Pharmacological Characterisation of Cysteinyl-Leukotriene Type 2 Receptors Stably Transfected into HEK-293 cells

W.T.H. Chow, A.M. Cazaly, S.J. Robinson, E.P. Healy, A.P. Sampson. Division of Infection, Inflammation & Repair, University of Southampton School of Medicine, Southampton General Hospital, Southampton, UK

Cysteinyl-leukotrienes (cysteinyl-LTs) are lipid mediators of inflammation derived from the 5-lipoxygenase pathway expressed in leukocytes. Cysteinyl-LTs act at two G-protein-coupled receptor types, namely CysLT1 receptors which mediate bronchoconstriction and eosinophilia in asthma, and CysLT2 receptors which may have additional inflammatory roles such as in atherogenesis. Lukast drugs which block CysLT1 are commonly used in asthma management, but no CysLT2 receptor antagonists have yet been developed for clinical use.

We have previously developed a line of human embryonic kidney cells (HEK-293) which stably express CysLT2 receptors conjugated with enhanced green fluorescent protein (EGFP), which can be used to visualize receptor expression. In this project, we aimed to characterize calcium signalling by this receptor in response to cys-LT agonists and its blockade by CysLT receptor antagonists.

HEK-293 cells stably transfected with CysLT2-EGFP were grown to 80-90% confluence and used as suspensions in culture medium between passages 6-14. Calcium signalling in response to agonists (leukotrienes C4 and D4) was measured by Indo-1 spectrofluorimetry, in the presence and absence of CysLT receptor antagonists. The results were compared with those in control cells transfected with EGFP alone. Leukotrienes and antagonists were from Cayman Chemical Co.

The cysteinyl-LTs LTC4 and LTD4 (2nM to 2000nM) both produced rapid and dose-dependent calcium responses in CysLT2-EGFP transfected cells (p=0.00006, n=11; and p=0.007, n=3, ANOVA), with EC50 values of approximately 63nM and 7nM respectively. A non-cysteinyl-LT (LTB4) had no effect at concentrations up to 2000nM. Control cells transfected with EGFP alone showed no response to LTD4, confirming that HEK293 cells do not constitutively express CysLT1 or CysLT2 receptors. In CysLT2-EGFP transfected cells, the dual CysLT1/CysLT2 antagonist BAY u9773 blocked responses to LTD4 (200nM) by over 75%, with an IC50 value <10 nM. As expected in the absence of CysLT1 receptors, the selective CysLT1R antagonist, MK-571, had no effect on responses to LTD4.

The HEK-293 cells stably-transfected with CysLT2-EGFP showed specific and dose-dependent signalling responses to cysteinyl-LTs similar to those observed in cells transiently-transfected with unconjugated CysLT2 (Heise et al. 2000). Conjugation of CysLT2 with EGFP and its stable transfection into HEK-293 cells does not appear to impair CysLT2 signalling or alter its dose-dependent blockade by CysLT2 antagonists. This stable cell-line may prove useful for screening novel CysLT2R antagonists, which may in turn be helpful in dissecting the role of CysLT2 receptors in cardiovascular disease in animal models and in humans.

Heise CE, et al. 2000 J Biol Chem 275(39):30531-30536.