gms | German Medical Science

Background: In rheumatoid arthritis (RA), synovial fibroblasts (SF) secrete pro-inflammatory cytokines and matrix degrading enzymes. Previous studies already demonstrated the functional expression of several transient receptor potential channels (TRP) such as TRPV1, TRPA1 and others in SF. Upon ligation, these receptors increase intracellular calcium but they have also been linked to modulation of inflammation. Additionally, activation of TRPA1, TRPV1 and TRPM8 permeates large cations such as the local anesthetic lidocaine or the cationic dye PoPo3 iodide through the plasma membrane. Similarly, the phytocannabinoid cannabidiol (CBD) has been found to trigger the uptake of chemotherapeutic agents into cells via TRPV2. Until now, this mechanism has not been exploited in the treatment of RA and, therefore, we investigated the effect of CBD on calcium release and on the uptake of the large cationic dye Po-Po3 iodide in SF.

Methods: Calcium flux and fluorescent dye uptake was monitored by a multimode reader.

Results: In first experiments, we investigated calcium flux in SF and found that CBD dose-dependently increased intracellular calcium which was enhanced by TNF pre-treatment. We identified TRPA1 to be responsible for the observed effects. Calcium flux was also increased when extracellular calcium was omitted and further experiments identified TRPA1 to be located in lysosomal membranes. We then investigated Po-Po3 uptake and found that CBD induced the uptake of this dye into SF. Similar to calcium mobilization this effect was enhanced by TNF-pretreatment. We found that activation of TRPM3 by pregnenolone sulfate inhibited Po-Po3 uptake into TNF pre-treated SF. In conclusion, we propose the following mechanism of dye uptake in SF: i) activation of lysosomal TRPA1 leads to an increase in intracellular calcium; ii) lysosomal calcium release triggers the sensitization of cell membrane-bound TRPM3; iii) opening of TRPM3 triggers Po-Po3 uptake; iv) pregnenolone sulfate desensitizes TRPM3 and precludes dye uptake.

Conclusion: This study might help to find a suitable delivery system to increase the concentration of anti-rheumatic drugs in cytokine-activated immune cells. In addition, the lysosomal localization of TRPA1 suggests an important role in autophagy and, therefore, TRPA1 might be an attractive therapeutic target to correct autophagy defects in e.g. lupus or RA.