endoplasmic reticulum Ca2+-ATPase

BBAGEN-28597; No. of pages: 7; 4C: Biochimica et Biophysica Acta xxx (2016) xxx–xxx

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Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase Sachiko Yamamoto a,⁎, Munenori Takehara b, Makoto Ushimaru a a b

Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan Department of Materials Science, The University of Shiga Prefecture, Hassaka, Hikone 522-8533, Japan

a r t i c l e

i n f o

Article history: Received 30 March 2016 Received in revised form 30 July 2016 Accepted 1 September 2016 Available online xxxx Keywords: Sarco/endoplasmic reticulum Ca2+-ATPase Primary fatty acid amide Signaling lipid Inhibitor Negative regulator Calcium homeostasis

a b s t r a c t 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 15–53 μM) and partially by oleamide (IC50 8.3–34 μM). 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 45 μM in linoleamide and from 1.6 to 55 μM 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. © 2016 Elsevier B.V. All rights reserved.

1. Introduction In eukaryotic cells, cellular calcium signaling governs many physiological functions, such as muscle contraction, secretion, fertilization, and proliferation [1]. The mechanisms that regulate cellular Ca2+ dynamics and homeostasis are critical to the control of many cellular functions and the viability of cells. Sarco/endoplasmic reticulum Ca2+ATPase (SERCA) is a crucial Ca2+ transport enzyme that is directly involved in intracellular Ca2+ handling [2,3]. This housekeeping transmembrane protein is located in the sarco/endoplasmic reticulum (SR/ER), and its main role is to sequester Ca2+ from the cytoplasm into the SR/ER lumen against the concentration gradient. Active Ca2+ transport by SERCA is achieved through coupling to the ATP hydrolysis reaction, and it contributes to replenishing the Ca2+ content of SR/ER

Abbreviations: [Ca2+]cyto, cytosolic Ca2+ concentration; ER, endoplasmic reticulum; PFAA, primary fatty acid amide; SR, sarcoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; rSERCA1a, rabbit SERCA isoform 1a; hSERCA2b, human SERCA isoform 2b; hSERCA3a, human SERCA isoform 3a. ⁎ Corresponding author. E-mail address: [email protected] (S. Yamamoto).

Ca2+ stores necessary for calcium signaling and to decreasing the Ca2+ concentration in the cytoplasm to basal levels [3,4]. In mammals, more than 10 different SERCA isoforms are encoded by three ATP2A1–3 genes [2]. The isoforms possess distinct enzymatic properties, such as differing affinity for substrate and velocity of Ca2+ uptake. The tissue- and development-specific distribution of one or more SERCA isoforms reflects the important role of SERCA in the proper regulation of Ca2+ dynamics of individual cells [2]. Abnormal alterations in SERCA activity are linked to several diseases such as muscular dystrophy [5] and skin disorder [6]. Endogenously produced biomolecules that modulate SERCA activity have recently received much attention owing to their close involvement in intracellular Ca2+ homeostasis [7,8] and/or their deleterious effects on human health [9,10]. Phospholamban [8] and sarcolipin [11] are the most well-known small proteins that specifically regulate SERCA activity, and other proteins have also been reported recently [12,13]. In addition to these proteins, it has been shown that the lipid bilayer composition regulates the enzymatic properties of SERCA. For example, an adequate lipid bilayer thickness with a chain length of C18–C22 is required to achieve maximum SERCA activity [14–16]. Other physicochemical properties, such as types of lipid headgroups and membrane

http://dx.doi.org/10.1016/j.bbagen.2016.09.001 0304-4165/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001

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S. Yamamoto et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx

fluidity [14], also significantly change the enzymatic turnover of SERCA and its apparent affinity for Ca2+: a serine or ethanolamine headgroup [14,17], or a monounsaturated bond in the acyl chain [14] has a stimulatory effect on SERCA activity. Primary fatty acid amides (PFAAs) are recognized as important signaling lipids that are ubiquitously distributed in the body [18,19]. These endogenously produced molecules have a simple chemical structure, R1-CO-NH2, and were first isolated and characterized from a biological source in 1989 [20]. The biological activities that have been attributed to PFAAs are dependent on the structure of their corresponding acyl group. Oleamide, for instance, consists of an 18-carbon acyl chain containing a sole cis-double bond at C9, and has been identified as an endogenous sleep inducer in the mammalian brain [21]. Among their many reported functions, PFAAs have been described to induce cannabinoid-like actions [22–24], to affect angiogenesis [25], to depress body temperature and locomotor activity [26], to inhibit gap junction communication [27,28], and to increase cytosolic Ca2+ concentration ([Ca2+]cyto) [29,30]. On the basis of the ability of oleamide and linoleamide to stimulate Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores [29,30], we predicted that these amides would have a negative modulatory action on SERCA activity, and thus that PFAAs might function as novel regulators of intracellular Ca2+ transport via the inhibition of SERCA activity. The aim of this study was therefore to evaluate whether PFAAs have regulatory activity toward SERCA proteins. Our findings characterize the inhibitory activity of oleamide and linoleamide toward three major SERCA isoforms. We discuss the inhibitory mechanisms of these amides and have attempted to comprehend the implications of this regulation of SERCA under physiological conditions.

2.3. Ca2+-ATPase activity The rate of Ca2+-dependent ATP hydrolysis was determined by measuring inorganic phosphate (Pi) liberated by the enzyme. The standard reaction mixture contained 10 mM HEPES (pH 7.0), 100 mM KCl, 3 mM ATP, 5 mM MgCl2, 0.5 mM EGTA, 0.45 mM CaCl2 (2.7 μM as free Ca2+), 20–80 μg/ml of microsomal proteins, and 2 μM calcium ionophore A23187. The reaction was carried out at 37 °C. The net ATPase activity was calculated by subtracting the value of ATPase activity determined in 50 mM EGTA (2.7 nM as free Ca2+) from that determined in the standard reaction mixture. To evaluate the effect of PFAAs on Ca2+ATPase activity, amides were dissolved in isopropyl alcohol (IPA) and added to the standard reaction mixture at b 3.5% IPA by volume. This concentration of solvent did not disturb the Ca2+-ATPase activity of SERCA proteins. The added PFAAs did not form insoluble aggregates in the reaction mixture under the concentration range employed in this study, while the turbidity of the mixture was slightly increased in the presence of ≥150 μM PFAAs. To measure the Ca2+ dependency of ATPase activity, various concentrations of Ca2+ were added to the standard reaction mixture at a fixed concentration of 0.5 mM EGTA. Free Ca2+ concentrations were determined by Maxchelator webware. 2.4. Miscellaneous assays Protein concentrations were determined by the method of Lowry et al. [32] with bovine serum albumin as a standard. Inorganic phosphate concentrations were determined by the methods of Fiske and Subbarow [33]. 2.5. Data fitting

2. Materials and methods 2.1. Reagents The following PFAAs were purchased: oleamide (Sigma–Aldrich, St. Louis, MO, USA), elaidamide (Toronto Research Chemicals Inc., North York, Canada), linoleamide (Abcam Biochemicals, Bristol, UK), palmitamide (TCI, Tokyo, Japan), stearamide (TCI), and erucamide (TCI). Calcium ionophore A23187 was obtained from Sigma–Aldrich.

2.2. Expression and preparation of SERCA enzyme Recombinant rabbit SERCA1a (rSERCA1a), human SERCA2b (hSERCA2b), and human SERCA3a (hSERCA3a) were expressed individually in COS-1 cells under the control of the CMV promoter by using an adenovirus expression system in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, CA). COS-1 cells were maintained in growth medium (Dulbecco's Modified Eagle Medium containing 100 units/ml penicillin–streptomycin and 10% fetal calf serum) under 5% CO2 at 37 °C, and were infected with recombinant adenovirus by replacing the growth medium of 100% confluent COS-1 cells with infection medium (growth medium containing recombinant adenovirus vector). Twelve hours after infection in 5% CO2 at 37 °C, the infection medium was replaced with fresh growth medium, and the cells were subsequently harvested at 72 h after infection. Microsomes were prepared as described previously [31] except that 10 mM HEPES was used instead of Tris–HCl. The microsomal preparation was used as SERCA enzymes throughout this study. The concentration of each SERCA in the preparations was 470 (rSERCA1a), 90 (hSERCA2b), and 250 (hSERCA3a) pmol per mg of microsomal protein. The amount of SERCA was quantified by measuring the maximum amount of phosphorylated enzyme intermediate accumulated in the presence of 10 μM [γ-32 P] ATP and saturated Ca2+ at 0 °C.

The value of half-maximal inhibition (IC50) was determined by modeling the ATPase activity profiles as a function of inhibitor concentration ([I]) using the following equation under the assumption that ATPase inhibition proceeds in a cooperative manner with respect to inhibitor binding [34]:
   activity ¼ V max þ ðV min −V max Þ½In = IC50 n þ ½In g where Vmax and Vmin are the maximal and the minimal activity, respectively, achieved under the experimental conditions, and n is the cooperativity coefficient. The Ca2+ dependence of the ATPase activity profile as a function of free Ca2+ concentration was modeled under the condition that Ca2+ causes activation at lower concentrations and inhibition at higher concentrations [34,35] using a modified Hill equation by assuming that ATPase activation and inactivation phases proceed in a cooperative manner with respect to Ca2+ dissociation: activity ¼

 h in1  h in1   h in2  h in2  V max Ca2þ = K m1 n1 þ Ca2þ − V max Ca2þ = K m2 n2 þ Ca2þ

where Vmax is the maximal activity achieved under the experimental conditions, Km1 is the Ca2+ dissociation constant for the activation phase, Km2 is that for the inactivation phase, and n1 and n2 are the Hill coefficients of the activation and inhibitory phases, respectively. 3. Results 3.1. Effect of PFAAs on SERCA activity To evaluate the effect of PFAAs on SERCA, the three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a were overexpressed individually in COS-1 cells via an adenovirus expression system and examined for their susceptibility to such amides. The Ca2+-ATP hydrolysis

Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001

S. Yamamoto et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx

Activity (% of control)

A 125 100 75 50 25 0 1

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B 125 Activity (% of control)

activity of microsomal proteins derived from COS-1 cells producing each SERCA isoform was measured in the presence of 150 μM PFAA [29,30], a concentration used in previous studies. As shown in Fig. 1, linoleamide (18:29,12-cis), a polyunsaturated PFAA of 18 carbons containing two cis-double bonds at C9 and C12, significantly inhibited the Ca2+-ATPase activity of the SERCA isoforms investigated: the Ca2+ATPase activity of hSERCA3a was completely inhibited, whereas that of rSERCA1a and hSERCA2b was reduced to b10% and 20%, respectively, of the control levels. Oleamide (18:19-cis), a monounsaturated fatty acid amide of 18 carbons containing a sole cis-double bond at C9, was also an effective inhibitor of hSERCA3a: the activity of the enzyme decreased to approximately 35% of the control. However, the inhibition of rSERCA1a and hSERCA2b by oleamide was weaker than the inhibition by linoleamide: the ATPase activity of rSERCA1a and hSERCA2b in the presence of oleamide remained above 70% of the control level, although this reduction was statistically significant (p b 0.01 versus control, Student's t-test). By contrast, the respective C18 and C16 saturated fatty acid amides, stearamide and palmitamide, showed no statistically significant effects on the inhibition of the Ca2+-ATPase activity of any SERCA isoform at the 150 μM concentration tested. In addition, at the same concentration, elaidamide (18:1 9-trans), a trans isomer of oleamide, and erucamide (cis-13-docosenoamide) hardly reduced the Ca2+-ATPase activity of hSERCA2b, although they significantly inhibited that of hSERCA3a.

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3.2. Concentration dependency of SERCA inhibition by linoleamide and oleamide To characterize the inhibitory actions of linoleamide and oleamide toward SERCA proteins, the Ca2+-ATPase activity of the three SERCA isoforms was measured in the presence of these amides at a concentration ranging from 0 to 300 μM (Fig. 2). The Ca2+-ATPase activity of each SERCA protein decreased as the concentration of linoleamide and oleamide increased. The inhibition profile of the two amides differed among the SERCA isoforms tested. For linoleamide (Fig. 2A), the Ca2+ATPase activity of hSERCA3a was inhibited at a lower concentration as compared with the other isoforms: 4–40 μM linoleamide suppressed hSERCA3a activity in a dose-dependent manner, whereas slightly

Activity (% of control)

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Fig. 1. Effect of primary fatty acid amides (PFAAs) on the Ca2+-ATPase activity of SERCA. The Ca2+-ATPase activity of rSERCA1a (solid bar), hSERCA2b (shaded), and hSERCA3a (open) was measured in the presence 150 μM PFAA. The concentration of SERCA in the Ca2+-ATPase reaction mixture was 33 μg/ml (rSERCA1a), 78 μg/ml (hSERCA2b), and 49 μg/ml (hSERCA3a). Values are expressed as a percentage of Ca2+-ATPase activity in the absence of PFAA as a control: 0.49 ± 0.01 (rSERCA1a), 0.14 ± 0.01 (hSERCA2b), and 0.21 ± 0.02 (hSERCA3a) μmol of inorganic phosphate (Pi) liberated per min per mg of microsomal protein was taken as 100% activity. Bars represent mean ± SD (n = 4). * Statistically significant (*, p b 0.01; **, p b 0.001, Student's t-test) in each group.

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Oleamide (µM) Fig. 2. Inhibition profile of two PFAAs on the Ca2+-ATPase activity of three SERCA isoforms. The Ca2+-ATPase activity of rSERCA1a (open circles), hSERCA2b (crosses), and hSERCA3a (squares) was measured in the presence of increasing concentration of (A) linoleamide and (B) oleamide. The concentration of SERCA in the Ca2+-ATPase reaction mixture was (A) 33 μg/ml (rSERCA1a), 78 μg/ml (hSERCA2b), and 49 μg/ml (hSERCA3a), and (B) 28 μg/ml (rSERCA1a), 84 μg/ml (hSERCA2b), and 40 μg/ml (hSERCA3a). Values are expressed as a percentage of Ca2+-ATPase activity in the absence of PFAA as a control: (A) 0.49 ± 0.04 (rSERCA1a), 0.13 ± 0.01 (hSERCA2b), and 0.28 ± 0.01 (hSERCA3a), and (B) 0.54 ± 0.03 (rSERCA1a), 0.12 ± 0.007 (hSERCA2b), and 0.31 ± 0.01 (hSERCA3a) μmol of Pi liberated per min per mg of microsomal protein was taken as 100% activity. Bars represent mean ± SD (n = 4).

higher concentrations (20–200 μM) were needed to reduce rSERCA1a and hSERCA2b activity. In addition, the activity of hSERCA2b was transiently increased by linoleamide at 2–10 μM. The IC50 value of linoleamide was estimated as 41 ± 3 μM, 53 ± 7 μM, and 15 ± 0.5 μM for rSERCA1a, hSERCA2b, and hSERCA3a, respectively. For oleamide (Fig. 2B), the ATPase activity of rSERCA1a and hSERCA3a was repressed at 2–10 μM, whereas the activity of hSERCA2b was reduced at concentrations higher than 20 μM. In contrast to linoleamide, the three SERCA isoforms maintained substantial Ca2+ATPase activity at higher concentrations of oleamide (≥50 μM). The residual activity at the maximum inhibition of SERCA was estimated to be approximately 66% for rSERCA1a and hSERCA2b, and 30% for hSERCA3a, consistent with the observations in Fig. 1; thus, 150 μM oleamide was sufficient to assess the maximum reduction in the Ca2+ATPase activity of SERCA proteins. The IC50 of oleamide was estimated to be 11 ± 2, 34 ± 4, and 8.3 ± 1 μM for rSERCA1a, hSERCA2b, and hSERCA3a, respectively. These observations suggest that, as compared with linoleamide, oleamide binds to all three SERCA proteins with higher apparent affinity, at least under the experimental conditions employed. However, linoleamide seems to be a more intense inhibitor of each SERCA isoform, especially rSERCA1a and hSERCA2b, as compared with oleamide. Thus, these amides might inhibit the Ca2+ATPase activity of the three major SERCAs in different manner with respect to the interaction.

Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001

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S. Yamamoto et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx

3.3. Binding affinity of linoleamide and oleamide to SERCA The Ca2+-dependency of the ATPase activity of the SERCA isoforms was measured in the presence of linoleamide and oleamide to elucidate how the two amides interact with and inhibit SERCA enzymes. The concentrations of the amides were based on the IC50 values determined above. The Ca2+-ATPase activity of each SERCA isoform showed a typical bell-shaped curve with respect to the concentration of free Ca2+ in the reaction mixture ([Ca2+]free) [34,35] irrespective of the addition of PFAA (Fig. 3). The ATP hydrolysis and calcium transport reactions of SERCA have been reported to proceed according to the E1/E2 scheme (see Fig. S1) [3,4]. In the case of Fig. 3, the left-hand side of the curve—that is, the phase of ATPase activation in response to increasing [Ca2+]free—was associated with an acceleration of Ca2+ binding from the cytoplasmic side to SERCA during the E2 to Ca2E1 transition state (Fig. S1), resulting in increase in the turnover rate of the enzyme. By contrast, the right-hand side of the curve—that is, the phase of ATPase inhibition due to the high concentration of [Ca2+]free—was related to the inhibition of Ca2+ release from SERCA to the luminal side via the Ca2E1P to E2P transition, causing a decrease in turnover rate. These observations suggest that, also in the presence of PFAAs, the ATP hydrolysis reaction should essentially proceed according to the typical Ca2+ transport cycle of SERCA proteins. In the presence of linoleamide (Fig. 3A–C, Table S1), the [Ca2+]free level giving apparent half-maximal activation (appKm1) and inhibition (appKm2) of ATPase activity was raised in accordance with the increment in amide dose for each SERCA isoform, indicating that linoleamide reduces the apparent Ca2+ affinity of SERCA. In addition, the apparent maximal Ca2+-ATPase activity (appVmax) was decreased in response to amide concentration. Dixon plots and Cornish-Bowden plots of the

A

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80 60

Activity (% of control)

ATPase activation phase (Fig. S2 A–C) were generated to determine the dissociation constant for inhibitor binding [36]. The intersection point of the former plot yields Ki1 for inhibitor binding to the substrate-free enzyme, and that of the latter yields Ki2 for inhibitor binding to the substrate–enzyme complex (Table 1). In the case of rSERCA1a and hSERCA3a, both plots gave an intersection point above and below the horizontal axis, respectively. In the case of hSERCA2b, by contrast, an intersection point was observed only in the Dixon plot, and not in the Cornish-Bowden plot. The Ki1 value obtained for linoleamide was in the micromolar concentration range and was indistinguishable across the three SERCA isoforms. The Ki2 value obtained for rSERCA1a and hSERCA3b was in the tens of micromolar range, and was almost 5-fold larger than Ki1. In the presence of oleamide (Fig. 3D and E, Table 2S), the appKm1 and app Km2 values of rSERCA1a and hSERCA2b significantly increased in response to amide concentration (p b 0.05). The increase in the constants was, however, considerably arrested above a certain concentration of amide (Fig. S3). This finding suggests that oleamide is not a typical competitive inhibitor of these enzymes (Fig. S4A). Accordingly, the dissociation constant (Ki3) for the interaction of oleamide with Ca2+-free form of rSERCA1a and hSERCA2b was calculated by assuming that oleamide negatively regulates the reaction of ATP hydrolysis (Eq. 2 in Fig. S4B [37]). The Ki3 value of the two isoforms indicated that oleamide has higher binding affinity for rSERCA1a than for hSERCA2b (Table 1), while the increment in the appKm1 value of hSERCA2b in response to oleamide concentration was approximately 2.5-fold larger than that of rSERCA1a (Fig. 3D and E, Fig. S3). In contrast, the appKm1 and appKm2 values of hSERCA3a (Fig. 3F and Table 2S) increased with the increment in oleamide concentration without the considerable arrest observed for rSERCA1a and hSERCA2b. Moreover, the appVmax value decreased in

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Free Ca2+ (M) Fig. 3. Ca2+ concentration-dependent inhibition of the ATPase activity of SERCA by PFAAs. In the presence of (A–C) linoleamide and (D–F) oleamide, the Ca2+-ATPase activity of (A, D) rSERCA1a, (B, E) hSERCA2b, and (C, F) hSERCA3a was measured in various concentrations of free Ca2+. The amide concentrations used are indicated in each panel. The concentrations of SERCA proteins in the Ca2+-ATPase reaction mixture were (A–C) 35 μg/ml (rSERCA1a), 74 μg/ml (hSERCA2b), and 51 μg/ml (hSERCA3a), and (D–F) 28 μg/ml (rSERCA1a), 81 μg/ml (hSERCA2b), and 44 μg/ml (hSERCA3a). Values are expressed as a percentage of maximum Ca2+-ATPase activity in the absence of PFAA as a control: (A–C) 0.56 ± 0.004 (rSERCA1a), 0.21 ± 0.001 (hSERCA2b), and 0.48 ± 0.009 (hSERCA3a), and (D–F) 0.75 ± 0.01 (rSERCA1a), 0.15 ± 0.007 (hSERCA2b), and 0.40 ± 0.02 (hSERCA3a) μmol of Pi liberated per min per mg of microsomal protein was taken as 100% activity. Bars represent mean ± SD (n = 4).

Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001

S. Yamamoto et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx Table 1 Dissociation constants for the interaction between PFAAs and SERCA isoforms. PFAA

SERCA isoform

Linoleamide

rSERCA1a hSERCA2b hSERCA3a rSERCA1a hSERCA2b hSERCA3a

Oleamide

Dissociation constant a (μM) Ki1 b

Ki2 b

Ki3 c

9.1 (7.6–11) 6.0 (5.6–6.3) 6.8 (5.4–8.8) ND ND 8.4 (5.9–12)

41 (31–57) –e 35 (26–52) ND ND 55 (49–61)

ND d ND ND 1.6 (1.3–2.0) 4.1 (2.8–5.3) ND

a Dissociation constant for inhibitor binding to the free enzyme (Ki1), inhibitor binding to the substrate–enzyme complex (Ki2), and regulator binding to the enzyme (Ki3). Ki1 and Ki2 were determined by using a Dixon plot and a Cornish-Bowden plot of the Ca2+-ATPase activation phase, respectively, in which the data points in Fig. 3A–C and F were re-plotted as shown in Fig. S2. Ki3 was calculated by data fitting of the values of the Ca2+-ATPase activation phase in Fig. 3D and E to (Eq. 2), as shown in Fig. S4. b Values are derived by the median of the intersection points of three or more straight lines, and are expressed as the best parameters. The numbers in parentheses correspond to the 68.3% confidence interval. c Values are expressed as the mean calculated from the best fits to the data in Fig. 3D and F. The numbers in parentheses correspond to the range of values. d Not determined. e The value was not detected in a Cornish-Bowden plot.

response to amide concentration. The Dixon and Cornish-Bowden plots of the ATPase activation phase gave two dissociation constants: the Ki2 value for the interaction between oleamide and hSERCA3a was 7-fold larger than the Ki1 value (Fig, S2D and Table 1), similar to the case for linoleamide. As summarized in Table 1, the binding affinity of linoleamide for SERCA (Ki1) was indistinguishable across the three isoforms, whereas that of oleamide was significantly higher for rSERCA1a (Ki3) than for hSERCA3a (Ki1). This tendency was not always consistent with the apparent binding affinity reflected in the IC50 values (Fig. 2), where hSERCA3a showed the highest affinity for both amides. The contradiction between these values can be accounted for by the experimental conditions employed: in the inhibition profiles (Fig. 2), the lower [Ca2+]free (2.7 μM) in the ATPase assay mixture would have led to a higher sensitivity of hSERCA3a to amides because hSERCA3a exhibited lower Ca2+ affinity as compared with the other two SERCA isoforms, as shown in Tables S1 and S2 (see appKm1 in the absence of amide). 4. Discussion This study has demonstrated that two mono- and di-unsaturated PFAAs of 18 carbons containing cis-double bond(s), oleamide and linoleamide, inhibit the Ca2+-ATPase activity of three major SERCA isoforms: rSERCA1a, hSERCA2b, and hSERCA3a. The dissociation constants for interaction with SERCA isoforms were 6–41 μM for linoleamide and 1.6–55 μM for oleamide. It is plausible to infer that these amides function as regulators of intracellular Ca2+ homeostasis via their inhibitory action on SERCA activity. PFAAs are known to be bioactive substances that are endogenously produced and exhibit various kinds of physiological effects such as sleep induction [21], modulation of gap junction communication [27, 28], influence of memory processes [38], and depression of locomotor activity [26]; however, the target proteins of these amides and the exact mechanisms underlying the induction of these various physiological effects are not well understood. In this context, we anticipated that SERCA, an enzyme promoting the transport of cytosolic Ca2+ to the SR/ER lumen, might be a potential target protein of oleamide and linoleamide on the basis of two studies reporting that exogenous loading of these amides cause a rise in [Ca2+]cyto levels in several epithelial mammalian cells [29,30]. It can be assumed that the increase in [Ca2+]2+ -transporting activity of SERCA cyto is in part due to inhibition of the Ca during treatment with linoleamide or oleamide. Indeed, we characterized the strong inhibitory effect of linoleamide on the Ca2+-ATPase activity of three major SERCA isoforms, as well as the moderate-to-weak

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effect of oleamide (Figs. 1 and 2). Although the inhibition of SERCA by oleamide was partial, the IC50 values (linoleamide, ≤53 μM; oleamide, ≤34 μM) determined here were highly consistent with the EC50 values (linoleamide, 50 μM [30]; oleamide, 20 μM [29]) previously reported for the amide-induced increase in cytosolic Ca2+ levels in several cell lines. At present, we also anticipate that SERCA might be a target molecule of the hypo-motility effect caused by peripheral administration of oleamide in rat [26] because transport of Ca2+ into SR stores via SERCA is essential in muscle contraction and relaxation [2,39]. The observation of two dissociation constants, Ki1 and Ki2, for the interaction of linoleamide and oleamide with the SERCA isoforms indicates how these PFAAs may inhibit the Ca2+-ATPase activity of SERCA (Table 1). The magnitude of Ki1 was distinguishable from that of Ki2 in the inhibition of rSERCA1a by linoleamide, and in the inhibition of hSERCA3a by linoleamide and oleamide. This observation indicates that each amide acts on the isoforms as a mixed-type inhibitor with respect to Ca2+. In particular, the Ki1 value was 4–7-fold smaller than Ki2, which means that the mixed-type inhibition might proceed predominantly via binding to Ca2+-free SERCA and by reducing the apparent Ca2+ affinity of these isoforms. On the other hand, the observation of Ki1 alone for the inhibition of hSERCA2b by linoleamide suggests that this amide inhibits hSERCA2b activity by lowering the apparent Ca2+ affinity. Although linoleamide and oleamide have a tendency to inhibit SERCA in a competitive manner with respect to Ca2+, these amides are likely to interact with another region of the enzyme aside from the Ca2+ binding site, based on their structure and the higher hydrophobicity of PFAA (log P N 6). Linoleamide and oleamide might interact with the transmembrane domains of SERCA, and might disturb dynamic conformational changes of these domains that are relevant to Ca2+ binding and transport, as observed for SERCA-specific inhibitors [40–42]. It has also been reported that the Ca2+ affinity of SERCA directly depends on membrane fluidity and the headgroup of the membrane lipid [14]. Notably, a competitive inhibitory mode also has been described for phospholamban and sarcolipin [8,43], two endogenously produced SERCA-specific regulatory proteins. These proteins reduce the apparent Ca2+ affinity of SERCA by binding to a region of the SERCA transmembrane domain away from the Ca2+-binding site [44–46]. They contribute to the strict regulation of cell- and development-specific intracellular Ca2+ homeostasis [8,43]. Therefore, we speculate that linoleamide and oleamide might provide additional regulation of SERCA activity and contribute to the overall control of Ca2+ homeostasis. Whether endogenously produced PFAAs play a modulatory role in SERCA and Ca2+ homeostasis should be the subject of future studies. Our findings suggested that oleamide has a negative regulatory action toward rSERCA1a and hSERCA2b: these enzymes seemed to retain their activity despite the amide interaction, even though their affinity for Ca2+ was significantly lowered. There are two points in support of this: first, a plateau region was observed in the inhibition profile of oleamide at higher concentrations (Fig. 2B); and second, the increase in the appKm1 and appKm2 values was arrested above certain concentrations of oleamide (Fig. 3D and E, Table S2). In accordance with the first point, oleamide might also possess negative regulatory action toward hSERCA3a; however, the second point was not observed for hSERCA3a under the oleamide concentration ranges employed (Fig. 3F). SERCA enzymes, particularly the type 1 and type 2 isoforms, are critical for maintaining Ca2+ stores in both muscle and/or nonmuscle cells, and regulate [Ca2+]cyto levels tightly to control many cellular functions; as a result, loss of their function leads to lethal disorders [2,47]. In contrast, disruption of the SERCA3 gene results in normal viable mutant mice [47,48]. Meanwhile, oleamide is an important signaling lipid that mediates many cellular functions and is distributed throughout body at relatively higher levels as compared with linoleamide [18,20]. The physiological levels of PFAA have been reported as 6.4 nmol/107 cells (sheep choroid plexus cells) and 160 nM (rat cerebrospinal fluid) for oleamide, and 0.25 nmol/107 cells (sheep choroid plexus cells) for linoleamide, although their levels in

Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001

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S. Yamamoto et al. / Biochimica et Biophysica Acta xxx (2016) xxx–xxx

other organ systems have not been documented [18,49]. In addition, oleamide was found to have substantially higher affinity for rSERCA1a (Ki3 = 1.6 μM) and hSERCA2b (Ki3 = 4 μM) than for hSERCA3a (Ki1 = 8 μM) (Table 1). Accordingly, rSERCA1a and hSERCA2b might intrinsically have an ability to prevent harmful inhibition by endogenous oleamide that is generated in abundance. This study has indicated the concentration ranges of linoleamide and oleamide that can exert SERCA inhibition. The effective ranges seem to be the same as those reported for other actions of oleamide or linoleamide [19,50,51]. It should be kept in mind, however, that determination of the exact effective concentration of these PFAAs toward SERCA is challenging for several reasons: for example, the PFAAs added into the reaction mixture might form micelles, the PFAAs might not be fully dissolved in the mixture at higher concentration (≥ 150 μM), or the PFAAs might be trapped and concentrated in the membrane lipids of microsomes. Although these issues make it difficult to determine not only the effective concentrations of PFAA but also how PFAAs interact with SERCAs, what is certain is that linoleamide and oleamide reduce the Ca2+-ATPase activity of each SERCA isoform in a different manner with different affinity. 5. Conclusions This study has revealed that linoleamide and oleamide function as novel inhibitors of SERCA proteins. These PFAAs were found to reduce the Ca2+-ATPase activity of three major SERCA isoforms, with estimated IC50 values of ≤ 53 μM for linoleamide and ≤ 34 μM for oleamide. Linoleamide and oleamide inhibit SERCA activity by lowering the apparent Ca2+ affinity. These observations lead to the suggestion that SERCA might be a target protein of linoleamide and oleamide to induce an increase in cytosolic Ca2+ levels in several human cell lines. Conflicts of interest The authors declare no conflicts of interest. Transparency document The Transparency document associated with this article can be found, in online version. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagen.2016.09.001. References [1] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signalling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11–21. [2] M. Periasamy, A. Kalyanasundaram, SERCA pump isoforms: their role in calcium transport and disease, Muscle Nerve 35 (2007) 430–442. [3] M. Brini, E. Carafoli, Calcium pumps in health and disease, Physiol. Rev. 89 (2009) 1341–1378. [4] J.M. East, Sarco(endo)plasmic reticulum calcium pumps: recent advances in our understanding of structure/function and biology (review), Mol. Membr. Biol. 17 (2000) 189–200. [5] A. Odermatt, K. Barton, V.K. Khanna, J. Mathieu, D. Escolar, T. Kuntzer, G. Karpati, D.H. MacLennan, The mutation of Pro789 to Leu reduces the activity of the fasttwitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) and is associated with Brody disease, Hum. Genet. 106 (2000) 482–491. [6] A. Sakuntabhai, V. Ruiz-Perez, S. Carter, N. Jacobsen, S. Burge, S. Monk, M. Smith, C.S. Munro, M. O'Donovan, N. Craddock, R. Kucherlapati, J.L. Rees, M. Owen, G.M. Lathrop, A.P. Monaco, T. Strachan, A. Hovnanian, Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease, Nat. Genet. 21 (1999) 271–277. [7] P. Vangheluwe, L. Raeymaekers, L. Dode, F. Wuytack, Modulating sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological implications, Cell Calcium 38 (2005) 291–302. [8] D.H. MacLennan, E.G. Kranias, Phospholamban: a crucial regulator of cardiac contractility, Nat. Rev. Mol. Cell Biol. 4 (2003) 566–577.

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Please cite this article as: S. Yamamoto, et al., Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.09.001