Cell Calcium 55 (2014) 231–237

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Effects of endogenous cannabinoid anandamide on cardiac Na+ /Ca2+ exchanger Lina T. Al Kury a , Keun-Hang Susan Yang e , Faisal T. Thayyullathil b , Mohanraj Rajesh a , Ramez M. Ali a , Yaroslav M. Shuba d , Frank Christopher Howarth c , Sehamuddin Galadari b , Murat Oz a,∗ a

Laboratory of Functional Lipidomics, Department of Pharmacology, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates c Department of Physiology, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates d Bogomoletz Institute of Physiology and International Center of Molecular Physiology, National Academy of Sciences of Ukraine, Kyiv 24, Ukraine e Department of Biological Sciences, Schmid College of Science and Engineering, Chapman University, One University Drive, Orange, CA 92866, USA b

a r t i c l e

i n f o

Article history: Received 3 December 2013 Received in revised form 17 February 2014 Accepted 23 February 2014 Available online 11 March 2014 Keywords: Anandamide Na+ /Ca2+ exchanger Whole-cell patch clamp technique

a b s t r a c t Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using wholecell patch clamp technique, we have investigated the effects of AEA on cardiac Na+ /Ca2+ exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 ␮M. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 ␮M) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 ␮M URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 ␮M) and AM630 (1 ␮M), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 ␮g/ml) treatment or following the inclusion of GDP-␤-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 ␮M AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Endocannabinoids are a class of polyunsaturated fatty acidbased compounds that mimic most of the effects of 9 tetrahydrocannabinol (THC), the active ingredient of the marijuana plant Cannabis sativa. N-arachidonoyl ethanolamide (AEA) or anandamide and 2-arachidonylglycerol (2-AG) are the most widely studied endogenous cannabinoids [1,2]. In recent years, extensive research focusing on the biological actions of endocannabinoid compounds indicated that they have important regulatory roles in several physiological and pathological conditions [1,3,4]. It has been shown that the endocannabinoid system consists of at least

∗ Corresponding author at: Department of Pharmacology, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666 Al Ain, Abu Dhabi, United Arab Emirates. Tel.: +971 3 7137523; fax: +971 3 7672033. E-mail address: murat [email protected] (M. Oz). http://dx.doi.org/10.1016/j.ceca.2014.02.017 0143-4160/© 2014 Elsevier Ltd. All rights reserved.

the endocannabinoid receptors (CB1 and CB2 cannabinoid receptors), the enzymes regulating the synthesis (such as phospholipaseD, and monoacylglycerol lipase), and the degradation (such as fatty-acid amide hydrolase and lipases) processes, and the proteins involved in their transport across the biological membranes [1,4]. CB1 receptors are located in the brain and several peripheral tissues including the heart and the vasculature [4,5]. The CB2 receptors, on the other hand, are expressed primarily in the immune system but recently their presence in the brain, myocardium, and smooth muscle cells has also been demonstrated [4]. Recent studies suggest that endocannabinoids have important modulatory roles on the function of the cardiovascular system under various pathological conditions, such as hypertension, myocardial infarction and heart failure [6,7]. AEA, the most studied endocannabinoid, has a complex set of actions on cardiac functions. Experiments with AEA performed in isolated Langendorff rat hearts and in isolated, electrically stimulated human atrial appendages [5,8] have revealed a negative inotropic effect which

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may underlie its ability to decrease cardiac output as observed in studies performed in vivo [9]. Moreover, AEA and other cannabinoids have been reported to have antiarrhythmic effects in in vivo animal models [10,11]. However, electrophysiological mechanisms underlying these cardiac actions of AEA remain largely unknown. We hypothesized that some of the actions of AEA on cardiomyocytes can be mediated by the modulation of NCX1 which plays a major role in Ca2+ homeostasis and generation of arrhythmias [12,13]. Thus, in the present study, using whole-cell patch clamp, we investigated the actions of AEA on NCX1-mediated currents (INCX1 ) in acutely dissociated rat ventricular myocytes and in HEK-293 cells expressing NCX1. 2. Materials and methods 2.1. Ventricular myocyte isolation The work was performed with approval of the Animal Research Ethics Committee of the College of Medicine and Health Sciences (Al Ain, UAE). Animals were bred at our own Animal Facility from the original stock. The animals were housed in polypropylene cages in climate and access controlled rooms (22–24 ◦ C; 50% humidity). The day/night cycle was 12 h/12 h. Food and water were provided ad libitum. Ventricular myocytes were isolated from adult male Wistar rats (250–270 g) according to previously described techniques [14]. Briefly, the animals were euthanized using a guillotine and hearts were removed rapidly and mounted for retrograde perfusion according to the Langendorff method. Hearts were perfused at a constant flow rate (8 ml/min/g weight of tissue) at 36–37 ◦ C with a solution containing (mM): 130 NaCl, 5.4 KCl, 1.4 MgCl2 , 0.75 CaCl2 , 0.4 NaH2 PO4 , 5 HEPES, 10 glucose, 20 taurine, and 10 creatine set to pH 7.3 with NaOH. Once the heart had stabilized, perfusion was continued for 4 min with Ca2+ -free isolation solution containing 0.1 mM EGTA, and then for 6 min with cell isolation solution containing 0.05 mM Ca2+ , 0.75 mg/ml collagenase (type 1; Worthington Biochemical Corp, USA) and 0.075 mg/ml protease (type X1 V; Sigma, Germany). Ventricles were excised from the heart, minced and gently shaken in collagenase-containing isolation solution supplemented with 1% BSA. Cells were filtered from this solution at 4 min intervals and re-suspended in isolation solution containing 0.75 mM Ca2+ . 2.2. Recordings of NCX1 currents in cardiomyocytes Currents through NCX1 were recorded from rat isolated ventricular myocytes using the patch clamp technique in whole cell configuration. Currents were filtered at 5 kHz and digitized using a Digidata 1322 interface. Briefly, INCX1 was recorded using a descending voltage ramp from +100 mV to −100 mV from a holding potential of −40 mV for 2 s duration. As described previously [15], INCX1 was measured as current sensitive to nickel (Ni2+ ), therefore, Ni2+ -insensitive components were subtracted from total currents to isolate INCX1 . External solution contained (in mM): 150 NaCl, 5 CsCl, 2 CaCl2 , 2 MgCl2 , 10 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES), 10 glucose (pH = 7.4). Nifedipine (10 ␮M), oubain (100 ␮M), and niflumic acid (30 ␮M) were used to block Ca2+ , Na+ - K+ -ATPase, and Cl− currents, respectively. 10 mM nickel chloride solution was used to block INCX1 . K+ currents were minimized by Cs+ substitution for K+ in both pipette and external solutions. The pipette solution contained (in mM): 120 CsCl, 20 NaCl2 , 10 tetraethylammonium chloride (TEACl), 2 MgCl2 , 1 CaCl2 , 10 HEPES, 1 MgATP and 10 1,2-bis(o-aminophenoxy)ethaneN,N,N ,N -tetraacetic acid (BAPTA) (pH = 7.2) with CsOH. The combination of 10 mM BAPTA and 1 mM Ca2+ in the pipette

solution gave a free [Ca2+ ]i of 20 nM (calculated with the “Maxchelator program”; WEBMAX v 2.10, Stanford, CA, USA, which was supplied by Dr. D. Bers). For experiments including pertussis toxin (PTX) pretreatment, cells were incubated with PTX (2 ␮g/ml) for 3 h at 37 ◦ C (control cells to this group were incubated in the same conditions with distilled water only). Changes of external solutions and application of drugs were performed using a multi-line perfusion system with a common outflow connected to the recording chamber. The pipettes were fabricated from filamented BF 150-86-10 borosilicate glass (OD = 1.5 mm, ID = 0.86 mm) (Sutter Instruments Co., CA, USA) on a horizontal puller (Sutter Instruments Co., CA, USA). Electrode resistance ranged from 2.0 to 4.0 M. Experiments were performed at room temperature (22–24 ◦ C). Electrophysiological data were analyzed using pClamp 10.2 (Molecular Devices, Union City, CA, USA) and Origin 7.0 (OriginLab Corp., Northampton, MA, USA) software. The amplitudes of the currents were normalized to the cell membrane capacitance to provide current densities (pA/pF). 2.3. Western blot analysis Ventricles were obtained from normal Wistar rats. Tissue samples were flash-frozen in liquid nitrogen and stored at −80 ◦ C. After thawing, tissue extracts were prepared by homogenization on ice with RIPA buffer (Pierce Biotechnology, IL, USA) supplemented with protease inhibitors (Roche, GmbH, IN, USA). Later, the extracts were clarified to remove the cellular debris by centrifugation at 13,000 r.p.m. for 15 min at 4 ◦ C. Protein content in the extracts was determined using the Lowry assay (BioRad). A measure of 50 ␮g protein was resolved in 12% SDS-PAGE and was transferred onto nitrocellulose membranes (GE Healthcare, UK). Blocking was performed for 2 h at room temperature with 5% nonfat skimmed milk powder prepared in phosphate buffer solution (PBS) containing 0.1% Tween 20 (Sigma, CA, USA). After washing with phosphate-buffered saline 0.1% Tween 20 (PBST), the membranes were probed with either rabbit polyclonal CB1 (Cayman Chemicals, 1:1000 dilution) or with an antibody raised against the last 15 residues of rat CB1 or CB2 antibody (Cayman Chemicals, 1:1000 dilution) overnight at 4 ◦ C. After washing with PBST, membranes were incubated for 1 h at room temperature in HRP (horseradish peroxidase)-coupled secondary antibody (goat antirabbit) (GE Biosciences, UK). Subsequently, the membranes were washed with PBST and were developed using chemiluminescence detection kit (Super Signal-West Pico Substrate, Pierce). To confirm uniform loading, the membranes were stripped and re-probed with ␤-actin (Chemicon, CA, USA). 2.4. Cell culture Human Embryonic Kidney 293 (HEK-293) cells were maintained in DMEM/Ham’s F-12 medium (50:50; Mediatech, Inc., Herndon, VA, USA) supplemented with 10% FBS. They were grown in a humidified atmosphere at 37 ◦ C and 5% CO2 . Twenty four hours after plating, cells were transiently transfected with cDNA for NCX1 using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Experiments were performed 48 h after transfection when cells were 70–80% confluent. 2.5. Image analysis Fluorescence signal was determined from the average pixel intensity within the cell using NIH-image-J. Cell surface pixel intensity was determined from the region of interest drawn inside and outside the cell surface. Pixel intensities were determined from the initial time point of YFP fluorescence (excitation, 488 nm; emission, 525–575 nm) and were compared after 10 min exposure to

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AEA. Values for the pixel intensities were expressed as arbitrary units provided by NIH-image-J. Fluorescent images were processed using Image J software (W. Rasband, NIH).

from Ascent Scientific (Cambridge, UK). All other materials used in this study (including PTX) were purchased from Sigma–Aldrich (St. Louis, MO, USA).

2.6. Recordings of NCX1 currents in HEK-293 cells

2.7. Data analysis

HEK-293 cells were bathed in an external solution containing the following (in mM): 130 NaCl; 5 CsCl; 1.2 MgSO4 ; 1.2 NaH2 PO4 ; 5 CaCl2 ; 10 HEPES; and 10 glucose (pH 7.4). Nifedipine (10 ␮M), oubain (100 ␮M), and niflumic acid (30 ␮M) were used to block Ca2+ , Na+ - K+ -ATPase, and Cl− currents, respectively. Patch Pipettes were filled with a solution containing the following (in mM): 120 Cs+ -glutamate; 10 mM NaCl; 1 MgCl2 ; 10 HEPES; 2 Na2 ATP; 10 BAPTA; 1 CaCl2 and adjusted to pH 7.2 with CsOH. Patch electrodes were pulled from borosilicate glass capillaries with a programmable Flaming-Brown micropipette puller (P-97; Sutter Instruments Co., Novato, CA, USA) and heat-polished to a final tip resistance of 2–4 M. Whole cell currents were recorded at room temperature (22–24 ◦ C), using an Axopatch 200B amplifier and the PClamp 8.1 software (Axon Instruments, Foster City, CA, USA). Currents were low-pass filtered at 5 kHz, stored on an IBM compatible computer and analyzed off-line by the Clampfit program (Axon). Cells were continuously superfused with bathing solution in the absence and presence of drugs. The cells were voltageclamped at a holding potential of −60 mV and a descending voltage ramp from +100 mV to −100 mV was applied for 2 s to elicit membrane currents. AEA, metAEA, AM251 and AM630 were purchased

The results of the experiments were expressed as means ± standard error of the means (S.E.M). Statistical analysis was performed using Student’s t-test or ANOVA using the computer software OriginTM (Originlab Corp. Northampton, MA, USA). On the graphs, * donates statistical significance with P < 0.05 between specified values, or if not specified, to the respective control.

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3. Results In initial studies, we have observed that after 15–20 min of AEA (10 ␮M) exposure, the passive membrane properties of the ventricular cells were not significantly altered. Resting membrane potentials were −77.3 ± 1.6 and −79.2 ± 1.8 mV in control (n = 21) and AEA treated (n = 18) myocytes, respectively. The mean cell capacitance in the control group was 121.3 ± 15.6 pF, whereas in the AEA treated cells was 117.5 ± 14.8 pF. The input resistance (measured close to the resting potential) was 79.6 ± 17.1 M in the control cells and 82.7 ± 15.9 M in AEA-treated cells. Currents mediated by NCX1 were elicited by descending voltage ramp pulses applied between +100 mV and −100 mV (dV/dt = 0.1 V/s) from a holding potential of −40 mV (Fig. 1A). In

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Voltage (mV) Fig. 1. Effect of AEA on INCX1 in rat ventricular myocytes: AEA inhibits INCX1 recorded using whole cell voltage clamp mode of patch clamp technique. (A) Time course of the effects of AEA on the inward and outward INCX1 recorded in a cardiomyocyte. INCX1 was elicited by 2 s voltage ramps from +100 mV to −100 mV every 15 s. Amplitudes of currents recorded at +100 mV and −100 mV were presented as a function of time in the figure. Drug application times were indicated by horizontal bars in the figure. Arrows correspond to the time points for the currents shown in panel B. (B) Current traces were recorded in control, after 5 min application of 10 ␮M AEA, and following 10 mM Ni2+ for 5 min. (C) Mean current density–voltage relationship of NCX1 in the absence and presence of 10 ␮M AEA. Ni2+ (10 mM) was added to the superfusing solution at the end of each experiment to obtain the Ni2+ -sensitive current, representing INCX1 , calculated by subtracting the currents recorded in Ni2+ from the current recorded without Ni2+ . Data points (mean ± S.E.M.) are from 7 cells. (D) Quantification of the extent of AEA inhibition of INCX1 at different membrane potentials. Data points (mean ± S.E.M.) are from 6 cells.

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order to verify that currents recorded in our experimental conditions, are mediated by the NCX1, we have used Ni2+ (10 mM). Bath application of Ni2+ for 5 min reversibly suppressed INCX1 , indicating that these currents are mediated by NCX1 in cardiomyocytes. Ni2+ (10 mM) was used routinely at the end of each experiment to determine the Ni2+ -sensitive NCX1 current (Fig. 1A and B). Command pulses were applied every 15 s and amplitudes of currents at +100 mV and −100 mV were plotted as a function of time (Fig. 1A). AEA largely attenuated both the outward and inward components of Ni2+ -sensitive current. The effect of AEA was detectable at 2–3 min and reached a steady-state level within 5 min (Fig. 1A). Within the time course of the experiment, the recovery was partial. Fig. 1B shows representative current traces in control solution, in the presence of AEA (10 ␮M) and in the presence of Ni2+ (10 mM). In our studies, AEA was dissolved in ethanol, maximal amplitudes of INCX1 were not altered by 10–15 min application of ethanol up to the concentration of 0.07% (V/V; n = 6, data not shown). Fig. 1C shows the mean current–voltage (I–V) relationships for Ni2+ -sensitive INCX1 in control and in the presence of 10 ␮M AEA. In order to determine whether or not AEA affected Ni2+ -sensitive INCX1 selectively, we tested the effect of AEA on Ni2+ -insensitive residual current. No significant alteration of residual current was observed (data not shown; n = 7). Evaluation of the AEA inhibition of INCX1 at different membrane potentials (Fig. 1D) indicated that AEA inhibits both outward and inward components of INCX1 equally. Collectively, these observations indicate that, AEA exerted an inhibitory effect on both inward and outward currents of NCX1. The effect of increasing AEA concentrations on the outward (measured at +100 mV) components of INCX1 was demonstrated in Fig. 2A. AEA inhibited INCX1 in a concentration-dependent manner with IC50 values of 4.7 ␮M. In the next series of experiments we have tested the effect of methanandamide (metAEA), a nonhydrolysable AEA analogue [16], to avoid likely confounding effects of degradation products and oxygenated metabolites on NCX1. At a concentration of 10 ␮M, metAEA also caused a significant inhibition of exchanger current (Fig. 2B). AEA is degraded mainly by fatty acid amide hydrolase (FAAH). In order to test whether the degradation products of AEA mediates its effect on NCX1, we have tested the effect of AEA in the presence of 1 ␮M URB597, a specific FAAH inhibitor. After incubation of cardiomyocytes with URB597 for 1 h, AEA continued to inhibit the function of NCX1 (Fig. 2C), further suggesting that intact AEA molecule, not the degradation products, mediates its effect on the exchanger. In order to confirm the expression of cannabinoid receptors in the ventricular tissue, Western blot analysis was performed. As shown in Fig. 3, cannabinoid receptors (CB1 and CB2 ) are expressed in ventricular tissue of Wistar rats. AEA has been shown to activate both CB1 and CB2 receptors which are coupled to PTX-sensitive Gi/o type G-proteins [4]. Therefore, it was likely that activation of cannabinoid receptors mediates the inhibitory effects of AEA on NCX1. However, in the presence of 1 ␮M AM251, a CB1 antagonist with Ki of 7.5 nM [17] and 1 ␮M AM630, a CB2 antagonist with Ki of 32.1 nM [18], AEA (10 ␮M) inhibition of INCX1 remained unaltered (n = 6–8, Fig. 4A and B). We have also tested whether the inhibitory effect of AEA on INCX1 can be modulated by PTX pretreatments. Our results show that the inhibitory effect of AEA on the maximal amplitudes of INCX1 was not affected by PTX pretreatment. In controls AEA caused 67 ± 5% and 71 ± 4% inhibition of forward and reverse mode of NCX currents, respectively (n = 6). In PTX-pretreated cells, AEA caused 64 ± 5% and 68 ± 6% inhibition of forward and reverse mode of NCX currents (n = 7), respectively. There was no statistically significant difference between these groups (P > 0.05, ANOVA). In positive control experiments, PTX, as it has been reported earlier [19], effectively attenuated the inhibitory actions of BRL-37344, a ␤3 adrenergic

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Fig. 2. Effects of increasing AEA concentrations and the effect of metAEA and fatty acid amide hydrolase inhibitor, URB597 on INCX1 in rat ventricular myocytes. (A) Concentration response curves of AEA on INCX1 . Ni2+ – sensitive current was measured at +100 mV. Data points (mean ± S.E.M.) are from 6 to 8 cells. (B) The effect of 10 ␮M metAEA on net INCX1 (after subtraction of current in10 mM Ni2+ ). Data points (mean ± S.E.M.) are from 5 cells. (C) The effect of 10 ␮M AEA on INCX1 in cardiomyocytes incubated with 1 ␮M URB597 for 1 h. Data points (mean ± S.E.M.) are from 6 cells.

Fig. 3. Expression of CB1 and CB2 receptors in rat heart. Expression of CB1 and CB2 receptors in the heart of control Wistar rats (n = 3) were analyzed by Western blotting.

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receptor agonist, on L-type voltage-gated Ca2+ currents recorded in ventricular myocytes (data not shown; n = 6). GDP-␤-S is also commonly used to inhibit the responses mediated by the activation of G-protein receptors [20]. For this reason we have tested the effect of AEA in the presence of GDP-␤-S in intracellular solution. After the inclusion of GDP-␤-S (1 mM) in pipette solution, AEA continued to inhibit NCX1 both in forward and reverse modes. In the presence of GDP-␤-S, AEA caused 61 ± 5% and 65 ± 6% inhibition of forward and reverse mode of NCX currents, respectively (n = 7). AEA (10 ␮M) effectively reduced the amplitudes of INCX1 in HEK-293 cells expressing NCX1 (Fig. 5A and B). AEA inhibited both inward and outward NCX1-mediated currents. YFP-tagged-NCX1 protein was also expressed in HEK-293 cells (Fig. 5C) and the cell surface expression was monitored by determining the pixel intensity of YFP signals in confocal microscopy images. The results of experiments indicated that pixel intensity of YFP-NCX1 on the cell surface was not altered by treatment with 10 ␮M AEA (Fig. 5D). 4. Discussion The results of this study indicate for the first time that AEA directly inhibits the activity of NCX1 in ventricular myocytes. Under physiological conditions, inhibition of NCX1 operating in reverse mode is expected to decrease Ca2+ entrance during the cardiac action potential, and induce negative inotropic actions. In fact, in several earlier studies, negative inotropic effects of AEA and other cannabinoids have been reported. Experiments with AEA

and the synthetic cannabinoid HU-210 performed in isolated Langendorff rat hearts and in isolated, electrically stimulated human atrial appendages [5,8] have revealed a negative inotropic effect of cannabinoids that may underlie the ability of AEA and HU-210 to decrease cardiac output as observed in studies performed in vivo [9]. Involvement of cannabinoid receptors in the negative inotropic and antiarrhythmic actions of cannabinoids has been investigated in several earlier studies [6]. However, the results of these investigations have not been conclusive [21–23]. Both cannabinoid receptor-dependent and independent mechanisms have been suggested [23]. Our findings suggest that neither CB1 nor CB2 receptors are involved in AEA inhibition of NCX in rat cardiomyocytes. Firstly, AEA inhibition was altered by neither CB1 nor CB2 receptor antagonists. Secondly, treatment with PTX or inclusion of GDP-␤-S in pipette solution did not affect the activity of NCX1 suggesting that G-proteins are not involved in AEA actions. Finally, in HEK293 cells which do not contain CB1 or CB2 receptors [24], AEA significantly inhibited NCX1-mediated currents. Collectively these results suggest that AEA interacts directly with NCX1 in ventricular myocytes in a manner that is independent of CB1 and CB2 receptors. In rat odontoblasts, cannabinoid-induced Ca2+ influx through TRPV1 was recently shown to be functionally coupled to NCXmediated Ca2+ extrusion [25]. It is unlikely that TRPV1 activation is involved in the effects of AEA observed in this study since adult cardiomyocytes do not express TRPV1 channels [26]. It was recently

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Fig. 5. Effects of AEA on NCX1 – mediated currents and the cell surface intensity of YFP-NCX1 expressed in HEK-293 cells. (A) Current traces were recorded in control, after 5 min application of 10 ␮M AEA, and following 10 mM Ni2+ for 5 min. Voltage protocol to elicit INCX1 was presented above the panel. (B) Mean net current density–voltage relationship of NCX1 – mediated currents in the absence and presence of 10 ␮M AEA. Ni2+ (10 mM) was added to the superfusing solution at the end of each experiment to obtain the Ni2+ – sensitive current, representing INCX1 . Data points (mean ± S.E.M.) are from 5 to 6 cells. * Indicates statistically different from the control values at the level of P < 0.05. (C) Confocal microscopy image of YFP-NCX1 protein expressed on the surface of HEK-293 cells. (D) The effect of 10 ␮M AEA on the pixel intensity of YFP-NCX1 expression on the surface of HEK-293 cells. Data points (mean ± S.E.M.) are from three separate experiments (17 cells).

reported that, under ischemic conditions, AEA inhibits NCX1 by CB2 receptor agonists via PTX-sensitive Gi/o proteins [27]. However, another recent study in endothelial cells demonstrated that AEA, in the concentration range used in our study, significantly inhibits the activity of NCX in a manner that is independent of G-protein receptors [20]. Metabolic degradation products of AEA, such as arachidonic acid and related fatty acids have been shown to regulate NCX1 function [28]. In our study, metAEA also inhibited INCX1 . Furthermore, in the presence of URB597, an inhibitor of enzyme (FAAH) that hydrolyzes AEA, INCX1 was suppressed to the same extent by AEA, suggesting that the degradation products of AEA were not involved in the inhibition of NCX1 in cardiac myocytes. In HEK-293 cells, cell surface expression, as determined from the intensity of YFP-NCX1 expression levels, was not altered after AEA applications suggesting that AEA is not likely to alter NCX1 trafficking to cell surface. AEA belongs to a group of fatty acid-based molecules called long-chain N-acylethanolamines (NAEs) which are produced abundantly in response to tissue necrosis and cellular stress [29,30]. In fact, accumulation of NAEs was first observed in experimental myocardial infarction induced by ligation of coronary arteries in canine heart [31–33]. It was demonstrated that NAE content increases up to 500 nmol/g (approximately 500 ␮M) in infarcted areas of canine heart during ischemia [31]. Although AEA constitutes a minor (1–3%) portion of total NAE levels [33], the partition coefficient of AEA is in the same order with that of arachidonic acid to biological membranes (2 − 9 × 104 ) [34]. Thus, the membrane concentration of AEA would reach much higher levels than those estimated for intracellular concentrations. Therefore, AEA, in the concentrations used in this study is likely to regulate the function of NCX1 and may have important implications regarding the

contractile and electrical responses of ventricular myocytes to ischemia and cellular stress [29,30,33]. We have previously reported that major NAEs species produced during ischemia have significant effects on the amplitudes and kinetics of action potentials and accompanying ionic currents that could account for the negative inotropic actions of these compounds on ventricular myocytes [35]. For example, functions of voltage-gated Ca2+ [35–40] and Na+ [35–41] channels have been demonstrated to be modulated by NAEs including AEA, suggesting that during cell stress these molecules can regulate the function of several ion channels and exchangers such as NCX. It is likely that, due to their high lipophilicity, AEA can alter the physico-chemical characteristics of the lipid environment or bind to hydrophobic sites on the ion channels and regulate the functional properties of these proteins ([42], for a review, [43]). Following their insertion into fluid membrane bilayer, ion channels assume an energetically minimal conformational state leading to a stable structure. Importantly, the binding of ligand leads to conformational changes associated with the alterations in the hydrophobic domains of the ion channels (for reviews, [44,45]). The energetic requirements of these conformational changes depend on the lipid environment in which they are immersed [46]. In addition to the NCX, SR Ca2+ -ATPase (SERCA2) also plays an important role in cardiac contraction and rhythmicity [47,48]. Under our experiment conditions, we cannot rule out the contribution of SERCA2 to the observed actions of AEA on the function of NCX in cardiomyocytes. However, AEA continued to inhibit the NCX1 currents in HEK-293 cells which, although express SERCA2 endogenously [49], is devoid of excitability. Furthermore, bath application of AEA does not alter intracellular Ca2+ levels in these cell lines [24]. In conclusion, the results of this study indicate for the first

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