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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Neonatology, Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California
Received for publication
August 2, 2006
Accepted
November 2, 2006.
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Abstract |
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). In lungs, PAF is a potent vasoactive mediator with different sites of action in different species (Toga et al., 1992; Argiola et al., 1995; Ibe et al., 1998). In the fetus, PAF plays an important physiological role in maintaining a high vasomotor tone in the pulmonary circulation (Ibe et al., 1998). In fetal lungs, PAFr binding and PAFr mRNA expression are high, so that a high level of pulmonary vasomotor tone can be maintained in utero (Ibe et al., 2000), whereas in lungs of the newly born lamb, PAFr binding and PAFr mRNA expression are low, suggesting a down-regulation of PAFr-mediated effects in vivo (Ibe et al., 1998, 2000). In addition, the effects of PAF can be completely abrogated by the enzymatic degradation of PAF by PAF acetylhydrolase (Kim et al., 2000), by specific PAFr antagonists (Argiolas et al., 1995; Ibe et al., 1998), or by agents that decrease PAFr protein expression (Ibe et al., 2000).
PAF evokes its effects by binding to its G protein-coupled receptor, which is a seven-transmembrane receptor (Carlson et al., 1996; Parent et al., 1996). Activation of G protein-coupled receptor by an agonist results in activation of signal transduction pathways (Ferguson et al., 1996), which may involve recruitment of intracellular second messengers such as cAMP, cGMP, inositol 1,4,5-triphosphate (IP3), and calcium (Lin and Rui, 1994; Rehring et al., 1996; Lee et al., 2001). cGMP and cAMP act via their endogenous receptors, cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively, to elicit smooth muscle relaxation (Abdel-Latif, 2001). Both cGMP and cAMP mediate relaxation of pulmonary vessels, but cGMP has been shown to be more effective than cAMP in producing relaxation of perinatal ovine pulmonary vessels (Dhanakoti et al., 2000). We recently showed that acute hypoxia up-regulates PAFr-mediated intracellular signaling in ovine fetal pulmonary vascular smooth muscle (Ibe et al., 2005). Therefore, chronic hypoxia in the perinatal period may result in abnormal up-regulation of PAFr protein expression, PAFr binding, and PAFr-mediated cell signaling, leading to increased pulmonary vasomotor tone and vascular remodeling, a key event in the onset of clinical disorders such as persistent pulmonary hypertension of the newborn (PPHN) (Caplan et al., 1990).
Pulmonary vascular resistance in utero is high, such that pulmonary blood flow is only 8 to 10% of total cardiac output (Heymann et al., 1990). At birth, with oxygenation, pulmonary vascular resistance falls dramatically, and blood flow to the lung increases to accommodate the total cardiac output (Fineman et al., 1995). We are interested in understanding the mechanisms of pulmonary vascular relaxation at birth. Our primary hypothesis is that with oxygenation at birth and the increased production of cGMP and cAMP in pulmonary vascular smooth muscle, PAFr-mediated cell signaling is down-regulated via cross-talk between the cyclic nucleotides and PAF-PAF receptor complex. We used ovine fetal intrapulmonary venous smooth muscle cells (SMC-PV) to study the effects of cGMP and cAMP on PAFr binding and PAFr-mediated cell signaling in both normoxia and hypoxia. We also studied the effects of cGMP and cAMP on PAFr expression and the effects of PAF on expression and activities of PKG and PKA proteins in these venous smooth muscle cells.
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Materials and Methods |
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Preparation of Pulmonary Vascular Smooth Muscle Cells
Intrapulmonary vessels were isolated from freshly killed term fetal lambs, and then smooth muscle cells were harvested from the freshly excised arteries and veins under sterile conditions as previously reported (Ibe et al., 2005). Cells were used at the fourth to sixth passage and identity of the smooth muscle cells at each passage was characterized with a smooth muscle cell-specific monoclonal antibody (Sigma Chemical). The smooth muscle cells were devoid of endothelial cells and fibroblasts. The cell phenotype did not change from the fourth to sixth passages as determined by the expression of 
-smooth muscle actin and myosin light chain kinase proteins.
Studies with Smooth Muscle Cells. All studies were done with smooth muscle cells harvested from intrapulmonary veins of term fetal lambs, because they exhibit more PAF binding than cells from arteries (Ibe et al., 2005). Also, we have previously reported that in the fetus, pulmonary veins often demonstrate significantly greater vasoactivity than pulmonary arteries, and veins contribute a significant fraction to pulmonary vascular resistance in utero (Kääpä et al., 1991; Toga et al., 1992).
Study Conditions. All studies were done with adherent cells in normoxia or in hypoxia.
Normoxia. Cells were studied in a humidified incubator at 37°C aerated with 5% CO2 in air. The oxygen concentration was monitored with a TED 60T percent oxygen sensor (Teledyne Analytical Instruments, City of Industry, CA). The incubator oxygen concentration was 21% and pO2 determined as in the hypoxia condition was normoxic at 80 to 100 torr.
Hypoxia. An incubator set at 37°C was first equilibrated for at least 1 h with a gas mixture of 2% O2, 10% CO2, and balance N2 to maintain incubator pO2 <40 torr, as determined on a Nova Stat Profile 3 blood-gas instrument (Nova Biomedical Corp., Waltham, MA) (Ibe et al., 2002,Ibe et al., 2002). Cells were then placed in this incubator for experimentation and were continuously aerated with the hypoxia gas mixture throughout the duration of the study.
Study of PAF Receptor Binding
General Protocol. Receptor binding assays were done during hypoxia and normoxia as we reported previously (Ibe et al., 2005). Briefly, cells were washed with calcium and magnesium-free PBS before use according to the specific study protocol. After a 30-min incubation in normoxia or hypoxia, unbound [3H]PAF was washed off with ice-cold PBS and then incubated on ice for 30 to 45 min in a saline-EDTA mixture containing 154 mM saline and 5 mM EDTA (Korth et al., 1995; Ibe et al., 2005). Receptor bound [3H]PAF was extracted on Whatman GF/C membrane filters using an in-line vacuum system as we reported previously (Ibe et al., 2005). Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Coulter, Inc., Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were preincubated with the agent before the addition of [3H]PAF and then incubated further according to the specific experimental protocol.
Specific Protocols. Effect of cGMP/cAMP on PAFr binding. Cells were preincubated for 30 min, in hypoxia or normoxia, with buffer for controls or 10 µM concentrations of each of the cell-permeable cyclic nucleotide analogs, 8-Br-cGMP or 8-Br-cAMP. Then 1 nM [3H]PAF was added, and the incubation was continued for 30 min more. In preliminary studies, we found no significant difference between inhibition of PAF binding produced by 10 or 100 µM concentrations of the cell-permeable cAMP and cGMP analogs. To study the effects of cGMP and cAMP on PAF receptor protein expression, cells were incubated for 3 h in normoxia only with 10 µM concentrations each of 8-Br-cGMP and 8-Br-cAMP or with 0.1% BSA for a baseline, and then protein was prepared for Western blotting of PAF receptor protein.
Effect of PKG and PKA inhibitors on PAFr binding. To study the effect of endogenous cyclic nucleotides and their receptor interaction on PAF binding, cells were preincubated in hypoxia or normoxia for 30 min with 10 µM concentrations each of the cGMP-dependent PKG antagonist Rp-8-Br-PET-cGMPS and the cAMP-dependent PKA antagonist Rp-cAMPS, and then 1 nM [3H]PAF was added and the incubation was continued for 30 min more. PAF bound to receptor was extracted and quantified.
Study of cGMP and cAMP Release by the Cells
Cells were seeded at 5 x 105 cells in 100-mm Petri dishes and allowed to attain confluence. The confluent cells were washed twice with protein-free PBS and preincubated for 15 min in normoxia or hypoxia at 37°C in Krebs bicarbonate buffer, pH 7.4, containing 119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 15 mM NaHCO3, 1.2 mM NaH2PO4, and 5.5 mM glucose and 0.1 mM 3-isobutyl-1-methyl-xanthine (IBMX) to inhibit phosphodiesterase-catalyzed breakdown of cAMP and cGMP produced during experimentation. After preincubation, cells were treated with 10 nM PAF and 0.1 mM concentrations each of forskolin and sodium nitroprusside (SNP) and incubated for 15 min more in normoxia and hypoxia. After incubation, the media were acidified with 0.1 ml of 10% trichloroacetic acid and aspirated into borosilicate glass tubes. The cells were treated with 0.5 ml of ice-cold ethanol, and the ethanolic cell suspension was combined with its incubation media. Ethanol content was evaporated with nitrogen in ambient temperature. The aqueous fraction was spun for 10 min at 2000 rpm to pellet cell debris. The resulting aqueous supernatant containing the cAMP and cGMP produced were extracted with diethyl ether saturated with water. The water fraction containing the cyclic nucleotides was lyophilized and then redissolved in 0.05 M sodium acetate. The amounts of cAMP and cGMP produced were determined by radioimmunoassay with a commercial kit (Biomedical Technologies, Cambridge, MA) following the instructions provided by the vendor. Amounts of cAMP and cGMP measured are presented as picomoles per milliliter.
Release of Inositol Phosphates. Labeling of cells with [3H]myoinositol. Cells were washed with protein-free, inositol-free medium and then fed with a culture medium containing 10% fetal bovine serum and 5 µCi/ml [3H]myo-inositol suspended in inositol-free media and incubated for 16 to 24 h in 5% CO2 in air as we reported previously (Ibe et al., 2005).
Stimulation of [3H]inositol phosphate release by cells. Each test stimulus was prepared in freshly made 10 mM LiCl buffer. Labeled cells were stimulated with PAF or other agents and incubated in hypoxia or normoxia for 20 min at 37°C to induce inositol phosphate production. Reactions were quenched by addition of 10 mM formic acid and incubation in an ice bath for 30 min, followed with neutralization with 15 mM ammonium hydroxide. The cell suspension was loaded on pre-equilibrated AG 1-X8 columns and washed with 2 ml of distilled water. The tritium-labeled inositol phosphate accumulated was eluted from the column with a mixture of 1.0 M ammonium formate and 0.1 M formic acid. [3H]Inositol phosphate radioactivity was quantified by scintillation spectrometry.
Specific Protocols. Effect of cGMP and cAMP on PAF-stimulated IP3 release. Cells prelabeled with [3H]inositol were preincubated for 20 min with buffer alone for control or 10 µM 8-Br-cGMP or 8-Br-cAMP after which 1 nM nonradiolabeled PAF was added, and the incubation was continued for 20 min more. Effect of PKG and PKA inhibitors on PAF-stimulated IP3 release. To study the effect of endogenous cyclic nucleotides on PAF-stimulated inositol phosphate release, cells were preincubated with buffer alone or with 10 µM concentrations each of cGMP and cAMP receptor antagonists Rp-8-pCPT-cGMPS and Rp-cAMPS. Then nonradiolabeled PAF was added, and the incubation was continued for 20 min more. The amount of inositol phosphate released was extracted and quantified.
Western Blotting. Preparation of proteins for Western blot analysis. Proteins were prepared from stimulated and unstimulated cells that were studied in normoxia or hypoxia as described above. Briefly, after incubation in hypoxia or normoxia, cells were washed with PBS and lysed with a modified 40 mM HEPES hypotonic lysis buffer, pH 7.4, containing the following: 1 mM EGTA, 4 mM EDTA, 2 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM PMSF, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µM 4-(2-aminoethyl) benzene sulfonyl fluoride, 200 mM sodium fluoride, 20 mM sodium pyrophosphate, 0.2 mM sodium vanadate, and 0.1 mg/ml trypsin inhibitor. Proteins were recovered from lysed cells by centrifugation at 1500g for 15 min in a refrigerated centrifuge, stored in 0.2-ml aliquots at –80°C, and used for Western blotting.
SDS-PAGE Electrophoresis. Proteins were subjected to Coomassie Blue quantitation before use in Western blotting with some modification of published methods (Ali et al., 1994; Ibe et al., 2005). The signals were developed for 1 min using an Amersham ECL Western Blot detection kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and then exposed to X-ray film. A band corresponding to PAFr protein was scanned with an EagleEye densitometer (Stratagene, La Jolla, CA) to quantify blot density.
PKG and PKA Activity in Extracts of Pulmonary Vascular Smooth Muscle Cells
Incubations with or without PAF. We are interested in understanding the postnatal effects of PAF on the pulmonary circulation. Therefore, we studied the effect of PAF on activities of PKG and PKA during normoxia only. Subconfluent cells were washed with PBS at 37°C and then incubated at 37°C with 0.1% BSA in Dulbecco's modified Eagle's medium for 210 min. Then, cells were stimulated with 1.0 or 10.0 nM PAF and incubated for 30 min more in normoxia. After incubation, media were removed, and cells were washed twice with ice-cold phosphate-buffered saline; then cells were detached and centrifuged at 400g for 5 min. The cell pellet was homogenized on ice in 50 mM Tris buffer, pH 7.4, containing 10 mM EDTA, 2 mM dithiothreitol, 1 mM IBMX, 0.1 mM nitro-L-arginine, 0.01 mM indomethacin, and 0.5 mM PMSF. After homogenization, the homogenate was sonicated for 30 s and recentrifuged at 14,000g for 10 min at 4°C. The protein concentration of the extracts was determined by the Bradford method using BSA (fraction V) as the standard. The supernatant was assayed for PKG and PKA activities as described previously (Dhanakoti et al., 2000) after assay standardization.
Assay of PKG and PKA Activities. Our preliminary data showed that the activity of PKG (picomoles per minute per milligram of protein) in the absence or presence of 5 µM cGMP was similar (34.1 ± 4.1 and 108.4 ± 7.3 versus 32.7 ± 1.8 and 100.9 ± 5.1) whether in fresh cell extract or in frozen extracts stored at –80°C. Also, in preliminary experiments, we found that PKG activity was linear up to 15 µg of protein extract and up to 15 min of incubation, and the activity was saturated with 5 µM exogenous cGMP. Similar results were obtained for PKA, except that the activity was saturated with 2 µM cAMP. Inclusion of a variety of protease inhibitors (leupeptin, pepstatin, antipain, and aprotinin) and phosphatase inhibitors (calyculin A, cypermethrin, and sodium vanadate) did not significantly enhance the activities of PKG (123 ± 10.3 versus 129 ± 8.4 pmol/mg/mg protein with 5 µM cGMP) or PKA (1063 ± 35 versus 1143 ± 86 pmol/mg/mg protein with 2 µM cAMP) and thus were excluded in our subsequent assays. PKG and PKA assays were done essentially as reported previously (Dhanakoti et al., 2000).
Data Analysis
All numerical data are means ± S.E.M. In all instances in which radioisotope was used, background radioactivity was subtracted before radioactivity was quantified. Data were analyzed with a two-tailed t test followed by analysis of variance (GraphPad Prism; GraphPad, San Diego, CA). Results were considered significant at p < 0.05.
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Because PAF stimulates release of inositol phosphates downstream from the PAF receptor, we investigated the possibility that cGMP and cAMP will attenuate PAF receptor-mediated inositol phosphate release via PKG and PKA. Figure 3 shows the effects of PAF, 8-Br-cGMP, and the cGMP/PKG antagonist, Rp-8-pCPT-cGMPS, on PAF-stimulated IP3 production. Under baseline conditions in hypoxia, IP3 released by cells (means ± S.E.M.) was 1150 ± 33 dpm/106 cells. During normoxia, baseline release of IP3 decreased by 22% (896 ± 38 dpm/106 cells). Thus, IP3 release by the cells was significantly reduced by normoxic conditions. Treatment of cells with 5 nM PAF increased IP3 release 7-fold during hypoxia to 8565 ± 314 dpm/106 cells and 6-fold during normoxia to 5418 ± 118 dpm/106 cells. PAF-stimulated inositol phosphate release was attenuated by normoxia.
Pretreatment of the cells with 8-Br-cGMP decreased PAF-stimulated IP3 release during hypoxia by 65% and during normoxia by 37%, compared with PAF treatment alone under the two conditions. When the cells were pretreated with Rp-8-pCPT-cGMPS to inhibit activity of endogenous cGMP, PAF-stimulated IP3 release increased 2-fold during hypoxia (19,172 ± 159 dpm/106 cells) and normoxia (10,392 ± 216 dpm/106 cells), and PAF-stimulated IP3 release was still down-regulated by conditions of normoxia. Thus, whereas the cell-permeable cGMP analog attenuated PAF-stimulated IP3 release, inhibition of endogenous cGMP activity with cGMP/PKG antagonist augmented PAF-stimulated IP3 release. The increase in IP3 release after inhibition of PKG activity suggests that the PKG effect on PAF-stimulated IP3 release occurred downstream from the PAF-PAF receptor interaction.
Figure 4 shows the effect of PAF, 8-Br-cAMP, and the cAMP/PKA antagonist, Rp-cAMPS, on PAF-stimulated IP3 release. Under baseline conditions in hypoxia, IP3 release (means ± S.E.M.) was 1150 ± 33 dpm/106 cells. During normoxia, baseline release of IP3 decreased by 22% (896 ± 38 dpm/106 cells). Treatment of cells with 5 nM PAF increased IP3 release 7-fold during hypoxia to 8565 ± 314 dpm/106 cells and 6-fold during normoxia to 5418 ± 118 dpm/106 cells. As with baseline conditions, normoxia down-regulated PAF-stimulated IP3 release by the cells. Pretreatment of cells with 8-Br-cAMP decreased PAF-stimulated IP3 release by 45%, both during hypoxia and normoxia: 4748 ± 298 and 3043 ± 112 dpm/106 cells, in hypoxia and normoxia, respectively, compared with PAF treatment alone under the two conditions. When the cells were pretreated with Rp-cAMPS to inhibit activity of endogenous cAMP at its receptor protein, PAF-stimulated IP3 release increased by 50% during hypoxia (13,104 ± 1480 dpm/106 cells) and by 60% during normoxia (8669 ± 169 dpm/106 cells), and PAF-stimulated IP3 release was still down-regulated by conditions of normoxia. Thus, whereas the cell-permeable cAMP analog attenuated PAF-stimulated IP3 release, inhibition of endogenous cAMP activity with cAMP/PKA antagonist augmented PAF-stimulated IP3 release. As with the PKG effect, the increase in IP3 release after inhibition of PKA activity suggests that the PKA effect on PAF-stimulated IP3 release occurred downstream from PAF-PAF receptor interaction.
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We investigated cGMP and cAMP production to show that the cells can synthesize these cyclic nucleotides in situ, thereby facilitating postnatal pulmonary vascular relaxation. Figure 5 shows cGMP (Fig. 5A) and cAMP (Fig. 5B) levels measured in the smooth muscle cells under unstimulated (baseline) and stimulated conditions. Baseline production of cGMP in hypoxia was 1.13 ± 0.06 pmol/ml and was not different from production in normoxia, which was 1.13 ± 0.04 pmol/ml. Cyclic GMP production by cells on treatment with PAF was 1.23 ± 0.05 pmol/ml, which decreased significantly to 0.90 ± 0.11 pmol/ml in normoxic conditions. Production of cGMP after forskolin treatment was not different from baseline values in hypoxia or normoxia. However, SNP treatment increased cGMP levels to 3.44 ± 0.05 pmol/ml in hypoxia, which increased to 4.75 ± 0.12 pmol/ml in normoxic conditions. Thus, PAF, the pulmonary vasoconstrictor, decreased cGMP synthesis during normoxia, whereas SNP, the nitric oxide-generating agent, increased synthesis of cGMP.
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PAF Attenuates PKG and PKA Protein Expression. We studied the effect of PAF on PKG and PKA protein expression to demonstrate that one mechanism by which PAF sustains high pulmonary venous vasomotor tone in utero is by down-regulation of expression of PKG and PKA proteins. Figure 6 shows representative Western blots of expression of PAFr, PKA and PKG proteins, and actin internal standard by the cells at baseline and after treatment with 10 nM PAF for a total incubation time of 120 min in normoxia or hypoxia (Fig. 6A) and the densitometry of the respective protein expression (Fig. 6, B, PAFr; C, PKA; and D, PKG. Hypoxia increased PAF receptor protein expression by 40%. Treatment with 10 nM PAF increased PAF receptor protein expression in both normoxia and hypoxia, but more so in hypoxia than normoxia.
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Cyclic Nucleotides cGMP and cAMP Attenuate PAF Receptor Protein Expression during Normoxia. We studied the effects of cGMP and cAMP on PAF receptor expression to demonstrate that one mechanism by which cGMP and cAMP facilitate postnatal pulmonary adaptation is via down-regulation of PAF receptor protein. Figure 7 shows a representative Western blot (inset) and densitometry of the effects of cGMP and cAMP on PAF receptor protein expression in normoxia. Treatment of cells with 10 µM 8-Br-cGMP attenuated PAF receptor protein expression by 80% compared with baseline conditions. Treatment of cells with 10 µM 8-Br-cAMP also decreased PAF receptor protein expression by 41%. Thus, both cGMP and cAMP attenuated PAF receptor protein expression during normoxia.
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; Argiolas et al., 1995; Ibe et al., 1998) maintain high vasomotor tone by increasing intracellular levels of IP3 and calcium. At birth, release of vasodilators such as cGMP and cAMP induce smooth muscle relaxation by acting through their respective protein kinases, PKG and PKA (Dhanakoti et al., 2000; Abdel-Latif, 2001). In ovine fetal SMC-PV, hypoxia up-regulates PAFr binding and PAFr-mediated intracellular IP3 and calcium release (Ibe et al., 2005); therefore, the fetal hypoxic environment facilitates PAFr binding, PAFr-mediated signaling, and maintenance of high pulmonary vasomotor tone in utero. The possibility that PAF may actively down-regulate vasodilator pathways in the pulmonary circulation of the fetal hypoxic environment has not been previously explored. Likewise, the decreased PAFr-mediated activity in the higher oxygen environment of the postnatal lung may involve down-regulation of PAFr-mediated cell signaling by other endogenous mediators such as cGMP and cAMP (Dhanakoti et al., 2000; Gao et al., 2004). In the present report, we investigated the interactions between cGMP/cAMP and PAF signaling pathways in fetal pulmonary vascular smooth muscle. We found that both cGMP and cAMP decrease PAFr binding in normoxia through their respective kinases, cGMP/PKG and cAMP/PKA. Cell-permeable analogs of cGMP and cAMP significantly attenuated PAFr protein expression, suggesting a translational regulation of PAFr protein expression. PAFr protein expression can be down-regulated by the protein synthesis inhibitor cycloheximide (Ibe et al., 2005). Thus, cGMP and cAMP, acting through their respective receptors PKG and PKA, do down-regulate PAF receptor protein expression by translational mechanisms. During normoxia, a physiological concentration of PAF in culture decreased PKG and PKA protein expression and activity. These results suggest that in the fetal hypoxic environment, PAF may be actively down-regulating cGMP- and cAMP-dependent signaling pathways and that postnatally cGMP and cAMP actively inhibit PAFr binding, PAFr expression, and PAFr-mediated signaling. This cross-talk between the two pathways will effectively maintain a high pulmonary vasomotor tone in utero and facilitate vasorelaxation at birth via down-regulation of PAFr-mediated signaling by the cyclic nucleotides. Scheme 1 explains our hypothesis for the interaction of PAF and the cyclic nucleotides in perinatal pulmonary adaptation. During hypoxia in utero, PAFr binding activates intracellular signaling pathways that decrease PKG and PKA activities, resulting in pulmonary vascular constriction. During normoxia postnatally, the cGMP and cAMP produced bind to their receptors PKG and PKA to inhibit PAFr expression and PAFr-mediated intracellular signaling.
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Cyclic Nucleotides and PAFr-Mediated Effects
Role of cGMP. Endothelium-derived nitric oxide produced under basal conditions or by a stimulus readily diffuses into the contiguous smooth muscle to activate soluble guanylyl cyclase, resulting in increased cGMP synthesis and smooth muscle relaxation (Ignarro, 1990; Nakahara et al., 2002; Gao et al., 2004). Nitric oxide (NO), a potent vasodilator in the pulmonary circulation, is important in the transition of the pulmonary circulation from fetal to postnatal life. Activation of soluble guanylyl cyclase activity by NO increases cGMP synthesis and relaxation of the pulmonary vasculature (Block et al., 1997). In this study, oxygen in physiological levels down-regulated PAFr binding. Also, 8-Br-cGMP, decreased PAFr binding in hypoxia and normoxia, suggesting that cGMP produced in vivo will counteract the vasoconstricting properties of PAF. Interestingly, when activity of endogenous cGMP was inhibited with an inhibitor of cGMP-dependent PKG, the endogenous cGMP receptor, the ability of cGMP to inhibit PAF binding was obliterated, showing that cGMP acts via its receptor to inhibit PAFr binding. The exact mechanism of this inhibition is not yet clear; however, our data show that the adverse effects of PAF can be ameliorated postnatally by the down-regulation of PAFr protein expression.
PAF-stimulated inositol phosphate release is augmented in hypoxia (Ibe et al., 2005), and the profile of release is similar to the effect of phorbol myristate acetate, a specific activator of PKC in the pulmonary circulation (Orton et al., 1990). This indicates that PKC/phospholipase B activation is one mechanism of PAF-stimulated inositol phosphate release in SMC-PV. Activation of PKC increases its affinity for calcium under physiological conditions (Standaert et al., 1997). Also activation of PKC inhibits cGMP production in vascular smooth muscle cells by inhibiting NO release (Nambi et al., 1987). Pretreatment of SMC-PV with the PKG antagonist, Rp-8-pCPT-cGMPS, increased PAF-stimulated inositol phosphate release. We can infer that in vivo, cGMP acting via its receptor, PKG, will inhibit PAF-stimulated inositol phosphate synthesis and decrease calcium release coupled with vasodilation. On the other hand, inhibition of cGMP release with a concomitant increase in the calcium level will result in increased vasoconstriction. The inhibitory effect of cGMP on PAF-stimulated inositol phosphate release was more pronounced under hypoxic conditions. In line with our findings, cGMP has been shown to inhibit endothelin-stimulated inositol phosphate release in pulmonary arteries of fetal lambs studied in an organ bath (Millard et al., 1998). Endothelin and PAF are potent endogenous vasoconstrictors in the pulmonary circulation. PAF inhibited cGMP production in normoxia by 26%, whereas SNP, an NO donor, augmented cGMP production, especially in normoxia. The physiological relevance of PAF inhibition of cGMP production is presently unclear, but we can surmise that increased levels of endogenous PAF postnatally can inhibit cGMP release, leading to elevated intracellular calcium levels and increased vasoconstriction.
Role of cAMP. cAMP production is linked to 
-adrenergic receptor-mediated activation of adenylyl cyclase (Gilman, 1995; Milligan and Kostenis, 2006). Inhibition of cGMP- and cAMP-dependent phosphodiesterases can also result in high cellular levels of cGMP and cAMP (Nakahara et al., 2002; Hicks et al., 2005). Although 8-Br-cAMP did not significantly alter PAF binding in hypoxia, inhibition of cAMP/PKA with Rp-cAMPS, obliterated the endogenous cAMP effect and significantly increased PAF binding in hypoxia, suggesting that cGMP and cAMP interact with PAFr after activation of their own receptors.
Similar to our observations on cGMP/PKG activity, inhibition of PKA with Rp-cAMPS led to increased inositol phosphate release, suggesting that the influence of PKG and PKA on PAF-mediated responses may be occurring downstream from the PAFr. This means that other endogenous mediators of inositol phosphate production such as diacyl glycerol may have been activated after inhibition of PKG and PKA activities.
Forskolin, a cAMP-elevating agent, increased cAMP production, suggesting that in vivo increased production of cAMP may activate PKA, resulting in inhibition of PAFr-mediated effects. This inhibition may constitute one mechanism whereby the vasodilator properties of cAMP are maintained postnatally.
Previous reports have shown that cGMP and cAMP signaling pathways may cooperate under certain conditions to regulate tissue physiological end points, for instance, decreases of cAMP and cGMP release by hypoxia (Taylor et al., 1998) and stimulation of NO release by cAMP in coronary microvessels of conscious pigs (Kudej et al., 2000) and by rat aortic endothelium (Ray and Marshall, 2006). The mechanisms of these stimulus-induced cAMP/cGMP interactions are not well understood, but may involve phosphorylation of endothelial nitric-oxide synthase by PKA (Ray and Marshall, 2006). Although cAMP and cGMP were not measured in the reports cited above, other studies (Gao et al., 1995) have shown that administration of NO or SNP does increase cGMP release. In this study, we measured cGMP release in forskolin-stimulated cells and cAMP in SNP-stimulated cells. Neither of the two stimuli produced a crossover effect on cyclic nucleotide release. Therefore, it is possible that cAMP activation of NO production does not occur in SMC-PV in culture. However, more studies are needed to address this paradigm.
PAF and Regulation of PKG and PKA Activities in Perinatal Pulmonary Adaptation
In normoxia, activities of PKG and PKA are high (Dhanakoti et al., 2000; Gao et al., 2004). The physiological implications of these findings are that up-regulation of PKG and PKA activities postnatally is one mechanism by which fetal high pulmonary vasomotor tone is down-regulated to facilitate postnatal pulmonary adaptation. Thus, favorable perinatal pulmonary vascular adaptation can be achieved by both down-regulation of mediators of pulmonary vascular constriction, such as PAF, and by up-regulation of mediators of pulmonary vasodilation, such as cGMP/PKG and cAMP/PKA. Endothelin (Fratz et al., 2004) and PKC (Johnson and Barman, 2004) are two other mediators that have been reported to evoke vasoconstriction in the perinatal pulmonary circulation. We report here that during normoxia PAF significantly down-regulates expression and activities of both cGMP/PKG and cAMP/PKA. Exposure of SMC-PV to PAF resulted in significant down-regulation of expression of PKG and PKA proteins with attendant up-regulation of PAFr protein. These data strongly indicate that congenial perinatal pulmonary adaptation entails a combination of down-regulation of PAFr-mediated effects via cyclic nucleotide-mediated pathways and up-regulation of cyclic nucleotide production as we have shown in this article. This is accomplished postnatally by down-regulation of translation of PAFr protein by cyclic nucleotides as well as by up-regulation of cGMP- and cAMP-mediated pathways as has been previously reported (Dhanakoti et al., 2000; Evgenov et al., 2004).
PPHN is a pathological condition with different etiologies. Neonates with PPHN have high PAF levels (Caplan et al., 1990), showing that persistence of high PAF levels postnatally leads to abnormal perinatal pulmonary adaptation. We speculate that inhibition of PKG and PKA activities by high PAF levels and the inability of cyclic nucleotides to down-regulate PAFr-mediated effects postnatally will also contribute to development of PPHN.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAF, platelet-activating factor; PAFr, PAF receptor; IP3, inositol 1,4,5-triphosphate; PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; PPHN, persistent pulmonary hypertension of the newborn; SMC-PV, intrapulmonary venous smooth muscle cells; 8-Br-cGMP, 8-bromoguanosine-3',5'-cyclic monophosphate; Rp-8-pCPT-cGMPS, Rp-8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate sodium; 8-Br-cAMP, 8-bromoadenosine-3',5'-cyclic monophosphate; Rp-cAMPS, Rp-adenosine-3',5'-cyclic monophosphorothioate; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IBMX, 3-isobutyl-1-methyl-xanthine; SNP, sodium nitroprusside; PAGE, polyacrylamide gel electrophoresis, NO, nitric oxide.
Address correspondence to: Dr. Basil O. Ibe, Division of Neonatology, Los Angeles Biomedical Research Institute, 1124 W. Carson St., RB-1, Torrance, CA 90502. E-mail: ibe{at}labiomed.org
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