Introduction
Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, is a phospholipid with a wide range of biological activities.Reference Prescott, Zimmerman, Stafforini and McIntyre1 PAF is a potent vasoconstrictor in the pulmonary circulation,Reference Argiolas, Fabi and del Basso2–Reference Toga, Ibe and Raj5 with different sites of action in the pulmonary vasculature. Endogenous PAF plays an important role in maintaining a high fetal pulmonary vasomotor tone and pulmonary vascular resistance in utero.Reference Ibe, Hibler and Raj6 PAF receptor (PAFR) binding to pulmonary vascular smooth muscle cells (SMC) and PAFR mRNA expression are higher in the lungs of fetal lambs,Reference Ibe, Sander and Raj7 whereas in the lungs of newly born lambs, <2 h of age, PAFR binding and receptor mRNA expressions are lower, suggesting a downregulation of the PAF binding and PAFR mRNA expression after birth. Furthermore, PAF level in plasma from pulmonary vasculature is higher in the fetus than in the newborn,Reference Ibe, Hibler and Raj6 and comparatively, SMCs from the pulmonary veins (PV) of fetuses produce more PAF than pulmonary arteries (PA) when both were studied by the remodeling and de novo pathways of PAF synthesis. However, rate of pulmonary vascular PAF catabolism by PAF acetylhydrolase (PAF-Ah) is slower in the fetus than in the newborn.Reference Ibe, Sander and Raj8, Reference Ibe, Portugal and Raj9 This means that in fetal pulmonary vasculature in utero, a higher PAF production coupled with a slower PAF catabolism will result in more PAF being available for binding to its receptors, in fetal pulmonary vasculature. This higher pulmonary PAF level is needed to induce the high pulmonary vascular tone necessary for the desired fetal pulmonary venous blood flow, which is 8–10% of total cardiac output. PAF is implicated in the pathogenesis of hypoxia-induced pulmonary hypertension in some animal models,Reference Chen and Chen10–Reference Bixby, Ibe and Abdallah12 as well as in persistent pulmonary hypertension of the newborn, where higher circulating plasma PAF level has been measured.Reference Caplan, Hsueh, Sun, Gidding and Hageman13 Thus, it is important to understand the physiological relevance of higher pulmonary PAF level in the fetus in contrast to the level in the newborn.
PAF evokes its effects by binding to its Gq protein isoform of G protein-coupled receptor (GPCR), which is a seven trans-membrane receptor.Reference Ibe, Hibler and Raj6, Reference Carlson, Chatterjee and Fisher14, Reference Parent, Le Gouill, Escher, Rola-Pleszcynski and Stankova15 Ligand binding to GPCR results in specific changes in the GPCR. These changes in the GPCR regulate signal transduction pathways by processes involving activation, desensitization, resensitization and inactivation.Reference Gaudreau, Le Gouill, Venne, Stankova and Rola-Pleszczynski16, Reference McDonald and Lefkowitz17 Earlier reports showed that PAF binds specifically to its receptors in the human umbilical vein endothelium and SMCs to evoke intracellular calcium flux,Reference Korth, Hirafuji, Benveniste and Russo-Marie18–Reference Renteria, Raj and Ibe20 and release of calcium was augmented by hypoxia.Reference Ibe, Portugal, Charturvedi and Raj19, Reference Renteria, Raj and Ibe20 PAF level in an organism will depend on the activities of PAF synthetic enzymes, for instance, lyso-PAF acetyl-S-Co-A acetyltransferase and the PAF catabolic enzyme, PAF-Ah.Reference Snyder21 As fetal lungs are normally exposed to a low-oxygen environment in utero (pO2 < 40 torr), we are interested in understanding the factors that control or modulate PAF binding to its receptors in the low-oxygen pulmonary vasculature of fetal lambs compared with normal oxygen environment (pO2 ∼80–100 torr) of the newborn lungs under baseline conditions. Our hypothesis is that in the fetal pulmonary vasculature in utero, PAFR gene, mRNA and protein expression are higher than in the newborn warranting a higher PAFR binding and maintenance of a high vasomotor tone in utero, and that activity of PAF-Ah is low resulting in a high PAF level in the fetal pulmonary vasculature. We studied PAF synthesis and catabolism by the fetal pulmonary vascular system [lung, vessels (arteries and veins) and SMCs] and compared it with the activity of PAF metabolic enzymes in the newborn lamb pulmonary vascular system. Our data show striking differences in PAF metabolism between the PAs and PVs, between the fetal lamb pulmonary vascular systems and those of the newborn lamb, indicating a heterogeneous PAF metabolism between the fetus and the newborn period that may serve to protect the newborn from PAF-induced persistent pulmonary hypertension of the newborn.
Materials and methods
Materials
All experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee of the Los Angeles Biomedical Research Institute. The authors do not have any financial conflict of interest with any of the vendors. Pregnant ewes (146–148 days of gestation, term being 150 days) and 6–12-day-old newborn lambs were purchased from Nebeker Farms, Santa Monica, CA. Authentic standards of PAF: hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C16-PAF); hexadecyl-sn-glyceryl-3-phosphorylcholine (Lyso-C16-PAF) were purchased from Biomol Research Laboratories, Plymouth Meeting, PA. Radiolabeled PAF standards and substrates were purchased from Perkin Elmer Lifer Sciences (Boston, MA). They are: hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, 1-O-[acetyl-3H(N)]-, (3H-acetyl-C16-PAF), 13.5 Ci/mmol (370 GBq/mmol); alkyl-sn-glyceryl-3-phosphorylcholine, 1-O-[alkyl-1′, 2′-3H] (3H-1-O-alkyl lyso-PAF), 60.00 Ci/mmol (2.2 TBq/mmol); [acetyl-1-14C]- 60 Ci/mmol (2.22 GBq/mmol). Ecolite(+) liquid scintillation cocktail was purchased from ICN Biochemicals (Irvine, CA). All other reagents and chemicals were purchased from Fisher Scientific, Tustin, CA.
Methods
PAF synthesis by perinatal lamb lung sections
Lungs were isolated from term fetal [146–148 days of gestation] and 6–12-day-old lambs and placed in Krebs’ buffer, pH 7.4, containing the following in mM: NaCl, 119; KCl, 4.7; CaCl2, 1.6; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 5.5. Lung vasculature was washed to remove intravascular blood cells. We first prepared 1-O-alkyl-sn-glycerol (alkylglycerol) by phospholipase C (PLC) catalyzed hydrolysis of 3H-alkyl lyso-PAF, using published method.Reference Ramesha and Pickett22 Briefly, 1.0 mM of 1-O-hexadecyl-lyso-PAF (C16-lyso-PAF) and 1.0 μCi of 3H-alkyl lyso-PAF and 30 U/ml of PLC from Bacillus cereus (Sigma-Aldrich) were dissolved in 0.25 ml of ether. The radiolabeled lyso-PAF was also used to monitor the extent of hydrolysis of the phosphocholine group by PLC.Reference Blank, Smith, Cress and Snyder23 The reaction was carried out for 2 h at 37°C in 50 mM sodium borate buffer, pH 8.0, containing 1.0 mM CaCl2 and 10.0 mM ZnCl2 in polypropylene plastic tubes. The [3H]-1-O-alkylglycerol hydrolysis product was extracted with ether and purified by silica gel thin layer chromatography (TLC) on hexane/ether (70:30). The [3H]-alkylglycerol had a retardation factor (R f) value of 0.3 in this system, and was stable in ethanol for 6 months at −80°C. Next, fresh lung explants from fetal lambs of 146–148 days of gestation and newborn lambs 6–12-day old were prepared and incubated with 100 nM of the [3H]-1-O-alkylglycerol for 24 h in 5% CO2 in air for tissue incorporation of 14C-labeled choline by endogenous cytidine-5′-diphospho(CDP)-choline:1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (CDP-choline: cholinephosphotransferase) and by endogenous acetate by lyso-PAF acetyl-S-CoA acetyltransferase enzymes present in the lung explants. In general, about 15% of radioactivity was incorporated into lung tissue in culture within the 24 h period. Unincorporated radioactivity was washed off the tissues, which were then homogenized together with 25 μg each of authentic non-radiolabeled PAF and lyso-PAF standards, which were added to aid in the identification of PAF bands on TLC plates. The experimental medium was subjected to lipid extraction.Reference Bligh and Dyer24 The [3H]-1-O-C16-alkyl-acetyl-14C-choline-PAF ([3H]-alkyl-[14C]-choline-PAF) synthesized was purified by TLC. TLC bands of the C-14-choline-PAF were scraped off, re-extracted and further purified by high-performance liquid chromatography, as we had reported previously,Reference Ibe, Portugal and Raj9 and quantified by scintillation spectrometry with tritium and carbon-14 windows (Beckman LS 6500). Functional assessment of the purified radiolabeled PAF synthesized from the perinatal lamb lung membrane proteins was tested for bioactivity by the serotonin release methodReference Pinckard, Farr and Hanahan25 with modifications, as we had recently reported,Reference Bae, Arteaga, Raj and Ibe26 and then normalized to tissue weight.
Preparation of membrane protein fractions from lung parenchyma and intrapulmonary arteries and veins
Lungs were isolated from term fetal (146–148 GA) and 6–12-day-old lambs as described above and placed in Krebs’ buffer, pH 7.4. Intrapulmonary arteries and veins, second to seventh passage, were isolated from the term fetal lambs and the 6–12-day-old newborns as previously reported.Reference Ibe, Portugal and Raj9 The vessels were homogenized separately in 46 mM KH2PO4 buffer, pH 7.4, containing 0.1 mM PMSF. The homogenate was centrifuged at 1200 g for 10 min, and 10% of supernatant was used to study acetylhydrolase activity. The remaining 1200 g supernatant was further centrifuged at 100,000 g for 1 h to pellet the membranes. The pellet was resuspended in 30 mM Tris buffer, pH 7.4, frozen in liquid nitrogen and stored at −80°C. Vessel membrane preparations were used to study PAF acetyl-S-CoA acetyltransferase activity. All procedures were carried out at 4°C. Protein concentrations of vessel homogenate and membranes were measured by Bradford method using bovine serum albumin (BSA) as the standard.
PAF synthesis by intrapulmonary arteries (PA) and veins (PV) of perinatal lambs
PAF synthesis by the vessel membrane proteins was studied by measuring the activity of lyso-PAF:acetyl-CoA acetyltransferase as we had previously reported.Reference Ibe, Portugal and Raj9 For each membrane protein, 25 μM aliquot of lyso-PAF and 250 μM [1-14C]-acetyl CoA was placed in a polypropylene tube and warmed to 37°C in a shaker bath. The reaction was initiated by adding 100 μg of membrane protein of each vessel type for a total volume of 1 ml of 30 mM Tris buffer, pH 7.4, containing 10 μM CaCl2, 1 mM dithiothreitol (DTT) and 0.025% BSA, and the mixture was incubated at 37°C for 10 min. After incubation, [14C]-acetyl-PAF synthesized was extracted, purified by TLC and HPLC and quantified by serotonin release bioassay.
Extraction and purification of PAF by TLC and HPLC
Before extraction, 25 μg each of authentic PAF and lyso-PAF standards are added to each tube to aid in establishing PAF bands on TLC. Then, PAF and lyso-PAF were extracted from samples, dried with nitrogen, resuspended in 200 μl of methylene chloride and then 10 μl of each sample was spotted on silica gel G-plates and developed in a solvent mixture containing methylene chloride–methanol–water (65:35:5) against authentic radiolabeled and non-radiolabeled PAF and lyso-PAF standards. The TLC spots corresponding in retardation factor (R f) values with the standard PAF were scraped off and further purified by HPLC as in the double-labeled study above. The HPLC peaks corresponding in retention time to authentic PAF standard was collected and PAF radioactivity was measured by scintillation spectrometry (Beckman Coulter, Fullerton, CA) and quantified, as we previously reported.Reference Ibe, Portugal and Raj9 Non-radiolabeled PAF standard was not added to TLC extractions and purification used in the serotonin bioassay quantification.
Measurement of activity of the purified PAF
PAF synthesized by perinatal lung parenchyma and pulmonary vessels was tested for biological activity after TLC and HPLC purification by the method of serotonin release using washed rabbit platelets with a modification of the method reported by Pinckard et al.,Reference Pinckard, Farr and Hanahan25 as we had previously reported.Reference Bae, Arteaga, Raj and Ibe26 In this quantification assay, authentic PAF standards were not added to the samples before lipid extraction or purification processes. Briefly, a calibration curve of 0.01 nM to 10.0 μM of authentic PAF in Tyrode's buffer was generated, and then the concentration of the purified PAF sample extracts in Tyrode's buffer (0.1 ml) was determined from the standard curve. The amount of serotonin released spontaneously was subtracted from the experimental values before the quantification of PAF concentration from the standard curve and presented as picomol (pmol) PAF/g tissue for lung culture study or pmol/μg protein for synthesis by vessel proteins.
Study of PAF-Ah gene expression by RT-PCR
Freshly isolated intrapulmonary PAs and PVs of the fetus and newborn lambs were quick frozen in liquid nitrogen. Then total RNA was isolated from the frozen vessels. An aliquot of RNA was reverse transcribed to synthesize cDNA for use as template for PCR. Using components of the First Strand cDNA Synthesis Kit (5 Prime–3 Prime Inc.), RNA was mixed with random hexamers, heat denatured at 65°C, allowed to anneal on ice, and then mixed with dNTPs, 10X buffer and MMLV Reverse Transcriptase. Then 20 μl of mixture was incubated at 42°C for 1 h, and then terminated by heating for 10 min at 95°C. PCR was performed in a 50 μl reaction, containing 5 μl and 0.5 μl of the cDNA components provided with the GeneAmp PCR Core Reagents Kit (Perkin Elmer Cetus Inc.), including 2.5 mM MgCl2, 0.25 units Taq DNA polymerase, 5X buffer, dNTPs and synthetic DNA primers designed to either ovine PAF-Ah, or ovine glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequence for both the ovine PAF-Ah and the GAPDH are as we previously reported and match PAF-Ah GenBank Accession U34247 at 395–418 and 857–834 bp. The 5′ sense sequence was 5′-TTTGCCCACAAGCCAGTGTGTTCC-3′ and the 3′-antisense sequence was 3′-CCCTATCAATAGAGTCCTCAGCT-5′. The primers used to amplify ovine GAPDH in order to standardize PAF-Ah expression match GenBank Accession AFO30943 at 108–131 and 399–375 bp. The 5′-sense sequence was 5′-ACCTGCCAACATCAAGTGGGGTGAT-3′ and the 3′-antisense sequence was 5′-GGACAGTGGTCATAAGTCCCTCCAC-3′. The PCR was carried out essentially according to our previous report.Reference Ibe, Sander and Raj8 PAF-Ah mRNA expression relative to GAPDH expression was quantified by image analysis of ethidium bromide fluorescence of PCR products using BioRad Fluor-S Multi-Imager.
cDNA library screening
We further examined PAF-Ah gene expression by ovine lungs in an attempt to characterize the gene regulation of PAF-Ah activity in perinatal lungs under baseline conditions. A cDNA library constructed in vector Lambda ZAP from fully oxygenated sheep lung RNA (Stratagene, San Diego, CA) was screened for a full-length cDNA to sheep lung PAF-Ah according to the manufacturer's instructions. The library was replica-plated onto nitrocellulose filters, and hybridized using stringent conditions to 32P-labeled radioactive probe prepared from the partial PAF-Ah clone described above. The cDNA clones that hybridize to the radioactive probe were initially screened for correct insert size. The full-length bovine PAF-Ah cDNA is ∼1500 bp, and sheep cDNA clones that are of this size were sequenced by City of Hope, Department of Biology, Core Sequencing Laboratory (Duarte, CA). The clone, which best matched the consensus sequence for PAF-Ah and appears to be full length, was purified and retained for functional analysis.
Cloning the PAF-Ah regulatory region
We inspected a restriction-site map of the full-length PAF-Ah cDNA to identify sites that cut close to the 5′end of the sequence. Genomic DNA was prepared from sheep lung and 10 μg aliquots of DNA were digested with each of the restriction enzymes. Then the digested DNAs were electrophoresed through a 1% agarose gel and then transferred to a nitrocellulose filter by capillary transfer. Southern hybridization was performed on a 5′ fragment of full-length sheep PAF-Ah as radioactive probe. DNA restriction fragments, which hybridized to the probe and which were the ones to several kilobases (Kb) in size, were gel purified and ligated to the PAF-Ah cDNA in plasmid pCAT Basic Vector (Promega) using the complementary restriction site. Clones constructed in this way were sequenced by the City of Hope, Department of Biology Core Sequencing Laboratory (Duarte, CA), to confirm that the correct reading frame has been retained throughout the cloning process. A control plasmid, which contains all of the PAF-Ah cDNA but without any 5′genomic sequence, was also constructed. Both constructs were transiently transfected into sheep lung PA SMCs using DEAE Dextrose, and allowed to recover for 48 h, RNA was isolated from these cultured cells and subjected to Northern analysis to assess expression of PAF-Ah mRNA levels, and to confirm that PAF-Ah transcription is being regulated by the cloned genomic DNA. Then the functional status of the clone with full-length DNA, the full-length recombinant PAF acetylhydrolase (rPAF-Ah) and the cloned genomic DNA [with functional site deleted (deletion rPAF-Ah)] were transfected into sheep lung PA SMCs, allowed to stay in culture for 48 h and then proteins were prepared from lysed cells to assess PAF-Ah activity. Catabolic activity of the deletion rPAF-Ah was compared with catabolic activity of full-length rPAF-Ah clone.
Study of PAF catabolism by PAF-Ah
PAF-Ah activity was studied in proteins from the fetal and newborn lamb PA and PV, in arterial (SMC-PA) and venous (SMC-PV) SMCs, by determining the rate of PAF catabolism, as we had previously reported.Reference Ibe, Portugal and Raj9 Proteins from each age group were incubated separately with [3H]-acetyl-C16-PAF (50 μM, 60 Ci/mmol) for 5 min, in 30 mM Tris buffer containing 0.01% BSA, after a time course (min) standardization of 1, 2.5, 5, 10 and 30 min incubations. The lipid extracts were concentrated with nitrogen and subjected to HPLC purification for PAF and lyso-PAF on Ultramex C18 column and quantified, as we had previously reported.Reference Ibe, Portugal and Raj9 One-minute HPLC fractions were collected and 3H-PAF radioactivity co-eluting with authentic non-radiolabeled PAF standard was quantified by scintillation spectrometry (Beckman-Coulter, Fullerton, CA). The extent of PAF catabolism was presented as picomolar (pM) lyso-PAF/min/μg protein).
Study of PAFR gene expression by real-time polymerase chain reaction (qPCR)
The SMC-PA and SMC-PV of fetal and newborn lambs were cultured in normal incubator conditions (5% carbon dioxide in air) without stimulation to study PAFR gene expression by qPCR. Total RNA was extracted with Qiagen's QIAshredder (cat# 79654) and Rneasy Plus Mini Kit (cat# 74134), according to the manufacturer's protocol (Qiagen, Valencia, CA). qPCR was performed on Step One Plus real-time PCR System (Applied Biosystems, Foster City, CA). Reverse transcription was accomplished with Applied Biosystems High-Capacity RNA-cDNA Master Mix (cat# p/n 4390715) program as follows: step 1, 25°C for 5 min; step 2, 42°C for 30 min; step 3, 85°C for 5 min; and step 4, hold at 4°C. The cDNA was subjected to PCR with SYBR-Green PCR Master Mix (cat# p/n 4309155) as fluorescent dye, with the following steps: step 1, 50°C 1 cycle; step 2, 95°C 10 min 1 cycle; step 3, 95°C 15 s, 59°C 1 min, 40 cycles (annealing/amplification); step 4, dissociation cure, one cycle, all carried out according to the manufacturer's protocol (Applied Biosystems) using primer pairs which are: PAFR primer #1, forward 5′-CCT GTG CAA CGT GGC TGG CT-3′, 98–117 bp, reverse 5′-GAG ATG CCA CGC TTG CGG GT-3′ 241–222 bp and PAFR primer #2, forward 5′-TCC TGT GCA ACG TGG CTG GC-3′, 97–116 bp, reverse 5′-GAG ATG CCA CGC TTG CGG GT-3′, 241–222 bp, both created by using NCBI's Primer-BLAST program (http://www.ncbi.nlm.nih.gov.tool/primer-blast/) by entering accession number AF099674.1. Both primer sets were authenticated by RealTimePrimers.com. The GAPDH primers sequence, GenBank Accession AFO30943, at 108–131 and 399–375 bp, forward 5′-ACC TGC CAA CAT CAA GTG GGG TGA T-3′, reverse 5′-GGA CAG TGG TCA TAA GTC CCT CCA C-3′ were synthesized by SIGMA-Aldrich (Saint Louis, MO). The negative control provided with the qPCR kit was used according to the manufacturer's protocol (Applied Biosystems). For quantification, the target gene (PAFR gene) was normalized to GAPDH housekeeping gene, which was used as the reference, and qPCR data are presented as ΔC t, means ± SD, where ΔC t = C tT − C tR: where C tT is the threshold cycle of target gene (PAFR gene); C tR is the threshold cycle of GAPDH gene.
Assay of PAFR binding in membrane proteins of SMC-PA and SMC-PV of fetal and newborn lambs
Studies were conducted essentially as we had previously reported.Reference Ibe, Portugal, Charturvedi and Raj19, Reference Renteria, Raj and Ibe20 Briefly, membrane proteins were prepared from the SMC-PA/PV at passage 4–6 cultured to sub-confluence in 5% CO2 in air without any exogenous treatments (baseline conditions) as we wanted to understand the receptivity of PAFR at baseline conditions. Membrane proteins were incubated with 1.0 nM [3H]-C18-PAF for 24 h at 4°C in normoxia with or without 500 nM non-radiolabeled PAF and with or without 1.0 μM of the specific PAFR antagonist CV-3988, which was added to authenticate PAF binding to its receptors. [3H]-PAF bound to receptor was extracted and quantified.Reference Ibe, Portugal and Raj9
Assay of PAF-mediated proliferation of SMC-PA and SMC-PV of fetal and newborn lambs
Functionality and differences in PAFR-mediated effect in perinatal SMCs was investigated on PAF-stimulated cell. Cell proliferation was conducted essentially as we previously reported.Reference Bixby, Ibe and Abdallah12 Briefly, cell proliferation was measured by 3H-thymidine incorporation into cell DNA. The SMC-PA and SMC-PV of the fetal and newborn lambs (fourth to sixth passage) were cultured in six-well plates until they attained 60–70% confluence. Growth was arrested in normoxia for 72 h by incubating in 0.1% fetal bovine serum (FBS) in DMEM followed by incubation for another 24 h in 10% FBS in normoxia with 1 μCi 3H-thymidine (Perkins-Elmer), with or without 10 nM non-radiolabed PAF (Biomol). Studies with 1 μM of the specific PAFR antagonist CV-3988 was used to test the specificity of PAFR-mediated cell proliferation. After 24 h in culture, unincorporated radioactivity was aspirated and DNA incorporation of radioactivity was determined and quantified.Reference Bixby, Ibe and Abdallah12 Direct cell count was carried out by hemocytometer to corroborate data from 3H-thymidine incorporation.
Data analysis
All numerical data are means ± s.e.m. In all instances where radioisotope was used, the background radioactivity was subtracted before quantifying the amount of radioactivity. Then all data were subjected to statistical analysis. A two-tailed t-test was used to compare data from normoxia and hypoxia within the same group. For multiple comparisons, we used ANOVA followed by Tukey's post-hoc test (GraphPad Prism program, San Diego, CA). Results were considered significant at P < 0.05.
Results
PAF synthesis by perinatal lungs
Amount of PAF synthesized by lung parenchyma of the perinatal lambs is shown in Fig. 1. Double-radiolabeled PAF (Fig. 1a) consisting of [3H]-O-alkyl- and [14C]-choline functional groups was measured from proteins from the two groups. The 144–146-days of gestation fetal (term fetal) lamb lungs synthesized 60% more PAF than the 6–12-day-old newborn (newborn) lungs. Amount of PAF synthesized by proteins from intrapulmonary arteries (PA) and veins (PV) of term fetal and 6–12-day-old newborn lambs is shown in Fig. 1b. PAF synthesis by the fetal lamb PVs was 40% greater than synthesis by fetal PA, but there was no difference in PAF production between the PAs and PVs of the newborn lambs. However, the PV of the fetal lambs produced more PAF than the PAs of the newborn lamb.
Fig. 1 (a) Platelet-activation factor (PAF) synthesis by lung membranes of term 142–146 days of gestation (term) fetal lambs and 6–12-day-old newborn lambs. Data are means ± s.e.m., n = 5. Washed lung tissues were cultured in normoxia for 24 h with [3H]-1-O-alkylglycerol and [14C]-choline as described in ‘Methods’ section. Radiolabed PAF was extracted and quantified. Lung tissues of fetal lambs synthesized more PAF than newborn lamb lung tissues. The statistics are: *P < 0.05, different term fetal lambs. (b) PAF synthesized by pulmonary arteries and veins of 142–146 days of gestation fetuses and 6–12-day-old lambs. Data are means ± s.e.m., n = 6. Fetal veins synthesized more PAF than fetal arteries, but there was no difference in PAF synthesis between arteries and veins of the newborn lambs. The statistics are: *P < 0.05, different from fetal arteries.
PAF catabolism by PAF-Ah in perinatal lungs
Rate of PAF catabolism by acetylhydrolase in the PAs and PVs of term fetal and the newborn lambs are shown in Fig. 2a. In fetal pulmonary vessels, PAF-Ah activity in the PVs of fetal lambs was 40% lower than activity in the PA. In the newborn, PAF-Ah activity in veins was 50% greater than activity in arteries. Comparing the PAF-Ah activity in fetal and newborn vessels, the PAF-Ah activity in newborn arteries was 20% lower than activity in fetal arteries; however, activity in the newborn veins was 2.5-fold greater than activity in the fetal veins. Thus, in the PVs of the newborn lamb, faster degradation of PAF will make less PAF available to produce PAF effect in the newborn pulmonary vasculature.
Fig. 2 (a) Rate of platelet-activating factor (PAF) catabolism by PAF-acetylhydrolase (Ah) in proteins of pulmonary arteries and veins of 142–146 days of gestation fetal and 6–12-day-old newborn lambs. Data are means ± s.e.m., n = 6. Fetal arteries catabolized PAF faster than fetal veins, but newborn veins catabolized PAF faster then newborn arteries. Catabolism by newborn arteries was not different than fetal arteries. The statistics are: *P < 0.05, different from arteries; #P < 0.05, different from fetal arteries and veins. (b) Comparison of bovine and ovine PAF-Ah gene clone maps. The ovine PAF-Ah gene with deletion of the hypoxia sensitive region is shown in the middle between the functional ovine PAF-Ah, upper map and full-length functional bovine PAF-Ah, lower map. (c) Catabolic activity of the ovine rPAF-Ah compared with rPAF-Ah with hypoxia sensitive region deleted. Data are means ± s.e.m., n = 4 different cell preparations. Smooth muscle cells (SMC) were transfected separately with each ovine rPAF-Ah and then proteins were assayed for catalytic activity. The statistics are: *P < 0.05, different from rPAF-Ah without site deletion; #P < 0.05, different from activities of both rPAF-Ah and rPAF-Ah deletion proteins in hypoxia. (d) PAF-Ah mRNA expression by term fetal and newborn 6–12-day-old lamb pulmonary arteries (PA) and veins (PV). Data are means ± s.e.m., n = 4 different vessel preparations. RNA from freshly isolated PAs and PVs of the two groups was subjected to Northern analysis with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression as the internal standard for quantification. The fetal PVs expressed more mRNA than the fetal arteries. The statistics is: *P < 0.05, different from fetal PA. (e) PAF-Ah activity in arterial (SMC-PA) and venous (SMC-PV) SMCs of term fetal and 6–12-day-old newborn lambs. Data are means ± s.e.m., n = 6 different cell preparations. SMC-PA and SMC-PV were cultured to sub-confluence in 5% carbon dioxide air. Proteins were isolated and assayed for PAF-Ah activity. In general, PAF-Ah activity in newborn proteins was greater than activity in fetal proteins. The statistics are: *P < 0.05, different from SMC-PA; #P < 0.05, different from PAF-Ah activities in the fetal SMC-PA and SMC-PV.
The DNA clone map of ovine recombinant PAF-Ah (rPAF-Ah) is shown in Fig. 2b, and compared with complete bovine PAF-Ah DNA, bottom map. The top map is the active PAF-Ah gene clone (rPAF-Ah), while the middle map is the PAF-Ah gene clone with deleted hypoxia sensitive active site, the deletion rPAF-Ah construct.
A comparison of catabolic activity of rPAF-Ah with the activity of the cloned deletion rPAF-Ah is shown is Fig. 2c. In the control incubation (incubation buffer without rPAF-Ah proteins), there was no difference between lyso-PAF measured from the buffer control. In hypoxia, to mimic fetal lung hypoxia environment (2% oxygen, pO2 < 40 torr), proteins extracted from SMCs transfected with rPAF-Ah showed fourfold increase in the activity of the enzyme that degrades PAF compared with control incubations. However, proteins from SMCs transfected with deletion rPAF-Ah construct produced no difference in PAF catabolism compared with control conditions. When the two SMC protein types (rPAF-Ah and deletion rPAF-Ah) were studied in normoxia (pO2 ∼80–90 torr), activity of rPAF-Ah increased threefold from activity in hypoxia. The activity of the deletion rPAF-Ah in normoxia also increased threefold from its activity in hypoxia or control conditions, but the catabolic activity of the deletion rPAF-Ah was still 4.5-fold less than that of the rPAF-Ah without deletion. Thus, there was successful cloning of the rPAF-Ah with deleted hypoxia-sensitive enzyme active site.
In Fig. 2d, we show PAF-Ah mRNA expression by term fetal lamb PAs and PVs compared with the expression by newborn PAs and PVs. In the term fetal lamb vessels, the PVs expressed 40% more PAF-Ah mRNA than the PAs. In newborn vessels, PAF-Ah mRNA expression by the PVs was not different from expression by PA. Compared with the expression by the fetal lamb lung vessels, PA of the newborn lambs expressed 30% more PAF-Ah mRNA than the fetal PA. However, PAF-Ah mRNA expression by the fetal PVs was not different than the expression by and newborn lamb PAs and PVs.
Rate of PAF catabolism by PAF-Ah present in proteins from SMCs of the fetal and newborn lungs is shown in Fig. 2e. PAF-Ah activity in proteins from the pulmonary artery SMCs (SMC-PA) and veins (SMC-PV) of the fetal and newborn lambs were different. With proteins from fetal SMCs, PAF-Ah activity in the SMC-PV was about fivefold greater than activity in the SMC-PA. With proteins from newborn cells, PAF-Ah activity in the SMC-PV was also greater (1.5-fold greater) than activity in the SMC-PA. Compared with the fetal pulmonary vascular cells, PAF-Ah activity was greater in newborn cells. For instance, PAF-Ah activity in newborn SMC-PA was sevenfold greater than activity in the fetal SMC-PA, and in the newborn SMC-PV, PAF-Ah activity was 2.5-fold greater than in the fetal SMC-PV.
PAFR expression and receptor-mediated function by perinatal lamb lungs
In Fig. 3a, we show PAFR mRNA expression by the fetal and newborn PAs and PVs. In fetal vessels, PAFR mRNA expression by veins was greater than expression by arteries. In addition, PAFR mRNA expression by the newborn PV was greater than expression by newborn PA. Compared with the fetal vessels, newborn PAs expressed 30% more PAFR mRNA than the fetal PAs, whereas the newborn PVs expressed 25% more mRNA than the fetal PVs.
Fig. 3 (a) PAF receptor (PAFR) mRNA expression by term fetal and newborn 6–12-day-old lamb pulmonary arteries (PA) and veins (PV) studied by Northern blotting. Data are means ± s.e.m., n = 4 different vessel preparations. RNA from freshly isolated PA and PV of the two groups was subjected to Northern analysis with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression as the internal standard for quantification. In general, newborn vessels expressed more mRNA than the corresponding fetal vessel. The statistics are: *P < 0.05, different from expression by PA; #P < 0.05, different from PAFR expression by fetal vessels. (b) PAFR gene expression by term fetal and newborn 6–12-day-old lamb smooth muscle cells (SMC)-PA and SMC-PV studied by qPCR. Data are means ± s.e.m., n = 4 different cell preparations. Freshly isolated RNA from the SMC-PA and SMC-PV from the two age groups was subjected to qPCR procedure. In general, SMC-PV of both groups expressed more PAFR than the corresponding SMC-PA. The statistics are: *P < 0.05, different from expression by SMC-PA; #P < 0.05, different from PAFR gene expression by SMC-PV of the fetus. (c) PAFR binding to membrane proteins from SMC-PA and SMC-PV of term fetus and 6–12-day-old lambs. Data are means ± s.e.m., n = 4 different cell preparations. Membrane proteins were isolated from the cultured cells and subject to [3H]-PAF ligand binding as described in methods. In general, PAFR binding to membrane proteins from SMC-PV of the fetus and newborn was greater than binding in SMC-PA of the two groups. Also binding to proteins from newborn cells was lower than for fetal cells. CV-3988 inhibited binding to fetal cells, but augmented binding to newborn cells. The statistics are: *P < 0.05, different from control SMC-PA and SMC-PV of both groups; #P < 0.05, different from PAFR binding to fetal cells.
PAFR gene expression by the SMC-PA and SMC-PV of the fetal and newborn lambs studied by quantitative qPCR is shown in Fig. 3b. In fetal cells, PAFR gene expression by the SMC-PV was greater than the expression by SMC-PA. In newborn cells, PAFR gene expression by SMC-PV was also greater than the expression by SMC-PA. Comparing fetal cells with newborn cells, there was no difference in PAFR gene expression by SMC-PA of the fetus and newborn. However, PAFR gene expression by the newborn SMC-PV was greater than PAFR gene expression by the fetal SMC-PV. Thus, in general, receptor gene expressions by the fetal and newborn SMC-PV are greater than the expression by the fetal and newborn SMC-PA.
The profile of specific PAFR binding to its receptors in membrane proteins from the fetal and newborn intrapulmonary SMC-PA and SMC-PV, respectively, is shown in Fig. 3c. PAF binding to membrane proteins of fetal SMC-PA was 110 ± 3.1 pmol/μg protein. Pretreatment of SMC-PA proteins with 1 μM of CV-3988 for 2 h attenuated PAFR binding by 50%. With proteins from the fetal SMC-PV, PAF binding was 442 ± 5.6 pmol/μg protein. As with SMC-PA, pretreatment of proteins with 1 μM of CV-3988 inhibited PAF binding by 55%. Compared with the SMC-PA, PAF binding in SMC-PV proteins was fourfold greater. Noteworthy is the finding that the CV-3988, the specific PAFR antagonist, inhibited PAF binding to membrane proteins from both the SMC-PA and SMC-PV of the fetal lungs. With respect to the newborn cells, binding in the SMC-PA was 37.8 ± 1.6 pmol/μg protein. Pre-treatment of SMC-PA with 1 μM CV-3988 led to an augmentation PAF binding by 20% (47.7 ± 1.8 pmol/μg protein). Also in the SMC-PV of the newborn, PAF binding was 83.2±5.7 pmol/μg protein, which was twofold greater than binding in the SMC-PA. Unlike with the fetal SMCs, 1 μM CV-3988 did not inhibit binding in the SMC-PA or SMC-PV of the newborn, instead in both cell types, CV-3988 stimulated PAF binding by 20% and 35%, respectively. In general, specific PAFR binding to membrane proteins from the fetal SMCs was greater than binding to membrane proteins from the newborn SMCs, with or without CV-3988 treatment, suggesting the possibility of different receptivity of PAFR in the SMCs of the fetus and newborn pulmonary vascular systems.
PAFR and perinatal SMC proliferation
The effect of PAF and the PAFR antagonist CV-3988 on proliferation of intrapulmonary SMCs of the perinatal lambs is shown in Fig. 4. In SMC-PA of the fetus, treatment of cells with 10 nM PAF increased cell proliferation by 15% compared with 10% FBS control. Co-incubation of cells with 10 nM PAF and 1 μM of CV-3988 significantly decreased cell proliferation by 45%. On the other hand, treatment of the fetal SMC-PV with 10 nM PAF increased the SMC-PV proliferation by 45% compared with 10% FBS. Co-incubation of the SMC-PV with 10 nM PAF and 1.0 μM of CV-3988 inhibited cell proliferation by 60% compared with effect of PAF alone and by 45% compared with effect of 10% FBS.
Fig. 4 Platelet-activation factor (PAF) stimulation of proliferation of smooth muscle cells-pulmonary arteries (SMC-PA) and SMC-pulmonary veins (SMC-PV) of the fetus and newborn lambs. Data are means ± s.e.m., n = 6 different cell preparations studies. Sub-confluent cells were serum starved with 0.1% fetal bovine serum (FBS) for 72 h and studied as described in methods. In fetal cells, PAF stimulation of cell proliferation was inhibited by CV-3988. In newborn, cell proliferation was attenuated by PAF treatment, and proliferation was significantly less than in fetal cells and then CV-3988 did not inhibit cell proliferation as observed in newborn cells. The statistics are: *P < 0.05, different from SMC-PA; **P < 0.05, different from proliferation in fetal FBS or PAF-treated cell; +P < 0.05, different from proliferation caused by 10% FBS in newborn SMC-PA; #P < 0.05, different from cell proliferation by SMC-PA and SMC-PV from the fetus.
In SMC-PA of the newborn, treatment of cells with 10 nM PAF decreased cell proliferation by 23% compared with 10% FBS alone. In SMC-PA, treatment of cells with PAF together with 1.0 μM of CV-3988 produced no significant inhibition in cell proliferation compared with effect of 10% FBS alone, but increased cell proliferation by 20% compared with effect of co-incubation with 10 nM PAF. Treatment of the newborn SMC-PV with 10 nM PAF increased SMC-PV proliferation by 43% compared with effect of 10% FBS control. As with SMC-PA, treatment of the SMC-PV cells with PAF and 1.0 μM CV-3988 increased the cell proliferation by 43% and 37% compared with effects of 10% FBS control and 10 nM PAF alone, respectively. Thus, although the PAFR antagonist CV-3988 had inhibitory effect on PAFR-mediated cell proliferation in fetal cells, its effect on newborn cell was generally stimulatory suggesting a different pathway of PAF-mediated responses in newborn and fetal pulmonary vascular SMCs.
Discussion
PAF binds specifically to its receptors in ovine fetal pulmonary vascular SMCs to activate intracellular signaling pathways leading to intracellular calcium flux and these effects are augmented by hypoxia.Reference Ibe, Portugal, Charturvedi and Raj19, Reference Renteria, Raj and Ibe20 In addition, in the fetus, pulmonary vascular plasma PAF level is high and the level falls dramatically after birth.Reference Ibe, Hibler and Raj6 PAF level in utero will depend on the activities of PAF synthetic enzymes, for instance, lyso-PAF acetyl-S-Co-A acetyltransferase and PAF catabolic enzyme, PAF-Ah,Reference Ibe, Portugal and Raj9, Reference Bixby, Ibe and Abdallah12 as well as the effect of oxygen tension on the activities of these enzymes. Although PAFR-mediated responses has been reported in ovine fetal pulmonary vascular SMCs, no studies have been reported to examine PAF synthesis, catabolic enzymes as well as PAFR expression and function in ovine fetal lamb lungs alongside with neonatal lamb lung systems. We have investigated synthetic, catabolic and PAFR expression and function by perinatal lambs using the PAs, PVs and the SMCs of arteries (SMC-PA) and veins (SMC-PV). In this report we show, for the first time, significant differences in PAF synthetic and catabolic enzyme activities as well as PAFR expression and function in ovine perinatal pulmonary vascular systems. Our major findings are: (a) PAF production in fetal lungs was higher than production in newborn lungs, but PAF catabolism by the newborn lung was greater than by fetal lungs, which indicates that, in the newborn lung, a decrease in PAF level results from a combined effect of decreased PAF synthesis and increased PAF catabolism. (b) Using cDNA library screening, we cloned, defined and confirmed the activity of the hypoxia-sensitive domain of ovine PAF-Ah. This suggests that the deletion rPAF-Ah may offer a method to increase PAF level in conditions where a high PAF level is physiologically beneficial. (c) Concerning PAFR expression and functionality, profile of PAFR gene expression was similar for both the fetus and the newborn, but PAF binding to its receptors in fetal cells was greater, and then binding in fetal cells was inhibited by the specific PAFR antagonist CV-3988. In newborn cells, CV-3988 augmented PAF binding, suggesting that, perhaps PAFR binding site in newborn lung may be different than in fetal lungs. (d) PAFR-mediated cell proliferation of fetal cells was also inhibited by CV-3988. However, in newborn cells, CV-3988 stimulated cell proliferation suggesting remarkable heterogeneity in PAF metabolizing enzymes and different functionality of PAFR in the newborn pulmonary vessels and SMCs.
Perinatal PAF synthesis
PAF synthesis occurs by the remodeling and de novo pathways in various cells and tissues.Reference Ibe, Portugal and Raj9, Reference Snyder21 The key enzyme in the remodeling pathway is lyso-PAF acetyl-CoA:acetyltransferase while the principal enzyme in the de novo pathway is the DTT-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT). PAF synthesis via the de novo and remodeling pathways has recently been shown in human leukocytes from healthy humans and those diagnosed with heart failure.Reference Detopoulou, Nomikos and Fragopoulou27, Reference Detopoulou, Nomikos and Fragopoulou28 PAF synthesis was seen to decrease with age of the leukocyte donors. This suggests that PAF synthetic enzyme activity may decrease with age of the model. In this report, we found that baseline PAF synthesis by newborn lungs was significantly less compared with synthesis by fetal lung. This means that the differences we observed represent the inherent enzyme activity. Greater production of PAF by fetal lungs suggests that PAF synthesis will be high in fetal lungs in utero, even in the absence of endogenous stimulus. This condition of higher PAF level in fetal lungs is necessary to maintain the desired high pulmonary vascular tone, which is needed for the successful left to right shunting of blood in the fetal pulmonary circulation.Reference Iwamoto, Teitel and Rudolph29, Reference Heyman, Creasy and Rudolph30 Differences in PAF synthesis between the fetal PVs and PAs may be related to the physiological functions of these two vessel types. In the PVs, greater production of the potent vasoconstrictor, PAF, is preferred to maintain the desired high venous vasomotor tone, whereas PAF synthesis by pulmonary artery is lower so as to maintain a lower arterial tone to permit the commensurate fetal and newborn pulmonary arterial blood flow. Regarding the mechanism of down-regulation of PAF effect in the newborn lung, the findings on fetal and newborn baseline PAF production does suggest that down-regulation of PAF synthesis after birth may be one method to maintain congenial basal pulmonary vascular tone in the newborn.
PAF catabolism by lungs of fetus and newborn lamb
Circulating level of PAF in utero will depend upon a joint regulation of its biosynthesis and catabolism, suggesting that the level of PAF in a system will be high in situations where the remodeling and de novo synthetic pathways are activated with concomitant decrease in catabolic pathways. During disease conditions, PAF clearance from site of action will be facilitated if PAF-Ah is activated. PAF-Ah is a family of enzymes present as both intracellular and plasma forms, but is specific in catalyzing the de-acetylation of the sn-2 position of PAF phospholipids.Reference Tjoelker and Stafforini31 In previous publications, we showed that PAF level is high due to a low activity of PAF-Ah, and that after birth, PAF level in the lung falls dramatically due to an up-regulation of PAF-Ah activity.Reference Ibe, Hibler and Raj6, Reference Ibe, Sander and Raj8 Other studies have also shown that pulmonary and systemic vascular endothelium exhibit PAF-Ah activity. In lungs, arterial SMCs,Reference Ibe, Pham, Kääpä and Raj32 alveolar type II cells and macrophagesReference Jahle, Schlame and Butter33 also possess significant PAF-Ah activity. It has been shown that normoxic condition up-regulates activity of PAF-Ah in arterial SMCs.Reference Ibe, Pham, Kääpä and Raj32 On the other hand, hyperoxia decreased the activity of PAF-Ah in macrophages,Reference Jahle, Schlame and Butter33 whereas in alveolar type II cellsReference Gao, Zhou and Raj3 PAF-Ah activity was un-affected by changes in oxygen tension. These show that depending on the cell type, oxygen tension produces different effects on PAF-Ah activity. Thus, PAF catabolic enzyme activity has continued to generate intense investigational interest.
In fetal lambs, PAF-Ah protein expression and PAF-Ah activity studied in normoxia and hypoxia showed that hypoxia significantly decreased both protein expression and catalytic activity.Reference Ibe, Portugal and Raj9 This is similar to the report by Yasuda et al.Reference Yasuda, Okumura and Okada34 where PAF-Ah activity was shown to correlate with higher PAF-Ah protein expression in pregnant uterus and myoma. We also show, in this report, that under baseline conditions, PAF-Ah activity is higher in the SMC-PV of the fetus in agreement with our earlier report.Reference Ibe, Portugal and Raj9 We further show, for the first time, that PAF-Ah protein expression and activity in the SMC-PV of the newborn lambs is higher than in SMC-PA. Noteworthy is the fact that PAF-Ah activities in SMC-PA/PV of newborn lambs are greater than the activities in fetal SMC. This implies that clearance of PAF in newborn vessels will be faster than in fetal vessels, thereby reducing vesicular PAF levels and decreasing PAF-induced vascular tone in newborn lungs. Although PAF-Ah mRNA expression in newborn vessels was higher than in fetal vessel, there was no difference between expression in newborn PA and PV, suggesting the existence of some post-transcriptional and perhaps post-translational modification of PAF-Ah protein anabolism, such as phosphorylation or dephosphorylation, which may add to modulate PAF-Ah activity in the newborn vascular system. This contention is further supported by our findings of PAF-Ah activity on proteins from arteries and veins of fetus and newborn in which fetal veins had lower activity, but significantly higher activity in newborn veins, in essence suggesting different regulatory mechanisms of PAF levels in vessels and SMCs. These findings also suggest that apart from a beneficial effect in fetal pulmonary physiology, in utero, PAF possesses other physiological functions, especially as a mitogen and an inflammatory mediator,Reference Henderson, Lu, Poole, Dietsch and Chi35, Reference Yost, Weyrich and Zimmerman36 functions that may be more manifested in postnatal life.
Our other goal in this study was to define a gene regulation of PAF-Ah activity in the fetus and newborn. In our PAF-Ah gene cloning studies, we defined the hypoxia sensitive region in our deletion construct. The catabolic activity of the rPAF-Ah deletion construct in normoxia is also minimal if at all. This construct offers a handle to examine the mechanism of PAF effects under physiological conditions, for instance, in physiological conditions where inhibition of PAF catabolism improves the expected outcome. PAF is an important endogenous lipid involved in in vitro fertilization technology.Reference Roudebush, Minhas, Ricker, Palmer and Dodson37–Reference Narahara, Tanaka and Kawano40 PAF produced in situ enhances ovulation, fertilization and pre-implantation.Reference Roudebush, Minhas, Ricker, Palmer and Dodson37–Reference O'Neill39 A decrease in activity of PAF-Ah has been shown to result in an increase in PAF level in follicular fluid and consequently contribute to a successful pregnancy outcome.Reference Narahara, Tanaka and Kawano40 PAF level during in vitro fertilization was improved by immobilizing PAF-Ah activity in culture mediaReference Ammit and O'Neill41 and perhaps inhibition of PAF-Ah present in platelet rich plasma grafts of musculoskeletal medicine may improve the expected outcome of a proliferative healing response, by increasing the level of PAF and therefore the mitogenic properties of PAF present in the platelet rich plasma.Reference Tate and Crane42 We speculate that our rPAF-Ah deletion construct will offer another non-pharmacologic method to inhibit PAF degradation where higher PAF level is desired,Reference Ibe, Hibler and Raj6, Reference Ibe, Sander and Raj7, Reference Ibe, Pham, Kääpä and Raj32, Reference Roudebush, Minhas, Ricker, Palmer and Dodson37–Reference Narahara, Tanaka and Kawano40 while our potent recombinant PAF-Ah construct will offer another method to decrease PAF level in conditions where high PAF levels are pathologicalReference Henderson, Lu, Poole, Dietsch and Chi35 such as in decreasing PAF levels in persistent pulmonary hypertension of the newborn.
PAFR expression and signaling
In earlier studies, it was shown that PAF binds specifically to its receptors in different types of cells.Reference Ibe, Portugal, Charturvedi and Raj19, Reference Renteria, Raj and Ibe20, Reference Korth43 Here, we show for the first time in this comparative study, that PAF binds distinctly to its receptors in ovine fetal and newborn SMC-PA and SMC-PV. In addition, Northern analysis and qPCR of both the PAs and PVs of the two ages expressed PAFR mRNA, with the expression in vein being greater than the arteries of the two ages. Interestingly, PAF binding to membrane proteins from the fetal SMC-PA and SMC-PV were significantly greater than for corresponding effects in proteins from newborn cells, whereas binding in fetal cells was attenuated by the specific PAFR antagonist CV-3988, this inhibitor augmented binding in newborn cells, suggesting some inherent difference in the translational processing of PAFR and its receptivity in the perinatal lungs. Furthermore, the decrease in newborn PAFR binding, indicates, perhaps, different PAFR-mediated function in the newborn period. PAF evokes its effects via activation of specific intracellular proteins such as phosphoinositide PLC,Reference Korth, Hirafuji, Benveniste and Russo-Marie18, Reference Ali, Fisher, Haribabu, Richardson and Snyderman44 MAPK, NF-kB and cyclin-dependent kinases.Reference Ibe, Abdallah, Portugal and Raj45 Test for the functionality of the PAFR in the perinatal lamb lungs, showed that PAF stimulation of cell proliferation in the SMC-PA and PV of the fetus was PAFR mediated, but not in newborn cells. The specific PAFR antagonist CV-3988 augmented PAFR-mediated cell proliferation especially in newborn SMC-PA in corroboration of the different effects of this inhibitor to specific PAFR binding to fetal and newborn cells.
In summary, considering perinatal pulmonary physiology, pulmonary vascular resistance in utero is high such that pulmonary blood flow is only 8–10% of total cardiac output and after birth, pulmonary blood flow increases to accommodate the total cardiac output.Reference Heyman, Creasy and Rudolph30 There are multiple mechanisms by which vasomotor tone is maintained high in the fetal pulmonary circulation, but low in the newborn pulmonary circulation. We have shown in this report that, in the fetus, pulmonary PAF synthesis, PAFR expression and PAFR-mediated responses are generally high, whereas pulmonary vascular PAF catabolism is generally low. On the other hand, in the newborn, the entire process of synthesis, receptor expression receptor-mediated and catabolism are reversed warranting low pulmonary vascular tone and successful perinatal pulmonary adaptation, failure of which will result in persistent pulmonary hypertension of the newborn, which present high morbidity and mortality of the newborn.
Acknowledgments
These studies are supported in part by Grant 513292 from the Los Angeles Biomedical Research Institute, Torrance, CA; grant 431231-6T-64440, subcontract from UCLA (Brian J. Koos, PI). The authors are grateful for the helpful suggestions, on the molecular studies by Dr Thomas Magee, Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and Ms Peng Xia, Manager of Molecular Biology Core Laboratory, Michael Opene, and Diana Guzmán Department of Pediatrics. Los Angeles Biomedical Research Institute.
Financial Support
This research received no specific grant from any funding agency, commercial or not-for-profit.
Conflicts of Interest
None.
Ethical Standards
None.
