Platelet-Activating Factor Modulates Activity of Cyclic Nucleotides in Fetal Ovine Pulmonary Vascular Smooth Muscle

ABSTRACT At birth, release of endogenous vasodilators such as nitric oxide and prostacyclin facilitate pulmonary vasodilation via the cyclic nucleotides, cGMP and cAMP. Interaction of cyclic nucleotides and platelet-activating factor (PAF)-mediated responses in pulmonary vascular smooth muscle is not known. We studied the effects of cGMP and cAMP on PAF-mediated responses in ovine fetal intrapulmonary venous smooth muscle cells. Studies were done in hypoxia or normoxia with buffer with 8-Br-cGMP (BGMP) and 8-Br-cAMP (BAMP), as well as cGMPdependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) inhibitors. All groups were treated with 1 nM PAF and incubated for 30 min for the binding assay or 20 min for measurement of inositol 1,4,5-phosphate (IP 3 ) production. BGMP and BAMP decreased PAF binding in normoxia by 63 and 14%, respectively. Incubations with the PKG inhibitor Rp-8-(4-chlorophenylthio)-guanosine-3Ј,5Ј-cyclic monophosphorothioate sodium and the PKA inhibitor Rp-adenosine-3Ј,5Ј-cyclic monophosphorothioate abrogated the inhibitory effects of BGMP and BAMP. PAF-stimulated IP 3 production was 8565 Ϯ 314 dpm/10 6 cells in hypoxia and 5418 Ϯ 118 dpm/10 6 cells in normoxia, a 40% decrease. BGMP attenuated PAFstimulated IP 3 production by 67 and 37% in hypoxia and normoxia, respectively; the value for BAMP was 44% under both conditions. Pretreatment with PKG or PKA inhibitor abrogated BGMP and BAMP inhibition of IP 3 release. PAF receptor (PAFr) protein expression decreased in normoxia, but pretreatment with 10 nM PAF up-regulated PAFr expression. Pretreatment with PAF decreased expression and activities of PKG or PKA proteins in normoxia and hypoxia. Our data demonstrate the existence of cGMP/cAMP-PAF cross-talk in pulmonary vascular smooth muscle cells, which may be one mechanism by which PAFr-mediated vasoconstriction is down-regulated at birth

Mechanism by which nuclear factor-kappa beta (NF-kB) regulates ovine fetal pulmonary vascular smooth muscle cell proliferation

Platelet activating factor (PAF) modulates ovine fetal pulmonary hemodynamic. PAF acts through its receptors (PAFR) in pulmonary vascular smooth muscle cells (PVSMC) to phosphorylate and induce nuclear translocation of NF-kB p65 leading to PVSMC proliferation. However, the interaction of NF-kB p65 and PAF in the nuclear domain to effect PVSMC cell growth is not clearly defined. We used siRNA-dependent translation initiation arrest to study a mechanism by which NF-kB p65 regulates PAF stimulation of PVSMC proliferation. Our hypotheses are: (a) PAF induces NF-kB p65 DNA binding and (b) NF-kB p65 siRNA attenuates PAF stimulation of PVSMC proliferation. For DNA binding, cells were fed 10 nM PAF with and without PAFR antagonists WEB 2170, CV 3988 or BN 52021 and incubated for 12 h. DNA binding was measured by specific ELISA. For NF-kB p65 siRNA effect, starved cells transfected with the siRNA were incubated for 24 h with and without 10 nM PAF. Cell proliferation was measured by DNA synthesis while expression of NF-kB p65 and PAFR protein was measured by Western blotting. In both studies, the effect of 10% FBS alone was used as the positive control. In general, PAF stimulated DNA binding which was inhibited by PAFR antagonists. siRNAs to NF-kB p65 and PAFR significantly attenuated cell proliferation compared to 10% FBS and PAF effect. Inclusion of PAF in siRNA-treated cells did not reverse inhibitory effect of NF-kB p65 siRNA on DNA synthesis. PAFR expression was inhibited in siRNA-treated cells. These data show that PAF-stimulation of PVSMC proliferation occurs via a PAFR-NF-kB p65 linked pathway