Multixenobiotic resistance in coelomocytes from three echinoderm species

Abstract

Multixenobiotic resistance (MXR) proteins are known to be present inmost living organisms, but only a few studies have been conducted onechinoderms and especially on their circulating cells, the coelomocytes.The objective of the present study was to investigate the presence ofMXR activity in coelomocytes of the sea urchin Strongylocentrotusdroebachiensis, the sea star Lept asterias polaris and the sea cucumberCucumaria frondosa. Cells were exposed to fluorescent substrates (1 mu MRhodamine B {[}RB] or 0.5 mu M calcein- AM {[}CAM]), with or withoutinhibitors (50 mu M verapamil {[}Ver], 5 mu M cyclosporin- A {[}CsA] and5 mu M MK571 {[}MK]), and single-cell fluorescence was measured by flowcytometry. The combinations RB + CsA and RB + Ver induced a fluorescencein crease in S. droebachiensis and L. polaris coelomocytes, as well asin S. droebachiensis vibratile cells. The combination RB + MK induced afluorescence increase in S. droebachiensis and C. frondosa coelomocytes.Finally, the combination CAM + MK induced a fluorescence diminution inL. polaris coelomocytes and S. droebachiensis vibratile cells. Thisdifference in fluorescence incorporation indicated an MXR-like activityin coelomocytes, probably due to the presence of a P-glycoprotein (Pgp)and a multidrug resistance-associated protein (MRP)-like transporter.Western blot analysis was also carried out (Ab C219 and Ab C9) in orderto detect potential MXR proteins using anti-MXR anti bodies. Both Pgpand MRP were detected, but could not be further discriminated. MXRactivity was clearly demonstrated in coelomocytes of S. droebachiensis,L. polaris and C. frondosa, although the identity of proteinsresponsible for this activity needs to be confirmed.