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Cardiovascular Research 1999 44(2):416-428; doi:10.1016/S0008-6363(99)00217-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Maturation of the response to bradykinin in resistance and conduit pulmonary arteries

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

aVascular Biology and Pharmacology Unit, Institute of Child Health, London WC1N 1EH, UK
bDepartment of Pharmacology, Royal Free Hospital School of Medicine, London NW3 2PF, UK

* Corresponding author: Tel.: +44-171-242-9789 (ext. 2348); fax: +44-171-813-8459 pboels{at}ich.ucl.ac.uk

Received 4 March 1999; accepted 11 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: Immaturity of the endothelial-dependent relaxation is thought to be characteristic of the newborn pulmonary elastic arteries. In adulthood, the reactivity of different pulmonary arterial segments varies. Therefore, we investigated the presence of endothelial heterogeneity in perinatal porcine pulmonary arteries and compared it with the adult by studying the bradykinin-, substance P- and acetylcholine-induced relaxations in different arteries. Methods: Three types of pulmonary arteries (large conduit elastic, distal branching and resistance-sized; in situ diameters 0.7–1.7, 0.3–0.5 and 0.1–0.2 mm, respectively) were isolated from lungs of adult (nine months), young (60–84 h), newborn (4 min) and near-term foetal pigs. They were mounted for isometric force recording, contracted first with K+=125 mmol/l (reference contraction). Cumulative concentration—response curves to acetylcholine, substance P or bradykinin were obtained from prostaglandin F2{alpha} (30 µmol/l) precontracted vessels. The effects of captopril and O2(95 or 8%) were also determined. Experiments were terminated by adding 100 µmol/l papaverine, obtaining maximal relaxation, which was used for normalising relaxations. Results: (i) Acetylcholine: In resistance arteries, relaxations were absent in the newborn and the adult. In conduit arteries, they were present from 60–84 h onward. (ii) Substance P: In resistance arteries, relaxations were only present in the adult. In the other two types of arteries, rudimentary relaxations were present from the mature foetal stage onward. (iii) Bradykinin: In resistance arteries, identical relaxations were present at all ages which, in the foetus and the adult, were insensitive to changes in O2 levels (95 to 8%). In conduit arteries, concentration-dependent relaxations were present from birth, increasing in amplitude with age and these were potentiated by captopril. Foetal conduit arteries relaxed to the single application of 0.1 µmol/l bradykinin, indicating age-dependent tachyphylaxis. Conclusions: (i) Bradykinin is unique among endothelium-dependent vasodilators in being able to relax all vascular segments, at all ages, subject to tachyphylaxis and bradykinin-breakdown but independent of the prevailing O2 concentration. (ii) Heterogeneity of the relaxations between conduit and resistance arteries is evident from the mature foetal stage onward. (iii) The type of agonist, the type of vessel and the age each independently determine the presence or absence of endothelial relaxations. Therefore, the perinatal pulmonary circulation is not immature with respect to endothelial-dependent relaxation; rather, the nature of this process changes within the perinatal period and between birth and adulthood.

KEYWORDS ACE inhibitors; Contractile function; Developmental biology; Endothelial factors; Prostaglandins


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Bradykinin (BK) is known to relax systemic and pulmonary arteries through activation of the endothelium [1–6] and has been reported to dilate the pulmonary circulation in the perinatal period. Working on porcine conduit intrapulmonary arteries, Liu et al. [7] reported the absence of BK-induced relaxation just after birth and Zellers and Vanhoutte [3] showed a prominent increase in the amplitude of the relaxation between three and ten days postnatally. However, studies on perfused porcine lungs indicated that BK-induced vasodilatation did not change between one and seven days after birth [8]. In the sheep, BK relaxed foetal pulmonary arteries both in the perfused lung and in vitro [9–11]. Thus, the first appearance of BK-induced relaxations in the perinatal period may depend on the species and the type of vessels studied. This first appearance could also depend on the functional maturation of the endothelium in the perinatal pulmonary circulation [3,7,12]. Development also could play a role as the potent pulmonary vasodilatation of BK in the ovine foetus is much reduced in the adolescent and adult [13,14].

The presence of acetylcholine (ACH)-induced relaxations has long been used as the benchmark for determining the functional integrity of the endothelium in vascular preparations in vitro and in vivo, although the functional significance of ACH relaxations in the pulmonary circulation is still unclear. In previous publications [3,7,12], conclusions regarding the functional immaturity of the endothelium in the newborn were partly based on the absence of ACH-relaxations in elastic pulmonary arteries. Although substance P (SP) is a powerful endothelium-dependent pulmonary vasodilator [15–17], its functional role in the pulmonary circulation is also enigmatic. The compound is also routinely used to assess endothelium-dependent vasodilation.

In vitro studies indicate considerable longitudinal heterogeneity of active responses between the more proximal and distal vessels isolated from the adult cat and rat lung [14,18–21]. These in vitro findings have been corroborated in part by lung perfusion studies. Different parts of the pulmonary circulation can react differently to vasoconstrictor and dilator stimuli [22–24], a phenomenon further influenced by age in the rabbit [25], sheep [24] and, possibly, pig [26,27].

In the transitional pulmonary circulation, BK might have a physiological role [13,28,29], but a systematic study of its effects, also in comparison with other vasodilators, has hitherto not been performed. The aims of the present study, using intrapulmonary porcine pulmonary arteries, were therefore: (i) to compare BK-induced relaxations between pulmonary arteries of different sizes; (ii) to compare BK-induced relaxations within different stages of perinatal life and between perinatal life and adulthood; (iii) to compare the presence of BK-induced relaxations in different segments and at different ages with those of the benchmark vasodilator agonist, ACH, and with those of another endothelium-dependent dilator, SP.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Piglets were produced by pregnant Large White sows, which were induced 24 h before term with Tiaprost (150 µg/100 kg, intramuscularly). All perinatal animals were sacrificed by means of a lethal intraperitoneal injection of sodium pentobarbitone (100 mg/kg). Animals were sacrificed immediately upon parturition [less than 4 min old (time required for the sodium pentobarbitone to become effective), lungs partially inflated because all animals did some breathing], and at the ages of 20–28 h and 60–84 h. Additional piglets (near-term foetuses, aged term minus five—six days and piglets aged 60–84 h) were obtained from Selborne Biological Services, Alton.

Foetal piglets were obtained after electrical stunning and exsanguination of pregnant sows. Adult material ({approx}nine months) was obtained from the local abattoir (Fresh Tissue Supplies, Horsham). The heart—lung bloc was removed, the lungs were isolated and rinsed thoroughly with chilled physiological salt solution (PSS; composition in mmol/l: NaCl 119, KCl 4.7, CaCl2 2.5, NaHCO3 25, Na2EDTA 0.026, KH2PO4 1.2, MgSO4 1.2, glucose 5.5, gassed with 95% O2/5% CO2). When material had to be transported, tissues were maintained on ice in a storage solution (composition in mmol/l: NaCl 145, KCl 5, CaCl2 2, MgSO4 1, NaH2PO4 1, Dextrose 5, pyruvate 2, EDTA 0.02, MOPS 3, pH 7.4). Tissues were available for further dissection within 4 h after sacrifice of the animal. The investigation conforms with the guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH-publication No 85-23, revised 1996).

2.2 Preparation of tissues
The main axial intrapulmonary artery (i.e. the main intrapulmonary artery of the lower lobe), distal branching arteries and resistance-sized arteries were studied. The main intrapulmonary artery was dissected between the first major lower intralobar branch and a distal symmetrical branching point. This encompassed the middle 3/5ths of the artery (elastic arteries, in situ diameter {approx}0.7–1.7 mm; see Fig. 1 for a representative arterial cross-section from a newborn and an adult animal). Vessels were cleaned of adhering connective tissue and cut into rings (see Table 1 for segment lengths). The rings taken from the main axial intrapulmonary artery had a branching order of four—six (main pulmonary artery, branching order, one). From some elastic arteries, the endothelium was mechanically removed, as described previously [30]. The effects of endothelium-removal on the relaxations induced by ACH and BK are described in Results. From the lungs of adult animals, an additional experiment was performed that was aimed at investigating the longitudinal heterogeneity within the stretch of artery studied. To this end, two rings were removed from the proximal and distal ends of the length of the axial artery and these segments were studied separately. All elastic arterial rings were suspended in water-jacketed (35°C) glass organ baths filled with 5 ml of Ca-free PSS and mounted between two tungsten hooks (120 or 500 µm diameter), one connected to a support and the other connected with a stainless steel wire to a Grass FT.03 force transducer.


Figure 1
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  		Fig. 1 Representative cross-sections of elastic arteries (A, B) and resistance-sized arteries (C, D) obtained from an adult (A, C) or a newborn (B, D). All vessels were mounted in organ baths and stretched to optimal dimensions (see Methods). After addition of papaverine, vessels were fixed in situ. Bars indicate 25 µm in C and D, and 100 µm in A and B.

 

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  		Table 1 In vitro characteristics of the pulmonary arterial preparations used

 
From the apical part of the lung, two types of arteries were isolated (approximate in situ diameters {approx}300–500 µm and {approx}80–200 µm; branching order, 9–11 and 12–14, respectively). The former will be referred to as distal branching arteries, the latter as resistance-sized arteries (see Fig. 1 for a representative cross-section of resistance-sized arteries obtained from a newborn and an adult animal). Two stainless steel wires of 40 µm diameter were inserted through the lumen of distal branching arteries and resistance-sized arteries and these vessels were mounted in a small vessel myograph in Ca-free PSS. Mechanical removal of the endothelium from resistance-sized arteries was performed with a human hair after mounting [30]. The effects of endothelium removal on the relaxations induced by ACH and BK are reported in Results and were also described previously [30]. As the elastic arteries were thick-walled (Fig. 1), possibly limiting adequate diffusion of O2 into the core of the preparation [31], all vessels were studied at 95% O2 (for exceptions, see Section 3.4).

2.3 Experimental protocols
After mounting, the solutions were exchanged with fresh pre-warmed and oxygenated PSS and all of the vessels were equilibrated for {approx}30 min. Signals were amplified, digitally converted at 1.7 Hz (MacLab for Macintosh LCII, AD-Instruments Ltd., UK) and recorded with Chart 3.3.6 for Macintosh.

2.3.1 Preliminary protocols
After the equilibration period, PSS was exchanged with fresh, prewarmed and preoxygenated Ca-free PSS. During this equilibration period, all vessels were transversely stretched to a level that corresponded to an effective transmural pressure of approximately 30 mmHg ({approx}10 mN of stable passive force). This configuration corresponded approximately to the optimal length for maximal isometric force development. From this configuration, the calculated in vitro diameter was derived (Table 1). For the proximal and distal rings from the adult elastic arteries, these in vitro calculated diameters were 7.3±0.4 and 2.8±0.2 mm, respectively (n=5). All preparations were subsequently challenged with K+=125 mmol/l (equimolar replacement of Na+) for 5 min, to obtain a reference contraction.

2.3.2 Effects of BK, SP and ACH
2.3.2.1 Initial protocol
After the wash-out of K+=125 mmol/l, all arteries were exposed to prostaglandin F2{alpha} (PGF2{alpha}; 10–30 µmol/l). After stabilisation of this contraction, ACH (1 or 10 µmol/l) was added to assess either the integrity of the endothelium or the efficiency of its removal. A second K+=125 mmol/l contraction was obtained after removal of the PGF2{alpha} and ACH. The response to K+=125 mmol/l proved to be very reproducible in all preparations. After the removal of K+=125 mmol/l, preparations were left for a second equilibration period of up to 30 min in PSS, until stabilisation of the baseline had occurred. When the experimental protocol required it, captopril (10 µmol/l) was subsequently added for 15–30 min, after which, all preparations were contracted with 30 µmol/l PGF2{alpha}. After this contraction had stabilised ({approx}20 min), BK (10 µmol/l to 1 µmol/l) was added cumulatively in log-unit increments. Alternatively, BK was added once to a final concentration of 100 nmol/l. After wash-out of PGF2{alpha} and BK, all preparations were exposed to papaverine (100 µmol/l), to determine the maximum possible extent of relaxation. As varying degrees of spontaneous tone were present in every preparation studied (Fig. 2), the application of papaverine also served to normalise the concentration—response curves obtained (see Methods, Section 2.6, Analysis of data).


Figure 2
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  		Fig. 2 Representative tracings of the effects of K+=125 mM, 30 µM PGF2{alpha}, cumulative additions of BK and 100 µM papaverine (pap) in a distal branching artery obtained from an adult and a 60–84-h-old animal and studied in a myograph-style organ bath. Calibration lines are 0.5 mN and 5 min for both tracings. Arrows indicate the exchange of PSS, either with K+=125 mM or with fresh PSS, as indicated; numbers above arrow-heads are –log of the concentrations of BK. Bars indicate the presence of the compound indicated and the dashed line indicates the level of relaxation reached by papaverine. Note the myogenic tone in the distal branching artery from the 60–84-h-old animal (distance between the dashed line and the tracing) and its virtual absence in the adult. Note also the large rebound upon wash-out of PGF2{alpha}/BK in the distal branching artery from the 60–84-h-old but not from the adult animal.

 
2.3.2.2 Protocol for the determination of the effects of ACH
It soon became apparent that some preparations in which the endothelium had not been removed did not respond or showed only a weak response to ACH but subsequently developed powerful relaxations to BK. Particularly in adult resistance-sized arteries, this was not due to damaged endothelium (Fig. 1C). The effects of ACH were therefore quantified in a more systematic way in a separate group of preparations. After the wash-out of the first contraction induced by K+=125 mmol/l, preparations were contracted with 30 µmol/l PGF2{alpha} and, after stabilisation of the contraction, a cumulative (log-increments) concentration—response curve to ACH was obtained (1 nmol/l to 10 µmol/l). Wash-out and papaverine-addition were as described in the previous section.

2.3.2.3 Modified protocol for the determination of the effects of BK and SP
Thus, the procedure of preliminary testing of endothelial function with ACH was abandoned. After obtaining the initial contraction to K+=125 mmol/l, we omitted the challenge with 10 µmol/l PGF2{alpha}, the addition of ACH and the second K+=125 mmol/l contraction. The preparations were equilibrated with or without captopril (see above), challenged to 30 µmol/l PGF2{alpha} and exposed to BK or SP. There was no difference in the BK-induced relaxations using this second, modified procedure as compared to the first procedure. SP was added cumulatively in log increments (10 pmol/l to 100 nmol/l).

2.3.2.4 Protocol involving reduced O2 concentrations
The effect of reducing the concentration of O2 bubbling the PSS in the organ bath (from 95 to 8%, 5% CO2, remainder N2) was tested in the foetal and adult resistance-sized arteries. Experiments were carried out using a paired protocol: this involved isolating two resistance-sized arteries from the same animal and obtaining the concentration—response relations to BK under 95 or 8% O2. In foetal vessels, 8% O2 was applied from the start of the experiment. In adult resistance-sized arteries, 8% O2 was applied 20 min before the challenge with 30 µM PGF2{alpha}.

2.4 Drugs
Bradykinin, acetylcholine—HCl, papaverine, substance P and captopril were purchased form Sigma. PGF2{alpha} (tromethamine salt) was obtained from Cayman Chemical. All drugs were dissolved in distilled water.

2.5 Histological studies
After the addition of papaverine, some preparations were fixed in 10% formalin (1 h at 35°C). Preparations were then dismounted and stored in formalin at 4°C. Prior to embedding, preparations were rinsed in calcium-free PSS and pre-embedded in a block of 7% agarose to facilitate further handling. Tissue was then embedded in paraffin. Transverse 5 µm sections were cut and stained with Miller's elastic van Gieson's stain.

2.6 Analysis of data
Because the degree of the myogenic tone, the ‘baseline’ from which the PGF2{alpha}-induced contractions were initiated, could become so pronounced that the BK-induced relaxations not only completely reversed the PGF2{alpha}-induced contractions but started to relax beyond the ‘baseline’ (Fig. 2), we felt justified in adding the amplitude of the myogenic tone to the amplitude of the contraction induced by PGF2{alpha} in order to quantify the total degree of contractile activation (Table 1). The extent of this myogenic tone was only fully uncovered by the addition of papaverine. As this myogenic tone was present when PGF2{alpha} was added to the preparations, the amplitude of the PGF2{alpha}-induced contractions, including the myogenic tone, was taken as the stable plateau from which the relaxations were initiated (Table 1). Amplitudes of the PGF2{alpha}-induced contractions were calculated as absolute tension (mN/mm, active force divided by segment length) or as relative force (% of K+ 125-induced contractions) (Table 1).

The effect of BK, ACH or SP was expressed as the % relaxation, taking as 0% the total active force prior to addition of the agonist (i.e. PGF2{alpha}-induced tone plus the spontaneous tone) and taking as 100% the level of relaxation achieved by papaverine (Fig. 2). All data are reported as mean±SEM. For each individual preparation, the data of the concentration—effect relation of BK were fitted to a logistic equation using Sigmaplot 3.0, which determined the range of extent of relaxation (Rmax, %), the steepness of the curve (slope) and the concentration of the half-maximal effect (ED50, mol/l) [30].

Within each age group, n indicates the number of different animals used. Statistical analysis of the dependent variables, ED50 and Rmax, was initially performed with ANOVA [general linear model, bifactorial (GLM-b), with the range of ages and the range of vessel types as the fixed factors]. The statistical package SPSS 7.5.1. was used. When GLM-b indicated a significant influence of a fixed factor on a dependent variable, further analysis (one way ANOVA, followed by post-hoc testing) on simplified subsets of the data was performed to determine which groups (characterised by both vessel type and age) were significantly different from each other. Every simplified subset contained the data of the entire range of the fixed factor of interest under study. When post-hoc comparisons were made with age as the fixed factor of interest, two post-hoc tests were performed. First, a two-sided Dunnett-test was performed with the adult as the control group and all the perinatal data were compared to the adult. Second, only the perinatal data were considered and a Bonferroni test was used. Post-hoc comparisons involving vessel type were carried out using the Bonferroni test. For some purposes (as indicated in the text), Student's t-test for paired observations or unpaired observations or simple one-way ANOVA were used. The level indicating statistically significant differences was set at P≤0.05 except for the post-hoc testing for age, in which case, P≤0.01 was used to compensate for the repeated testing (i.e. Dunnett and Bonferroni).


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Dimensions and active force
The internal circumference of resistance-sized arteries was similar at different ages. The internal circumference of distal branching and conduit elastic arteries increased with age (Table 1). Although the preparations were obtained from lungs that differed considerably in size, the wall structure of the vessels, specifically in the group of the resistance-sized vessels, was identical between the different ages (Fig. 1).

The absolute tension induced by 30 µmol/l PGF2{alpha} was significantly dependent on both age and the type of artery (Table 1). The elastic arteries developed more PGF2{alpha}-induced tension than the resistance-sized arteries (label x, Table 1), but the statistical significance of this difference disappeared in the adult. Distal branching arteries transiently developed more PGF2{alpha}-induced active tension at birth. The relative contractile force of the PGF2{alpha}-induced contractions (% of K+=125 mmol/l) decreased with age but was comparable within each age-group (Table 1).

3.2 ACH-induced relaxation
The response to a single application of ACH (1 µmol/l) was tested in nine adult resistance-sized arteries before studying the responses to BK in an attempt to assess the functional integrity of the endothelium (see Methods, 3.2.1). The vasodilator effect of ACH (1 µmol/l) amounted only to 11±9%. Therefore, complete concentration—response relations to ACH (1 nmol/l to 10 µmol/l) were obtained in the three types of vessels from the adult (Methods, 3.2.2, Fig. 3). In the resistance-sized arteries, no relaxations were observed and transient contractile responses were noted at 10 µmol/l ACH. A small relaxant response was noted in the distal branching arteries, which disappeared at higher doses of ACH. As was previously reported [7], relaxations were observed in the elastic arteries (Fig. 3). Removal of the endothelium in 38 elastic arteries obtained from 19 adult animals, inhibited the ACH-response, uncovering a small contraction, increasing the preceding stable active force (10 µM PGF2{alpha}) by 3±2%.


Figure 3
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  		Fig. 3 Effect of ACH in resistance-arteries (bullet), distal branching arteries ({circ}) and large elastic arteries ({blacktriangleup}) of the adult (n=20, 15 and 6, respectively), the 60–84-h-old (n=12, 11 and 9, respectively), the newborn (n=9, 8 and 10, respectively) and the foetus (n=4, 5 and 5, respectively). All preparations were precontracted with PGF2{alpha} (30 µM). Data within each panel are unpaired. Y-axes have been drawn using different scales, with negative values (above the dotted line) indicating contractions.

 
The response to ACH in the perinatal animals was highly variable, depending on age and vessel type (Fig. 3). In the foetus (Fig. 3), ACH relaxed distal branching arteries and resistance-sized arteries but not elastic arteries. In the resistance-sized arteries from the newborn, ACH caused concentration-dependent transient contractions, thus augmenting the contraction induced by 30 µmol/l PGF2{alpha} (Fig. 3). In the larger arteries of the newborn, ACH had no significant effects (Fig. 3). In all of the vessels studied from animals aged 60–84 h, ACH caused concentration-dependent relaxations (Fig. 3) that were of a larger amplitude in resistance-sized and small distal branching arteries than in the elastic arteries.

Using a single application of ACH (1 µmol/l, Methods, 3.2.1) in intact resistance-sized arteries from animals aged 60–84 h, the relaxation was 55±10% (n=4) and this was reduced to 12±3% in paired preparations in which the endothelium had been removed. The relaxant response to 1 µmol/l ACH in conduit elastic arteries with endothelium from animals aged 60–84 h was 46±8% (n=5). In paired denuded elastic arteries, this response was turned into a small contraction that increased the preceding PGF2{alpha} contraction by 4±4%.

3.3 BK-induced relaxations
3.3.1 Adult resistance arteries
BK relaxed the PGF2{alpha}-precontracted resistance-sized arteries near maximally (sensitivity of {approx}5 nmol/l). Small contractions preceded the relaxant responses to BK in two out of 26 preparations. The relaxant response to 1 µmol/l BK was 78±4% (n=6) in intact preparations, whereas in paired denuded preparations, this response was inhibited to 10±7%. Inclusion of captopril increased sensitivity (ED50) but not the maximal effect of BK-induced relaxations (Fig. 4A).


Figure 4
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  		Fig. 4 Effect of captopril (10 µmol/l, panel A, n=5) and oxygen (8%, panel B, n=4) on the relaxations induced by BK in adult resistance-sized arteries. Observations in each panel are paired and all preparations were precontracted with PGF2{alpha} (30 µmol/l). Open symbols indicate control values, closed symbols paired experimental values, *indicates statistically significant difference with the paired and corresponding control value (paired t-test).

 
Reducing the O2 concentration from 95 to 8% had no effect on the amplitude of the PGF2{alpha}-induced contractions (46±11 to 51±6%, n=4) or on the baseline preceding the challenge with PGF2{alpha}. BK-induced relaxations were not influenced by the reduction in O2 concentration (Fig. 4B).

3.3.2 Adult elastic and distal branching arteries
These vessels also showed a concentration-dependent relaxant response to BK. In comparison with the resistance-sized arteries, both the ED50 and the maximal relaxation to BK were similar in small distal branching arteries but were significantly less in the large elastic arteries (Table 2, Fig. 5).


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  		Table 2 Characteristics of BK-induced relaxations

 

Figure 5
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  		Fig. 5 Effect of BK in resistance-arteries (bullet), distal branching arteries ({circ}) and large elastic arteries ({blacktriangleup}) of the adult, the 60–84-h-old, the newborn and the foetus. All preparations were precontracted with PGF2{alpha} (30 µmol/l). See Table 2 for further details.

 
In order to study the effect of size on BK-induced relaxations in more detail, paired vessels with a similar elastic wall structure but different size (see Methods, Section 2) were examined. Both proximal and distal rings of the same axial pulmonary artery developed similar relative forces in response to 30 µmol/l PGF2{alpha}, i.e., 87±6 and 81±13%, respectively (n=5). They also relaxed similarly to ACH, i.e., 29±10 and 27±7%, respectively. The pED50 of the BK-relaxations was also similar, 7.4±0.3 and 7.5±0.3 mol/l, respectively. The extent of the relaxations at 1 µmol/l BK was significantly greater (paired t-test) in the small distal elastic arteries (61±10%) compared with the large elastic arteries (40±8%), emphasising that the smaller the vessel, the greater the relaxation to BK.

In the elastic arteries, endothelium-removal attenuated the relaxation to BK significantly (from 60±4 to 15±3%, n=19). Captopril (10 µmol/l) significantly increased the slope (from 0.71±0.09 to 1.3±0.1, paired t-test; n=7, Fig. 7) and reduced the pED50 (from 7.6±0.3 to 9.0±0.3 mol/l, paired t-test).


Figure 7
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  		Fig. 7 Effect of captopril (10 µmol/l) in elastic arteries of the adult (n=7), the 60–84-h-old (n=5), the newborn (n=8) and the foetus (n=3). Symbols as in Fig. 4. All preparations were precontracted with 30 µmol/l PGF2{alpha}. Data in each panel are paired.

 
Captopril shifted the concentration—response relations in elastic and resistance-sized arteries to a similar degree, but the differences in sensitivity and extent of relaxation between the two vessel types in response to BK remained significant, even after captopril treatment (unpaired t-test, compare Figs. 4 and 7Go).

3.3.3 Perinatal resistance-sized arteries
BK caused relaxations of equal amplitude in the resistance arteries from all perinatal animals (Fig. 5, Table 2). Denudation significantly attenuated the relaxant effects of BK in vessels from animals aged 60–84 h (at 1 µmol/l, 9±18%, n=4), compared to 70±3% in paired control vessels and from newborn animals (at 1 µmol/l, 20±11%, n=5), compared to 90±5% in paired control vessels.

The only difference in the response to BK in perinatal and adult resistance-sized arteries was the incidence of the transient contractions that preceded relaxation. This increased from 0/11 in the foetuses, to 4/9 in the newborn, 2/6 at 20–28 h and 3/17 in animals aged 60–84 h. At 1 µmol/l BK, these contractions were {approx}25% of the stable PGF2{alpha}-induced contraction. The level of oxygenation did not influence the relaxations to BK in foetal resistance-sized arteries (Fig. 6).


Figure 6
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  		Fig. 6 Effect of of oxygen (8%, n=4) on the relaxations induced by BK in foetal resistance-sized arteries. Observations in each panel are paired, and symbols are the same as in Fig. 4.

 
3.3.4 Perinatal elastic and distal branching arteries
Both distal branching and elastic arteries showed a maturational response for the BK-induced relaxations (see Section 3.3.5). In the newborn, two distinct populations of elastic arteries were discerned, one that showed a small degree of concentration-dependent relaxation to BK (60% of the arteries studied) and the other, which failed to do so (Table 2). The amplitude of the maximal relaxations was greater at 60–84 h of age than at the newborn stage, being already comparable to those seen in adult life. The significant maturation of the BK-response in the elastic arteries contrasted sharply with the absence of such maturation in the resistance-sized arteries (Fig. 5). Given the sudden increase in the BK-induced relaxation between birth and 60–84 h of age, we investigated the BK-induced relaxations of elastic pulmonary arteries from piglets aged 20–28 h of age. Out of six preparations, two failed to show any relaxation whereas the four others relaxed by 26±5%, which was similar to the extent of relaxation in the elastic arteries that responded at birth (Table 2). For the six preparations taken together, the extent of relaxation was 17±7%, which was also comparable to the extent of relaxation obtained at birth.

The absence of BK-induced relaxations in the elastic arteries of the foetus and the newborn was further investigated with two different protocols involving (i) the study of the influence of captopril on the concentration—response relation and (ii) the assessment of the impact of a singular application of 100 nmol/l BK. Because we only wanted to determine if the absence of bradykinin-induced relaxations in the perinatal conduit arteries was due to angiotensin converting enzyme (ACE)-activity, we did not repeat these experiments in the smaller vessels, as these already showed BK-induced relaxations that were indistinguishable from those of the adult (Fig. 5, Table 2). Captopril caused small relaxations to BK to appear at intermediate doses in the foetus and augmented the relaxations in the newborn and the 60–84 h group (Fig. 7) but the age-dependency of BK-induced relaxations in the foetal, newborn and 60–84 h old group remained after captopril.

In the second series of experiments, the relaxant effect of a single concentration of 100 nmol/l BK was evaluated in elastic arteries of the foetus (n=11), the newborn (n=5), the 20–28-h-old (n=8), the 60–84-h-old (n=6) and the adult (n=8). Relaxations thus obtained were 34±9, 27±4, 50±8, 60±6 and 55±8%. There was a significant age-dependency (one-way ANOVA) with the amplitude of relaxation being comparable to the one obtained in concentration—response relationships from the age of 60–84 h onward. By contrast, in the three youngest age-groups, the amplitude of the relaxation after a single application of 100 nmol/l BK was larger than the maximal relaxation after a study with cumulative concentration—responses (compare with Table 2). These results indicate tachyphylaxis in the elastic arteries of the younger, but not of the older animals.

3.3.5 General characteristics and comparison between adult and perinatal vessels
BK relaxed all vessels obtained from animals in the perinatal period in a concentration-dependent fashion, except the large elastic arteries from foetal animals (Fig. 5, Table 2). Vessel type and age significantly influenced Rmax and ED50 and there was also a significant interaction between age and vessel type (GLM-b, Table 2). For the factor vessel type, resistance-sized arteries had significantly higher sensitivities and maximal relaxations than distal branching arteries, which, in turn, had higher sensitivities and potencies than elastic arteries (GLM-b followed by the Bonferroni test). For the factor age, in general, sensitivity was lower in the newborn than in the adult. The parameter Rmax was significantly higher in the adult than at the perinatal stage (GLM-b followed by the Dunnett t-test) and, within the perinatal stage, it was larger in the group of 60–84-h-old animals than in the younger animals (GLM-b followed by the Bonferroni). The significant interaction between age and vessel type for the parameter Rmax indicated that the difference between the Rmax values in the various arteries became smaller with increasing age.

Subsequent univariate analysis on Rmax showed that this parameter increased significantly with age only in the elastic arteries (labels a,b and c in Table 2). In every age group studied, Rmax was always greater in the distal branching arteries and resistance-sized arteries than in the elastic arteries (labels x and y in Table 2). In the perinatal period, maximal BK-induced relaxation in the age group 60–84 h was significantly larger than at younger ages for the elastic arteries only, indicating a significant maturation process in these vessels.

Univariate analysis on the ED50 indicated that, in all age-groups, the sensitivity was always less in the elastic arteries than in the resistance-sized arteries and distal branching arteries (labels x and y in Table 2). The significant overall difference (with GLM-b, see above) between the ED50 of resistance-sized arteries and small distal branching arteries was largely due to the differences found during the foetal period. The ED50 of the foetal distal branching arteries was significantly smaller than corresponding values in the adult.

The slope of the concentration—response relation was not significantly different between the three preparations (Table 2, Fig. 5).

3.4 Substance P
As the results using ACH and BK yielded two completely divergent sets of results, we tested the relaxations to a third endothelium-dependent vasodilator, SP. Relaxations to SP were completely absent in the foetal and newborn resistance-sized arteries and only rudimentary relaxations at the highest concentration employed were present in the small branching and elastic conduit arteries (Fig. 8). By 60–84 h of age, both conduit and small arteries showed moderately potent concentration-dependent relaxations to SP, whereas resistance-sized arteries still only showed a rudimentary, rapidly reversing relaxation at 10 nmol/l of SP. In the adult, the three preparations were fully and concentration-dependently relaxed by SP, without differences in either amplitude or sensitivity (Fig. 8).


Figure 8
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  		Fig. 8 Effects of SP in conduit elastic, distal branching and resistance-sized arteries of the foetal, newborn, the 60–84-h-old and the adult pig. Symbols as in Fig. 3. n=3 for foetal and newborn preparations, n=4 for 60–84-h-old and adult preparations.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
The in vitro relaxant effects of BK on precontracted porcine pulmonary arteries show that important differences exist between conduit elastic arteries, small distal branching arteries and resistance-sized arteries during perinatal life and between perinatal life and adulthood. The most important differences are (i) the presence, in the near-term foetal stage, of potent BK-induced relaxations in resistance-sized but not in elastic arteries; (ii) the maximal relaxation, sensitivity and susceptibility to tachyphylaxis of BK-induced relaxations. In comparison with ACH and SP, BK appears to be unique in that all of the vessels at all of the ages studied were relaxed by BK.

4.1 BK-induced relaxation in different types of pulmonary arteries with respect to age
The ages of the young animals used in this study reflect three important points during perinatal adaptation. The mature but non-functional pulmonary circulation of the foetus and the fully adapted pulmonary circulation of the 60–84-h-old [32]. The pulmonary circulation of the newly born is in the process of adapting to extra-uterine life and reflects pulmonary arterial reactivity immediately after parturition.

Previous reports on endothelium-dependent relaxation of porcine elastic pulmonary arteries described a pronounced postnatal maturation of the response to ACH, A23187 [GenBank] and BK [3,7,12]. Thus, pulmonary endothelial immaturity was thought to contribute to the high pressure of the pulmonary circulation before and immediately after birth [7]. However, in these in vitro experiments, the contribution of smaller arteries with different wall structures was not considered. The relaxation to BK in the distal branching and elastic arteries took until birth and well into the postnatal period of adaptation, respectively, to become comparable to those obtained in adult, mature arteries, partially confirming earlier data obtained in the presence of indomethacin [3]. The response appeared to be already mature in the resistance arteries of the near-term foetus, confirming observations made in the foetal lamb [9,11]. Thus, the maturation of the endothelium-dependent relaxation to BK varies in different segments of the pulmonary arterial tree. The absence of any change in the response to BK in isolated porcine lungs aged 24 h and seven days [8] can thus be explained by the contribution of smaller arteries. Although BK-induced relaxations in late foetal life appear to be comparable in ovine and porcine pulmonary arteries, subsequent postnatal development is different. In the sheep, BK relaxations in elastic arteries become less pronounced between two and eleven days of age [33], while they are absent in the resistance-sized arteries of the mature sheep [14].

In order to find out if differences in the response to BK seen in elastic and resistance-sized arteries could be due to differences in the metabolism of the peptide in the vessel wall, we exposed the preparations to the ACE inhibitor, captopril. ACE-inhibitors can upregulate the relaxant actions of BK [34]. Although the addition of captopril increased the amplitude and sensitivity of the BK-induced relaxations, it did not alter the age-dependency or the dependency on vessel type. These experiments show that ACE is functionally active and relevant in perinatal pulmonary arteries but that the activity of this enzyme does not account for the absence of BK-induced relaxations in foetal or newborn elastic arteries, or for the smaller responses of the elastic arteries in comparison with the resistance-sized arteries from both the adult and the 60–84-h-old groups.

The absence of BK-induced relaxations in perinatal conduit arteries of the foetus, the newborn and the 20–28-h-old piglet could also be partially explained by the presence of pronounced tachyphylaxis at these ages, which abated by the age of 60–84 h. However, even with an experimental protocol more favourable to detecting BK-induced relaxations, there was still an age-dependency, suggesting a delayed maturation of the elastic arteries for the relaxant effect of BK. A genuine longitudinal heterogeneity for endothelium-dependent relaxations of BK thus exists in the porcine pulmonary circulation and it is already established in the perinatal period.

The presence of BK-induced relaxations in the foetal resistance-sized arteries could also, in an as yet unclarified way, be induced by the high levels of in vitro O2. However, BK-relaxations in foetal and adult resistance-sized arteries were not influenced by the prevailing level of O2. Thus, BK-relaxations are not due to in vitro artefacts, but these experiments emphasize species differences as these results contradict earlier findings obtained in foetal lambs [9]. Despite the increase in the maximal relaxation (Rmax) of BK with progression from proximal to distal elastic arteries (vessels with a similar type of wall structure), longitudinal differences probably do not originate from geometrical factors, as SP-induced relaxations in the adult were comparable between the three types of arteries. Thus, other factors, such as the density of BK-receptors, their subtypes [35], the efficacy of the various EDRFs and the sensitivity of the smooth muscle to these EDRFs could contribute to the longitudinal heterogeneity observed for BK [14,18–21]. Heterogeneity of contractile activation did not play a role as there was no correlation between the amplitude of the PGF2{alpha}-induced contraction and the BK-induced relaxation (compare Tables 1 and 2Go).

4.2 Comparison of the responses to BK with those to ACH and SP
In foetal porcine, as in ovine [9,11], resistance-sized arteries, ACH- and BK-relaxations were comparable (Figs. 3 and 5Go). However, studying further postnatal development in the porcine vessels uncovered important differences. While the relaxant response to BK was either present at birth or matured progressively thereafter, according to vessel type, the ACH-response showed a complex dependence on both age and type of artery (Figs. 3 and 5Go). The absence of ACH-induced relaxations in the adult was not related to ACH-induced release of vasoconstrictor prostaglandins overriding relaxation [36]: inclusion of indomethacin does not alter the effects of ACH (Boels, unpublished). An endothelial lining was moreover present in the newborn and adult resistance-sized arteries (Fig. 1). The insensitivity of pulmonary resistance-sized arteries to ACH has also been described in adult rat [21] and sheep [14]. Furthermore, postnatal development and growth also reduces the amplitude of ACH-induced relaxations in ovine distal branching arteries [33]. However, the ACH-relaxations in porcine large elastic arteries, once established at 60–84 h of age, continue to be present in the adult, as in ovine elastic arteries [37]. The absence of ACH-induced relaxations in any type of pulmonary artery just after birth could be explained by birth-related events having an attenuating effect on the pulmonary endothelial function, such as the rapid change of shape of pulmonary arterial cells after birth [38,39]. The fact that BK-induced relaxations were not affected could be related to the documented capacity of the peptide to relax even in the presence of endothelial injury [40], such as after ischaemia/reperfusion insults [40,41].

The pattern of relaxation by SP in different types of vessel at different ages contrasted with that of BK and ACH. Resistance-sized arteries were relaxed with a potency and a sensitivity that was less or, at best, equal to those in the larger vessels (contrasting with the pattern of BK). In this, we were not able to confirm previous experiments by Liu et al. [42] on rabbit pulmonary arteries. Moreover, the largest and most potent relaxations were found in the adult. These results suggests that SP, as a mediator of neurogenic inflammation and endothelial growth, does not have a significant role in the postnatal adaptation to extra-uterine life.

More generally, we conclude from these experiments that the concept of longitudinal heterogeneity can be extended to SP- and ACH-induced endothelium-dependent relaxations. The important differences between BK and ACH suggest that the suitability of the latter as a universal tool for testing endothelial integrity in pulmonary arteries should be questioned. Finally, the concept of a general endothelial immaturity in the perinatal pulmonary circulation is not supported by our present data: we propose that this concept should be reformulated relating to the nature of the stimulus used, the vascular segment under study and the perinatal age or stage. Our experiments do not suggest that the endothelium of resistance-sized arteries is more mature than in larger arteries: this explanation is refuted by the experiments involving SP.

4.3 Functional implications
The presence of a mature vasodilator response to BK only in porcine pulmonary resistance-sized arteries throughout the perinatal period supports the contention that endogenous BK [29] may be important in facilitating and maintaining vasodilation of the peripheral pulmonary bed during extra-uterine adaptation. The extent to which these findings in the perinatal healthy piglet extend to the newborn human infant remains to be determined.

Time for primary review 19 days.


   Acknowledgements
 
This study was made possible through grant RG/97007 from the British Heart Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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