Rho Kinase inhibitors and hypoxia modulate expression of platelet activating factor (PAF) receptor and gene expression in fetal ovine pulmonary vascular smooth muscle cells.

Abstract

Hypoxia and PAF augment intracellular calcium flux in fetal ovine pulmonary vascular smooth muscle cells (PVSMC) indicating that high pulmonary vascular tone in fetal pulmonary vessels in utero is consequent on combined effects of hypoxia and PAF receptor (PAFR)‐linked vascular responses. PAFR internalization is said to inactivate PAF effects in PVSMC in vitro. Hypoxia increases PAFR expression, but Rho kinase (ROCK) inhibitors decrease the expression suggesting PAFR internalization may be one pathway to downregulate PAFR‐mediated responses in PVSMC. These findings also suggest that ROCK may act in synergy with PAF to modulate PAFR‐mediated pulmonary vascular responses. We studied effects of PAF, hypoxia and other modulators of pulmonary vascular reactivity in arterial (PA) and venous (PV) smooth muscle cells (SMC) to aid in understanding the signaling pathways of PAFR‐mediated fetal ovine pulmonary vascular reactivity in utero. We hypothesized that ROCK inhibitors attenuate PAFR expression in hypoxia leading to increased expression of the contractile proteins. Cells were cultured in normoxia or hypoxia, with and without 10 nM PAF or ROCK inhibitors (Y27632 and HA 1077, each 10 μM). Proteins were probed for PAFR, and Calponin, MLCK and Desmin, which are contractile protein modulators of PVSMC activity. PAF binding, PAFR protein and gene expression by qRT‐PCR were also determined. In normoxia, PAFR density (Bmax) in PA and PV SMC were 487±37 and 624±17 respectively, which increased by 30–35% in hypoxia, and higher in PV than PA in normoxia and hypoxia. The KD for PAF binding to PASMC was higher than for PVSMC, and then hypoxia decreased the KD of PAFR binding in both cell types. ROCK inhibitors attenuated PAFR binding with comparable effect to cycloheximide, but unlike PAFR inhibitor, CV3988, cycloheximide had no effect on pre‐synthesized proteins. ROCK inhibitors decreased PAFR expression, but increased expression of MLCK2, Calponin and Desmin in PASMC especially in hypoxia, with different effects in PVSMC. Generally, PAFR gene expression studied by qRT‐PCR was higher in PVSMC under all conditions. This study shows that hypoxia in conjunction with ROCK inhibitors modulate PAFR‐mediated signaling in PVSMC in vitro with different effects on cells from PA and PV. Increased expression of the contractive proteins in hypoxia indicates involvement of these proteins in PAF‐induced contractions in PVSMC.Support or Funding InformationLABiomedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.