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Am J Physiol Lung Cell Mol Physiol 295: L201-L213, 2008. First published May 9, 2008; doi:10.1152/ajplung.00264.2007
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Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

Anne-Christine Peyter,1 Vincent Muehlethaler,1 Lucas Liaudet,2 Mathieu Marino,1 Stefano Di Bernardo,1 Giacomo Diaceri,1 and Jean-François Tolsa1

1Neonatal Research Laboratory, Department of Pediatrics, and 2Division of Critical Care, Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M3 muscarinic receptor was implicated in the relaxing action of ACh and M1 muscarinic receptor (M1AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M1AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M1AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M1AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M1AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

muscarinic receptors; phosphodiesterases; endothelial nitric oxide synthase; pulmonary artery; acetylcholine

ADVERSE EVENTS in utero such as maternal hypertension and growth retardation are associated with the occurrence of diseases in adulthood, including coronary artery disease, systemic hypertension, stroke, and non-insulin-dependent diabetes mellitus (4). These observations support the concept that a perinatal insult may result in long-term physiological modifications predisposing to a pathological response later in life. The endothelium has been shown to play a key role in these disease patterns (17, 67). Limitation of nutrient and/or oxygen supply to the fetus are linked to the development of "programmed" diseases; however, the cellular and molecular mechanisms of the programming process are unknown (26). Several lines of evidence have indicated that chronic pulmonary vascular diseases and abnormal pulmonary vascular reactivity in adulthood may be associated with a hypoxic insult occurring around birth (28, 43, 63). In particular, human and animal studies have demonstrated that individuals born in a hypoxic environment show later in life an exaggerated pulmonary hypertensive response following a reexposure to hypoxia (10, 30–32, 63). The cellular and molecular mechanisms involved in the dysregulation of adult pulmonary vasomotor tone secondary to a transient perinatal hypoxic insult have not been elucidated so far.

At birth, the gas exchange transition from the placenta to the lungs requires dramatic changes in the pulmonary circulation. The fetal pulmonary circulation is characterized by low perfusion and high vascular resistances, which rapidly fall at birth, allowing pulmonary blood flow to increase nearly 10-fold. Pulmonary vascular tone is regulated by a complex and intricate group of mechanisms (5). In particular, the nitric oxide (NO)-cGMP pathway plays a key role in establishing relaxation of the pulmonary vessels during the neonatal transition as well as later in life (1, 45). NO is an endothelium-derived factor produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS) (45, 52). It diffuses into surrounding smooth muscle cells, where it activates soluble guanylate cyclase (sGC), resulting in the production of cGMP. The accumulation of cGMP leads to smooth muscle relaxation after the activation of cGMP-dependent protein kinase (PKG). Degradation of cGMP is performed by phosphodiesterases (PDEs). In pulmonary vessels, the NO-cGMP pathway can be activated, among others, after stimulation of muscarinic receptors by ACh.

ACh is an important endogenous neurotransmitter that regulates pulmonary vascular tone via binding to different muscarinic receptors. Until now, five isoforms of muscarinic receptors, named M1–M5, have been identified (37). In the pulmonary circulation, vascular tone is regulated mainly by isoforms M1–M3 in a tone-dependent manner (36). On one hand, ACh induces relaxation in constricted vessels by the activation of the M3 muscarinic receptor (M3AChR) and production of NO following the activation of endothelial NOS (eNOS). On the other hand, in relaxed vessels, ACh induces vasoconstriction by the activation of isoforms M1 and M2 (M1AChR and M2AChR, respectively). The M2AChR seems to be implicated in the retro control of ACh-mediated signaling by inhibition of the release of ACh at presynaptic terminations (36). However, the specific isoforms implicated in pulmonary vasoconstriction or vasodilatation vary in different animal species (3, 13, 33, 57). Muscarinic receptors are G protein-coupled receptors. Isoforms M1 and M3 are coupled to Gq/11, which activates phospholipase C, resulting in inositol triphosphate (IP3) and diacylglycerol production (39). IP3 induces increases in the cytosolic calcium concentration by release from the intracellular calcium pool. Isoform M2 is coupled to Gi/o, which inhibits adenylyl cyclase activity (39).

We postulated that a hypoxic insult during a critical period of development of the lung vasculature around birth could influence the expression and/or activity of proteins implicated in pulmonary vascular tone regulation, particularly those involved in the NO/cGMP pathway. This could represent a risk for the subsequent development of pulmonary vascular pathologies in adulthood, such as pulmonary hypertension.

The present study was designed to identify, in a murine experimental model, the long-term effects of a transient exposure to hypoxia in the perinatal period on the lung vasculature. Hemodynamic measurements were performed in anesthetized mice to assess modifications in the pulmonary circulation. Pulmonary arterial reactivity to endothelium-dependent and -independent relaxing agents was tested by isolated vessel tension experiments, and modifications at the molecular level were investigated.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model

C57BL/6 mice from Harlan (Horst, The Netherlands) were used. They were all fed ad libitum and exposed to day-night cycles. All experimental procedures were approved and carried out in accordance with the Swiss Veterinarian Animal Care Office. Perinatal hypoxia was induced as previously described (51). Pregnant mice were placed in hypoxic conditions (13% O2) 5 days before delivery (day 21) and left under hypoxia with their litter for 5 days after birth. Pups were then raised in normoxia (21% O2) until adulthood. The timing of exposure to hypoxia was chosen to cover the lung vasculogenesis period, during which the functional units of gas exchange develop (74). The degree of hypoxia was chosen according to previous studies in rodents (31, 32). Pups born and grown in normoxic conditions were used as controls. Female mice were studied as adults before week 25 (20 ± 3 wk, mean ± SD). This timing was chosen for technical reasons, in particular to allow reproducible hemodynamic measurements.

Hemodynamic Experiments

Adult mice were anesthetized with ketamine-xylazine (100 and 10 mg/kg ip, respectively). Closed-chest measurements were performed using a Millar mikro-tip 1.4-Fr catheter (Millar Instruments, Houston, TX) to record right ventricular (RV) pressure (RVP) (6). RVP was used as an estimation of the pulmonary arterial pressure (20). Baseline RVP was recorded in normoxic conditions (21% O2) and under acute hypoxia (12% O2). During the measurements, mice were placed on a heating board to prevent hypothermia and under a small Plexiglas hood to control oxygen administration. The gas mixture was controlled using an oxygen monitor mono2 (Roche, Biolelectronics, Switzerland). Body temperature was continuously recorded using a rectal probe (ADInstruments, Hastings, UK).

Isolated Vessel Tension Experiments

Adult mice were administrated a lethal dose of pentobarbital (1 g/kg ip), and the main pulmonary artery (PA) was immediately harvested, resulting in a ring of 1.5- to 2.0-mm length and 0.5- to 1.0-mm diameter. The vessel ring was suspended in organ chamber filled with 10 ml of modified Krebs-Ringer bicarbonate solution [containing (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose] maintained at 37°C and aerated with 95% O2-5% CO2 (pH 7.4) (51, 69). Each ring was suspended by two stirrups (0.1-mm diameter) passed through the lumen. One stirrup was anchored to the bottom of the organ chamber, and the other connected to a strain gauge (PowerLab/8SP, ADInstruments) for the measurement of isometric force. Vessels were brought to their optimal resting tension after 2 stretches of 0.5 g. After equilibration, NG-nitro-L-arginine (L-NNA; 10–4 M, Alexis, San Diego, CA) and/or indomethacin (10–5 M) were added to exclude the possible interference of endogenous NO and prostanoids, respectively. Experiments with ACh or A23187 [GenBank] were performed, unless otherwise specified, in the absence of L-NNA. Vessels were then contracted with phenylephrine (Phe; 10–5 M) at a level corresponding at least to the maximal response to potassium (100 mM KCl). Finally, pharmacological responses of isolated PAs were evaluated in the presence of increasing concentrations of endothelium-dependent relaxing agents [ACh or calcium ionophore A23187 [GenBank] (Alexis)] and endothelium-independent agents (gaseous NO or the cell-permeant cGMP analog 8-bromo-cGMP). NO was prepared as previously described (69). Changes in tension induced by vasodilators were expressed as percentages of the initial contraction induced by Phe. In a set of experiments, ACh-induced relaxation was tested in the presence of 10–4 M L-NNA, an inhibitor of NOS activity (53); 10–5 M 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a preferential antagonist of the M3AChR (11, 39); 10–7 M pirenzepine or 10–10 M telenzepine, two preferential inhibitors of the M1AChR (11, 39); or 10–5 M vinpocetine, an inhibitor of calcium-dependent PDE1 (2, 29). The specificity of pirenzepine and telenzepine is dose dependent. Pirenzepine and telenzepine display high affinity for M1AChR and low affinity for the other isoforms of muscarinic receptors, in particular M3AChR. In our model, dose-response experiments determined 10–7 M as the dose of pirenzepine that allowed better relaxation to ACh with minimal inhibition of the M3AChR (Fig. 1A). For telenzepine, the most appropriate dose appeared to be 10–10 M (Fig. 1B).


Figure 1
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  		Fig. 1. Pharmacological responses of isolated pulmonary arteries (PAs) preconstricted with phenylephrine (Phe) to ACh in the presence of increasing concentrations of pirenzepine (Pir) or telenzepine (Tel). Cumulative dose responses to ACh (10–8–10–4 M) were tested on the main PA of adult mice born in normoxia in the absence or presence of increasing concentrations of Pir (10–9–10–5 M, n = 3–7 animals; A) or Tel (10–11–10–5 M, n = 2–7 animals; B), two preferential inhibitors of the M1 muscarinic receptor (M1AChR). Results are expressed as means ± SE of percentages of changes in tension induced by ACh.

 
To investigate the effects of perinatal hypoxia on the vasoconstrictive capacity of the adult main PA, cumulative concentrations of KCl or Phe were also tested in isolated vessel tension experiments in the presence of indomethacin and L-NNA.

Semiquantitative RT-PCR Analysis of mRNA Expression

Specific mRNA expression in adult lungs was tested by RT-PCR after extraction of total RNA using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. RT was performed from 2 µg total RNA using the Omniscript RT Kit from Qiagen (Valencia, CA) (final volume: 20 µl) in the presence of a mixture of random primers and oligodT (Promega, Madison, WI). PCR was then performed from 1/20 of the volume of the RT product using the HotStarTaq MasterMix Kit from Qiagen and the following primers (Microsynth): M1AChR, sense 5'-GCTGTACTGGCGCATCTACC-3' and antisense 5'-GCCTGTGCCTCAGGATCTAC-3' (12); and 18S RNA, sense 5'-TTAAGCCATGCATGTCTAAGTAC-3' and antisense 5'-TGTTATTTTTCGTCACTACCTCC-3' (GeneBank Accession No. AF397158). PCR conditions were as follows: 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, with 37 cycles for M1AChR and 25 cycles for 18S RNA. RT-PCR products were analyzed after electrophoresis on 2% agarose gel using 18S RNA as the standard for normalization. Two RT-PCR runs were performed for each animal, and the products of each RT-PCR run were analyzed by two electrophoreses.

To investigate specific mRNA expression in the main PA, a nested RT-PCR analysis was necessary because of the low amount of mRNA contained in a single PA. Total RNA was extracted from each PA using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. RNA was then submitted to RT using the Omniscript RT Kit from Qiagen in the presence of a mixture of random primers and oligodT (Promega). The first PCR was then performed from 1/20 of the volume of the RT product using the HotStarTaq MasterMix Kit from Qiagen and the following primers (Microsynth) for M1AChR: sense 5'-TAGTTGGGGAGCGGACAGTG-3' and antisense 5'-GGCCAGTTGTTCCTTCCCTC-3', designed on the basis of cDNA sequences of mouse M1AChR (GeneBank Accession No. NM007698). These primers were chosen, respectively, above and below the primers used for detection in the lungs, which were used for the second PCR described below. For 18S RNA, the primers were the same as for detection in total lungs because a single RT-PCR was sufficient according to the high expression level of this RNA. PCR conditions were as follows: 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, with 35 cycles for M1AChR and 30 cycles for 18S RNA. A second PCR of 35 cycles was performed for M1AChR from 1/10 of the volume of the first-round PCR product using the same primers as for detection in the lungs. RT-PCR products were analyzed after electrophoresis on a 2% agarose gel using 18S RNA as the standard for normalization. Two nested PCR were performed for each animal, and the products of each nested PCR were analyzed by two electrophoreses.

Western Blot Analysis of Protein Expression

The relative expression of specific proteins was investigated by Western blot analysis in adult lungs homogenates and in PA extracts. Flash-frozen lungs were crushed in a cryogenic mortar and homogenized in 1 ml lysis buffer {50 mM HEPES, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM DTT, 5 µg/ml pepstatin, 3 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1 mM sodium vanadate, 50 mM NaF, and 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}. Homogenates were then centrifuged for 10 min at 3,000 g at 4°C. The protein concentration of the supernatant was quantified using a BCA protein assay kit (Pierce, Rockford, IL). Samples were diluted in Laemmli buffer and heated for 10 min at 95°C before being loaded on a 7.5% polyacrylamide gel. Proteins were fractioned by SDS-PAGE (35 min at 200 V) and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) during 2 h at 100 V. Blots were blocked overnight at 4°C in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) containing 5% nonfat dry milk. Membranes were then immunoblotted for 1 h at room temperature using specific antibodies targeted against eNOS (BD Transduction Laboratories, Franklin Lake, NJ), PKG (Stressgen Bioreagents, Ann Arbor, MI), M1AChR (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma), diluted 1:1,000, 1:1,000, 1:100, and 1:250, respectively, in TBS-T containing 5% nonfat dry milk. After being washed in TBS-T, blots were incubated with ECL horseradish peroxidase-linked anti-mouse antibody (for eNOS detection) or anti-rabbit antibody (for the detection of the other proteins) (Amersham Biosciences, Buckinghamshire, UK), diluted 1:10,000 in TBS-T. After membranes had been washed in TBS-T, specific proteins were detected by chemiluminescence using the ECL Western blot analysis system (Pierce) and exposition to X-ray film. The expression of specific protein was quantified using UN-SCAN-IT gel software (Silk Scientific, Orem, UT) and normalized to β-actin content. The relative protein content in homogenates of mice born in hypoxia was reported to the content measured in controls. The use of β-actin as an internal control in our model was validated as previously described (51) (data not shown).

Specific protein expression in isolated PAs was determined as described above with some modifications. Briefly, for eNOS protein detection, 10 PAs per sample were pooled and homogenized in 100 µl lysis buffer. For PKG protein detection, each main PA was crushed in a cryogenic mortar and homogenized in 12 µl lysis buffer. After centrifugation, 10 µl of supernatant were diluted in Laemmli buffer and heated for 3 min at 95°C before being loaded on an electrophoresis gel. Samples were then processed as described above except that detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

cGMP Production in PAs

cGMP production was investigated as previously described (46) with some technical modifications. Briefly, PAs were dissected as for isolated vessel tension experiments (see the description of the technique in Isolated Vessel Tension Experiments) and incubated in modified Krebs-Ringer bicarbonate solution maintained at 37°C and bubbled with 95% O2-5% CO2. All measurements were performed in the presence of indomethacin (10–5 M) and the nonspecific PDE inhibitor IBMX (10–3 M) to prevent the degradation of cGMP. In some experiments, L-NNA (10–4 M), 4-DAMP (10–5 M), and/or pirenzepine (10–7 M) were also used. After a 20-min equilibration period, Phe (10–5 M) was added in the bath, and vessels were incubated for 1 h before the addition of ACh (10–5 M) to reproduce the experimental conditions used in isolated vessel tension experiments. After a 3-min incubation period with ACh, PAs were quickly frozen in liquid nitrogen and homogenized in 0.1 N HCl. cGMP production was also tested after a 3-min incubation period with the NO donor diethylamine (DEA)/NO (10–4 M, Alexis) instead of ACh. The cGMP content was then tested in homogenates using the Direct cGMP Enzyme Immunoassay Kit from Sigma according to the manufacturer's instructions. Samples treated with ACh, in the absence or presence of pirenzepine, or with DEA/NO were tested using the classical procedure, whereas samples with low cGMP content, such as in the absence of ACh or DEA/NO (basal level of cGMP production) or in the presence of L-NNA or 4-DAMP, were tested using the acetylation procedure.

Drugs

Unless otherwise specified, all drugs were purchased from Sigma.

Indomethacin was prepared in equal molar Na2CO3 (69). IBMX was dissolved in ethanol, A23187 [GenBank] and vinpocetine were dissolved in DMSO, and DEA/NO was dissolved in 10 mM NaOH. The other drugs were prepared using distilled water.

Data Analysis

Statistical analyses were performed using InStat 3.0 (GraphPad Software, San Diego, CA). Data are expressed as means ± SE, and n represents the number of animals per group. The effect of acute hypoxia on RVP in each group was tested using a paired Student's t-test, whereas the effects of perinatal hypoxia on all studied parameters were tested using an unpaired Student's t-test with the Welch correction. Two-way ANOVA was performed using Prism 4 (GraphPad Software) to compare the dose-response curves in isolated vessel tension experiments.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Anatomic Data

Perinatal hypoxia did not influence adult body or heart weights, nor the heart-to-body weight ratio (Table 1). The RV-to-left ventricle (LV) plus septum (S) ratio [RV/(LV + S) ratio] was used as an index of RV hypertrophy. This was a direct consequence of the increased pulmonary vascular resistance present in pulmonary vascular pathologies. In adult mice, the RV/(LV + S) ratio was similar in both groups (Table 1).


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  		Table 1. Anatomic data

 
Hemodynamic Experiments

Hematocrit was similar in adult mice born in normoxia or in hypoxia (48.0 ± 0.5% and 47.7 ± 0.4%, respectively, means ± SE, n = 12–13).

RVP was used as an estimation of the pulmonary arterial pressure (20). Basal RVP measured in normoxic conditions (21% O2) was significantly higher in adult mice born in hypoxia compared with controls (Table 2). Exposure to acute hypoxia (12% O2) during hemodynamic measurements resulted in a significant increase of RVP in both groups (Table 2). This increase was twofold higher in animals born in hypoxia than in controls (Table 2), suggesting that their pulmonary vessels react more to acute hypoxia later in life. A transient exposure to hypoxia around birth appears then to have long-term effects on the pulmonary circulation.


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  		Table 2. RVP in normoxia or acute hypoxia

 
Vasoconstriction of Isolated Main PAs

Isolated adult PAs were constricted in a dose-dependent manner by cumulative concentrations of KCl or Phe in the presence of indomethacin and L-NNA. No significant differences were shown in vasoconstrictive responses of PAs from both groups (data not shown). The vasoconstriction induced by KCl was maximal at 100 mM. This concentration was used to verify the reactivity of all vessels at the beginning of each vessel tension experiment. The maximal response to Phe was obtained with 10–5 M. This concentration was used to precontract the PA before vasorelaxant agents were tested in the isolated vessel tension experiment described below. Interestingly, the maximal increase in tension induced by 10–5 M Phe in absence of L-NNA, a condition used when testing endothelium-dependent vasorelaxant agents, was similar in both groups (0.21 ± 0.02 g in controls and 0.22 ± 0.03 g in mice born in hypoxia, n = 6–7) but was significantly lower than in the presence of L-NNA (0.39 ± 0.04 g in controls and 0.44 ± 0.05 g in mice born in hypoxia, n = 8, P < 0.01 compared with contraction induced without L-NNA by unpaired t-test). This suggests that NOS contributes significantly to the vascular tone homeostasis in the murine main PA.

ACh and Muscarinic Receptors

Isolated vessel tension experiments. Pulmonary vascular reactivity was tested in isolated PAs preconstricted with Phe in the presence of ACh, an endothelium-dependent relaxing agent, or NO, an endothelium-independent agent. PAs of mice born in hypoxia showed an important reduction in the relaxant response to ACh (Fig. 2A) compared with PAs of mice born in normoxia. The maximal relaxation (Emax) induced by ACh in PAs of mice born in hypoxia was significantly decreased by ~25% compared with controls (57.61 ± 4.74% and 75.52 ± 4.67%, respectively, means ± SE, n = 6–7, P < 0.05 by unpaired t-test). In contrast, endothelium-independent relaxation induced by NO was similar in the perinatal hypoxia and control groups (Fig. 2B), with equal Emax (88.79 ± 4.79% and 80.73 ± 2.66%, respectively, means ± SE, n = 8–10). Therefore, perinatal hypoxia appears to impair the NO/cGMP pathway at the endothelial but not smooth muscle cell level. However, additional modifications in smooth muscle cells cannot be excluded. Compensatory mechanisms could also be implicated, resulting in a global unmodified NO-induced relaxation.


Figure 2
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  		Fig. 2. Pharmacological responses of isolated PAs preconstricted with Phe to endothelium-dependent (ACh) and endothelium-independent [nitric oxide (NO)] relaxing agents. Cumulative dose-responses to ACh (10–8–10–4 M, n = 6–7 animals; A) or NO (10–8–10–5 M, n = 8–10 animals; B) were tested on the main PA of adult mice born in normoxia (Ctr) or perinatal hypoxia (PH). Cumulative dose responses to ACh (10–8–10–4 M) were also tested in the presence of 4-diphenylacetoxy-N-piperidine methiodide (4-DAMP; 10–5 M, n = 10 animals; C), NG-nitro-L-arginine (L-NNA; 10–4 M, n = 8 animals; D), Pir (10–7 M, n = 5 animals; E) or Tel (10–10 M, n = 8–9 animals; F). Results are expressed as means ± SE of percentages of changes in tension induced by the vasodilator. *Significant difference between Ctr and PH groups (P < 0.05 by unpaired t-test).

 
The tone-dependent action of ACh on PAs explains the pattern of the pharmacological response to ACh (Fig. 2A). Low doses of ACh induce the relaxation of precontracted arteries, whereas higher doses result in the constriction of relaxed vessels. This pattern has also been observed in different species (23, 38, 46, 73). In our murine model, ACh-induced relaxation was completely inhibited in the presence of 4-DAMP (10–5 M), a selective antagonist of M3AChR (11, 39) (Fig. 2C) or in the presence of L-NNA (10–4 M), an inhibitor of NOS (53) (Fig. 2D). These results confirm that ACh-induced relaxation of the murine PA is mediated by M3AChR and NOS. In the presence of 10–7 M pirenzepine, a selective antagonist of M1AChR (11, 39), the vasorelaxation induced by ACh was not modified in controls (Emax = 72.54 ± 5.54% and 75.52 ± 4.67% with or without pirenzepine, respectively, means ± SE, n = 5–7; Fig. 2E). In contrast, under these conditions, PAs from mice born in hypoxia reacted as well as PAs from controls, showing a significant increase in Emax compared with ACh without pirenzepine (Emax = 77.07 ± 1.19% and 57.61 ± 4.74% with or without pirenzepine, respectively, means ± SE, n = 5–6, P < 0.05 by unpaired t-test; Fig. 2E). Similar results were obtained using telenzepine, another preferential inhibitor of M1AChR (39) (Fig. 2F). In the presence of 10–10 M telenzepine, ACh-induced relaxation was not modified in controls (Emax = 79.64 ± 2.77% and 75.52 ± 4.67% with or without telenzepine, respectively, means ± SE, n = 7–8), whereas PAs from mice born in hypoxia reacted as well as PAs from controls, showing a significant increase in Emax compared with ACh without telenzepine (Emax = 73.31 ± 2.99% and 57.61 ± 4.74% with and without telenzepine, respectively, means ± SE, n = 6–9, P < 0.05 by unpaired t-test). Therefore, the M1AChR seems to be a major molecular component implicated in the alteration of ACh-induced vasorelaxation following perinatal hypoxia.

Muscarinic receptor expression. M1AChR mRNA expression was tested in both the whole lung and PA. Similar results were obtained in both tissue extracts (Fig. 3). M1AChR mRNA expression was significantly increased in both the lungs and PAs of mice born in hypoxia compared with controls (Fig. 3). This is consistent with the functional results obtained in the absence or presence of pirenzepine or telenzepine. Western blot analysis of the relative M1AChR protein content showed no differences between the lungs of mice born in normoxia or hypoxia (data not shown). However, M1AChR protein could not be detected in isolated PAs using classical Western blot analysis. The low level of M1AChR protein content and the weak quantity of tissue and proteins present in isolated murine PAs made it necessary for us to pool vessels of several mice to obtain a sufficient protein concentration and to use a more sensitive technique of immunodetection [biotinylated secondary antibody and ABC reactive of Vectastain ABC kit Elite (Vector Laboratories, Burlingame, CA)]. However, the latter technique led to the apparition of two strong unspecific signals, one of which displayed a molecular weight close to M1AChR and masked its weak specific signal.


Figure 3
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  		Fig. 3. RT-PCR analysis of M1AChR mRNA expression. Semiquantitative analysis of M1AChR mRNA content was performed in the total lung or main PA of adult mice born in normoxia (Ctr) or PH. Results are expressed as means ± SE (n = 4 animals) of relative M1AChR mRNA expression after normalization by 18S RNA content. *Significant difference between Ctr and PH groups (P < 0.01 by unpaired t-test).

 
A23187 and eNOS Expression

A23187. The calcium ionophore A23187 [GenBank] induced dose-dependent relaxation in isolated PAs preconstricted with Phe (Fig. 4A). PAs of mice born in hypoxia showed a significant reduction in the relaxant response to 3 x 10–6 M A23187 [GenBank] (Fig. 4A) compared with PAs of mice born in normoxia. In the presence of the NOS inhibitor L-NNA (10–4 M), A23187 [GenBank] -induced vasorelaxation was highly, but not completely, inhibited in both groups (Fig. 4B). Interestingly, PAs of mice born in hypoxia reacted as controls in the presence of L-NNA, suggesting that the difference observed in the absence of L-NNA could be due to a decrease in NOS activity in adult PAs following a perinatal hypoxic insult.


Figure 4
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  		Fig. 4. Pharmacological responses of isolated PAs preconstricted with Phe to the endothelium-dependent relaxing agent A23187 [GenBank] and Western blot analysis of endothelial NO synthase (eNOS) protein. Cumulative dose responses to the calcium ionophore A23187 [GenBank] (10–9–10–5 M) were tested on the main PA of adult mice born in normoxia (Ctr) or PH in the absence (A; n = 5–6 animals) or presence (B) of L-NNA (10–4 M, n = 3–4 animals). Results are expressed as means ± SE of percentages of changes in tension induced by the vasodilator. *Significant difference between Ctr and PH groups (P < 0.01 by unpaired t-test). C: Western blot analysis of eNOS protein relative content was performed in total lung homogenates (n = 4 animals) or PA extracts (n = 3 pools of 10 PA) of adult mice born in normoxia (Ctr) or PH. Results are expressed as means ± SE of relative eNOS protein content after normalization by β-actin protein content. *Significant difference between Ctr and PH groups (P < 0.05 by unpaired t-test).

 
eNOS expression. eNOS protein expression was tested in both the whole lung and main PA. The eNOS relative protein content was not influenced in adult lungs by perinatal exposure to hypoxia, whereas it was significantly decreased in isolated PAs of mice born in hypoxia compared with controls (Fig. 4C). This is consistent with the altered relaxation to ACh and A23187 [GenBank] observed in mice born under hypoxic conditions.

cGMP and PDEs

cGMP production in isolated PAs. cGMP production was investigated in isolated main PAs of mice born in normoxia or hypoxia in the presence of Phe and indomethacin to reproduce the conditions used in isolated vessel tension experiments (Fig. 5). The nonselective PDE inhibitor IBMX (10–3 M) was added to avoid the degradation of cGMP produced. Basal cGMP production was similar in both groups. The addition of ACh resulted in a significant enhancement of cGMP production, which, however, did not differ between control PAs and PAs of mice born in hypoxia. ACh-induced cGMP production was significantly decreased in both groups in the presence of L-NNA or 4-DAMP. cGMP accumulation in the presence of ACh and 4-DAMP did not differ from basal cGMP production, whereas cGMP production in the presence of ACh and L-NNA was significantly lower than in the presence of ACh and 4-DAMP and became almost undetectable. In the presence of pirenzepine, ACh-mediated cGMP production in mice born in hypoxia was similar to that in control PAs. These results suggest that ACh-induced cGMP production is mediated by M3AChR and NOS, whereas ACh-induced activation of M1AChR did not interfere with cGMP production, at least in the presence of IBMX (10–3 M). However, in the absence of IBMX, the cGMP content was dramatically low, even under ACh stimulation (data not shown). This suggests the presence of highly active PDEs in these vessels. Interestingly, the addition of DEA/NO resulted in a strong stimulation of cGMP production in both groups, which was almost twice in PAs of mice born in hypoxia than in controls (Fig. 5). Therefore, sGC protein expression and/or activity appears to be higher in PAs of mice born under hypoxic conditions than in controls.


Figure 5
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  		Fig. 5. cGMP production in isolated PAs preconstricted with Phe. Basal or ACh (10–5 M)-induced cGMP production was assessed in the isolated main PA of adult mice born in normoxia (Ctr) or PH in the absence or presence of L-NNA (10–4 M), 4-DAMP (10–5 M), or Pir (10–7 M). cGMP production was also measured after induction by the NO donor diethylamine (DEA)/NO (10–4 M). Data are means ± SE (n = 4–10 animals) of the quantity of cGMP produced and are expressed as picomoles cGMP per milligram of protein. *Significant difference compared with basal cGMP production (P < 0.05 by unpaired t-test); {dagger}significant difference between ACh-induced cGMP production in the presence or absence of inhibitor (P < 0.05 by unpaired t-test); {ddagger}significant difference between both conditions (P < 0.05 by unpaired t-test).

 
Isolated vessel tension experiments. Pharmacological dose responses of isolated PAs to the cell membrane-permeant cGMP analog 8-bromo-cGMP were similar in mice born in normoxia or hypoxia (Fig. 6). This cGMP analog is resistant to hydrolysis by PDEs (76). This suggests that perinatal hypoxia does not influence cGMP-induced relaxation.


Figure 6
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  		Fig. 6. Pharmacological responses of isolated PAs preconstricted with Phe to the endothelium-independent relaxing agent 8-bromo-cGMP. Cumulative dose responses to the cell-permeant cGMP analog 8-bromo-cGMP (10–8–10–4 M) were tested on the main PA of adult mice born in normoxia or PH in the presence of L-NNA (10–4 M, n = 6–9 animals). Results are expressed as means ± SE of percentages of changes in tension induced by the vasodilator. No significant difference was observed between both groups (unpaired t-test).

 
The absence of differences between mice born in normoxia or hypoxia observed in cGMP production experiments in the presence of IBMX and in pharmacological responses to the PDE-resistant analog of cGMP suggests that PDEs could contribute to the decrease of ACh-induced relaxation in PAs of mice born in hypoxia.

Interestingly, in the presence of indomethacin and absence of L-NNA, the addition of IBMX (10–3 M) in organ chambers prevented Phe-induced contraction of PAs, whereas the addition of IBMX (10–3 M) in preconstricted PAs induced complete vasorelaxation (data not shown). This also suggests the presence of highly active PDEs.

As M1AChR activation is known to result in IP3 production and a cytosolic calcium concentration increase (39), we tested the effect of selective inhibition of calcium-dependent PDE1. Cumulative concentrations of the PDE1 inhibitor vinpocetine induced similar dose-dependent relaxations in preconstricted PAs of mice born in normoxia or hypoxia (Fig. 7A). This relaxation was totally reversed by the addition of L-NNA (10–4 M; data not shown), suggesting that vinpocetine-induced relaxation is completely mediated by basal NOS activity. Interestingly, the presence of 10–5 M vinpocetine did not significantly modify the pharmacological response to ACh in control PAs (Emax = 68.70 ± 4.40% and 75.52 ± 4.67% with or without vinpocetine, respectively, means ± SE, n = 6–7) but resulted in a significant increase of the relaxant response to ACh in PAs of mice born in hypoxia (Emax = 71.51 ± 3.94% and 57.61 ± 4.74% with and without vinpocetine, respectively, means ± SE, n = 6, P < 0.05 by unpaired t-test). In fact, the addition of 10–5 M vinpocetine totally abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation (Fig. 7B). This suggests that PDE1 contributes to M1AChR-mediated alteration of ACh-induced relaxation in PAs of mice born in hypoxia.


Figure 7
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  		Fig. 7. Pharmacological responses of isolated PAs preconstricted with Phe in the presence of the phosphodiesterase 1 inhibitor vinpocetine (Vinpo). A: cumulative dose responses to Vinpo (10–8–10–4 M, n = 8 animals) were tested on the main PA of adult mice born in normoxia (Ctr) or PH. B: cumulative dose responses to ACh (10–9–10–4 M) were tested after the addition of Vinpo (10–5 M, n = 6 animals). C: cumulative dose responses to A23187 [GenBank] (10–9–10–5 M) were tested after the addition of Vinpo (10–5 M, n = 6–7 animals). Results are expressed as means ± SE of percentages of changes in tension induced by the vasodilator. No significant difference was observed between both groups (unpaired t-test).

 
Interestingly, 10–5 M vinpocetine also abolished the alteration of A23187 [GenBank] -induced relaxation observed in PAs of mice born in hypoxia compared with controls (Fig. 7C). Vinpocetine did not influenced A23187 [GenBank] -induced relaxation in controls but significantly improved the relaxant response to A23187 [GenBank] in PAs of mice born in hypoxia, which then relaxed as well as control PAs.

Figure 8 summarizes the effects of the various inhibitors on endothelium-dependent relaxation induced by ACh (3 x 10–6 M) or A23187 [GenBank] (3 x 10–6 M) in PAs of mice born under normoxic or hypoxic conditions.


Figure 8
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  		Fig. 8. Pharmacological responses of isolated PAs preconstricted with Phe to the endothelium-dependent relaxing agents ACh or A23187 [GenBank] in the absence or presence of various inhibitors. A and B: summary of relaxant responses to ACh (3 x 10–6 M; A) or A23187 [GenBank] (3 x 10–6 M; B) observed in the main PA of adult mice born in normoxia (Ctr) or PH in the absence or presence of L-NNA (10–4 M), 4-DAMP (10–5 M), Pir (10–7 M), Tel (10–10 M), or Vinpo (10–5 M) (n = 3–10 animals). Results are expressed as means ± SE of percentages of relaxation induced by the vasodilator. *Significant difference between Ctr and PH groups (P < 0.05 by unpaired t-test); {dagger}significant difference compared with ACh- or A23187 [GenBank] -induced relaxation in the absence of inhibitor (P < 0.05 by unpaired t-test).

 
PKG protein expression. Perinatal exposure to hypoxia did not influence relative PKG protein content in adult whole lung homogenates but induced a slight but statistically significant increase in PAs (Fig. 9).


Figure 9
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  		Fig. 9. Western blot analysis of PKG-1{alpha} protein expression. Western blot analysis of the relative PKG-1{alpha} protein content was performed in the total lung (n = 4 animals) or main PA (n = 12 animals) of adult mice born in normoxia (Ctr) or PH. Results are expressed as means ± SE of relative PKG-1{alpha} protein content after normalization by β-actin protein content. *Significant difference between Ctr and PH groups (P < 0.05 by unpaired t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present findings show that a transient exposure to hypoxia during a critical period of development induces long-term adverse effects on the pulmonary circulation, even in the absence of a new hypoxic stimulus. Although adult mice exposed to perinatal hypoxia did not phenotypically differ from controls, their regulation of the pulmonary circulation was significantly altered. Mice born in hypoxia displayed, as adults, higher RVP in basal conditions compared with controls. This elevated RVP observed in mice born in hypoxia was not due to an increase in blood viscosity, since their hematocrit did not differ from controls. This strongly suggests a higher resistance state in the pulmonary circulation of adults following perinatal hypoxia, although the RV/(LV + S) ratio was not significantly increased in young adult animals. However, the development of RV hypertrophy later in life cannot be excluded and remains to be investigated. When exposed to acute hypoxia in adulthood, mice having suffered from perinatal hypoxia showed a larger increase in RVP compared with the response of control mice, suggesting an increased sensitivity of the lung vasculature to hypoxia. Such observations are consistent with previous studies in animals (10, 30–32, 68). In humans, several lines of evidence have also indicated that chronic pulmonary vascular diseases and disturbed pulmonary vascular reactivity in adulthood may be associated with a perinatal insult (28, 63). The mechanisms underlying the exaggerated pulmonary vasoconstrictor responsiveness and the resulting altered lung circulation in adulthood following a perinatal hypoxic event have not been elucidated so far. Indeed, whereas the effects of chronic or acute hypoxia on the regulation of the pulmonary circulation have been extensively studied, the long-term effects of perinatal hypoxia on the pulmonary circulation were mainly described in terms of morphological or hemodynamic modifications, with limited investigations of the regulatory processes of the pulmonary circulation. In adults, diseases that have been potentially linked to a perinatal event, such as systemic hypertension, ischemic heart disease, or non-insulin-dependent diabetes mellitus, are clinical conditions associated with endothelial dysfunction (14, 19, 27). In our model of perinatal hypoxia, the altered pulmonary vascular responsiveness appeared to be also in relation to some degree of endothelial dysfunction, as suggested by the decrease of the relaxant response to the endothelium-dependent agents ACh and A23187 [GenBank] , whereas endothelium-independent relaxation induced by NO or 8-bromo-cGMP was not modified. Such a decrease in endothelium-dependent relaxation of isolated PAs has been previously observed, for example, in adult rats (9) or piglets (71) exposed to chronic hypoxia as well as in isolated rabbit PAs submitted to moderate hypoxia (42). Isolated vessel tension experiments showed that perinatal hypoxia did not modify the vasoconstrictive responses of adult main PAs to KCl or Phe, whereas endothelium-dependent relaxation of pulmonary vessels induced by ACh was significantly reduced in adult mice exposed to perinatal hypoxia. Interestingly, such a decrease in ACh-induced relaxation has also been observed in systemic vessels following either chronic moderate hypoxia (61) or food restriction (7, 35) during pregnancy. A23187 [GenBank] is a calcium ionophore that induces endothelium-dependent vasorelaxation by the activation of the calcium-dependent NOS isoform eNOS via an increase in the cytosolic calcium concentration. The inability of A23187 [GenBank] to induce vasoconstriction in vessels with an intact endothelium could be explained by the presence in smooth muscle cells of the sodium/calcium exchanger, which extrudes calcium (8). The decreased response to A23187 [GenBank] observed in PAs of mice born in hypoxia compared with controls suggests a decrease in eNOS protein expression and/or activity, since the residual relaxation induced by A23187 [GenBank] in the presence of L-NNA was similar in both groups. The decreased eNOS relative protein content observed in PAs of mice born in hypoxia compared with controls is consistent with the altered response to A23187. [GenBank] The effects of hypoxia on eNOS protein expression have been extensively investigated and appear to be controversial, depending on the studied species, type of vessel, duration of hypoxia, and timing of exposure (47). Whereas eNOS protein was found to be upregulated by chronic hypoxia in the lungs of adult rats (48, 58, 60, 65, 66) or mice (44, 49) or in mice with severe hypoxia-induced pulmonary hypertension (16), reduced eNOS protein expression has been observed, for example, in pulmonary vessels of patients with pulmonary hypertension (24) as well as in PAs of newborn piglets with chronic hypoxia-induced hypertension (34), in whole lung homogenates of piglets exposed to chronic hypoxia (18), or in the lungs of fetal lambs exposed to chronic intrauterine pulmonary hypertension (72). However, it is not excluded that activation mechanisms of eNOS could also be impaired following perinatal hypoxia. Decreased eNOS activity resulting from an abnormal interaction between eNOS and its regulatory proteins has been described in isolated PAs of adult rats with chronic hypoxia-induced pulmonary hypertension (54). The total inhibition of ACh-induced vasorelaxation observed in both experimental groups in the presence of the NOS inhibitor L-NNA showed that, in mice, ACh-induced relaxation of the adult main PA was completely mediated by NOS, as previously demonstrated (70). Therefore, the decreased NOS activity suggested by the A23187 [GenBank] dose-response curve and the diminished eNOS protein content in isolated PAs of mice having suffered from perinatal hypoxia could explain at least partially the alteration of ACh-induced relaxation. However, functional and molecular experiments further demonstrated that alterations of the relaxant response of the main PA from adult mice born in hypoxia were mainly due to increased expression and/or activity of M1AChR. In fact, pirenzepine or telenzepine, two preferential antagonists of M1AChR, abolished the adverse effect of perinatal hypoxia on ACh-induced relaxation, and M1AChR mRNA expression was increased in pulmonary tissues of mice born under hypoxic conditions. The resulting imbalance in muscarinic receptors could explain the decrease of the relaxant effect of ACh on pulmonary vessels due to a reduced availability of ACh to M3AChR. Indeed, the ACh-induced relaxation of PAs appeared to be mediated by M3AChR, whereas M1AChR seemed to be implicated in the vasoconstrictive effects of ACh, as demonstrated by the addition of inhibitors of M3AChR or M1AChR, respectively. The M1AChR-mediated decrease of ACh-induced relaxation observed in PAs of mice born in hypoxia could be due either to an increased counteracting contractility of the vessel or to a direct interaction of the M1AChR-stimulated signaling pathway with the NO/cGMP relaxing pathway. cGMP production measurements in PAs were performed to investigate this hypothesis. Basal cGMP production as well as ACh-induced cGMP synthesis were similar in both groups. As expected, L-NNA or 4-DAMP inhibited ACh-induced cGMP production in PAs of both groups. This confirms that ACh-induced relaxation is mediated by NOS and M3AChR. In the presence of ACh and 4-DAMP, cGMP production was similar to the basal level, suggesting that ACh-induced cGMP production is completely mediated by M3AChR and not by other mechanisms. In the presence of ACh and L-NNA, cGMP production was significantly and almost completely inhibited compared with ACh and 4-DAMP, suggesting that NOS activity is necessary for ACh-mediated cGMP production. Interestingly, DEA/NO-induced cGMP production was significantly higher in PAs of mice born in hypoxia than in controls, suggesting increased sGC activity in adult PAs following perinatal hypoxia. The effects of hypoxia on sGC protein expression and/or activity in the lungs described in the literature are controversial. Chronic hypoxia has been found to result in increased sGC protein expression and/or activity in the adult lungs of rats (50) or mice (49), whereas others have shown either no influence (40, 44) or an inhibitory effect (58) of chronic hypoxia on adult pulmonary sGC protein expression and/or activity. The absence of differences in ACh-induced cGMP production between PAs of mice born in hypoxia or normoxia despite increased sGC activity suggests that eNOS activity was probably reduced in vessels of the perinatal hypoxia group. Moreover, the absence of differences in NO-stimulated relaxation between isolated PAs of both groups, despite the increased production of cGMP observed in mice born in hypoxia under these conditions, suggests that PDE activity should be also higher in pulmonary vessels of mice born in hypoxia than in controls. In the presence of pirenzepine, ACh-induced cGMP production was similar in both groups. Such results suggest that the M1AChR-mediated signaling pathway did not directly interfere with cGMP production, at least in the presence of the nonspecific PDE inhibitor IBMX. However, it is not excluded that ACh-induced activation of M1AChR interferes with the NO/cGMP pathway in the absence of IBMX. Indeed, M1AChR stimulation induces the production of IP3 and an increase in the intracellular calcium concentration. Several isoforms of PDEs participate in the degradation of cGMP in pulmonary vessels, among which PDE1 is activated by calcium (62). M1AChR activation could therefore result in the stimulation of cGMP degradation by PDE1. Increased expression and/or activity of M1AChR and/or PDE1 in PAs of mice born in hypoxia could then contribute to the decreased ACh-induced relaxation. cGMP production assays performed in the absence of IBMX and isolated vessel tension experiments in the presence of IBMX suggest that PDEs are highly active in the main PA and seem to play an important role in vascular tone homeostasis. Isolated vessel tension experiments using the selective inhibitor of PDE1 vinpocetine demonstrated that basal PDE1 activity seems to be similar in both groups. However, the complete restoration of ACh-induced relaxation observed in mice born in hypoxia in the presence of vinpocetine argues in favor of PDE1 as a mediator of the M1AChR-induced alteration of endothelium-dependent relaxation. This hypothesis is strengthened by the abolishment of the altered response to A23187 [GenBank] observed in mice born in hypoxia in the presence of vinpocetine, suggesting that the decrease in A23187 [GenBank] -induced relaxation could be due to enhanced PDE1 activity resulting from a transient increase in the calcium concentration in smooth muscle cells. Interestingly, PDE1 has been recently found to be upregulated in human patients and animal models with pulmonary arterial hypertension (64) as well as in PA smooth muscle cells from patients with pulmonary hypertension (55). Taken together, such observations could be of therapeutical interest. For example, inhibition of PDE1 by vinpocetine has been recently found to augment the pulmonary vasodilator response to inhaled NO in awake lambs with acute pulmonary hypertension (15). Further investigations using specific inhibitors and antibodies could therefore be of interest to better characterize which isoforms are implicated in the alteration of endothelium-dependent relaxation.

In our murine model, perinatal exposure to hypoxia did not modify PKG-1{alpha} protein content in the whole lung homogenate but induced a slight but significant increase in relative PKG-1{alpha} protein expression in adult PAs. However, relaxation induced by the cell-permeable cGMP analog 8-bromo-cGMP was similar in both groups, suggesting that either global PKG activity was not modified despite the increase in protein content or that there are additional downstream modifications resulting in limited signal transduction. In the literature, the effects of oxygen tension on PKG-1{alpha} in the pulmonary vasculature appear also to be controversial, depending, among others, on the animal model and type of vessel considered as well as on the timing and duration of exposure to hypoxia. For instance, chronic hypoxia was shown in adult rats to attenuate the pulmonary vasodilatory response to the cGMP analog 8-bromo- cGMP (40, 41) despite a significant increase in PKG-1 protein expression and activity (41). In fetal lambs, sustained exposure to normoxia induced a significant increase in both PKG-1{alpha} mRNA and protein expression (21, 59) as well as in PKG activity and 8-bromo-cGMP-induced relaxation (21). In contrast, chronic intrauterine pulmonary hypertension decreased both PKG-1{alpha} mRNA and protein expression and attenuated the effects of sustained normoxia in PAs of fetal lambs (59). Chronic high-altitude hypoxia in utero significantly decreased PKG-mediated relaxation and PKG activity in PAs of fetal lambs despite an increased expression of PKG-1 protein (22). Acute hypoxia has been shown to reduce 8-bromo- cGMP-induced relaxation as well as PKG protein expression and activity in pulmonary vessels of fetal lambs (56). There are, however, no data on the long-term effects of perinatal hypoxia on PKG-1 expression and/or activity in the pulmonary vasculature.

Concerning the specific proteins tested throughout the study, their relative content in lung homogenates was similar in both groups, in contrast to the observations made in PA extracts for eNOS and PKG-1. However, the whole lung homogenate contains also other cell types, in particular bronchial epithelial and smooth muscle cells, that express these proteins. Immunohistochemical staining would be necessary to differentiate vascular and bronchial expression.

The long-term adverse effects of perinatal hypoxia on the developing lung vasculature appear to be dependent on alterations of muscarinic receptors or muscarinic receptor-mediated signaling pathways in adults. We therefore postulate that an imbalance in muscarinic cholinergic receptors secondary to a perinatal insult could play an important role in diseases related to vascular dysfunction with developmental origins. Diseases regrouped in the so-called metabolic syndrome (syndrome X), like hypertension, stroke, insulin resistance, Type 2 diabetes, and dyslipidemia, have been shown to have potential perinatal origins (25). Whether physiological dysfunctions linked to syndrome X could be observed in our model of perinatal hypoxia remains to be studied. In particular, it would be interesting to investigate whether muscarinic receptors alterations also play a role in systemic vessels. Moreover, it remains to be demonstrated whether such muscarinic receptor imbalance persists throughout life. In particular, it could be of interest to determine whether the alteration is permanent or progressive. Potential interactions with other regulatory mechanisms of pulmonary vascular tone need also to be investigated. We recently demonstrated that the expression and activity of voltage-gated and calcium-dependent potassium channels are increased in PA smooth muscle cells from adult mice born in hypoxia compared with controls (51). Whether such modifications in potassium channel subpopulations are implicated in the altered ACh-induced relaxation observed in this study remain to be further investigated.

In summary, we showed that a transient exposure to hypoxia during a critical period of development of the lung vasculature results in an alteration of the regulation of pulmonary vascular tone later in life. Therefore, individuals having suffered from perinatal hypoxia could be at risk to have an altered regulation of their pulmonary circulation and to develop pulmonary hypertensive pathologies in adulthood. This could be particularly possible during reexposure to clinical conditions in humans associated with hypoxia, like sleep apnea, chronic and acute pulmonary diseases, or when going at high altitude.

More generally, we observed, like others (61, 75), that, although most epidemiological studies have linked cardiovascular diseases occurring in adulthood to fetal malnutrition, limitation of oxygen supply during the perinatal period could also lead to some vascular dysfunction characterized by impairment of endothelium-dependent relaxation. This pulmonary vascular dysfunction appears to be strongly associated with alterations in muscarinic receptors. In particular, muscarinic receptor signaling pathways seem to have important interactions with PDEs, which need to be further investigated and could be of therapeutical interest. We also hypothesize muscarinic receptors could be biological markers of pulmonary diseases with vascular dysfunction in relation to perinatal programming.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Swiss National Foundation for Scientific Research Grants 3200-067046 and PP-00B-68882/1, the Leenaards Foundation, the Eagle Foundation, the Emma Muschamp Foundation, and the Fern Moffat Foundation.


   ACKNOWLEDGMENTS
 
We are grateful to Profs. Michel Dolivo and Jean-Léopold Micheli for helpful suggestions and critical review of the manuscript. We thank also Profs. Adrien Moessinger and Sergio Fanconi as well as the Department of Pediatrics of the University Hospital Center of Lausanne for support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-F. Tolsa, Neonatal Research Laboratory, Division of Neonatology, Dept. of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne 1011, Switzerland (e-mail: Jean-Francois.Tolsa{at}chuv.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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