Platelet Activating Factor Receptor Binding and Contractile Protein Expression by Ovine Fetal Pulmonary Vascular Smooth Muscle Cells: Role in Rho Kinase Mediation of PAF Receptor-Linked Physiological Responses

Abstract

We studied effects of hypoxia and some modulators of PAFR–mediated signaling in fetal ovine arterial and venous smooth muscle cells to aid in explaining PAF regulation of fetal pulmonary hemodynamics in utero. PAFR, a G protein coupled receptor, is characteristically measured in membrane proteins. Profile of binding to soluble and nuclear fraction proteins from SMC have not been studied. Rho kinase (ROCK) inhibitors decrease PAFR-linked smooth muscle growth, suggesting that PAFR internalization may not be only the pathway to downregulate PAFR-mediated responses. We hypothesized that PAF binds to SMC subcellular protein fractions and together with ROCK contributes to physiological effects of PAF. Protein fractions were prepared from cells cultured in normoxia or hypoxia. PAF binding, PAFR protein and gene expression were studied. In normoxia, PAFR density (Bmax) in arterial and venous SMC were 487±37 and 624±17 respectively, which increased by 30-35% in hypoxia, and was greater in venous than arterial SMC in normoxia and hypoxia. The KD for PAFR binding to arterial cells was higher than for venous, and then hypoxia decreased the KD of PAFR binding in both cell types. PAFR binding to subcellular fractions were detectable, but were significantly lower than for membrane fractions in all conditions; e.g., normoxia: venous versus membrane, soluble fraction, 40%; nuclear fraction, 20%. ROCK inhibitor (Y-27632) attenuated PAFR binding after 24 hr incubation comparable to effects of cycloheximide and CV-3988. Unlike CV-3988, Y-27632 had no effect on PAF binding to pre-synthesized receptors within 30 min incubation. Y-27632 and HA-1077 increased MLCK and Calponin expression, although oxygen-related effects in venous SMC were different. PAFR gene expression was higher in hypoxia and effect of PAF and CV-3988 treatment on gene expression was paradoxically higher in arterial SMC supporting a translational control of PAFR activity in lung SMC. This study shows that PAFR binding to the SMC is not exclusive to membranes and that hypoxia in conjunction with ROCK modulate PAFR-mediated signaling in pulmonary vascular SMC in vitro with different effects on cells from arteries and veins. Different effects of ROCK on contractile proteins expression in hypoxia suggest involvement of these proteins in PAF-induced lung SMC reactivity in utero.