Dexfenfluramine inhibits hypoxic-induced activation of p38 MAP kinase in pulmonary arterial fibroblasts from mice over-expressing the 5-HT transporter

Yvonne Dempsie1, Lynn Loughlin1, David J Welsh2, Andrew J Peacock2, Margaret R MacLean1
1University of Glasgow, Glasgow, United Kingdom, 2Scottish Pulmonary Vascular Unit, Glasgow, United Kingdom

Ingestion of the anorexigenic drug dexfenfluramine (Dfen) is a risk factor for development of pulmonary arterial hypertension (PAH). We have previously shown that Dfen induces PAH in wildtype (WT) mice whilst protecting against the exaggerated hypoxia-induced PAH observed in mice over-expressing the 5-HT transporter (5HTT+ mice; Dempsie et al., 2005; Morecroft et al., 2005). In the present study we have investigated the effects of both hypoxia and Dfen on proliferation and expression of p38 MAP kinase and extracellular signalling related kinase (Erk1/Erk2) in pulmonary arterial fibroblasts (PAFs) from WT and 5-HTT+ mice.

Mice (C57BL/6, 5-6 months, 25-35g, n=4) were euthanised using sodium pentobarbitone (0.7g/kg), pulmonary arteries (PAs; 750-1000μm i.d.) dissected out, and PAFs cultured as described previously (Welsh et al., 2001). PAFs were then serum starved for 24 hours prior to exposure to either normoxia or hypoxia (5%O2) in the presence of Dfen (0.3-10μM), p38 MAP kinase inhibitor SB203580 (5μM) or Erk1/Erk2 inhibitor U0126 (1μM for 24 hours. Proliferation was measured by [3H]thymidine incorporation. Soluble cell extracts were prepared and then fractionated by SDS-PAGE for immunoblotting with phosphorylated p38 (1:500) and phosphorylated Erk1/Erk2 (1:1000) antibodies (Cell Signalling, USA). Data is shown as mean±s.e.mean. Statistical comparisons were made by one-way ANOVA with a Tukeys multiple comparison test or a Students t-test where appropriate.

Dfen had no effect on proliferation of WT PAFs under either normoxia or hypoxia. Neither did Dfen have any effect on proliferation of 5-HTT+ PAFs under normoxic conditions. However, hypoxic exposure led to proliferation of PAFs from 5-HTT+ mice, an effect which was inhibited in a concentration-dependent manner by Dfen. Hypoxic-induced proliferation of 5-HTT+ PAFs was abolished in the presence of SB203580, but not U0126. In line with this, hypoxia induced an increase in phosphorylated p38 MAP kinase but not phosphorylated Erk1/Erk2 MAP kinase in 5-HTT+ PAFs. Dfen completely inhibited the hypoxia-induced increase in phosphorylated p38 MAP kinase observed in 5-HTT+ PAFs. Neither p38 MAP kinase nor Erk1/Erk2 were affected by either hypoxia or Dfen in WT PAFs.

In conclusion, we have shown that hypoxic-induced proliferation of 5-HTT+ PAFs is mediated via activation of p38 MAP kinase. P38 MAP kinase has previously been shown to play an important role in hypoxic-induced proliferation (Welsh et al., 2001). Dfen inhibits the hypoxic-induced increase in p38 MAP kinase, thereby inhibiting hypoxic-induced proliferation of 5-HTT+ PAFs. This may provide the mechanism by which Dfen protects against hypoxic-induced PAH in 5-HTT+ mice.

Welsh, D.J, et al., (2001) Am. J. Respir. Crit. Care 164: 282-289
Dempsie, Y. et al., (2005) Proceedings of the British Pharmacological Society at http://www.pa2online.org/volume/abstracts/volume3Issue4abst140P.html

Morecroft, I. et al., (2005) Proceedings of the British Pharmacological Society at http://www.pa2online.org/volume/abstracts/volume3Issue4abst141P.html