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Inhibition of rho-kinase blocks pulmonary vascular effects of 5HT in mice over-expressing the 5HT transporter 5HT plays a role in the pathophysiology of pulmonary arterial hypertension (PAH; MacLean et al., 2000). Rho Kinase (ROCK) may play a role downstream of 5HT in both vasoconstriction (Nagaoka et al., 2003) and remodelling of pulmonary arteries (PAs; Lui et al., 2004). Furthermore, the extracellular signal-related kinase (ERK) pathway, has been shown to be involved in 5HT-induced proliferation of pulmonary cells (Suzuki et al., 2003). Therefore, effects of the ROCK inhibitor Y27632 on 5HT-induced constriction of PAs from both normoxic and hypoxic mice over-expressing the 5HT transporter (5HTT) gene and their wildtype (WT) controls were investigated. We also examined the effects of Y27632 on 5HT-induced proliferation and 5HT-induced ERK activation in Chinese hamster lung fibroblasts (CCL-39 cells). Female mice (C57BL/6 x CBA, WT and 5HTT+, 5 to 6 months, 20-30g) were exposed to 14 days hypoxia (10% O2) or normoxia. Isolated PAs (~250 μm i.d.) were set up on wire myographs and concentration response curves constructed to 5HT (1nM-100 μM) in the presence or absence of the ROCK inhibitor Y27632 (10 μM). Statistical analysis was by one-way analysis of variance with Newman-Keuls post-test. The effects of Y27632 (5 μM) on 5HT-induced proliferation and 5HT-induced ERK activation were examined in CCL-39 fibroblast cells. Cells were serum-starved for 24 hours before addition of 5HT (0.01 μM-10 μM). Proliferation was determined by monitoring [3H]-thymidine incorporation and ERK activation detected by immunoblotting using a phospho-specific ERK antibody. Statistical analysis was students unpaired t-test. Results are expressed as mean ± S.E.M. Y27632 attenuated the 5HT contractile response in PAs from both WT and 5HTT+ mice by approx. 70% (Emax values: WT: 96.9±2.6% n=9 to 26.2±1.8% n=10, P<0.001; 5HTT+: 91.9±6.2% n=5 to 28.0±2.3% n=8, P<0.001). Y27632 also reduced the 5HT contractile response in PAs from chronic hypoxic mice by approx. 70% (Emax values: WT: 117.09±3.9% n=7 to 31.1±2.5% n=6, P<0.001; 5HTT+: 119.6±8.0% n=7 to 39.3±2.4% n=7, P<0.001). Treatment with Y27632 markedly decreased 5HT-induced proliferation (to 36.55±2.12% of maximum effect, n=3 P<0.001) and significantly attenuated 5HT-induced ERK activation (1 μM 5HT: 95.62±3.6% to 66.77±6.9%, P<0.05; 10 μM: 99.4±0.6% to 53.42±12.7%, n=3 P<0.05) in CCl-39 cells. ROCK functions downstream of 5HT to cause contraction of mouse PAs. The vasoconstrictive effect of ROCK is unchanged by over-expression of the 5HTT, or by hypoxia. ROCK is also involved in 5HT-induced ERK activation and 5HT-induced proliferation of fibroblasts.
Liu et al., (2004) Circ Res 95 :579-586. |
