({circ},{square}). For N and statistical analysis of differences, see Table 2.

 

Figure 6
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  		Fig. 6 Effect of joint application of L-NAME (100 µmol/l) and SKF525a (100 µmol/l), {diamondsuit} or L-NAME (100 µmol/l) with SKFS25a (100 µmol/l) and indomethacin (10 µmol/l, {bigtriangleup}) compared with untreated controls {diamond} on BYK-relaxations in new-born (3) and adult (6) RPA.

 
SKF525a had no effects on BYK-induced relaxations in new-born and adult EPA (Fig. 5). In new-born and adult RPA, SKF525a reduced the Rmax of BYK (Table 2). In foetal RPA, SKF525a significantly reduced the pD2 of the BYK–CCRR without affecting Rmax (Table 2). The overall estimated contribution of a SKF525a-sensitive metabolite to the relaxant process (BYK(SKF525a/EDHF)) was {approx}0.37 in foetal RPA, 0.26 in new-born RPA and 0.11 in adult RPA.

Joint application of SKF525a and L-NAME to new-born and adult RPA severely attenuated (Fig. 6, left panel) BYK–CCRR to an extent greater than when each inhibitor was applied seperately. Application of L-NAME, SKF525a and indomethacin resulted in a similar degree of attenuation (Fig. 6, right panel).

3.3 Acetylcholine
ACH at lower concentrations relaxed both EPA and RPA, whereas at higher concentrations (≥10 µmol/l), contractions were recorded (Figs. 8 and 9GoGo). The age-dependency of the relaxant effects of ACH corroborated earlier findings [1]: although relaxations were prominently present in new-born RPA, they were completely absent in adult RPA (see also Section 2.2).


Figure 8
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  		Fig. 8 Effects of L-NAME (100 µmol/l, {blacksquare}) on ACH-relaxations in PGF2{alpha}-contracted new-born EPA (5), new-born RPA (5) and adult EPA (10). Asterisk indicate significant differences (General Linear Model Repeated Measures) between control ({square}) and L-NAME.

 

Figure 9
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  		Fig. 9 Effects of combined application of L-NAME and indomethacin (bullet) on ACH-induced relaxations in adult EPA (7) as compared with paired controls ({circ}). Asterisk indicate significant differences (General Linear Model Repeated Measures).

 
Indomethacin did not have any significant effects on the relaxations induced by ACH in new-born RPA or in new-born and adult EPA (results not shown).

Addition of L-NAME completely abolished ACH-relaxations in new-born EPA and RPA (Fig. 8), but had no effects in adult EPA (Fig. 8). Combined application of L-NAME and indomethacin significantly attenuated the ACH-induced relaxations in adult EPA but did not abolish them (Fig. 9).

3.4 Substance P
The amplitude of the relaxant effects of SP increased between new-born and adult EPA and RPA (Tables 3 and 4Go). New-born EPA and RPA were relaxed by a single application of 0.1 µmol/l SP in order to obtain a significant, although still highly transient, relaxation (Methods, Section 2).


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  		Table 3 Effect of L-NAME (100 µmol/l) on relaxations induced by substance P

 

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  		Table 4 Effect of L-NAME (100 µmol/l) and indomethacin (10 µmol/l)) on relaxations induced by substance P

 
Indomethacin did not alter the CCRC to SP in adult EPA and RPA, nor the relaxations to a single application of SP (0.1 µmol/l) in new-born EPA and RPA (results not shown). Addition of L-NAME (100 µmol/l) attenuated the relaxations to SP comparably in new-born and adult EPA and RPA (Table 3) but a significant degree of relaxation still occurred which did not differ between the four groups of arteries tested. No change to this attenuation was observed when SP-induced relaxations were obtained in the presence of both L-NAME and indomethacin (Table 4).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
Note added in proof
 References
 
We have shown here that BYK utilises different endothelium-derived relaxing factors (EDNO, EDPG, EDHF) depending on the stage of lung maturation and growth and the type of pulmonary artery. These dependencies are unique for BYK as they are not mimicked by other endothelium-dependent vasodilators. The various perinatal stages which were compared with the adult state reflect the pulmonary circulation of the non-breathing but mature foetus, the pulmonary circulation immediately after birth at the onset of breathing and of extra-uterine adaptation and after the first rapid phase of extra-uterine adaptation has been completed in the pig [7]. The choice of vessels at these different stages was governed by the criterion of comparable locations: RPA, by virtue of their similar in vitro diameters at different ages (Table 1), thus were obtained from comparable sites upstream from the alveoli; similarly, EPA had comparable locations downstream for the main pulmonary artery. Estimating, through isometric force, the contribution of various endothelium-derived relaxing factors requires firstly that it is established to what extent these factors are released in absence of BYK. This was done by evaluating the effects of L-NAME, indomethacin and SKF525a on the amplitude of spontaneous and PGF2{alpha}-induced tone [31,32]. We found that NO modifies both spontaneous and agonist-induced tone, depending on age and arterial size/function. Possible mechanisms for this could be: (i) receptors for PGF2{alpha} [33] may be distributed differently on the endothelium, depending on age and vessel size/function; (ii) a varying and age-dependent excitability of EPA and RPA, possibly related to different electrophysiological properties [19,34]. It is thus possible that NO, already released in absence of BYK in quantities sufficient to modulate isometric force, could further potentiate the relaxant effects of BYK. This remains to be resolved.

Spontaneous or PGF2{alpha}-induced vasoconstrictor PG-release was indicated by the indomethacin-induced attenuation of basal tone in neonatal and new-born EPA and the attenuation of PGF2{alpha}-induced contractions in foetal RPA. The latter can be explained by the autocrine release of PG by PGF2{alpha} [35]. The converse occurred in new-born EPA where indomethacin potentiated the response to PGF2{alpha} indicating that at this age, a basal release of vasoconstrictor PG is accompanied by a PGF2{alpha}-induced release of vasodilator PG. However, PG-release in absence of BYK, although dependent on age and arterial size, is not likely to contribute mechanically to BYK-relaxations as this release has only small mechanical effects and consists mainly of vasoconstrictor PG's.

The attenuation by SKF525a of the PGF2{alpha}-induced contractions in foetal and adult RPA remains currently unexplained. SKF525a has inhibitory effect on KCa-channels [36], but KCa-channel blockade is usually associated with increased contractions [37] and furthermore, KCa-channels are only sparsely present in pulmonary myocytes [19,34].

The contribution of EDPG to BYK-relaxations had the following characteristics: (i) it only occurred postnatally; (ii) it explained typically {approx}20% of the relaxant effects in RPA but up to {approx}50% in EPA and (iii) it was delayed in EPA until the completion of the first rapid phase of extra-uterine adaptation. These conclusions do not corroborate earlier results obtained from sheep where indomethacin attenuated BYK-relaxations in foetal small arteries [2] and where these attenuating effects became progressively less with age in large elastic arteries [10] (this latter observation is, in itself, difficult to reconcile with BYK, releasing progressively increasing amounts of prostacyclin with age [26]). Different oxygen tensions employed in Ref. [2] and in our study might influence experimental outcomes although BYK–CCRR in porcine foetal RPA were not influenced by the prevailing oxygen tension [1] as was the case for ovine foetal arteries [2]. EDPG, released by BYK, appeared only to have a minor auxiliary role for BYK-relaxations in RPA, but more so in EPA as the phenomenon of EDNO/EDPG-synergy (see below) was prominent in the latter. A further significant contribution of EDPG's to endothelium-mediated vasodilatation seems to be absent in all porcine pulmonary arteries as indomethacin did not influence ACH- or SP-induced relaxations.

In RPA, BYK-relaxations became progressively less sensitive to L-NAME with increasing age despite characteristics of relaxations in the absence of L-NAME (pD2 and Rmax) being identical at all the stages of lung development studied. This decrease of the primary (i.e. significant attenuation by sole application of L-NAME) dependence on EDNO for BYK-relaxations in RPA is the opposite of that in EPA. Here, the primary dependence on EDNO is substantial ({approx}50%) and further raises in postnatal EPA, much larger than in corresponding RPA (Figs. 2 and 7Go). These divergent dependencies on EDNO occur during a period in which overall NOS-levels increase in porcine lungs [24]. Except for the mature foetal stage, EPA relied more on EDNO for endothelium-dependent relaxation than RPA. This characteristic, described for adult, mature vessels [14,15] has here shown to be present in pulmonary arteries from birth onward. However, the increasing dependence with postnatal age on EDNO in EPA and decreasing dependence on EDNO in RPA is unique for BYK. For ACH-induced relaxations in EPA, dependence on EDNO, as judged from the decreasing attenuating effects of L-NAME (Fig. 8) decreased with age. Furthermore, in new-born RPA, ACH-relaxations were completely dependent on EDNO (Fig. 8) and this at a time when the relaxations to BYK were almost completely independent of EDNO (compare Fig. 2 with Fig. 8). A further degree of heterogeneity was shown by the effects of SP which did not show age-dependency for the attenuating effects of L-NAME and indomethacin and in which these attenuating effects were identical in RPA and EPA.

The persistence of BYK-relaxations in presence of L-NAME and indomethacin in new-born and adult RPA does not imply that EDNO is not released anymore by BYK at these ages. Rather, BYK stimulates the release of two, mutually redundant endothelium-derived relaxing factors through duplicate pathways (Fig. 6), a process which emerges postnatally in RPA only. This conclusion is based on the experiments with SKF525a and K+=125 mmol/l. Similar to L-NAME and indomethacin in new-born and adult RPA, SKF525a on its own failed to attenuate BYK-relaxations. The prominent attenuation of BYK-relaxations after combined NOS- and cytochrome P450-inhibition suggests that in RPA, upon inhibition of the synthesis of NO or of a P450 metabolite, each factor alone could almost maintain BYK-induced vasodilatation at control levels and only the combined inhibition of both pathways could disrupt relaxations. The postnatal developmental aspects of the P450 isozyme responsible for the synthesis of EDHF (P450 2C [38]) have not been studied but the expression of P450 isozymes appears to be increasing postnatally [39,40]. The persistence of ACH-induced relaxations in adult EPA and of SP-induced relaxations in new-born and in adult after combined application of L-NAME and indomethacin indicate that the localisation of a P450/EDHF pathway is not restricted to RPA.

The hyperpolarising nature of this P450 metabolite was confirmed with K+=125 mmol/l as the contractile agonist, which inhibited BYK-relaxations in RPA from the new-born stage onward. The emergence of this inhibition correlated with the primary dependence on L-NAME becoming greatly reduced. The ineffectiveness of SKF525a on its own on BYK-relaxations made us anticipate that K+=125 mmol induced depolarisation would attenuate rather than inhibit the response to BYK in RPA but it has been shown in porcine EPA that part of the NO effects on smooth muscle can be mediated through K-channel dependent hyperpolarisation [13,27]. The postnatal emergence of a P450 metabolite-EDHF-contributing to the relaxant effects of BYK in RPA could thus be suggested.

A further difference between EPA and RPA was the synergy of NO and PG, present in neonatal new-born and adult EPA [29,41]. The labile nature of NO may necessitate synergy as a more efficient way of myo-endothelial coupling in thick walled vessels, but it remains puzzling why such a phenomenon is only activated by BYK and not by ACH or SP. The opposite, amplification (neonatal RPA and foetal EPA, Section 3.2.3) remains equally unexplained.

The use of BYK, and to a lesser extent ACH, in these developmental studies has revealed a novel phenomenon existing in myo-endothelial coupling. Whereas previously the relative contributions of EDPG, EDNO or EDHF to BYK-induced endothelium-dependent relaxations were thought to be a static property depending on species differences (see Ref. [3] for a discussion), differences between vascular beds [42] (compare also responses in porcine pulmonary arteries with those in porcine coronary arteries [43]) or arterial size/function (see Introduction), it now becomes apparent that such changes can be much more dynamic and can fluctuate within days in the same arterial segment. The precise molecular arrangement and control of these multiple pathways, remains to be investigated.

A further level of flexibility in which the EDNO-, EDPG- and EDHF-pathways are being activated was uncovered by the experiments which compared, at similar ages and in similar vessel types, the mechanisms of BYK-induced endothelium-dependent relaxations with those induced by ACH and SP. These comparisons were very limited as BYK is the only endothelium dependent vasodilator which relaxes all types of pulmonary arteries at all ages. SF-induced relaxations, completely absent in the foetus and the neonate, become more prominent with increasing age (Tables 3 and 4Go) whereas the relaxant responses to ACH, which are present perinatally, have completely disappeared in adult RPA, even upon application of a single concentration of ACH (to bypass possible ongoing desensitisation processes). From these comparisons, it appeared that BYK, in the porcine pulmonary arteries, was the only endothelium-dependent vasodilator to (i) activate EDPG-pathways in RPA and EPA; (ii) display the phenomenon of synergy between EDNO and EDPG in EPA; (iii) decrease its primary dependency on EDNO with increasing maturation in RPA and (iv) lack the capacity to stimulate a non-EDNO/non-EDPG pathways in postnatal EPA. How different endothelial receptors can be selectively coupled to different intracellular pathways, depending on development and vascular function needs further investigation.

In conclusion, the mechanisms of BYK-relaxations are unique for this vasodilator and are further characterised by a spatial and temporal heterogeneity. Thus, it cannot be assumed that the mechanisms of agonist-induced endothelium-dependent vasodilatation are similar in the newly born and in the adult. BYK appears to be an extremely adaptable endotheliumdependent vasodilator, perhaps well suited to accommodate the rapid changes in the regulation of pulmonary blood flow that are required to take place upon adaptation to extrauterine life.


   Note added in proof
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Note added in proof
References
 
While in proof, further experiments on foetal RPA have revealed that the relaxations to SP (bolus-application) and ACH (CCRC) are completely attenuated by L-NAME as are the relaxations to SP (bolus-application) in foetal EPA (Boels and Deutsch, unpublished). These results further illustrate the significant changes of the mechanisms of pulmonary, endothelium-dependent vasodilators brought about by age, type of vessel and agonist.

Time for primary review 29 days.


   Acknowledgements
 
This study was made possible through a grant of the British Heart Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 Note added in proof
 References
 

  1. Boels P.J, Deutsch J, Gao B, Haworth S.G. Maturation of the response to bradykinin in resistance and conduit pulmonary arteries. Cardiovasc. Res. (1999) 44:416–428.[Abstract/Free Full Text]
  2. Wang Y, Coceani F. EDRF in pulmonary resistance vessels from fetal lamb: stimulation by oxygen and bradykinin. Am. J. Physiol. (1994) 266:H936–H943.[ISI][Medline]
  3. Frantz E, Soifer S.J, Clyman R.I, Heymann M.A. Bradykinin produces pulmonary vasodilation in fetal lambs. J. Appl. Physiol. (1989) 67:1512–1517.[Abstract/Free Full Text]
  4. Davidson D, Eldemerdash A. Endothelium-derived relaxing factor: evidence that it regulates pulmonary vascular resistance in the isolated neonatal guinea pig lung. Pediatr. Res. (1991) 29:538–542.[ISI][Medline]
  5. Perreault T, De Marte J. Maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation. Am. J. Physiol. (1993) 264:H302–H309.[ISI][Medline]
  6. Zellers T.M, Vanhoutte P.M. Endothelium-dependent relaxations of piglet pulmonary arteries augment with maturation. Pediatr. Res. (1991) 30(2):176–180.[ISI][Medline]
  7. Haworth S.G, Hislop A.A. Adaptation of the pulmonary circulation to extra-uterine life in the pig and its relevance to the human infant. Cardiovasc. Res. (1981) 15:108–119.[Abstract/Free Full Text]
  8. Theis J.G, Toyoda O, Coceani F. Effect of endothelium removal on prostaglandin and nitric oxide function in pulmonary resistance arteries in the lamb. Can. J. Physiol. Pharmacol. (1998) 76(2):182–187.[CrossRef][ISI][Medline]
  9. Davidson D, Eldemerdash A. Endothelium-derived relaxing factor: presence in pulmonary and systemic arteries of the newborn guinea pig. Pediatr. Res. (1990) 27(2):128–132.[ISI][Medline]
  10. O’Donnell D.C, Tod M.L, Gordon U.B. Developmental changes in endothelium dependent relaxations of pulmonary arteries: role of EDNO and prostanoids. J. Appl. Physiol. (1996) 81:2013–2019.[Abstract/Free Full Text]
  11. Gao Y, Tolsa U.-F, Raj J.U. Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs. Pediatr. Res. (1998) 44:723–729.[ISI][Medline]
  12. Glasgow R.E, Buga G.M, Ignarro U, Chaudhuri G, Heymann M.A. Endothelium derived relaxing factor as a mediator of bradykinin-induced perinatal pulmonary vasodilatation in fetal sheep. Reprod. Fertil. Dev. (1997) 9:213–216.[CrossRef][Medline]
  13. Félétou M, Girard V, Canet E. Different involvement of nitric oxide in endotheliumdependent relaxation of porcine pulmonary artery and vein: influence of hypoxia. J. Cardiovasc. Pharmacol. (1995) 25:665–673.[ISI][Medline]
  14. Garland C.J, Plane F, Kemp B.K, Cocks T.M. Endothelium-dependent hyperpolarization: a role in the control of vascular tone. Trends Pharmacol. Sci. (1995) 16:23–30.[CrossRef][Medline]
  15. Nagao T, Illiano S, Vanhoutte P.M. Heterogeneous distribution of endothelium dependent relaxations resistant to NG-nitro-L-arginine in rats. Am. J. Physiol. (1992) 263:H1090–H1094.[ISI][Medline]
  16. Kemp B.K, Smolich J.J, Cocks T.M. Evidence for specific regional patterns of responses to different vasoconstrictors and vasodilators in sheep isolated pulmonary arteries and veins. Br. J. Pharmacol. (1997) 121:441–450.[CrossRef][ISI][Medline]
  17. Leach R.M, Twort C.H.C, Cameron I.R, Ward J.P. A comparison of contractile function in large and small pulmonary arterial vessels of the rat. Q. J. Exp. Physiol. (1989) 74:947–950.[Abstract/Free Full Text]
  18. Madden J.A, Dawson C.A, Harder D.R. Hypoxia-induced activation in small isolated pulmonary arteries from the cat. J. Appl. Physiol. (1985) 59:113–118.[Abstract/Free Full Text]
  19. Archer S.L, Huang J.M.C.X, Reeve H.L, et al. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ. Res. (1996) 78:431–442.[Abstract/Free Full Text]
  20. Leach R.M, Twort C.H, Cameron I.R, Ward J.P. A comparison of the pharmacological and mechanical properties in vitro of large and small pulmonary arteries of the rat. Clin. Sci. Colch. (1992) 82:55–62.[Medline]
  21. Brannon T.S, MacRitchie A.N, Jaramillo M.A, et al. Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung. Am. J. Physiol. (1998) 274:L66–L71.[ISI][Medline]
  22. Shaul P.W. Ontogeny of nitric oxide in the pulmonary vasculature. Semin. Perinatol. (1997) 21:381–392.[CrossRef][ISI][Medline]
  23. North A.J, Star R.A, Brannon T.S, et al. Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung. Am. J. Physiol. (1994) 266:L635–L641.[ISI][Medline]
  24. Hislop A.A, Springall D.R, Buttery L.D.K, Pollock J.S, Haworth S.G. Abundance of endothelial nitric oxide synthase in the newborn intrapulmonary arteries. Arch. Dis. Child Fetal Neonatal-Ed. (1995) 73:F17–F21.
  25. Parker T.A, Le Cras T.D, Kinsella J.P, Abman S.H. Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung. Am. J. Physiol. (2000) 278:L202–L208.[ISI]
  26. Brannon T.S, North A.J, Wells L.B, Shaul P.W. Prostacyclin synthesis in ovine pulmonary artery is developmentally regulated by changes in cyclooxygenase-1 gene expression. J. Clin. Invest. (1994) 93:2230–2235.[ISI][Medline]
  27. Cohen R.A, Vanhoutte P.M. Endothelium-dependent hyperpolarization. Beyond nitric oxide and cyclic GMP. Circulation (1995) 92:3337–3349.[Free Full Text]
  28. Hecker M, Bara A.T, Bauersachs J, Busse R. Characterisation of endotheliumderived hyperpolarising factor as a cytochrome P450-derived arachidonic acid metabolite in mammals. J. Physiol. (Lond.) (1994) 481:407–414.[Abstract/Free Full Text]
  29. Gambone L.M, Murray P.A, Flavahan N.A. Synergistic interaction between endothelium-derived NO and prostacyclin in pulmonary artery: potential role for K+ATP channels. Br. J. Pharmacol. (1997) 121:271–279.[CrossRef][ISI][Medline]
  30. Cocks TM. 10. Endothelium-dependent vasodilator mechanisms. In: Garland CJ, Angus JA, editors. Pharmacology of vascular smooth muscle. Oxford, UK: Oxford University Press, 1996, pp. 233–251.
  31. Frew J.D, Paisley K, Martin W. Selective inhibition of basal but not agonist-stimulated activity of nitric oxide in rat aorta by NG-monomethyl-L-arginine. Br. J. Pharmacol. (1993) 110:1003–1008.[ISI][Medline]
  32. Steeds R.P, Thompson J.S, Channer K.S, Morice A.H. Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade. Br. J. Pharmacol. (1997) 122:99–102.[CrossRef][ISI][Medline]
  33. Chen J, Champa-Rodriguez M.L, Woodward D.F. Identification of a prostanoid FP receptor population producing endothelium-dependent vasorelaxation in the rabbit jugular vein. Br. J. Pharmacol. (1995) 116:3035–3041.[ISI][Medline]
  34. Evans A.M, Osipenko O.N, Haworth S.G, Gurney A.M. Resting potentials and potassium currents during development of pulmonary artery smooth muscle cells. Am. J. Physiol. (1998) 275:H887–H899.[ISI][Medline]
  35. Yousufzai S.K, Ye Z, Abdel-Latif A.A. Prostaglandin F2{alpha} and its analogs induce the release of endogeneous prostaglandins in iris and ciliary muscles isolated from cat and other mammalian species. Exp. Eye Res. (1996) 63:305–310.[CrossRef][ISI][Medline]
  36. Alvarez J, Montero M, Garcia-Sancho J. High affinity inhibition of Ca2+-dependent K+ channels bycytochrome P450 inhibitors. J. Biol. Chem. (1992) 267(17):11789–11793.[Abstract/Free Full Text]
  37. Cook N.S. Effect of some potassium channel blockers on contractile responses of the rabbit aorta. J. Cardiovasc. Pharmacol. (1989) 13(2):299–306.[ISI][Medline]
  38. Fissithaler B, Popp R, Kiss L, et al. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature (1999) 401:493–497.[CrossRef][Medline]
  39. Tornquist S, Sundin M, Moller L, Gustafsson J.A, Toftgard R. Age dependent expression of cytochrome P450b and metabolism of the potent carcinogen 2-nitrofluorene in the rat lung. Carcinogenesis (1988) 9(12):2209–2214.[Abstract/Free Full Text]
  40. Eltom S.E, Schwark W.S. CYP1A1 and CYP1B1, two hydrocarbon-inducible cytochromes P450, are constitutively expressed in neonate and adult goat liver, lung and kidney. Pharmacol. Toxicol. (1999) 85(2):65–73.[ISI][Medline]
  41. Shimokawa H, Flavahan N.A, Lorenz R.R, Vanhoutte P.M. Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig. Br. J. Pharmacol. (1988) 95:1197–1203.[ISI][Medline]
  42. Toda N, Bian K, Akiba T, Okamura T. Heterogeneity in mechanisms of bradykinin action in canine isolated blood vessels. Eur. J. Pharmacol. (1987) 135:321–329.[CrossRef][ISI][Medline]
  43. Kühberger E, Groschner K, Kukovetz W.R, Brunner F. The role of myoendothelial cell contact in non-nitric oxide-, non-prostanoid-mediated endothelium-dependent relaxation of porcine coronary artery. Br. J. Pharmacol. (1994) 113:1289–1294.[ISI][Medline]

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