Effects of hypoxia on 5-HT induced pulmonary arterial vasoconstriction in mice over-expressing the 5-HT transporter: role of the 5-HT1B receptor and rho-kinase

Kevin White, Marta Baranowska, Yvonne Dempsie, Margaret R. MacLean

University of Glasgow, Glasgow, UK

A polymorphism resulting in over-expression and increased activity of the 5-HT transporter (SERT) has been associated with the development of pulmonary arterial hypertension (PAH). We have previously shown that mice over-expressing the SERT (SERT+ mice) develop elevated pulmonary pressures and increased hypoxia-induced PAH (MacLean et al., 2004). Here we investigate the role of the 5-HT1B receptor and rho-kinase (ROCK) on the increased pulmonary pressures observed in these mice.

Wildtype and SERT+ mice (C57BL/6xCBA, female 5-6 months, 25-35g) were exposed to 14 days of hypoxia using a hypobaric chamber set to 550mbar (equivalent to 10% O2). Normoxic controls were also used. Mice were then euthanised using sodium pentobarbitone (500 mg kg-1) and the heart & lungs removed. The pulmonary resistance arteries (PRA’s, ∼200μm i.d) were dissected free of tissue and mounted on a wire myograph in Krebs solutions (37°C) and cumulative concentration response curves for 5-HT were constructed (1nM-0.1mM). Protein expression of the 5-HT1B receptor, ROCK-1 and ROCK-2 were investigated in first and second generation pulmonary arteries from normoxic and hypoxic wildtype and SERT+ mice using Western blotting. Statistical analysis was performed using a two-way ANOVA followed by Bonferronis post-test. Data are expressed as mean ± SEM.

Normoxic SERT+ mice exhibited a 50% reduction in protein expression of the 5-HT1B receptor compared to wildtype littermates. In line with this, the 5-HT1B receptor antagonist GR55562 (1μM) had no effect on 5-HT mediated contraction observed in SERT+ PRA’s (pEC50 values: 5.46±0.10 n=12 and 5.47±0.09 n=10; p>0.05). However LY93358 (100nM), an inhibitor of both SERT and the 5-HT1B receptors, markedly reduced the potency of 5-HT in PRA’s from SERT+ mice ( pEC50 values: 5.46±0.10 n=12 and 4.54±0.12 n=6; p<0.001). Following exposure to hypoxia, ROCK-2 (but not ROCK-1) expression was increased in both wildtype (fold increase p<0.001) and SERT+ (fold increase p<0.05) mice. Inhibition of ROCK by Y27632 completely abolished 5-HT induced contraction of PRA’s from both normoxic and hypoxic wildtype and SERT+ mice.

When SERT is over-expressed, inhibition of both the 5-HT1B receptors and SERT is required to attenuate 5-HT mediated pulmonary vascular contraction, suggesting interdependence between their respective signaling pathways. Also, we consider ROCK to function downstream of 5-HT and play an important role in pulmonary vasoconstriction. Specifically, the ROCK-2 isoform is over-expressed in mice exposed to hypoxia and this may contribute to the increased 5-HT contractile response observed in these mice.

MacLean, M.R. et al. (2004) Circulation 109: 2150-2155