ELSEVIER Biochimica et BiophysicaActa 1258(1995) 90-94 Biochi~ic~a et Biophysica Acta Inhibition of microsomal cholesterol ester hydrolase by okadaic acid in isolated rat hepatocytes Maria J. Martinez, Mafia L. Hernandez, Olatz Fresnedo, Mercedes Lacort, Begofia Ochoa * Department of Physiology, Faculo' of Medicine, Unicersity of the Basque Country, P.O. Box 699, 48080-Bilbao, Spain Received 15 March 1995; accepted 27 April 1995 Abstract Okadaic acid, a potent and specific inhibitor of protein phosphatases 1 (ICsn 10-20 nM) and 2A (ICso 0.05-2 nM) caused early and sustained inhibitions of microsomal cholesterol ester hydrolase activity in hepatocyte suspensions. The changes in the kinetic properties of the esterase and its response to exogenous alkaline phosphatase and cyclic AMP-dependent protein kinase after cell exposure to 1 /xM or 1 nM okadaic acid differed markedly among themselves, which suggests the involvement of both protein phosphatases 1 and 2A in the regulation of the microsomal hydrolysis of cholesterol esters. Furthermore, the inhibitory effect of okadaic acid is likely to be independent of the dibutyryl-cyclic AMP promoted cell events leading to stimulation of esterase activity. Keywords: Okadaic acid; Cholesterol ester hydrolase; Phosphatase; Hepatocyte: (Rat) 1. Introduction In the complex metabolism of cholesterol in the liver, the hydrolysis of cholesterol esters by cholesterol ester hydrolases (CEH) (E.C. 3.1.1.13) could be an important point of regulation. Neutral cholesterol ester hydrolases located in cytosol and the microsomal fraction release free cholesterol from intracellular cholesterol esters, the storage form of hepatic cholesterol [1,2]. The major neutral hydro- lytic activity has been traditionally attributed to the soluble enzyme, although microsomes obtained from liver crude extracts elicit high CEH activity [3,4]. Cytosolic CEH has been purified and it is rather well characterized [5-7]. However, little information has been obtained on the bio- chemistry, the function and the physiological role of the microsomal enzyme. Microsomal CEH from rat liver is located in the luminal side of the rough membranes [8,9], shows a diurnal variation [10], is under hormonal control by sex steroids [3] and its activity is markedly enhanced by the divalent cations Ca 2+ and Mg 2+ [1 1]. Phosphoryla- Abbreviations: CEH, cholesterol ester hydrolase; PP1, protein phos- phatase type 1; PP2A, protein phosphatase type 2A; db-cyclic AMP, dibutyryl-cyclicAMP. * Corresponding author. Fax: + 34 4 4648152. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0005-2760(95)00103-4 tion-dephosphorylation of microsomal CEH is one pro- posed mechanism for the regulation of the enzyme activity. A recent in vitro cell-free study from our laboratory has reported that marked stimulation of microsomal CEH was elicited by cyclic AMP-dependent protein kinase or CaZ+/calmodulin, whereas enzyme deactivation was de- pendent on phosphatase action [12]. However, it is not known what, if any, effect cellular phosphoprotein phos- phatase activities had on microsomal CEH, to date. Okadaic acid is a polyether derivative of 38-carbon fatty acid implicated as the causative agent of diarrhetic shellfish poisoning, which was recently demonstrated to be a potent and specific inhibitor of protein phosphatases 1 (PP1, ICs0 10-20 nM) and 2A (PP2A, ICs0 0.05-2 nM) [13,14]. Thus, while PP2A is completely inhibited by 1 nM okadaic acid PPI is unaffected at this concentration but is inhibited by l /zM okadaic acid. This compound is recognized to stimulate rapidly the phosphorylation of a number of pro- teins involved in glucose and lipid metabolism in intact hepatocytes and adipocytes [13]. The objective of this study was to examine the effect of okadaic acid on micro- somal CEH activity in freshly isolated rat liver cells. The results are the first to indicate that the activity of liver protein phosphatases 1 and 2A may be involved in the regulation of the microsomal hydrolysis of cholesterol esters.