Original research article Effect of calcium channel blockers on paraoxonase-1 (PON1) activity and oxidative stress Cu ¨ neyt Tu ¨ rkes ¸ a , Hakan So ¨yu ¨t b , S ¸ u ¨ kru ¨ Beydemir a, * a Department of Chemistry, Faculty of Science, Atatu ¨rk University, Erzurum, Turkey b Department of Primary Education, Faculty of Education, Bayburt University, Bayburt, Turkey Introduction The human serum paraoxonase-1 (PON1) enzyme (arylesterase, EC 3.1.8.1, hPON1), which is synthesized in the liver, is an ester hydrolase. This enzyme, which is calcium dependent, is associated with HDL and has a molecular weight of 43–45 kDa [11,38,53,58]. Due to its physiological effects, paraoxonase was previously thought to be a lactonase. It has now been proven to have many substrates [2,46]. However, the physiological substrate and the biological function of this enzyme have not yet been elucidated. Free oxygen radicals are produced continuously during normal cell metabolism and are neutralized by the antioxidant defense system. However, the antioxidant defense system is suppressed when excessive amounts of free oxygen radicals are produced or when there is a significant reduction in the antioxidant defense and oxidative stress occurs [7,18,20,47,48,61]. PON1 neutralizes free oxygen radicals, including hydrogen peroxide [9] and inhibits lipid peroxidation through the hydrolysis of cholesteryl linoleate hydroperoxides. In addition, PON1 inactivates the formation of oxidized phospholipids in oxidized LDL and neutralizes the effects of atherogenic lipid peroxides, which indicates the protective effect of cell membranes [10,58,70,75]. Therefore, PON1 exerts an anti-oxidative stress effect through the hydrolysis of lipid peroxides and the prevention of the oxidation of LDH. In addition, PON1 inhibits not only the oxidation of LDL but also the oxidation of HDL [12,55]. Paraoxonase exerts a continuous reverse choles- terol transport function through the protection of HDL from oxidation. This situation slows the development of foam cell formation and atherosclerosis by preventing the accumulation of cholesterol in macrophages [30]. It has been reported that the increased oxidative stress in patients with cardiovascular diseases, such as diabetes mellitus, chronic renal failure, hyperthyroidism, and age-related macular degeneration, decreased the serum PON1 activity [1,14–16,23,54,55,57,60,68]. PON1 serves as an antioxi- dant enzyme by protecting low-density lipoproteins (LDHs) and high-density lipoproteins (HDLs) from oxidative stress, which is known to be associated with many different vascular diseases, including atherosclerosis [11]. PON1 enzymes from various sources have been purified using different methods. Usually, the purification folds found in our laboratory are close to each other [6,27–29,45]. Based on the Pharmacological Reports 66 (2014) 74–80 A R T I C L E I N F O Article history: Received 7 January 2013 Received in revised form 1 July 2013 Accepted 2 August 2013 Available online 3 February 2014 Keywords: Paraoxonase Nifedipine Nitrendipine Isradipine Amlodipine besylate A B S T R A C T Background: In this study, we investigated the in vitro effects of calcium channel blockers (nifedipine, nitrendipine, isradipine, and amlodipine besylate) on the activity of paraoxonase-1 (PON1). Methods: PON1 was purified from human serum using simple chromatographic methods, including DEAE-Sephadex anion-exchange and Sephadex G-200 gel filtration chromatography. Results: The calcium channel blockers decreased the in vitro PON1 activity. The inhibition mechanism of amlodipine besylate was noncompetitive, whereas nifedipine, nitrendipine, and isradipine were competitive inhibitors. Conclusions: Our results showed that calcium channel blockers exhibit inhibitory effects on PON1 at low concentrations. The IC 50 values for nifedipine, nitrendipine, isradipine, and amlodipine besylate were determined to be 0.121 mM, 0.130 mM, 0.255 mM, and 0.304 mM, respectively, and the K i constants were calculated to be 0.222  0.049 mM, 0.151  0.067 mM, 0.286  0.137 mM, and 0.321  0.002 mM, respectively. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; Mw, molecular weight; PON1, paraoxonase 1; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. * Corresponding author. E-mail address: beydemir@atauni.edu.tr (S ¸ . Beydemir). Contents lists available at ScienceDirect Pharmacological Reports jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/p h arep 1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.007