Pharmacological characterisation of the 5-HT-stimulated phosphoERK response in mouse activated hepatic stellate cells

VJ Barrett1, F Chowdhury2, WG Dunn3, DA Hall1, JE Coote2, JC Denyer1. 1Respiratory TAU, GlaxoSmithKline, Stevenage, Herts, UK, 2PTS, GlaxoSmithKline, Stevenage, Herts, UK, 3Faculty of Medicine, Glasgow University, Glasgow, Scotland, UK

Introduction: Hepatic stellate cells (HSC) have a central role in the pathogenesis of liver fibrosis (1). In the injured liver, these cells activate (aHSCs), transforming into myofibroblast-like cells which are proliferative, persistent, express smooth muscle αα-actin (αSMA), secrete extracellular matrix and transforming growth factor-β1 (TGF-β1). There is evidence that 5-hydroxytryptamine (5-HT) influences HSC biology, and expression of 5-HT1B, 5-HT2A and 5-HT2B receptors are induced upon activation (2). 5-HT stimulation of HSCs signals through mitogen activated kinase 1 (ERK1/2) and transcription factor Jun D, to activate TGF-β1 expression and HSC fibrogenic activity (3). Pharmacological characterisation of the 5-HT receptor responsible for stimulating the ERK response in mouse aHSC was undertaken using 5-HT2A antagonists ketanserin and volinanserin and 5-HT2B antagonists GSK1606260A (6-((4,4-difluoropiperidin-1-yl)sulfonyl)-1-(piperidin-4-yl)-1H-indole hydrochloride) and RS-127445.

Methods: HSCs, isolated from C57BLK/6 mouse livers were grown on plastic to induce activation. aHSCs were plated out at 10000 cells per well in serum-free medium and incubated overnight at 37°C. Medium was replaced with HBSS and the cells incubated with antagonists or vehicle (0.1% DMSO) for 30 min before stimulating with 5-HT for 10 min. Medium was removed, the cells lysed and lysate was analysed for phosphoERK (pERK) and total ERK using an MSD assay as per kit instructions.

Results: 5-HT produced a concentration dependent increase of pERK. The 5-HT2B antagonists had only very weak effects despite being used at approximately 1000-fold their pKi’s at the mouse receptor (determined in cells recombinantly expressing the receptor). (Table 1). However, the 5-HT2A antagonists produced a marked but insurmountable antagonism of the 5-HT response (Table 2). All data shown is the mean ±± SEM, n>2.

Table 1 5-HT pEC50 values obtained in the presence and absence of GSK1606260A and RS-127445.

Table 2 5-HT CRC fitting parameters in the absence and presence of ketanserin and volinanserin.

Summary: The pharmacological profile of the 5-HT-induced pERK response is most consistent with the involvement of a 5-HT2A receptor.

References:

(1) Friedman SL, Toxicology 254:120, 2008

(2) Ruddell RG et al, Am J Pathol 169:861, 2006

(3) Ebrahimkhani MR et al, Nat Med 17:1668, 2011