Fig. 2.

In vitro characterisation of GPR4 activity.

A. Increases in cAMP induced by a water-soluble forskolin analogue NKH 477 and detected using Glosensor assay are stable across the physiologically relevant pH range. 20μM NKH 477 was applied to naïve HEK293 cells (not GPR4 transfected). Data represent mean ± s.e.m (n = 4 of triplicates). Differences are not statistically significant (repeated measures one-way ANOVA).

B. pH-dependent cAMP accumulation in GPR4 expressing HEK293 cells depends on the quantity of DNA used for transfection and by implication on the level of the expression of GPR4. Data represent mean ± s.e.m. for averages of triplicates (n = 7 of triplicates for 0.1 μg/μl, n = 3 of triplicates for 0.01 and 0.001 μg/μl). High levels of expression shifts the activation curve towards the alkaline range. Activation profile with 0.001 μg/μl DNA leads a profile which resembles response of HUVEC (see panel 2C) which express GPR4 natively.

C. cAMP accumulation in HUVEC at different pH. Increase in cAMP without NE 52-QQ57 at pH 7.1 and 6.8 is statistically significant (n = 6 of triplicates, repeated measures one-way ANOVA shows p < 0.01)

D. Concentration-response curve for cAMP production evoked using non-selective β-adrenoceptor agonist isoprenaline to illustrate that HUVEC cells can mount a robust cAMP response to a different Gs-coupled GPCR. Isoprenaline activates natively expressed β2 adrenoceptors in a concentration-dependent manner (n = 3 of triplicates). -∞ indicates zero concentration of the drug.