**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 aggre‐ gation [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 vaso‐ constrictor [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 intra‐ cellular 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 PAFRmediated 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 PAFRmediated responses at birth.
