Microcystin uptake and inhibition of protein phosphatases: effects of chemoprotectants and self-inhibition in relation to known hepatic transporters.
The microcystins (Mcyst) are cyclic peptide hepatotoxins produced by cyanobacteria. They are chemically very stable and represent a public health threat when they occur in water bodies used for human consumption. Mice injected ip with Mcyst (LD50 50-100 micrograms/kg) accumulate Mcyst in the liver and die within 2-4 hr with massive intrahepatic hemorrhage. Pretreatment of mice with cyclosporin A (CP), rifamycin (Rif), trypan blue (TB), and trypan red (TR) protected the animals from a lethal dose of the toxin. The studies reported here using the freshly isolated rat hepatocyte model were undertaken in order (1) to evaluate the contribution of Mcyst transport in hepatocytes to the mechanism of chemoprotection for Mcyst in vivo toxicity by CP, Rif, TB, and TR and (2) to better characterize the hepatic Mcyst transporter in this model and determine its relationship to other bile acid/organic anion transporters that have already been fully described. Incubations with 125I-Mcyst were used to measure Mcyst uptake and accumulation in hepatocytes. It has been shown that at the cellular level Mcyst binds to and inhibits protein phosphatases 1 and 2A (PP) at nanomolar concentrations. Lethal doses of Mcyst in mice resulted in rapid profound inhibition of hepatic PP activity. PP activity was also inhibited in hepatocytes incubated with 100-500 nM Mcyst. PP inhibition in these studies was used as a marker of metabolic effects of the toxin. The chemoprotectants CP (5 microM), Rif (50 microM), TR (20 microM), and TB (20 microM) decreased accumulation of Mcyst (320 nM, a toxic concentration) after 30 min incubation to 37, 26, 30, and 66%, respectively, of that of cells treated with Mcyst only. Inhibition of PP activity in these cells was decreased. Inhibition of PP activity in hepatocytes was also decreased by known inhibitors of Mcyst transport: 50 microM of the bile acids cholate and taurocholate (TC) and 50 microM sulfobromophthalein. For all compounds tested the amount of Mcyst accumulated in the hepatocytes correlated qualitatively with the extent of PP inhibition. From these results it can be concluded that inhibition of Mcyst uptake by hepatocytes is the most likely mechanism of chemoprotection for Mcyst in vivo toxicity for TR, TB, CP, and Rif. Uptake of Mcyst was unaffected by changes in the ionic composition of the uptake buffer but was significantly decreased when PP activity of hepatocytes was inhibited by preincubation with Mcyst.(ABSTRACT TRUNCATED AT 400 WORDS)
Runnegar
M
M
Department of Medicine, University of Southern California School of Medicine, Los Angeles, USA.
Berndt
N
N
Kaplowitz
N
N
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008387:Marine Toxins; D051379:Mice; D052998:Microcystins; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000493:pharmacokinetics* / Q000633:toxicity; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013656:Taurocholic Acid / Q000378:metabolism
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D052998:Microcystins; D010080:Oxazoles; D010456:Peptides, Cyclic; D013656:Taurocholic Acid; C078588:microcystin; C059041:calyculin A; D016572:Cyclosporine; D010749:Phosphoprotein Phosphatases