Figure 3

OAG-induced Ca²⁺ increase is inhibited by TRPV1 blockers in heterologous expression system. (a) RT-PCR analysis showed that in untransfected HEK 293 cells, there was no endogenous expression of TRPV1, TRPC3, TRPC6 or TRPC7. TRPV1 expression was detected in TRPV1-transfected HEK 293 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was positive control on RT PCR. (a) Ratiometric Ca²⁺ imaging loaded with fura-2AM revealed activation of TRPV1 by 100 μM OAG. (b) OAG application evoked Ca²⁺ transients that were blocked by the pretreatment with 10 μM CZP. Capsaicin-induced Ca²⁺ transients confirmed expression of functional TRPV1 in the transfected HEK 293 cells. (c) Concentration-response relationship of OAG response in TRPV1-expressing HEK 293 cells obtained from ratiometric Ca²⁺ imaging. (d, e) The OAG-induced Ca²⁺ transients persist in the presence of 1 μM Thapsigargin (Thap). (f) The summary bar graph was generated from the peak Ca²⁺ responses of OAG-induced and capsaicin-induced Ca²⁺ transients in the absence and presence of 1 μM thapsigargin. Although the capsaicin-induced Ca²⁺ transient was slightly decreased, there was no difference in OAG-induced-Ca²⁺ transients between both conditions.