Multixenobiotic resistance in coelomocytes from three echinoderm species

Abstract

Multixenobiotic resistance (MXR) proteins are known to be present in most living organisms, but only a few studies have been conducted on echinoderms and especially on their circulating cells, the coelomocytes. The objective of the present study was to investigate the presence of MXR activity in coelomocytes of the sea urchin Strongylocentrotus droebachiensis, the sea star Leptasterias polaris and the sea cucumber Cucumaria frondosa. Cells were exposed to fluorescent substrates (1 µM Rhodamine B [RB] The combination RB + MK induced a fluorescence increase in S. droebachiensis and C. frondosa coelomocytes. Finally, the combination CAM + MK induced a fluorescence diminution in L. polaris coelomocytes and S. droebachiensis vibratile cells. This difference in fluorescence incorporation indicated an MXR-like activity in 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 order to detect potential MXR proteins using anti-MXR antibodies. Both Pgp and MRP were detected, but could not be further discriminated. MXR activity was clearly demonstrated in coelomocytes of S. droebachiensis, L. polaris and C. frondosa, although the identity of proteins responsible for this activity needs to be confirmed. KEY WORDS: Multixenobiotic resistance · MXR · Echinoderms · Coelomocytes · P-glycoprotein · Pgp · Multidrug resistance-associated proteins · MRPsResale or republication not permitted without written consent of the publisher Aquat Biol 12: 81-96, 2011 Pgp is known to be the primary active transporter of unmodified xenobiotics (Zaja et al. 2007), transporting mostly large and moderately hydrophobic cations (Litman et al. 2001). It is widely distributed in tissues and has a variety of physiological roles in healthy cells, such as lipid transport, intracellular cholesterol trafficking and the cytotoxic activity of NK and T cells (Johnstone et al. 2000). Among the family of MRPs, MRP1 facilitates the extrusion of numerous glutathione, glucuronate and sulphate conjugates. MRP1 is a ubiquitous protein taking part in various physiological functions including defence against xenobiotics and endogenous toxic metabolites, leukotrienemediated inflammatory responses, as well as protection from the toxic effect of oxidative stress (Bakos & Homolya 2007).A combination of substrates and inhibitors is often used to indicate MXR activity (Neyfakh 1988, Cornwall et al. 1995, Marin et al. 2004). Since some substrates and inhibitors are more specific to one MXR protein than to another, it is sometimes possible to discriminate the proteins involved. For example, Rhodamine B (RB) is often used as a Pgp substrate (Minier & Moore 1996, Reungpatthanaphong et al. 2003, Zaja et al. 2006) and a similar molecule, Rhodamine 123, is used as a Pgp-and MRP-like substrate (Hollo et al. 1996, Daoud et al. 2000. Calcein-AM (CAM) is also known as a substrate for both Pgp-like and MRP-like proteins,...

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach

“…FC fractionation of cell types in fluids is regularly performed in many species, although its use has been limited in members of the Echinodermata. In a previous study with FC done in the asteroid Leptasterias polaris only a single population of coelomocytes was observed (28). In specimens of Asterias rubens injected with artificial sea water, a main cluster, attributed to amoebocytes, was originally described, which was further sub-divided in three cellular groups that were differentially affected by bacterial challenge (29).…”

A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging

“…A maximum of 20% of these cells are osmotically compromised, displaying lower DRAQ5 incorporation/fluorescence values -P2low in Figure 1A. In a previous study with FC done in the asteroid Leptasterias polaris only a single population of coelomocytes was observed (18). In specimens of Asterias rubens injected with artificial sea water, a main cluster, attributed to amoebocytes, was originally described, which was further sub-divided in three cellular groups that were differentially affected by bacterial challenge (19).…”