**4. Discussion**

produced a 2-fold increase in cell proliferation compared to 10% FBS control and 37% increase compared to effect of 10nM PAF. The effect of the sham siRNA control was not different from

**Figure 3.** Effects of cAMP and cGMP on PAF receptor protein expression in normoxia and hypoxia. Data are means ±SEM, n= 3. Cells were incubated in for 24 hr in normoxia or hypoxia with buffer alone, or with 10µM of 8-Br-cAMP, figure 3a, or with 10µM 8-Br-cGMP, figure 3b. PAF receptor (PAFR) expression was measureb by Western blotting and normalized to expression of beta-actin standard. Rp-8-pCPT-cGMPS, then 5nM PAF was added as needed and incu‐ bated for 20 min more. Treatment of cell with 8-Br-cAMP or 8-Br-cGMP surpressed PAFR protein expression in nor‐

**Figure 4.** Effects of cAMP and and siRNA to PKA-cα on PAFR binding and PAF stimulation of cell proliferation. Data are means ± SEM, n= 5. Studies were done as described in methods section. PAFR binding (figure 4a) and cell prolifera‐ tion were determined. PAFR binding was attenuated by 8-Br-cAMP, but siRNA to PKA-cα increased PAFR binding. Effect of sham siRNA was not different from PAF alone. In figure 4b, 8-Br-cAMP decreased cell proliferation compared to effect of PAF alone. siRNA to PKA cα increased cell proliferation. Effect of sham siRNA was not different from 10% FBS or 8-br-cAMP. \*p <0.05, different from PAF alone of 10% FBS control; +p <0.05, different from PAF alone; #p <0.05,

moxia and hypoxia. \*p <0.05, different from control; +p <0.05, different from normoxia.

effect of 10% FBS control.

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different from all other condition.

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**

*Role of cAMP:* cAMP production is linked to β-adrenergic receptor-mediated activation of adenylyl cyclase [38, 39]. Inhibition of cAMP- and cGMP-dependent phosphodiesterases (PDEs) can also result in high cellular levels of cAMP and cGMP [38, 40], but the effect of cAMP and cGMP on PAFR-mediated signaling in PASMC has not been reported. Unlike in pulmo‐ nary venous smooth muscle cells, the cell permeable analog of cAMP, 8-Br-cAMP, decreased PAFR binding in normoxia and hypoxia. Inhibition of cAMP-dependent PKA, the endogenous receptor of cAMP, with Rp-cAMPS, reversed the inhibitory effect of endogenous cAMP and significantly increased PAFR binding. Also, when PKA was inhibited with Rp-cAMPS, PAF binding in hypoxia was increased beyond the effect of addition of exogenous PAF alone. This suggests that cAMP interacts with PAFR after activation of its own receptor, PKA.

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.

*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 an increase in cGMP synthesis and smooth muscle relaxation [37, 38]. Nitric oxide is a potent vasodilator in the pulmonary circulation and it is important in the transition of the pulmonary circulation from fetal to postnatal life. In this study, 8-Br-cGMP blunted PAFR binding to PASMC in physiologic oxygen level (normoxia) and in hypoxia. This suggests that cGMP produced in vivo will counteract the vasoconstricting properties of PAF. Interestingly, inhibition of endogenous cGMP activity with the inhibitor of cGMP-dependent PKG, the endogenous receptor of cGMP, resulted in abrogation of the ability of cGMP to inhibit PAF binding to its receptors, similar to the effect of PKA receptor antagonist. This shows that cGMP acts via its receptor to inhibit PAFR binding.

cGMP has been shown to inhibit endothelin-stimulated inositol phosphate release in pulmo‐ nary 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 down‐ regulation 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 inhibi‐ tion 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.
