Figure 9

Delayed TNF-α induced increases in TRPV1 and TRPV4. Immunocytochemistry reveals an increase of TRPV1 and TRPV4 in human SW982 synoviocytes stimulated with 1 ng/ml TNF-α over time. (A) No staining is evident in peptide blocked controls: anti-TRPV1 pre-adsorbed with P-19 peptide (B) TRPV1 immunostaining in control cultures. An example of TRPV1 protein accumulation in a long cellular process is evident in one cell. (C) TRPV1 immunoreactivity is elevated after 8 hr incubations with TNF-α the TRPV1 staining has a cytoplasmic and perinuclear distribution indicating its synthesis in the rough endoplasmic reticulum. (D) TNF-α stimulated increase in TRPV1 staining was greatly reduced by co-incubation with TNF-α inhibitor (CAY10500, 5 μM, 8 hr). (E) No staining is evident in peptide blocked controls: anti-TRPV4 pre-adsorbed with peptide 853–871 (1:1, w/w, for 30 min). (F) TRPV4 immunostaining in control cultures. (G) At 8 hr, TRPV4 immunoreactivity is not increased. (H). TRPV4 immunostaining is enhanced after 12 hr of TNF-α stimulation compared to untreated cell cultures. (I) TRPV1 stain density was significantly increased at 8 hr. Other time points were observed visually, but not quantified. (J) TRPV4 stain density was significantly increased at 12 hr and significantly decreased at 16 hr. (K) The bar graph indicates that the synoviocyte cell areas are significantly increased at the same time point when TRPV4 staining is increased (12 hr). * indicates p < 0.05 compared to normal synoviocytes. # indicates p < 0.01 compared to normal synoviocytes. Data is representative of n = 3 experiments.