Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin
Introduction
Microcystins are naturally occurring toxins produced by freshwater cyanobacteria and can be found in lakes, ponds, and rivers that are often used for recreational activities (Chorus et al., 2000, Watanabe et al., 1996). Thus, besides menacing terrestrial and aquatic wildlife and livestock (Carmichael, 1994, Fischer and Dietrich, 2000, Schwimmer and Schwimmer, 1968), cyanobacteria also represent a threat to human health (Jochimsen et al., 1998, Teixeira et al., 1993). In 1996, a hemodialysis unit in Caruaru, Brazil, used water for dialysis from a reservoir, which was contaminated with blue-green algae and contained microcystins. Sixty of the 126 intoxicated patients died of severe microcystin-induced neurotoxicity or toxic liver failure (Jochimsen et al., 1998, Pouria et al., 1998). Microcystins act by inhibiting Ser/Thr protein phosphatase-1 and -2A (Mackintosh et al., 1990), which leads to disruption of the cytoskeleton and subsequent cell death (Eriksson et al., 1989).
Target organs of microcystin are mainly the liver and the brain. This requires uptake of microcystin across the sinusoidal (basolateral) plasma membrane of hepatocytes and its transport across the blood–brain barrier. In rat hepatocytes, basolateral uptake of microcystin was inhibited by cholate, taurocholate (TC), and bromosulfophthalein (BSP) (Eriksson et al., 1990, Runnegar et al., 1995). All latter compounds are substrates of the organic anion transporting polypeptide (rodent: Oatps; human: OATPs) superfamily of membrane transporters (Hagenbuch and Meier, 2003, Hagenbuch and Meier, 2004) that are classified within the SLCO family of solute carriers (Human Gene Nomenclature Committee Database) (Hagenbuch and Meier, 2004). Oatps/OATPs mediate Na+-independent uptake of a wide variety of amphipathic organic compounds including bile salts, organic anionic dyes, steroids and steroid conjugates, drugs, various peptides, and toxins (Hagenbuch and Meier, 2003). Oatps/OATPs that are expressed in the brain (blood–brain barrier, choroid plexus) as well as in the liver include rat Oatp1a1 (Jacquemin et al., 1994), rat Oatp1a4 (Abe et al., 1998, Noé et al., 1997), human OATP1A2 (Kullak-Ublick et al., 1995), and human OATP2B1 (Kullak-Ublick et al., 2001). Rat Oatp1b2 (Cattori et al., 2000), human OATP1B1 (Abe et al., 1999, Hsiang et al., 1999, König et al., 2000b), and human OATP1B3 (König et al., 2000a) are primarily expressed in the liver. In the present study, we used the radiolabeled dihydromicrocystin-LR epimer [L-MeAla7]microcystin-LR (Meriluoto et al., 1990), which was previously shown to have similar characteristics as native microcystins (Meriluoto et al., 1990). The study served to investigate whether the hepato- and neurotoxicity observed after microcystin intoxication (Jochimsen et al., 1998, Pouria et al., 1998) could be explained by microcystin transport mediated by members of the Oatp/OATP superfamily that are expressed in hepatocytes and in the blood–brain barrier.
Section snippets
Animals
Female Xenopus laevis were purchased from the African Xenopus facility c.c, Noordhoek, Rep. South Africa.
Chemicals
[3H]-dihydromicrocystin-LR (0.5 mCi/mmol) was prepared as described (Meriluoto et al., 1990). [3H]Estrone-3-sulfate (53 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA), [γ-32P]ATP (3000 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK).
Uptake studies in X. laevis oocytes
Complementary RNA was transcribed in vitro from linearized cDNA using the mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX). X. laevis
Transport of microcystin by liver Oatps/OATPs
Several Oatps/OATPs are expressed in hepatocytes where they mediate uptake of numerous organic compounds from blood plasma. To determine whether one of these transporters could mediate uptake of microcystin-LR into hepatocytes, we expressed these transporters in the X. laevis oocyte expression system and measured uptake of radiolabeled microcystin-LR. As demonstrated in Fig. 1, rat Oatp1b2-expressing oocytes accumulated microcystin-LR about 2-fold. Furthermore, human OATP1B1 and OATP1B3 showed
Discussion
The cyanobacterial toxin microcystin-LR, a cyclic heptapeptide, has been shown to exhibit hepato- and neurotoxicity in patients of a dialysis unit in Caruaru, Brazil, when the water was contaminated by blue-green algae (Pouria et al., 1998). In this study, we provide evidence that the organotropism of microcystin toxicity can be explained by microcystin transport mediated by OATPs that are expressed in the liver and at the blood–brain barrier.
Liver-specific rat Oatp1b2, human OATP1B1, and human
Acknowledgments
Supported by grants 31-59204.99, 3100A0-100786/1 (to B.H.) and 31-045536.95, 31-64140.00 (to P.J.M.) from the Swiss National Science Foundation and by a grant from the European Commission DG VII INCO-Copernicus ERBIC15CT961010 (to D.R.D.)
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