Abstract
Platelet-activating factor (PAF) plays an important physiological role of maintaining a high vasomotor tone in fetal pulmonary circulation. At birth, endogenous vasodilators such as nitric oxide and prostacyclin are released and facilitate pulmonary vasodilation via cAMP-dependent protein kinase (cAMP/PKA) and cGMP-dependent protein kinase (cGMP/PKG) pathways. Interaction between the cyclic nucleotides and PAF receptor (PAFR)-mediated responses in pulmonary arterial smooth muscle is not well understood. To further understand the interactions of PAF-PAFR pathway and the cyclic nucleotides in ovine fetal pulmonary arterial smooth muscle cells (FPASMC), effects of cAMP and cGMP on PAFR-mediated responses in pulmonary arterial smooth muscle cells (PASMC) were studied. Ovine FPASMC were incubated with 10μM cAMP or cGMP in normoxia (5% CO2 in air, pO2~100 Torr) or hypoxia (2% O2, 5% CO2, pO2~30-40 Torr). Proteins were prepared and subjected to Western blotting. Effect of cell permeable cAMP and cGMP on PAFR binding was also studied and effect of cAMP on cell proliferation was also studied by RNAi to PKA-Cα. cAMP and cGMP significantly decreased PAFR binding and protein expression in normoxia and hypoxia, more so in hypoxia, when PAFR expression was usually high. PKA-Cα siRNA demonstrated that inhibition of PAFR-mediated responses by the cyclic nucleotides occurred through PKA. These data suggest that the normally high levels of cyclic nucleotides in the normoxic newborn pulmonary circulation assist in the downregulation of postnatal PAFR-mediated responses and that under hypoxic conditions, increasing the levels of cyclic nucleotides will abrogate PAF-mediated vasoconstriction thereby ameliorating PAF-induced persistent pulmonary hypertension of the newborn.
Keywords
- Pulmonary artery
- PAFR binding
- cyclic nucleotides
- siRNA
1. Introduction
Platelet-activating factor (PAF) is an endogenous phospholipid which evokes a wide range of biological activities, such as vasoconstriction and systemic hypotension [1], mainly under pathophysiological conditions. The discovery of PAF, its cellular origin, and biological actions were first reported by Benveniste and Associates [2, 3]. Following these reports, investigations of the physiological effects of PAF involved its roles in fetal lung maturation and lung function [4-9], and its role in reproduction where it is involved in implantation of embryos, among other effects [10, 11]. PAF produces a myriad pathological effects in vivo, including platelet aggregation [12-14], mediation of immune response and bronchoconstriction [15, 16], and smooth muscle contraction [17-21], which hinges on its role as an inflammatory mediator and vasoconstrictor [17]. In the fetus, PAF plays an important physiological role in maintaining a high level of vasomotor tone in the pulmonary circulation [22]. Therefore, the high PAF receptor (PAFR) binding in fetal lamb lungs supports the existence of a high level of pulmonary vasomotor tone in utero [23]. On the other hand, in lungs of the newborn lamb, PAFR binding and receptor mRNA expression are low, suggesting a down regulation of PAFR-mediated effects in vivo [22, 23].
PAF acts by binding to its Gq protein isoform of G protein-coupled receptors, which is a seven transmembrane receptor [24]. Activation of G protein-coupled receptors by an agonist results in activation of signal transduction pathways [25], which may involve recruitment of intracellular second messengers such as cAMP, cGMP, inositol 1,4,5-triphosphate (IP3), and calcium [26, 27]. cAMP and cGMP act via their endogenous receptors, cAMP-dependent protein kinase (cAMP/PKA) and cGMP-dependent protein kinase (cGMP/PKG), respectively, to elicit relaxation of smooth muscle, and cAMP and cGMP mediate relaxation of pulmonary vessels, but cGMP has been shown to be more effective than cAMP in producing relaxation of perinatal ovine pulmonary vessels [28, 29]. Acute hypoxia upregulates PAFR-mediated intracellular signaling in fetal ovine pulmonary vascular smooth muscle [30]. Chronic hypoxia in the perinatal period may result in abnormal upregulation 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) [31].
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 cAMP and cGMP in pulmonary vascular smooth muscle, PAFR protein expression and PAFR-mediated cell signaling may be inhibited via cross-talk between the cyclic nucleotides and PAFR-mediated responses. This hypothesis was investigated in ovine fetal pulmonary vascular smooth muscle cells using cAMP and cGMP on PAFR binding and PAFR-mediated cell signaling in both normoxia and hypoxia. After birth, during normoxia, pulmonary levels of cAMP rise significantly, perhaps contributing to the decrease in PAFR binding, PAFR protein expression and the ensuing fall in pulmonary vascular resistance. We employed siRNA to the catalytic domain of PKA to define the role of PKA in decreased pulmonary PAFR-mediated responses at birth.
2. Materials and methods
2.1. Materials
The study was approved by the Institutional Animal Care and Use Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Pregnant ewes (146-148 d gestation, term being 150 d) were purchased from Nebekar Farms (Santa Monica, CA). Authentic standards of PAF (C16-PAF) as well as 8-Br-cAMP, Rp-cAMPS, 8-Br-cGMP, Rp-8-pCPT-cGMPS were purchased from Biomol, Plymouth Meeting, PA. Radiolabeled PAF standards and substrates: hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, 1-O-[acetyl-3H-(N)]-, (3H-acetyl-C16-PAF), 21.5 Ci/mmol (370 GBq/mmol), and 3H-thymidine were purchased from Perkin Elmer Life Sciences (Boston, MA). siRNA to PKA-Cα and its control were purchased from Cell Signaling Technologies (Carlsbad, CA). Phenylmethysulfonyl fluoride, leupeptin, pepstatin, as well as bovine serum albumin, were purchased from Sigma Chemical Company (St. Louis, MO). Antibody to PKA and PKG were purchased from Cell Signaling, while PAFR antibody was purchased from Cayman Chemical Company (Ann Arbor, MI). Studies were done with freshly made reagents. Ecolite(+) liquid scintillation cocktail was purchased from MP Biochemicals (Irvine, CA).
2.2. Methods
Arterial intrapulmonary vessels were isolated from freshly killed term fetal lambs and then smooth muscle cells were harvested from the excised arteries and veins under sterile conditions as previously reported [20, 32]. Cells were used at the 3rd to 10th passage. Cell phenotype did not change from 1st to 10th passage as determined by the expression of α-smooth muscle actin and myosin light-chain kinase proteins.
2.3. Study conditions
Studies were done with adherent cells in normoxia and in hypoxia.
2.4. Study of PAFR binding
2.4.1. General protocol
Receptor-binding assays were performed during hypoxia and normoxia as we previously reported [30]. Briefly, after incubation in normoxia or hypoxia, unbound 3H-PAF was washed off with ice-cold phosphate buffer saline, and then incubated on ice for 30 to 45 min in saline/EDTA mixture containing 154 mM saline and 5 mM EDTA. Receptor bound 3H-PAF was extracted on Whatman GF/C membrane filters using inline vacuum system. Then culture flasks or dishes were washed with calcium-free 0.25% bovine serum albumin-containing Tyrodes buffer, pH 6.4. Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were pre-incubated with the agent before the addition of 3H-PAF, and then incubated further according to the specific experimental protocol.
2.4.2. Specific protocols
2.5. Western blotting
2.6. Data analysis
All numerical data are mean ± SEM. In all instances where radioisotope was used, background radioactivity was subtracted before quantifying radioactivity. Data were analyzed with two-tailed
3. Results
Figure 1 shows the effect of 8-Br-cAMP and 8-Br-cGMP on PAF binding to its receptors in pulmonary arterial smooth muscle cells (PASMC) (fmol/106 cells). For both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM,
Effect of cyclic nucleotides cAMP and cGMP on PAF stimulation of fetal pulmonary arterial smooth muscle cells (FPASMC) growth (3H-thymidine DPM × 103) is shown in Figure 2. For both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM,
Figure 3 shows representative Western blots of effect of cAMP and cGMP on PAFR protein expression. For both 8-Br-cAMP and 8-Br-cGMP, data are mean ± SEM,
We then investigated the effect of PKA siRNA PAFR binding and PAF stimulation of FPASMC proliferation in normoxia only. For both binding and proliferation, data are mean ± SEM,
4. Discussion
In fetal pulmonary circulation, vasomotor tone is maintained high by multiple mechanisms such as elevated calcium, PAF, and thromboxane A2 [22, 34, 35]. At birth, endogenous vasodilators induce smooth muscle relaxation via increased intracellular concentrations of the cyclic nucleotides, cAMP and cGMP, acting through their respective receptors PKA and PKG [27]. We have published previously that in ovine fetal pulmonary vascular smooth muscle cells, hypoxia upregulates PAFR binding and PAFR-mediated intracellular IP3 and calcium release [30], suggesting that the hypoxic environment of the fetus facilitates PAFR binding and PAFR-mediated signaling and the maintenance of a high pulmonary vasomotor tone in utero. The possibility that vasoconstrictors such as PAF may actively downregulate vasodilator pathways in the hypoxic environment of fetal pulmonary circulation is being actively explored. Similarly, the decreased PAFR-mediated activity in the higher oxygen environment of the postnatal lung may involve the downregulation of PAFR-mediated cell signaling by other endogenous mediators such as cAMP and cGMP [36, 37]. The present report investigates the interaction between cyclic nucleotides, cAMP and cGMP, and PAF signaling pathway in FPASMC. We have found that in ovine fetal pulmonary vascular smooth muscle cells, both cAMP and cGMP decrease PAFR binding in normoxia through the actions of their respective kinases, PKA and PKG. Addition of PAF in physiologic concentrations to pulmonary venous smooth muscle cells decreased PKA and PKG protein expression and kinase activities during normoxia and hypoxia, suggesting that in the hypoxic environment of the fetal lungs, PAF may be actively downregulating cAMP- and cGMP-dependent signaling pathways and that postnatally, in the normoxic environment, cAMP and cGMP actively inhibit PAF binding to its receptors 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.
4.1. Cyclic nucleotides inhibit PAFR binding and PAFR-mediated cell proliferation
Effect of cAMP downstream from PAFR in the nucleus of PASMC is not clear. 8-Br-cAMP enhanced cell growth in normoxia, with no change in cell growth in hypoxia compared to PAF effect in hypoxia. However, when cells were pulsed with 8-Br-cAMP and exposed to PAF, cell proliferation was significantly decreased in normoxia and hypoxia, suggesting that cAMP effect occurs after activation of its receptor. This relationship is relevant physiologically because it suggests that postnatally, cAMP will stimulate growth of PASMC and under this condition, the presence of PAF will be detrimental cell growth and pulmonary vascular development. Thus, we can speculate that cAMP/PKA-mediated inhibition of PAF effects, in vivo, may constitute one mechanism whereby the postnatal vasodilator properties of cAMP are maintained. Our studies with PKA siRNA demonstrate that cAMP acts at its receptor, PKA, to inhibit postnatal adverse PAFR-mediated responses in the pulmonary circulation of the newborn lamb lung.
cGMP has been shown to inhibit endothelin-stimulated inositol phosphate release in pulmonary artery of fetal lambs studied in organ bath [41]. Inositol phosphate is released downstream from PAFR effect. Both endothelin and PAF are potent endogenous vasoconstrictors in the pulmonary circulation. This shows that increased levels of endogenous PAF under normoxic conditions can inhibit cGMP effect and as a corollary, increased levels of cGMP after birth can inhibit PAF effect in normoxia, leading to increased vasodilation. Our data show that cAMP and cGMP produce different effects on cell proliferation. During normoxia, cGMP inhibited PAF stimulation of cell proliferation, but the effect in hypoxia seemed to be stimulatory. This physiological significance of this effect is not clear, but may indicate a protective role of cGMP against unwarranted cell growth in the presence of PAF.
We can infer that in vivo, activation of PKA will result in inhibition of PAFR-mediated effects such as stimulation of inositol phosphate release, calcium mobilization, and vasoconstriction. As with cGMP effect, cAMP/PKA-mediated inhibition of PAF effects, in vivo, may constitute one mechanism whereby the postnatal vasodilator properties of cAMP are maintained.
4.2. PAF and regulation of PKG and PKA activity and role in perinatal pulmonary adaptation
Previous reports have shown that the activities of PKG and PKA are upregulated by normoxic condition [36, 37, 42]. The physiologic implications of these findings are that upregulation of PKG and PKA activities by normoxia, after birth, is one mechanism by which fetal high pulmonary vasomotor tone is downregulated to facilitate postnatal pulmonary adaptation. It can be deduced that favorable perinatal pulmonary vascular adaptation can be achieved by both downregulation of mediators of pulmonary vascular constriction, such as PAF, and upregulation of mediators of pulmonary vasodilation such as cGMP/PKG and cAMP/PKA. Endothelin [43] and protein kinase C (PKC) [44] are two other mediators that have been reported to evoke vasoconstriction in the perinatal pulmonary circulation. In this report, we show that during normoxia, PAF significantly downregulates the activities of both cGMP- and cAMP-dependent protein kinases. We also found that exposure of the smooth muscle cells to 8-Br-cAMP and 8-Br-cGMP for 30 hr in normoxia and hypoxia, resulted in significant downregulation of PAFR in line with attenuation of PAFR binding to the cells. These findings strongly indicate that congenial perinatal pulmonary adaptation entails a combination of downregulation of PAFR-mediated effects by cyclic nucleotide-mediated pathways as we have shown in this report, as well as by upregulation of cGMP- and cAMP-mediated pathways as has been previously reported [31, 36, 37, 42].
PPHN is a pathological condition with different etiologies. High PAF levels have been reported in neonates with PPHN [31, 45], suggesting that persistence of high PAF levels postnatally may lead to abnormal perinatal pulmonary adaptation. In addition, we speculate that inhibition of PKG and PKA activities by high levels of PAF and the inability of the cyclic nucleotides to downregulate PAFR-mediated effects postnatally will also contribute to the development of PPHN.
Acknowledgments
This work was supported in part by a Bridge Grant (513292) from Los Angeles Biomedical Research Institute, Torrance, CA.
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