Fig. 4.

LL inhibits cAMP accumulation induced by acidosis in a concentration-dependent manner in (A) HEK293 and (B) HUVEC cells.

All values are normalised to control at pH 8.0 for each triplicate measurement. Two-way repeated measures ANOVA revealed a significant effect of pH (p < 0.0001) and LL (p < 0.05) on cAMP levels in HEK293 cells. Post-hoc Sidak's multiple comparisons test shows confirmed significant differences between effects of 0 and 10mM LL (p < 0.05 at pH 7.4 and 6.8, p < 0.01 at pH 7.1) and between 100 μM and 10 mM (p < 0.05), but the difference between 1 and 10mM LL was not significant (p = 0.53 at pH 7.1, p = 0.78 at pH 6.8). In HUVECs, the same analysis revealed a significant effect of pH (p < 0.0001) and LL (p < 0.05) and a significant interaction effect (p < 0.001). The post-hoc test shows significant differences between 0 and 10mM (p < 0.05 at pH 7.1, p < 0.0001 at pH 6.8), between 100 μM and 10 mM (p < 0.05 at pH 7.1, p < 0.0001 at pH 6.8), and between 100 μM and 1 mM (p < 0.05 at pH 6.8).n = 4–5 of triplicates for each data point in (A) and (B), apart from n = 3 for 1μM NE 52-QQ57 curve in HUVEC. 1μM NE 52-QQ57 antagonises pH dependent activation of GPR4, the block is overcome at acidic pH in HEK293 cells.