Characterisation of anandamide uptake in resting and activated murine cells

Sammanfattning: Modifying the metabolism of the body’s own endocannabinoids is a novel approach for analgesia. Two key catabolic enzymes are fatty acid amide hydrolase (FAAH) and inflammation-inducible cyclooxygenase 2 (COX-2). The cellular uptake of the key endocannabinoid anandamide (AEA) has been found to be regulated by its FAAH-catalysed intracellular degradation, but COX-2 has not been investigated in this respect. We aimed to find out whether or not COX-2 in an in vitro inflammation setting would be able to gate AEA uptake. To achieve this, C6 cells and Raw 264.7 cells were stimulated with LPS/INF-γ and lysates then analyzed by immunoblot in order to verify COX-2 expression. AEA cellular uptake was quantified using a radioassay with [3H]-AEA. It was found that COX-2 was not inducible in C6 cells using the LPS/INF-γ conditions studied, while it was inducible in Raw 264.7 cells. AEA uptake in the COX-2-induced Raw 264.7 cells was not reduced by inhibitors of this enzyme. FAAH appeared to be down-regulated in the stimulated Raw 264.7 cells, and this was reflected in an overall lower AEA uptake. Our interpretation of the data points to FAAH as gating AEA uptake. Additional experiments are required to validate our findings by verifying significance.