Rho A‐Rho Kinase (RK) Pathway and Ovine Fetal Pulmonary Vascular Smooth Muscle Cell (PVSMC) Proliferation

Rho is a part of the Ca 2+ sensitization mechanism in cell signaling and RK, the downstream effector of Rho, is important in Ca 2+ release. A role for RK in proliferation of PVSMC is not known. Sub‐confluent fetal ovine PVSMC were starved for 24 h with 1% FBS in 5% CO 2 in air [normoxia (NMX)]. Cells were then cultured for 24 h more in NMX or in 2% O 2 [hypoxia (HPX)] in 10% FBS with 1:Ci 3 H‐Thymidine, with and without 10nM PAF and 10μM Y27632 (an inhibitor of RK). Cells cultured in FBS alone were used as controls. Cell proliferation was quantified and is reported as cell numberx10 3 /well, means ± SEM, n=6. Cell number was 138.0±6.53 in NMX and 220.9±8.5 in HPX, showing that hypoxic exposure for 24 h augments cell growth by 60%. Addition of 10nM PAF further increased cell growth by 15% in NMX and by 100% in HPX‐an augmentation of 24% over the effect of HPX alone. The RK inhibitor Y‐27632 attenuated cell growth by 42% in NMX, and abolished the stimulatory effect of HPX on cell growth. To test whether RK activation is involved in PAF‐stimulation of cell growth, cells were pretreated with Y‐27632 for 2 h followed by addition of 10nM PAF for 24h, in NMX and HPX. Pretreatment with Y‐27632 in NMX followed by addition of PAF inhibited cell growth by 44%, which was not different than the effect of Y‐27632 alone. Thus the small stimulatory effect of PAF on cell growth in NMX was abolished. However, when PAF was added in HPX, even in the presence of Y‐27632, cell growth increased by 116% compared to PAF plus Y‐27632 in NMX. Thus the cell growth stimulatory effect of PAF in HPX was not abolished by inhibition of RK. Our data suggest that RK activation is important for cell growth in NMX and is critical for the growth stimulatory effect of HPX alone. However, PAF‐stimulated cell growth in HPX appears to be independent of the RK pathway. Supported by Grant#HL077819.