Figure 6

Accumulation of cAMP by cells exposed to acidic pH. (A) HEK293T cells were transfected with pIRES-GFP-TDAG8 or pIRES-GFP plasmids for 36 hours. Transfected cells were exposed to pH7.4 and 6.4 buffers for 30 minutes at 37°C in the presence of RO201724 for detection of intracellular cAMP. All values were normalized with pH 7.4 vector control (as 100%). Data are obtained from at least three independent experiments performed in duplicate. Comparison between vector- and TDAG8-transfected cells was by t test, *p < 0.05. (B) The accumulation of cAMP in TDAG8-transfected HEK293T cells pre-treated with PTX for 4 hours or U73122 for 15 minutes at 37°C before exposure to pH 6.4 buffer for 30 minutes. All values were normalized with pH 7.4 control (as 100%). (C) The pH-dependent cAMP accumulation in DRG primary cultures. Primary DRG cultures were exposed to pH7.4, 6.8, 6.4 and 6.0 buffers for 30 minutes at 37°C in the presence of RO201724 for detection of intracellular cAMP. The cAMP levels at pH 7.4 were a control (= 100%). (D) The cAMP levels in DRG cultures from CFA-injected mice after exposure to pH 6.4 buffer for 30 minutes at 37°C in the presence of RO201724. The cAMP levels from contralateral DRG were a control (100%). (E) Intracellular [Ca²⁺] change in DRG cultures from CFA-injected mice after transient exposure to pH 6.4 buffer. Peak values of [Ca²⁺] response (approximately 20 seconds after the addition of agonists) are presented as histograms. All data are presented as mean ± SEM (n = 12 cells). (F) Intracellular [Ca²⁺] change in DRG cultures from CFA-injected mice. DRG cultures were transiently exposed to pH 6.4 buffer after pre-treatment with PTX for 2 hours. Peak values of [Ca²⁺] response are presented as histograms. All data are presented as mean ± SEM (n = 7–12 cells).