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Am J Physiol Heart Circ Physiol 278: H1168-H1176, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1168-H1176, April 2000

Platelet-activating factor receptors in lamb lungs are downregulated immediately after birth

Basil O. Ibe, Fred C. Sander, and J. Usha Raj

Department of Pediatrics, Harbor-University of California at Los Angeles Medical Center, Torrance, California 90502


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Platelet-activating factor (PAF) is a phospholipid with diverse biological functions mediated by a G protein-coupled receptor. We determined PAF receptor binding in lung membranes of four groups of perinatal lambs. Membrane protein (100 µg/ml) was incubated for 60 min at 30°C with 0.5-24 nM of acetyl-[3H]PAF in 30 mM Tris buffer, pH 7.2, containing 0.25% BSA, 10 mM MgCl2, and 125 mM choline chloride. PAF bound to membrane was isolated and quantified by scintillation spectrometry, followed with Scatchard analysis for receptor density (Bmax). The Bmax (means ± SE, fmol/mg protein) were 445.8 ± 12.3, 244.2 ± 3.3, 250.6 ± 3.6, and 419.9 ± 8.6 for the fetal, 90-min-old, <1-day-old, and 6- to 12-day-old lambs, respectively. The Bmax for the 90-min-old and <1-day-old lambs were not different but were significantly lower than those of either the term fetal or 6- to 12-day-old lambs. These data show a significant decrease in PAF binding to its receptor and in PAF Bmax in lung membranes of immediate newborn lambs. The dissociation constants (KD, nM) were 7.7 ± 0.52, 11.5 ± 0.34, 6.9 ± 0.48, and 5.0 ± 0.53 for fetal, 90-min-old, <1-day-old, and 6- to 12-day-old newborn lamb lungs, respectively. The KD of the 90-min-old lamb was the highest of all. PAF receptor gene measured by RT-PCR showed a significant downregulation of PAF receptor gene mRNA in lungs of lambs <1 day old, suggesting a transcriptional regulation of PAF receptor gene expression in the immediate newborn period. We speculate that decreased PAF receptor binding immediately after birth will facilitate the fall in pulmonary vascular resistance in the immediate newborn period.

reverse transcriptase-polymerase chain reaction; pulmonary circulation; receptor gene expression; perinatal


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PULMONARY CIRCULATION is modulated by a number of endogenous mediators such as eicosanoids (12, 34), endothelin (33), nitric oxide (1), and platelet-activating factor (PAF; see Ref. 16). PAF is a group of structurally related compounds synthesized from membrane lipid precursors, after a variety of stimuli (38). PAF induces a broad spectrum of biological activities (16, 18). Administration of exogenous PAF in the lung in the nanomolar concentration produces a series of biological responses in the vasculature, including dilation, constriction, and changes in permeability (2, 17, 21). We have recently shown that PAF plays an important role in the maintenance of vasomotor tone in both the pulmonary and systemic circulations in the perinatal period (24). Thus, apart from being a potent inflammatory mediator, PAF has an important physiological function in the perinatal pulmonary circulation. Previous studies have demonstrated that PAF acts by interacting with specific membrane receptors that have been identified and cloned (26, 31) and has been shown to exist as a G protein-coupled receptor with seven transmembrane domains (10, 11, 32). Specific PAF-binding sites have been demonstrated in a variety of cells and tissues, including circulating blood cells and the lung (13, 26). The sheep is often used to investigate the role of endogenous and exogenous compounds in the modulation of pulmonary hemodynamics (11, 24), especially fetal and newborn lambs, which are used as models to study human fetal and neonatal pulmonary functions (7, 11, 24). The role of PAF in pulmonary hypertension of the neonate has been investigated in lambs and in other species (3). It is known that the biological effects of PAF on lungs are inhibitable by specific PAF-receptor antagonists (7, 11). This suggests the existence of specific PAF receptors in this organ. However, PAF receptors in lamb lungs have not been characterized. In this report, we studied PAF binding to its receptors in lamb lungs during the perinatal period. Our primary objective was to elucidate the profile of PAF binding to its receptors in the perinatal lamb lungs as an aid in determining the role of PAF in the modulation of pulmonary vascular tone during the perinatal period.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Pregnant ewes (142-148 days gestation, term being 150 days), newborn lambs 4-16 h old (<1 day old), and newborn lambs 6-12 days old were purchased from Nebekar Farms (Santa Monica, CA). Term fetuses were delivered and ventilated for 90 min and then were killed and studied as immediate newborn lambs 90 min old. Authentic standards of PAF [hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C16-PAF); hexadecyl-sn-glyceryl-3-phosphorylcholine (Lyso-C16-PAF); and octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C18-PAF)] were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Radiolabeled PAF standards and substrates were purchased from NEN (Boston, MA). They are hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine,1-O-[acetyl-3H(N)]PAF (acetyl-C16-[3H]PAF; 10 Ci/mmol; 370 GBq/mmol) and hexadecyl-sn-glyceryl-3-phosphorylcholine,1-O-[alkyl-1',2'-3H]PAF (Lyso-C16-[3H]PAF; 60 Ci/mmol; 2.2 TBq/mmol). Phenylmethylsulfonyl fluoride (PMSF), BSA, leupeptin, and pepstatin A were purchased from Sigma Chemical (St. Louis, MO). Stock solution of the lipids was made in ethanol and stored at -70°C. Dilutions for use in each experiment were made fresh each day in the receptor assay buffer. Ecolite(+) liquid scintillation cocktail was purchased from ICN Biochemicals (Irvine, CA). Glass G4 filter circles and all other solvents and reagents were purchased from Fisher Scientific (Santa Clara, CA).

Methods

Study groups. Lungs from the following groups of lambs were used to prepare membranes employed in the receptor binding assays.

GROUP 1 (N = 5). Lungs of fetal lambs 142-148 days gestation were studied. The pregnant ewes were sedated with intramuscular injection of ketamine (25 mg/kg) and acepromazine (0.5 mg/kg). After caesarean section, the fetuses were prevented from breathing and were killed by an intravenous overdose of pentobarbital sodium (100 mg/kg). The estimated fetal weight of 3.0 kg was based on previous experiences.

GROUP 2 (N = 5). Lungs of newborn lambs 90 min old were studied. Pregnant ewes at 148 days gestation were sedated as in group 1 animals. After delivery of the fetal head and neck through a uterine incision, an endotracheal tube was tied in the trachea. Next, the fetuses were prepared for mechanical ventilation with a Healthdyne Infant Ventilator, as previously described (24). After 90 min of ventilation, lambs were killed with an intravenous overdose of pentobarbital sodium (100 mg/kg).

GROUP 3 (N = 5). Lungs of newborn lambs 4-6 h (<1 day) old were studied. Two lambs were mechanically ventilated for 4 h before death as in group 2 animals. The other three were from spontaneous delivery and were maintained in our vivarium before being killed by intravenous injection of pentobarbital sodium (100 mg/kg).

GROUP 4 (N = 5). Lungs of newborn lambs 6-12 days old were studied. The lambs were killed by intravenous injection of 100 mg/kg of pentobarbital sodium.

In all instances, the study protocols were approved by the institutional animal care and use committee. The study groups were chosen on the basis of previous observations of the effect of PAF on the lung during the perinatal period (24, 33, 35).

Membrane preparation. Lungs from the four groups of lambs, i.e., 142- and 148-day gestation fetal lambs, newborn lambs 90 min old, newborn lambs <1 day old, and newborn lambs 6-12 days old, were isolated, vasculature washed, quickly frozen in liquid nitrogen, and then homogenized separately at 4°C in 9 vol buffer/g lung tissue. The homogenization buffer was a 50 mM Tris buffer, pH 7.2, containing 0.1 mM PMSF (20). The homogenate was centrifuged at 1,200 g for 5 min to rid the homogenate of tissue debris, after which the supernatant was recentrifuged at 40,000 g for 20 min. The resulting pellet was resuspended in 50 mM Tris buffer containing 5 mM MgCl2, 125 mM choline chloride, 0.1 mM PMSF, 0.1 µg/ml leupeptin, and 1 µg/ml pepstatin at a ratio of 1 vol buffer per gram original lung weight. Protein concentration of the lung membrane suspension was determined by the method of Lowry et al. (29) using BSA as the standard. Membrane preparations were frozen in liquid nitrogen in 0.5-ml aliquots and stored at -80°C. Storage at this temperature did not affect the binding profile of PAF to the membranes being studied; however, thawed protein sample was not refrozen for further use.

Radioligand binding assay. Direct radioligand binding assays were done in 50 mM Tris buffer, pH 7.2, containing 5 mM MgCl2, 125 mM choline chloride, and 0.25% BSA (20). The concentration of BSA used did not hamper PAF binding to its receptor but was necessary to enhance PAF solubility in the aqueous buffer. All assays were done in triplicate.

Assay standardization. Preliminary studies were done with 25-1,000 µg of membrane protein from each group to determine the concentration of protein to use in the rest of the studies. Assays were done with 0.5 nM of acetyl-C16-[3H]PAF in 1 ml of the assay buffer at 4°C for 4 h and at 30°C for 1 h. Binding of the acetyl-C16-[3H]PAF to its receptor was linear between 50 and 500 µg of membrane protein at both 4 and 30°C, although binding at 4°C was significantly lower. We elected to conduct all studies with 100 µg protein/ml of the incubation buffer. We then performed kinetic studies of PAF binding to its receptor to determine the optimum duration of incubation. Studies were done with 100 µg protein/ml and 0.5 nM acetyl-C16-[3H]PAF for 1, 3, and 6 h at 4°C and for 30 min, 1 h, and 90 min at 30°C. Analysis of PAF receptor binding at these conditions showed that incubations for 1 h at 30°C produced threefold more binding in the four groups than incubations done for 6 h at 4°C. Acetylhydrolase activity on PAF was measurable in membranes of the four groups at 4°C for 6 h and 30°C for 60 min; however, the hydrolysis product [3H]acetate was not significantly bound to the filters as to influence the profile of PAF binding in the four groups. We then elected to conduct the rest of the studies under the conditions of 1 h at 30°C because this condition not only produced better binding of PAF to its receptor but also allowed us to conduct an array of similar experiments in 1 day.

Effect of PAF Concentration on PAF Binding to its Receptor

All assays were done in 1 ml of the 50 mM Tris buffer using 100 µg of membrane protein from each lung. Binding assays of PAF to membrane receptors were conducted in the presence of 10 µM nonradiolabeled acetyl-C16-PAF and varying concentrations (0-24 nM) of acetyl-C16-[3H]PAF. A nonspecific binding study was done at each concentration of the acetyl-C16-[3H]PAF. Ligand binding to the receptor was terminated by filtration of incubation media through a Whatman fiberglass GF/B filter on a Whatman filtration manifold (Whatman, Clifton, NJ). Each filter was washed three times with 4 ml of the assay buffer. Next, [3H]PAF radioactivity retained on the membrane filters was extracted with 5 ml of Ecolite liquid scintillation cocktail (ICN Biochemicals), after which the radioactivity was measured by liquid scintillation spectrometry (Beckman, Fullerton, CA).

Effect of WEB-2086 on PAF Binding to its Receptor

Assays with the specific PAF-receptor antagonist [3H]WEB-2086 were conducted in the presence of 100 µM of acetyl-C16-PAF. Because there was no difference in PAF binding to its receptors in the 90-min- and <1-day-old lamb lung membranes, lung membrane from 90-min-old lambs was studied as <1 day old. Studies were done and processed as described for the PAF binding study. Specific binding of the agonist or antagonist was calculated as the difference between binding in the presence or absence of membrane.

Effect of CV-6209 and SQ-29548 on PAF Binding to its Receptors

To assess the specificity of PAF binding to its receptors, the effect of CV-6209, a competitive PAF-receptor antagonist, and SQ-29548, a thromboxane A2-receptor antagonist, was tested on lung membranes from the perinatal lambs. Membrane protein (100 µg) was pretreated for 30 min at 4°C with different concentrations of each receptor antagonist or with buffer alone for control. Next, 0.5 nM of acetyl-C16-[3H]PAF was added to the membranes and incubated for an additional 6 h at 4°C. Acetyl-C16-[3H]PAF bound to its receptors was processed as described above. Inhibition of PAF binding to its receptor was calculated as percent control.

Study of Ovine PAF Receptor Gene Expression

RNA preparation. Total RNA was prepared from lung tissue of the term fetal, 4- to 16-h-old (<1 day), and 6- to 12-day-old newborn lambs by the acid-phenol extraction method (15).

Cloning of fragment of ovine PAF receptor gene. Lamb lung cDNA was synthesized from total RNA using random hexamer primers and Moloney murine leukemia virus RT according to the manufacturer's instructions (5 Prime-3 Prime, Boulder, CO). The GenBank database was searched for previously cloned PAF receptor genes. Comparison of cDNA nucleotide sequences for PAF receptor from human, rhesus monkey, and guinea pig (GenBank accession nos. M76674, Z18856, and X56736, respectively) showed good species-to-species homology (85% human to guinea pig). PCR primers that match nucleotides 170-191 (sense strand) and nucleotides 667-646 (antisense strand) of the published human cDNA sequence (26) were designed and synthesized by Biotech Diagnostic Institute (Laguna Niguel, CA). The 5' sense primer sequence was 5'-TTCAATGAGATAAAGATCTTCA-3' and matched base pairs 170-191. The antisense primer sequence was 5'-GAGCAAGGTACGGATGATGACC-3' and matched base pairs 667-646. These two primers were used for PCR using reverse-transcribed sheep mRNA as template. An ~485-bp fragment of sheep PAF receptor cDNA was cloned and sequenced, and the identity was confirmed.

RT-PCR procedures. Reverse transcription was carried out for 1 h at 42°C in 50 mM Tris buffer, pH 8.3, containing 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol essentially as previously described (41). The total volume of incubation is 20 µl and contains 0.5 mM each of dNTP, 20 units of RNasin, 25 pmol of oligo(dT) primer, and 200 units of Moloney murine leukemia virus RT (Life Technologies). At the end of the incubation, the reaction was stopped by heating at 90°C for 5 min. PCR amplification was performed in a 75-µl final reaction volume that contains the cDNA mixture generated from fetal, <1-day-old, and 6- to 12-day-old lambs diluted with the reaction buffer (10×) to a final composition of 10 mM Tris buffer, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 100 µM dNTPs, 2.5 units of Taq polymerase, and 55 pmol of each primer. The cDNA is adjusted to equal concentrations as assessed by the PCR of the constitutively expressed housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of GAPDH product synthesized from each cDNA template, fetal, <1 day-old, and 6- to 12-day-old lamb lung tissues, was visualized by ethidium bromide-stained agarose gel electrophoresis and was found to be equal in all three reaction groups. PCR was performed for expression of PAF receptor in the three tissues applying the linear region of the input/output plot. The input template cDNAs were all equal to the output PCR product, as judged by synthesis of GAPDH and as described above. For synthesis, the tubes were placed in an Ericomp Thermal Cycler that was programmed as follows: incubation at 95°C for 4 min for the initial melt; 35 cycles at 94°C for 30 s (melt), 55°C for 1 min (anneal), and 72°C for 2 min (extend). For final extension, tubes were incubated at 72°C for 7 min. The PCR product was purified by 1.5% agarose gel electrophoresis and sequenced by the City of Hope at the Department of Biology Core Sequencing Laboratory (Duarte, CA).

Data analysis. PAF receptor binding isotherms in the presence or absence of WEB-2086 were analyzed with a nonlinear regression for a one-site binding model (PRISM program). In all instances, the nonspecific binding was subtracted from the binding in the presence of membrane protein, and the data are presented as specific PAF or WEB-2086 binding in femtomoles per milligram of membrane protein. PAF receptor density (Bmax) for each group of lungs was calculated from the linear regression analysis (Scatchard analysis) of the binding characteristics of PAF at the different concentrations studied. All data (means ± SE) for Bmax and the dissociation constant (KD) of the binding isotherms for each group of lungs (fetus, 90-min-old newborn, and <1-day-old and 6- to 12-day-old lamb lung membranes) were subjected to statistical analysis. To test for differences in PAF Bmax between two groups, e.g., fetus and newborn 90 min old, a two-tailed Student's t-test was used. For multiple comparisons to detect differences among more than two experimental groups, for example, data from fetus, newborn 90 min old, and newborn <1 day old, an ANOVA with Tukey's test was used. A P value <0.05 was accepted as a statistically significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PAF Binding to Perinatal Lamb Lung Membrane

Figure 1 shows a representative [3H]PAF isotherm binding to its receptor in lung membrane of the four groups of lambs and representative Scatchard plots for the binding isotherms of the four groups of lamb lungs (inset). PAF was bound to its receptors in lung membranes in a dose-dependent manner. [3H]PAF binding to its receptors in lung membranes of 90-min-old and <1-day-old lambs were similar. Also, [3H]PAF binding to its receptors in lung membranes of fetal and 6- to 12-day-old lambs was similar. However, binding isotherms of the 90-min-old and <1-day-old lambs were lower than those of the fetus and 6- to 12-day-old lambs. The Scatchard analysis of receptor binding of 90-min-old and <1-day-old lambs was different from that of the fetus and the 6- to 12-day-old lambs.


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  		Fig. 1.   Representative specific platelet-activating factor (PAF) binding isotherms and Scatchard plot (inset) of fetal, 90-min-old newborn, 4- to 16-h-old newborn (<1 day old), and 6- to 12-day-old lamb lung membranes. PAF was significantly bound to lung membrane receptor at each age. Specific binding of PAF to lung membrane receptors of the newborn lambs 90 min and <1 day old was lower than the specific PAF binding to lung membrane receptors of the fetal or newborn 6- to 12-day-old lambs.

Figure 2 shows the calculated Bmax of PAF binding to its receptors in the perinatal lamb lungs in femtomoles per milligram protein. In lung membrane of the fetus and 6- to 12-day-old newborn lambs, the receptor densities were 435.8 ± 12.3 and 418.9 ± 9.5 fmol/mg protein, respectively. There was no difference between the PAF Bmax in lungs of the fetal and 6- to 12-day-old lambs. In lungs of newborn lambs 90 min and <1 day old, the Bmax were 244.2 ± 3.3 and 250.3 ± 9.5 fmol/mg protein, respectively. There was no difference between the PAF receptor Bmax in lungs of the newborn lambs 90 min old and <1 day old. However, the PAF receptor Bmax in lung membranes of the 90-min and the <1-day-old newborn lambs were significantly lower than the amounts in lungs of the fetal and 6- to 12-day-old newborn lambs.


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  		Fig. 2.   Comparison of the PAF receptor densities (Bmax) of lung membranes of fetal and of newborn lambs 90 min, <1 day, and 6-12 days old. Data are means ± SE; n = 5 experiments. The receptor densities in the membranes of the 4 groups of lungs ranged from 244 fmol/mg protein in the 90-min-old lambs to 445 fmol/mg protein in the fetal lamb lungs. *P < 0.05, different from either near-term fetal or newborn 6- to 12-day-old lungs.

Figure 3 shows a comparison of the KD (nM) of PAF binding to its receptors in the four groups of lamb lungs. In fetal lungs, the KD of PAF binding was 7.7 and was significantly different from the value of 11.5 in the 90-min-old lambs. The values for the <1-day-old and 6- to 12-day-old lamb lungs were 6.9 and 5.0, respectively. The KD of PAF binding to 6- to 12-day-old newborn lamb membranes was the least and was significantly different from binding to lung membranes of the fetal and 90-min- and <1-day-old lambs.


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  		Fig. 3.   Comparison of dissociation constants (KD) of PAF binding to its receptors in the 4 groups of lamb lungs. Data are means ± SE; n = 5. The KD ranged from 4 to 11 nM, with significant differences among the 4 groups of lungs. * P < 0.05, different from either near-term fetal, <1-day-old, or 6- to 12-day-old newborn lamb lungs. # P < 0.05, different from fetal, 90-min-old, or <1-day-old newborn lamb lungs.

WEB-2086 Binding to Ovine PAF Receptor

Figure 4 shows a representative isotherm of [3H]WEB-2086 binding to PAF receptors in lung membrane of the three groups of lambs and a representative Scatchard plot for the binding isotherms of the three groups of lambs (inset). Binding of [3H]WEB-2086 to PAF receptors in lung membrane was similar in the three groups of lungs. The Scatchard analyses of [3H]WEB-2086 binding to PAF receptor in the three groups of lungs were also similar.


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  		Fig. 4.   Competitive binding isotherms and Scatchard plot (inset) of different concentrations of [3H]WEB-2086 and PAF (100 µM) to PAF receptors in fetal, <1-day-old newborn, and 6- to 12-day-old lambs. Data are means ± SE; n = 5. [3H]WEB-2086 displaced PAF binding to membrane receptors in a concentration-dependent manner.

Figure 5 shows the comparison of the Bmax of [3H]WEB-2086 binding to lung membrane PAF receptors of fetal, <1-day-old newborn, and 6- to 12-day-old lambs. The receptor densities in membranes of the three groups of lungs ranged from 730 fmol/mg protein in the fetal lambs to 850 fmol/mg protein in the 6- to 12-day-old lambs. There was no difference in Bmax of WEB-2086 to PAF receptor among the three groups of lungs.


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  		Fig. 5.   Comparison of the Bmax of [3H]WEB-2086 binding to PAF receptors in fetal, <1-day-old newborn, and 6- to 12-day-old lambs. Data are means ± SE; n = 5. The receptor densities in membranes of the 3 groups of lungs ranged from 750 fmol/mg protein in the fetal lambs to 1,000 fmol/mg protein in the 6- to 12-day-old lambs. There was no difference in Bmax of inhibitor binding to PAF receptor among the 3 groups of lungs.

Figure 6 shows the comparison of the KD of [3H]WEB-2086 binding to PAF receptors in lung membranes of fetal, <1-day-old newborn, and 6- to 12-day-old lambs. The KD values of WEB-2086 binding were higher than the KD values for PAF binding to the receptors and ranged from 50 to 175 nM. However, there was no difference in the KD of the three age groups.


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  		Fig. 6.   Comparison of the KD of [3H]WEB-2086 binding to PAF receptors in lung membranes of fetal, <1-day-old newborn, and 6- to 12-day-old lambs. Data are means ± SE; n = 5. The KD values ranged from 50 to 175 nM; however, there was no difference in the KD of WEB-2086 binding to lung membranes of the 3 age groups.

Figure 7 shows the effect of different concentrations of CV-6209 and SQ-29548 on PAF binding to its receptors in the perinatal lamb lung membranes. When membranes were treated with 10-12 M CV-6209, the competitive PAF-receptor antagonist, binding of PAF to its receptor was 75-80% in the three groups of lung membranes (a 20-25% inhibition). Treatment with a 10-9 M concentration of this inhibitor significantly reduced the PAF binding further to 33-43% of control (a 57-67% inhibition) in the three groups of lung. When treated with a 10-6 M concentration of inhibitor, binding of PAF to its receptor was 18% in fetal and 6- to 12-day-old lambs but only 5% in <1-day-old lamb lung membranes. Thus CV-6209 produced a concentration-dependent inhibition of PAF binding to its receptor in the three groups of lamb lung membranes.


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  		Fig. 7.   Comparison of the inhibition, by CV-6209 and SQ-29548, of PAF binding to its receptors in lung membranes of fetal, <1-day-old newborn, and 6- to 12-day-old lambs. Data are means ± SE; n = 4. Lung membrane suspension from the 3 groups of lambs was preincubated with the inhibitor for 30 min before the addition of 0.5 nM of acetyl-C16-[3H]PAF. CV-6209 produced a concentration-related inhibition of PAF receptor binding. SQ-29548 was ineffective in causing a concentration-related inhibition of PAF receptor binding in the 3 groups of lungs. * P < 0.05, different from inhibition by 10-12 M. # P < 0.05, different from inhibition by 10-12 and 10-9 M of CV-6209.

Treatment of membranes with a 10-12 M concentration of SQ-29548, a thromboxane A2-receptor antagonist, resulted in 75, 70, and 65% binding of PAF to its receptor in fetal, <1-day-old, and 6- to 12-day-old lamb lung membranes, respectively. With a 10-9 M concentration of SQ-29548, PAF binding in the three groups ranged from 65 to 79%, and with a 10-6 M concentration, the PAF binding was 72-85%. Hence, inhibition of PAF binding to its receptors by SQ-29548 was not concentration related, nor was the inhibition profile consistent within a group.

Ovine PAF Receptor Gene Expression

Figure 8 shows an agarose gel electrophoresis of RT-PCR measurement of PAF receptor gene expression in fetal, <1-day-old newborn, and 6- to 12-day-old lamb lungs. Each pair of bands shows the product amplified by using 2.0 and 0.2 µl of cDNA. Figure 8, bottom, shows RT-PCR measurement of the expression of the housekeeping gene GAPDH with the same amount of cDNA but with fewer PCR cycles. GAPDH gene expression was similar in the three groups. Qualitative assessment of receptor gene expression showed high expression in fetal lung membranes, very low expression in lung membranes of lambs <1 day old, but high expression in lung membranes of 6- to 12-day-old lambs. PAF receptor gene expression was semiquantified by densitometric measurement with a Stratagene Eagle Eye II Still Video System (Stratagene) using the PCR products from the 0.2 µl cDNA. Blot density was normalized to the blot density of the corresponding GAPDH gene expression from the 0.2 µl of cDNA. PAF receptor gene expression by fetal lungs was 100%, receptor gene expression by <1-day-old lambs was 5%, and expression by lungs of newborn lambs 6 to 12 days old was 18%.


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  		Fig. 8.   Agarose gel electrophoresis showing RT-PCR measurement of PAF receptor (PAF-R) gene expression in lungs of 142- to 148-day-old fetal lambs, newborn lambs 4 h to 16 h old (<1 day old), and newborn lambs 6-12 days old. Each pair of bands shows product amplified by using 2.0 and 0.2 µl of cDNA. Bottom: RT-PCR measurement of the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with same amount of cDNA.

Figure 9 shows the sequence of the sheep PAF receptor cDNA fragment obtained by RT-PCR. The PAF receptor sequence is conserved, showing complete homology with the human leukocyte PAF receptor gene sequence at the nucleotide level (26), except the two nucleotides in bold type (nos. 2 and 305). This sequence has been deposited with Genebank with accession no. AF099674.


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  		Fig. 9.   Comparison of the nucleotide sequence of the ovine and human (26) PAF receptor cDNAs. The ovine sequence shows significant homology to the human sequence. This ovine PAF receptor partial cDNA sequence has been deposited in Genebank with accession no. AF099674.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PAF is an important phospholipid with a wide range of physiological and pathological effects in vivo, including action as a vasoactive mediator in the circulation (2, 10, 16, 17, 24). Because of its potent action on blood vessels, it has been implicated in some abnormalities of fetal and neonatal circulations, such as neonatal pulmonary hypertension (3, 9, 16). However, its role in modulating normal fetal and neonatal circulations still needs further study. PAF plays a role in the maintenance of pulmonary and systemic vasomotor tone during the perinatal period (24), suggesting that PAF performs beneficial functions in the circulation under physiological conditions. The lamb is used extensively to study pulmonary physiology and pharmacology during the perinatal period (2, 5, 7, 24). PAF is a potent modulator of the pulmonary circulation of sheep, which is a widely used model to study the vascular effects of PAF (2, 5, 7, 24). However, little is known about PAF receptors in this species, especially during the perinatal period.

In the present study, we have used direct radioligand binding and RT-PCR methods to describe specific binding of PAF to its receptors in lung membranes of lambs during the perinatal period. The results show that PAF binds specifically to its receptors in lung membranes of the four groups of lambs. In a previous report, we showed that PAF functions to maintain a high pulmonary vascular tone in utero in the fetus and that, after birth, a fall in PAF level correlated with the fall in pulmonary vascular tone that must occur for normal transition of the newborn lamb lung to an air-breathing organ (24). The binding of PAF to its lung membrane receptor at each age group showed relatively high affinity, with a KD range of 4.95 nM in the lungs of 6- to 12-day-old newborns to 11.53 nM in the 90-min-old newborns, and was also saturable with different Bmax among the groups. Importantly, the binding of PAF to its receptor in lung membrane at each age was displaced by the specific PAF-receptor antagonist WEB-2086 in a concentration-dependent manner. This shows that PAF binds to its specific receptor and suggests that PAF binding to the receptor is reversible, an important requirement for a receptor-mediated event (27). Even though the characteristics of PAF binding to its receptor may be similar in various tissues, different binding characteristics have been reported in some tissues and cells, both under similar and different experimental conditions (14, 23, 40, 42). For instance, in human platelets, the KD of PAF binding to platelet membrane receptors varied from 0.05 to 37 nM (41). In asthmatics, a KD of 0.38 nM was reported compared with 0.26 nM in nonasthmatic volunteers that have different Bmax (6). These data show that PAF binding affinity to its receptor and the Bmax can be regulated by physiological and pathological conditions. Our observation of different PAF binding affinity to its receptor in perinatal lamb lungs demonstrates a novel control mechanism of PAF receptor binding and suggests an important regulatory mechanism of PAF effects in vivo. Thus the combination of a downregulation of Bmax and a decrease in binding affinity in lamb lungs during the immediate newborn period will result in a decrease in pulmonary vascular resistance and thereby facilitate the transition of the newborn pulmonary circulation from a high-resistance to a low-resistance circulation.

To confirm that [3H]PAF was bound to its receptors, we studied the binding of [3H]WEB-2086 to PAF receptors in the three groups of lamb lungs in the presence of 100 µM of nonradiolabeled PAF. [3H]WEB-2086 competed favorably for binding to PAF receptors in the lamb lung membranes, with significantly higher KD than the binding of PAF. There was no difference in the affinity of WEB-2086 binding to the PAF receptors in the perinatal lambs, suggesting a very close similarity in the PAF receptors in these perinatal lungs. Furthermore, WEB-2086 binding to the PAF receptors was saturable, with Bmax of 732, 812, and 843 fmol/mg protein in fetal, <1-day-old newborn, and 6- to 12-day-old lamb lungs, respectively. These values are over twofold higher than the Bmax of PAF binding to its receptor. Thus, in keeping with its properties as a specific PAF-receptor antagonist, WEB-2086 binding to PAF receptor in the perinatal lamb lungs showed a high KD and saturable binding with a high Bmax (14, 23, 42). Some other studies (14, 40) have used binding of [3H]WEB-2086 alone to characterize PAF receptors on the premise that use of direct binding of PAF produces a high nonspecific binding, possibly a result of the inclusion of BSA in the assay buffer. However, assays of direct [3H]PAF binding to its receptor have also been reported (6, 20). By using a direct radioligand binding method, we have demonstrated a downregulation of PAF binding to its membrane receptors in lamb lungs during the perinatal period. This phenomenon is critical in our understanding of the factors regulating the pulmonary circulation during the transition from the fetal to newborn period. Accordingly, it is obvious that use of [3H]WEB-2086 binding assays alone in this study would have failed to discover this important difference in PAF binding to perinatal lamb lung membranes. We further tested the specificity of the PAF receptor in lamb lungs by examining the displacement of PAF receptor binding in lung membranes of the fetus and newborn lambs <1 day old and 6-12 days old by other receptor antagonists. The membranes were subjected to competitive binding with CV-6209, a non-benzodiazepine-related PAF-receptor antagonist with close structural similarity to PAF, and SQ-29548, a thromboxane A2-receptor antagonist, in the presence of 100 µM of nonradiolabeled PAF. Both PAF and thromboxane A2 are vasoconstrictors in the pulmonary vasculature. Our aim in this study was to examine the profile of inhibition of PAF binding to its receptor rather than characterize affinity of PAF receptor in lung membranes from each age group. The competitive PAF-receptor antagonist CV-6209 exhibited a concentration-related displacement of PAF binding to its receptor in the three groups of lungs. For instance, 10-12 M of CV-6209 inhibited PAF binding to its receptor in fetal and <1-day-old lamb lung membrane by 25% and in 6- to 12-day-old lambs by 20%. The percent inhibition in the three groups increased significantly at 10-9 M of CV-6209 and further to 82-95% inhibition at 10-6 M of CV-6209 for the three groups. Thus, as with WEB-2086, CV-6209 competed in a concentration-dependent manner to inhibit PAF binding to its receptors. On the other hand, SQ-29548 was ineffective in producing a concentration-related displacement of PAF binding to its receptors. Inhibition of PAF binding to its receptors by SQ-29548 at the highest concentration of SQ-29548 studied was as follows: 21, 15, and 28% for the fetal, <1-day-old newborn, and 6- to 12-day old lambs, respectively. Ideally, a complete binding profile (using 10-12 to 10-3 M concentrations) of these and other receptor antagonists should be examined in these perinatal lamb lungs. However, we can conclude from these studies that specific PAF receptors are present in perinatal lamb lungs and that the differences in PAF binding among the four groups of lambs studied are not due to ectopic binding of PAF to receptors, such as benzodiazepine receptors or thromboxane A2 receptors present in the lung. At present, it is not clear whether PAF receptors in these lamb lungs possess multiple conformationally controlled binding sites or signify the presence of receptor subtypes. However, we can speculate that multiple receptor subtypes exist in these groups of lungs to account for the difference in PAF binding to its receptor in the three groups of lungs. Furthermore, we cannot judiciously speculate the cell locale of these receptors, although it is conceivable that the receptors will be present in endothelium and smooth muscle cells. Our primary goal in this study was to identify PAF receptor in the perinatal lamb lungs and to discover a probable difference in PAF binding profile to the receptors in the immediate newborn period.

In previous studies, PAF receptor gene expression in various tissues and organs was studied by Northern analysis (25, 30, 31, 37). However, the technique of RT-PCR has also been used to localize PAF receptor gene to a specific region of the hamster oviduct (41), and the RT-PCR technique has also been used to demonstrate the expression of PAF receptor in various tissues under different conditions (19, 36, 39). In this report, we used the semiquantitative techniques of RT-PCR to demonstrate PAF receptor gene expression in lungs of perinatal lambs. Our data show that PAF receptor gene was expressed in lungs of these perinatal lambs and that PAF receptor gene expression in lungs of the newborn lamb <1 day old was decreased compared with the expression in either lungs of the near-term fetuses or the 6- to 12-day-old newborn lambs. This suggests that the PAF receptor gene is transcriptionally regulated in the immediate newborn period and may also suggest the existence of inhibitory factors, and perhaps receptor subtypes, that may control PAF receptor gene expression at this critical period of newborn life. Previous studies have shown that expression of PAF receptor can be modulated by physiological and pathological factors (14, 25, 30, 36, 37, 39). For instance, PAF receptor is upregulated in lungs of asthmatics (25, 37), showing that the increased PAF binding to its receptors in lungs of asthmatic patients correlates with the increased expression of PAF receptors in lungs of asthmatic patients. Upregulation of PAF receptors has also been demonstrated in endometrial cells treated with sex steroid hormones (36). On the other hand, downregulation of PAF receptor genes has been demonstrated in lungs of cigarette smokers (37) and in human macrophages treated with oxidized low-density lipoprotein, a condition that was reversible on discontinuation of treatment (39). In our study, with the use of GAPDH as the standard, PAF receptor gene expression was high in fetal lungs but decreased in the immediate newborn lung. However, expression of the PAF receptor gene returned to a higher level in the lungs of older newborn lambs 6-12 days old compared with newborn lambs <1 day old. Our RT-PCR results corroborate our data from direct radioligand binding assays indicating a high PAF binding to its receptor in lungs of term fetuses and newborn lambs 6-12 days old but significantly decreased binding in lungs of the immediate newborn lambs. This indicates that the decrease in PAF binding to its receptors in lungs of lambs <1 day old is due, in part, to the downregulation of PAF receptor gene expression at this period. We have previously shown that production of PAF by lamb lungs within the age of 6-12 days old is low (35). In this situation, it is possible that occupancy of PAF receptors will be low so that the effect of PAF on the pulmonary circulation should be low. During states of inflammation when the synthesis of PAF is high (8, 22), it can be said that the upregulation of receptor gene expression will make more receptors available for PAF binding to propagate and/or sustain the inflammatory condition.

The pulmonary circulation is modulated by a variety of endogenous agents such as nitric oxide, eicosanoids, and PAF (4, 19, 24, 28, 34). In the fetus, pulmonary vascular resistance is high due to a greater production of mediators of pulmonary vasoconstriction (16, 21). PAF is one of the endogenous compounds that maintains a high pulmonary vascular resistance in lungs of the near-term fetus (24). At birth, with oxygenation, pulmonary vascular resistance falls dramatically so that blood flow to the lungs increases. Our data show a high PAF binding to its receptor together with a high PAF receptor gene expression in lungs of the near-term fetuses and a significant decrease in PAF binding to its receptor coupled with a downregulation of PAF receptor gene expression in lungs of newborn lambs <1 day old. We speculate that downregulation of PAF receptor at this age coupled with a decrease in Bmax in the immediate newborn period is related to the fall in pulmonary vascular resistance in the immediate newborn period.


    ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health Grants HL-47804, HL-38438, and HD-29713.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. O. Ibe, Harbor-UCLA Medical Center, 1124 W. Carson St., RB-1, Torrance, CA 90502 (E-mail: ibe{at}prl.humc.edu).

Received 9 April 1999; accepted in final form 26 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 278(4):H1168-H1176
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