Sachiko Yamamoto1, Munenori Takehara2, Makoto Ushimaru3. 1. Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan. Electronic address: s_yamamoto@ks.kyorin-u.ac.jp. 2. Department of Materials Science, The University of Shiga Prefecture, Hassaka, Hikone 522-8533, Japan. 3. Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Abstract
BACKGROUND: SERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. METHODS: Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. RESULTS: The Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15-53μM) and partially by oleamide (IC50 8.3-34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. CONCLUSIONS: Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. GENERAL SIGNIFICANCE: Our findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity. Copyright Â
BACKGROUND: SERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acidamides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. METHODS: Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. RESULTS: The Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15-53μM) and partially by oleamide (IC50 8.3-34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. CONCLUSIONS:Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. GENERAL SIGNIFICANCE: Our findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity. Copyright Â
Authors: Muhammad Abdullah; Joe N Kornegay; Aubree Honcoop; Traci L Parry; Cynthia J Balog-Alvarez; Sara K O'Neal; James R Bain; Michael J Muehlbauer; Christopher B Newgard; Cam Patterson; Monte S Willis Journal: Metabolites Date: 2017-07-29