Inhibitory actions of bisabolol on α7-nicotinic acetylcholine receptors

Accepted Manuscript Inhibitory actions of Bisabolol on α7-nicotinic acetylcholine receptors Syed Nurulain, Tatiana Prytkova, Ahmed M. Sultan, Olexandr Ievglevskyi, Dietrich Lorke, Keun-Hang Susan Yang, Georg Petroianu, Frank C. Howarth, Nadine Kabbani, Murat Oz PII: DOI: Reference:

S0306-4522(15)00743-5 http://dx.doi.org/10.1016/j.neuroscience.2015.08.019 NSC 16506

To appear in:

Neuroscience

Accepted Date:

10 August 2015

Please cite this article as: S. Nurulain, T. Prytkova, A.M. Sultan, O. Ievglevskyi, D. Lorke, K.S. Yang, G. Petroianu, F.C. Howarth, N. Kabbani, M. Oz, Inhibitory actions of Bisabolol on α7-nicotinic acetylcholine receptors, Neuroscience (2015), doi: http://dx.doi.org/10.1016/j.neuroscience.2015.08.019

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Bisabolol inhibits α7-nACh receptors

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Inhibitory actions of Bisabolol on 7-nicotinic acetylcholine receptors

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Syed Nurulain, 2Tatiana Prytkova, 1Ahmed M. Sultan, 1Olexandr Ievglevskyi, 3Dietrich Lorke,

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Keun-Hang Susan Yang, 3Georg Petroianu, 4Frank C. Howarth, 5Nadine Kabbani, and 1Murat Oz

Laboratory of Functional Lipidomics, Departments of 1Pharmacology and 4Physiology, College of Medicine and Health Sciences, UAE University, Al Ain, UAE; 3Department of Cellular Biology & Pharmacology, College of Medicine, Florida International University, Miami, FL 33199, USA; 2

Department of Biological Sciences, Schmid College of Science and Technology, Chapman

University, One University Drive, Orange, CA 92866, USA. 5Department of Molecular Neuroscience, Krasnow Institute for Advanced Study, George Mason University, 4400 University Drive, Fairfax, VA 22030, USA;

Corresponding author: Murat Oz, M.D., Ph.D. Department of Pharmacology and Therapeutics, Laboratory of Functional Lipidomics Faculty of Medicine and Health Sciences, UAEU Abu Dhabi, Al Ain, UAE Phone: (971)-03-713-7523 Fax: (971)-03-767-2033 E-mail: [email protected]

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Abstract Bisabolol is a plant-derived monocyclic sesquiterpene alcohol with antinociceptive and antinflammatory actions. However, molecular targets mediating these effects of bisabolol are poorly understood. In this study, using a two-electrode voltage-clamp and patch clamp techniques and live cellular calcium imaging, we have investigated the effect of bisabolol on the function of human α7 subunit of nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes, interneurons of rat hippocampal slices. We have found that bisabolol reversibly and concentration dependently (IC50=3.1 µM) inhibits ACh induced α7 receptor mediated currents. The effect of bisabolol was not dependent on the membrane potential. Bisabolol inhibition was not changed by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free solution containing Ba2+, suggesting that endogenous Ca2+-dependent Cl- channels are not involved in bisabolol actions. Increasing the concentrations of ACh did not reverse bisabolol inhibition. Furthermore, the specific binding of [125I] -bungarotoxin was not attenuated by bisabolol. Choline-induced currents in CA1 interneurons of rat hippocampal slices were also inhibited with IC50 of 4.6 µM. Collectively, our results suggest that bisabolol directly inhibits α7-nAChRs via a binding site on the receptor channel.

Keywords: Nicotinic acetylcholine receptor; nicotine; bisabolol; Xenopus oocyte; hippocampus neurons

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Introduction (–)-α-bisabolol, a monocyclic sesquiterpene alcohol, is a naturally occurring volatile constituent found in the essential oil of several plants from the Asteraceae family (McKay and Blumberg 2006). Bisabolol containing plants such as chamomile have been used in the traditional medicine of Europe and East Asia for hundreds of years (McKay and Blumberg 2006; Kamatou et al. 2008). In Germany, the chamomile flower is licensed as a standard medicinal tea for the treatments of gastrointestinal spasms and inflammatory diseases of the gastrointestinal tract (McKay and Blumberg 2006). In addition, anti-nociceptive and anti-inflammatory actions of bisabolol have been reported in recent studies (Leite et al., 2011; 2012; Rocha et al., 2011a). The molecular and cellular mechanisms mediating the pharmacological action of bisabolol remain unknown. Studies indicate that inhibition of peripheral nerve conduction (Alves et al., 2010), blockade of Ca2+ (de Siqueira et al., 2012) and K+ channels (Bezerra et al., 2009), opening of the mitochondrial permeability transition pore (Cavalieri et al., 2009), and an inhibition of lipid peroxidation (Rocha et al., 2011b) are all part of the pharmacologic action of bisabolol. Homomeric α7nicotinic acetylcholine receptors (nAChR) are expressed in central and peripheral nervous tissue. This class of receptors is distinguished by their rapid desensitization and high permeability to Ca2+ (Albuquerque 2009). In contrast to the well-defined functions of nAChRs in synaptic transmission at muscle end plates and in autonomic ganglia, neuronal nAChRs such as α7-nAChRs are located on presynaptic terminals or at extrasynaptic sites on soma and dendrites. Together with their capacity for engaging various cellular signaling pathways (Kabbani et al., 2013), these receptors exert an important modulatory influence in the nervous system and have been shown to have roles in inflammation and nociception (for reviews Umana et

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al., 2013; Papke, 2014). Since bisabolol has also been reported to have anti-nociceptive and anti-inflammatory actions, we hypothesized that the interaction of bisabolol with α7-nAChR can mediate some of its cellular effects. In the present study, we have tested the effect of bisabolol on the functional properties of human α7-nAChRs expressed in Xenopus oocytes, native nicotinic receptors in interneurons of rat hippocampus. Our findings show that bisabolol modulates the function of α7-nAChRs.

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Material and methods Recordings from oocytes: Mature female frogs (Xenopus laevis) from Xenopus Express (Haute-Loire, France), were maintained in dechlorinated water in a room with temperature adjusted to 18 °C. Animals were fed food pellets supplied by Xenopus Express Inc (Brooksville, FL, USA). Experiments conducted in this study were in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) of the National Institutes of Health (Bethesda, MD) and approved by the Institutional Animal Ethics Committee at the UAEU. Ovarian lobes were surgically removed under benzocaine-induced anesthesia (Sigma, St. Louis, MO; 0.15 % w/V). The lobes were cut into small pieces and digested with collagenase A (Worthington Biochemicals, Freehold, NJ; 0.2 % w/V) with constant stirring at room temperature for 2 h. Oocytes were dissected manually in Ca2+ free solution containing (in mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; MgSO4, 0.8; HEPES, 10 (pH 7.5). Stage V and VI oocytes were injected with 10 ng α7 nAChR subunit cRNA in 50 nL volume using a Nanoject Automatic Oocyte Injector (Drummond, Broomall, PA), then incubated 2-7 days at 18 °C in modified Barth's solution (MBS) containing (in mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; CaCl2, 2; MgSO4, 0.8; HEPES, 10 (pH 7.5), supplemented with sodium pyruvate, 2 mM, penicillin 10,000 IU/L, streptomycin, 10 mg/L, gentamicin, 50 mg/L, and theophylline, 0.5 mM. Electrophysiological recordings were performed as described previously (Singhal et al., 2007). Briefly, oocytes were placed in a 0.2 ml recording chamber and continuously perfused with the bathing solution containing (in mM): NaCl, 95; KCl, 2; CaCl2, 2; and HEPES 5 (pH 7.5) at the flow rate of 4-5 ml/min. Oocytes were impaled with two glass microelectrodes filled with a 3 M KCl (1-3 MΩ) and voltage clamped at a holding potential of -70 mV using a GeneClamp-500

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amplifier (Axon Instruments Inc., Burlingame, CA). Current-voltage relationship of ACh-responses was determined by changing membrane potentials ranging from -100 to -20 mV for 30 sec to1 min and then returned to -70 mV. In experiments evaluating the effects of drugs, baseline conditions were defined by three control applications of 100 µM (5 min intervals) acetylcholine (ACh) made before the experimental applications. Test compounds applied into the bath using the gravity flow via a glass pipette positioned about 2 mm from the surface of the oocyte. All the chemicals and reagents used in experiments were purchased from Sigma-Aldrich (St. Louis, MO). Bisabolol was obtained from Sigma (St. Louis, MO). BAPTA (50-100 nL, 100 mM) was prepared in Cs4-BAPTA (Oz et al., 1998) and injections were performed 1 hr prior to experiments using microsyringe pump (Micro4, WPI, Inc. Sarasota, FL). Stock solution of bisabolol was prepared in ethanol at a concentration of 10 mM. cDNA plasmids for human α7-nicotinic acetylcholine receptor expression were kindly provided by Dr. J. Lindstrom (University of Pennsylvania, PA). Capped cRNA transcripts were synthesized in vitro using a mMESSAGE mMACHINE kit from Ambion (Austin, TX) and analyzed on a 1.2 % formaldehyde agarose gel to check the size and quality of the transcripts. Radioligand binding studies: Oocyte membranes were prepared using a previously published method (Oz et al., 2004b). Briefly, oocytes (200-300 oocytes per assay) injected with 10 ng human α7-nicotinic acetylcholine receptor cRNA were suspended (in 20 μl/oocyte) in a homogenization buffer containing HEPES 10 mM, EDTA 1 mM, 0.02% NaN3, 50 μg/mL bacitracin, and 0.1 mM PMSF (pH 7.4) at 4 C on ice and homogenized using a motorized Teflon homogenizer (six

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strokes, 15 sec each at high speed). The homogenate was centrifuged for 10 min at 800 x g. The pellet was resuspended in homogenization buffer and centrifuged at 800 x g for 10 min. Supernatants were combined and centrifuged for 1 hr at 36000 x g. Subsequently, pellet of the oocyte membrane was suspended in homogenization buffer and used in the binding studies. Binding experiments were conducted in 500 μL of 10 mM HEPES (pH 7.4) containing 50 μL of oocyte preparation and 0.1-5 nM [125I] -bungarotoxin (2200 Ci/mmol; Perkin-Elmer, Inc. Waltham, MA). Membrane preparations were incubated with [125I] -bungarotoxin in the absence and presence of drugs, for 1 hr at room temperature (22-24 °C). Nonspecific binding was determined in the presence of 10 μM -bungarotoxin. The radioligand was separated by rapid filtration onto GF/C filters presoaked in 0.2% polyethyleneimine. Subsequently, filters were washed with three 5 ml washes of ice-cold HEPES buffer, and the radioactivity of the filters was determined by Beckman Gamma-300 -counter. Whole-cell patch clamp recordings in rat hippocampal slices: Male Sprague-Dawley rats (10-30 days old) were killed by CO2 narcosis followed by decapitation. Their brains were removed in ice-cold artificial cerebrospinal fluid (ACSF), which was composed of (in mM): NaCl, 125; NaHCO3, 25; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 2, MgCl2, 1; and glucose, 25. Slices of 350-400 µm thickness were cut using a Vibratome and stored at room temperature in an immersion chamber containing ACSF bubbled with 95% O2 and 5% CO2. Whole-cell patch-clamp experiments were performed on visually identified CA1 stratum raditum (SR) interneurons of rat hippocampal slices (Singhal et al., 2007; Mahgoup et al., 2013). Whole-cell currents were recorded from the soma of SR interneurons according to the standard patch-clamp technique using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). The slices were superfused with ACSF at 2ml/min

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in the presence of the tetrodotoxin (1 µM), the muscarinic antagonist atropine (0.5 µM), the 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), the 2-amino-5-phosphonovaleric acid (APV, 10 µM), and the bicuculline (10 µM). Choline (1 mM) was applied every 2 min to interneuron cell bodies via a quartz tube (i.d., 300 µm) positioned about 150-200 µm from the cell; drug delivery was controlled by a computer-driven valve system. Other drugs were applied via bath superfusion. Signals were filtered at 5 kHz and recorded by a microcomputer using the pCLAMP8.1 program (Axon Instruments, Foster City, CA). Patch pipettes were pulled from borosilicate glass capillary (1.2-mm outer diameter), and when filled with internal solution had resistance between 3 and 5 MΩ. At -70 mV holding potential, the leak current was generally between 100 and 250 pA, and when it exceeded 250 pA, the data were not included in the analysis. The internal pipette solution contained (in mM): ethylene-glycol bis(b-amino-ethyl ether)-N-N0-tetraacetic acid, 10; HEPES, 10; Cs-methane sulfonate, 130; CsCl, 10; MgCl2, 2; and lidocaine N-ethyl bromide (QX-314), 5 (pH adjusted to 7.2 with CsOH; 330 mOsm). All recordings were done at room temperature (20-22 °C). Only a single neuron was studied in a given slice, therefore the number of neurons represents the number of hippocampal slices analyzed.

Docking studies: We used recently identified NMR structure of transmembrane domain of human α7 acethylcholine receptor with pdb code 2MAW (Bondarenko, Mowrey et al. 2014) Docking of bisabolol (zinc id :01609418) to structural model was made by Autodock Vina program (RRID:OMICS_01595) (Trott and Olson 2010). Ligand and receptor files were prepared based on procedure described in program Autodock Tools (ADT) (Morris, Huey et al. 2009) documentation.

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ADT assigned polar hydrogens, united atoms Kollman charges and solvation parameters, prepared files were saved in PDBQT format. Affinity grid maps of 20x20x20Å with spacing 0.375Å were added. Grid center was designated x,y,z dimensions: -8.73,-11.56 and -20.68. These coordinates correspond to recently identified binding site for ketamine in transmembrane domain of human α7 acetylcholine receptor (Baxter 1981). Autodock Vina was used for docking using protein, ligand and grid box information saved to configuration file. Docking calculations were performed using the Lamarckian genetic algorithm (LGA)(Morris, Goodsell et al. 1998). The position with lowest binding free energy was aligned with receptor for further analysis of interactions. Data analysis: Average values were calculated as the mean ± standard error means (S.E.M.). Statistical significance was analyzed using Student's t test or ANOVA as indicated. Curves for concentration-response relationship were determined by fitting the data to the logistic equation, y = Emax/(1+[x/EC50]-n), where x, y, and Emax are concentration, response, and the maximal response, respectively. EC50 is the half-maximal concentration, and n is the slope factor.

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Results Bisabolol inhibits α7-nAChR activity The effects of 10 min bath application of bisabolol (5 μM) on the maximal amplitudes of α7-nAChR mediated currents are shown in Fig. 1A. A time-course of the bisabolol action on the amplitudes of ACh-induced currents is presented in Fig. 1B. The vehicle solution alone did not change ACh-induced currents. However, application of bisabolol (5 μM) caused a significant inhibition of ACh-induced currents. The inhibitory effect of bisabolol was partially reversed during a washout period of 10 to 15 min (Fig. 1B). In initial control experiments, ACh, at the highest concentration used in this study (1 mM) did not induce detectable currents in oocytes injected with distilled water (n=9) (data not shown). Bath application of ACh (100 μM) for 3 to 4 sec caused activation of fast inward currents that desensitized rapidly in oocytes injected with cRNA transcribed from cDNA encoding the α7-subunit of human nAChR. In addition, ACh-induced inward currents were inhibited completely with 100 nM α-bungarotoxin (n=6, data not shown), indicating that these currents are mediated by the α7-nAChR. Bisabolol alone (100 µM) did not cause any alteration in holding currents (n=8). At the highest bisabolol concentration vehicle (ethanol 0.01% v/v) did not alter the maximal amplitudes of ACh-induced currents (n=9). It is notable that the inhibitory effect of bisabolol developed gradually in a time-dependent manner. Without pre-incubation, a co-application of ACh (100 μM) and bisabolol (10 μM) did not change the maximal amplitudes of ACh-induced currents (Fig. 2A). However the extent of bisabolol inhibition was significantly increased with prolonged incubation times reaching a maximum level within 5 min (Fig. 1B). Plotting the incubation time versus bisabolol effect

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indicated that the inhibition occurs with the time constant of τ1/2 = 0.7 min (Fig. 2A). Since the extent of bisabolol inhibition was time-dependent, bisabolol was routinely applied for 10 min to ensure equilibrium conditions. The effect of bisabolol on α7-nAChR was concentration-dependent with IC50 and slope values of 3.1 ± 2.1 µM and 1.4, respectively (Fig. 2B). α7-Nicotinic acetylcholine receptors are highly permeable to extracellular Ca2+ (for a recent review: Uteshev, 2012). This property of the nicotinic channel allows entry of sufficient Ca2+ to activate endogenously expressed Ca2+-activated Cl- channels in Xenopus oocytes. Therefore, we have determined whether the effect of bisabolol was exerted directly on nAChR-mediated currents or Ca2+ activated Cl- currents by replacing extracellular Ca2+ with Ba2+. Although Ba2+ can pass through α7-nAChRs, it does not induce activation of Ca2+-dependent Cl- channels (Sands et al., 1993). In earlier reports, a small Ca2+-dependent Cl- current observed in the presence of Ba2+ has been shown to be eliminated by the injection of the Ca2+ chelator BAPTA (Sands et al., 1993). We investigated the actions of bisabolol in a solution containing 2 mM Ba2+ in BAPTA-injected oocytes. Magnitude of bisabolol (10 µM)-induced inhibition remained the same (66 ± 5 in controls versus 63 ± 4 in BAPTA-injected oocytes; ANOVA, P>0.05) when BAPTA-injected oocytes were recorded in 2 mM Ba2+ containing solutions (Fig. 3A) suggesting that Ca2+-activated Cl- channels are not significantly involved in bisabolol inhibition of nicotinic receptors. We examined whether inhibition of α7-nAChRs by bisabolol was altered by changes in the membrane potential. As indicated in Fig. 3B, bisabolol (5 µM) was found to inhibit ACh (100 µM)-induced currents at all tested potentials and therefore was independent of voltage. Indeed, the current-voltage relationship (Fig. 3C) shows that the extent of bisabolol inhibition does not change significantly at different holding potentials (P>0.05, n=7, ANOVA).

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It is possible that bisabolol exerts its effect by competing with binding of ACh to the nAChR at the same binding site and acting as a competitive antagonist. We plotted concentration-response curves for ACh in the absence and presence of 5 µM bisabolol and found that the affinity of ACh for the nAChR remained unchanged in the presence of bisabolol (EC50 values of 68 ± 9 μM versus controls 74 ± 11 μM; P>0.05, ANOVA, n=6-8). Maximal ACh response was inhibited to about 43 ± 5 % of controls (n=6) however, suggesting that bisabolol acts on nAChRs in a non-competitive manner (Fig. 4A). We also conducted radioligand binding experiments to investigate the effect of 10 μM bisabolol on the specific binding of [125I] -bungarotoxin. Equilibrium curves showing the specific binding of [125I] -bungarotoxin, in the presence and absence (controls) of bisabolol are presented in Fig. 4B. Specific binding of [125I] -bungarotoxin was not significantly altered in the presence of 10 µM bisabolol. Maximum binding activities (Bmax) of [125I] -bungarotoxin (2.3 ± 0.5 and 2.1 ± 0.4 pM/mg for controls and bisabolol-treated preparations, respectively) were not significantly different in the presence of bisabolol (Fig. 4B). The apparent affinity (KD) of the receptor for [125I] -bungarotoxin was 0.8 ± 0.3 and 0.9 ± 0.2 pM for controls and bisabolol, respectively (P>0.05, ANOVA, n=5-6). The α7-nAChR belongs to the cys-loop family of ligand-gated ion channels. Therefore, we have also investigated the actions of bisabolol on the activity of the other members of cys-loop family of ligand gated ion channels. Bisabolol (10 µM) caused a modest inhibitory action (approximately 20% inhibition) on the amplitudes of currents mediated by serotonin type 3 receptors and caused a small (less than 10 %) potentiation of glycine α1-receptor mediated currents (Figure 5).

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We also tested the effect of bisabolol on α7-nAChRs in the CA1 region of stratum radiatum interneurons in rat hippocampal brain slices. In whole cell patch clamp mode, focal application of 10 mM choline, a selective agonist for α7-nAChR (Albuquerque et al., 2009) for a short duration (0.5 to 1 sec) caused a rapidly activating and fast desensitizing inward currents that were completely inhibited by the bath application of 1 µM methyllycaconitine, a selective antagonist for α7-nAChR (data not shown, n=4). Choline-induced currents were significantly inhibited by 10 min bath application of 10 µM bisabolol (Fig. 6A). Concentration -response studies indicated that bisabolol inhibits choline-induced currents with IC50 of 4.6.µM (Figure 6B).

Modeling the Bisbolol binding pocket within α7-nACh receptors We performed docking simulations of (–)-α-bisabolol to the recently resolved NMR structure (Bondarenko, Mowrey et al. 2014) of the human nAChR transmembrane domain. In this binding simulation the site for ketamine was identified. We hypothesize that bisabolol has similar binding site as ketamine. Results of the docking simulations are presented in Figure 7. As shown in Figure 7A and B, bisabolol binds the channel cavity at a low part of the transmembrane domain within a hydrophobic pocket between helices. The position of bisabolol appears stabilized by hydrophobic interaction with several surrounding hydrophobic residues. A conformation of the best position with binding energy -5.7 kcal/mol is presented in Figure 7. An analysis of protein ligand interactions reveals that the cyclohexane group of bisabolol is sandwiched between the aromatic sidechains of Phe453 and Phe230. The Phe453 residue plays an important role here similar to ketamine binding (Bondarenko and Mowrey et al. 2014), and the methyl group interacts with the backbone oxygen of Cys449. The isopropyl group of bisabolol interacts with Val268 and 308 with

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Val268 being located on helix TM2. It has been shown that helix TM2 plays an important role in gating of acetylcholine receptors (Unwin and Fujiyoshi, 2012). According to Unwin and Fujiyoshi (2012), the pore-lining helix TM2 is bent towards the pore in the closed conformation. However, in the open state, helix TM2 loses its bent shape and straightens. The hydrophobic cavity - where inhibitors such a ketamine and bisabolol bind - is located behind helix TM2 in-between transmembrane domain helices. In the closed conformation, when helix TM2 is bent towards the pore, the hydrophobic cavity between helices will have a larger volume. Thus, the closed conformation will be more favorable for binding of receptor inhibitors such as bisabolol and ketamine. In contrast, the open conformation with a straight helix TM2 will have smaller volume. An inhibitor bound to the closed conformation will prevent helix TM2 from straightening, thus, making the closed conformation more favorable. This mechanism of inhibition is supported by the observation that the inhibitory effect of bisabolol depends on the application mode in that maximal inhibition occurs when the compound is pre-applied not co-applied. Thus, we hypothesize that bisabolol binds to the closed conformation of the receptor, which then prevents helix TM2 from straightening upon ACh binding to open the pore.

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4.1 Discussion Using electrophysiological, biochemical and imaging methods we provide first evidence that bisabolol can inhibit α7-nAChRs in a reversible and concentration -dependent manner. Increases in the concentration of ACh did not reverse bisabolol inhibition indicating that bisabolol acts on α7-nAChRs in a noncompetitive manner. The findings present the possibility that the therapeutic actions of this natural compound may at least in part derive from its ability to bind nAChRs in cells. It is important to consider the role of bisabolol interaction with α7 nAChR in inflammation. Previous work shows that α7 agonists are reported to be active in models of pain and inflammation (Alsharari et al., 2013) while the current study provides a first demonstration for an inhibitory effect of this compound on α7 receptors in non-immune cells. How this property of bisbalol can contribute to nAChR activity in immune cells is unclear and future studies in immune cells will be required to address the role of this compound on cholinergic immunity. It is plausible that the inhibitory actions of bisabolol on α7-nAChR channels may promote an intracellular anti-inflammatory signaling response by α7-nAChR in a non-ionotropic ways (Clark et al., 2014; Papke et al., 2014). Our electrophysiological studies suggest that bisabolol interacts with the closed state of the receptor while published studies by others have shown that immune cells do not display nAChR currents (for a review, Papke, 2014). Activation of α7-nAChRs has been shown to cause sufficient Ca2+ entry to activate endogenous Ca2+-dependent Cl- channels (Sands et al., 1993). The extent of bisabolol inhibition remained unaltered in oocytes injected with BAPTA and recorded in a solution containing 2 mM Ba2+, suggesting that Ca2+-dependent Cl- channels were not involved in bisabolol inhibition of nicotinic receptors. Furthermore, since Ca2+-activated Cl- channels are highly sensitive to

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intracellular Ca2+ levels (for reviews: Hartzell et al., 2005; Marin, 2012), changes in intracellular Ca2+ levels would cause alterations in the holding current under voltage-clamp conditions. However, bisabolol, even at the high concentrations (30 µM) used in this study, did not alter holding currents, suggesting that intracellular Ca2+ concentrations were not changed by bisabolol. Open-channel blockade of ligand-gated ion channels is a widely used model (Hille, 2001). However, this model does not seem to be consistent with the results of the present study. For an open channel blocker, the presence of the agonist is required to allow the blocker to enter the channel after an agonist-induced conformational change. In contrast to open channel blockers, preincubation of bisabolol enhances its effect on α7-nAChR activity, suggesting that bisabolol interacts with the closed state of the receptor. In addition, inhibition by bisabolol is not voltage sensitive, suggesting that the bisabolol-binding site is not affected by the transmembrane electric field. Furthermore, changing the application frequency of ACh (from every 5 minute to every 10 min; unpublished results) did not change the extent of bisabolol inhibition further suggesting that bisabolol exerts its blocking effects by the hydrophobic pathway, reaching its receptor site independently of the activation state of the channel. Bisabolol, with a logP(octanol/water) value of 5.07, is a lipophilic compound and it is expected to diffuse into plasma membrane. In addition, computer modeling studies locate the binding site of bisabolol on the transmembrane regions of the nAChR in the lipid bilayer. Furthermore, inhibition by bisabolol occurs relatively slowly (τ1/2=0.7 min) suggesting that bisabolol needs to partition in lipid membrane to reach its binding site on nAChR. Relatively slow time course of bisabolol action would also suggest involvement of second messenger pathways. Based on our BAPTA experiments, intracellular Ca2+ activated second messengers are not likely to be involved in bisabolol actions. However, this possibility

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was not investigated further for other second messenger pathways in the present study. In electrophysiological studies, although the efficacy of ACh was significantly inhibited by bisabolol, the potency of ACh was not altered indicating that bisabolol did not compete with the ACh binding site on the receptor. In agreement with these results, radioligand binding experiments showed that the specific binding characteristics of [125I] -bungarotoxin were not affected in the presence of bisabolol, further suggesting that bisabolol does not act on the ACh binding site. Similar conclusions were also reached by our modeling data revealing that bisabolol binds at receptor residues Phe453, Phe230 and Val268 (Fig. 7). Collectively, these findings indicate that bisabolol acts as an allosteric inhibitor of α7-nAChR; i.e., bisabolol binds to site(s) topographically distinct from the ACh binding sites on α7-nAChR. In earlier studies, bisabolol was shown to inhibit smooth muscle contractions induced by phenylephrine and KCl with IC50 values of 201 and 23 µM, respectively (De Siqueira et al., 2014). Similarly, bisabolol, in the concentration range of 30-200 µM, was shown to cause relaxation of various smooth muscle preparations (De Siqueira et al., 2012; Roberts et al., 2013) and was suggested to mediate hypotensive effects of bisabolol. At significantly higher concentrations (500 µM-10 mM), bisabolol has been reported to inhibit compound action potentials and sodium channels (Alves et al., 2010). Bisabolol, in the concentration range used in this study, also has been shown to act directly on mitochondrial permeability transition pore (5 µM; Cavalieri et al., 2009). Our results provide the first demonstration that bisabolol directly inhibits the function of α7-nAChR at pharmacologically relevant concentrations in both oocytes expressing human α7-nAChR (IC50=3.1 µM) and rat hippocampal neurons (IC50=4.6 µM). The antagonistic action of bisabolol on α7-nAChR is a somewhat surprising finding since

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Bisabolol inhibits α7-nACh receptors

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agonists of α7-nAChR are usually associated with anti-inflammatory and anti-nociceptive effects. However, in recent studies it has been suggested that ion conduction by α7-nAChR may not be related to anti-inflammatory actions of ligands that bind to this receptor (Clark et al., 2014). Adding to the complexity, there have been reports suggesting that agonists of α7-nAChR have deleterious effect on lung inflammation (Matsunaga et al., 2001; Giebelen et al., 2009; Lafargue et al., 2012). Recently, some of the biological effects of α7-nAChR have been shown to be mediated by the activation of G-proteins (Nordman and Kabani, 2012; 2014). Bisabolol may interfere with coupling of α7-nAChR with G-proteins or may promote a closed state of the receptor (see for a review Papke et al., 2014) that mediates pharmacological actions of bisabolol. Regardless of mechanisms, our results indicate that bisabolol, at relatively low concentrations, interacts with α7-nAChR.

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Acknowledgements This study was supported by grants from CMHS, UAE University and a SEED Grant Award from George Mason University to NK. Research in our laboratory is also supported by LABCO partner of Sigma-Aldrich. The authors thank Dr. Jon Lindstrom for providing cDNA clones of the human α7-nicotinic acetylcholine receptor subunit.

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Figure legends Figure 1. Effect of bisabolol on α7-nicotinic acetylcholine receptor. (A) Records of currents activated by acetylcholine (ACh, 100 μM) in control conditions (left), during co-application of 5 μM bisabolol and acetylcholine after 10 min pretreatment with 5 μM bisabolol (middle), and 15 min following bisabolol washout (right). (B) Time-course of the effect of bisabolol (5 μM) on the peaks of the acetylcholine-induced currents. Each data point represents the normalized mean ± S.E.M. of 5 to 7 experiments. Duration of drug application is shown by the horizontal bar in the figure. Figure 2. Time and concentration-dependence of bisabolol effect on α7-nicotinic acetylcholine receptor. (A) Prolongation of bisabolol per-incubation time enhances that inhibitory effect of bisabolol (10 µM) on the α7-nicotinic acetylcholine receptor. Each data point represents the mean ± S.E.M. of 7 to 8 oocytes. (B) Bisabolol inhibits α7-nicotinic acetylcholine receptor function in a concentration-dependent manner. Each data point represents the mean ± S.E.M. of 5 to 7 oocytes. The curve is the best fit of the data to the logistic equation described in the methods section.

Figure 3. Inhibition of acetylcholine-induced currents by bisabolol is independent of Ca2+-activated Cl- channels and membrane potential. (A) The effects of bisabolol on α7-nicotinic acetylcholine receptor expressing oocytes injected with 50 nl distilled water and recorded in 2 mM Ca2+ containing MBS solution (control) or injected with 50 nl of BAPTA (100 mM) and recorded in 2 mM Ba2+ containing MBS solution (BAPTA). Bars represent the means ± S.E.M. of 6 to 7 experiments. (B) Current-voltage relationships of acetylcholine-activated currents in the absence and presence of bisabolol (5 μM). Normalized currents activated by 100 μM acetylcholine before

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Bisabolol inhibits α7-nACh receptors

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(control,) and after 15 min treatment with bisabolol (). Each data point presents the normalized means and S.E.M. of five to six experiments. (C) Quantitative evaluation of the effect of bisabolol as percent inhibition at different voltages.

Figure 4. Concentration-response curves for acetylcholine-induced currents and binding of [125I] -bungarotoxin in control and in the presence of bisabolol. (A) Effect of bisabolol on the acetylcholine concentration-response relationship. In controls, currents were activated by acetylcholine (1 μM to 3 mM). Subsequently, bisabolol (5 µM) was bath-applied for 10 min and acetylcholine dose-response curve was plotted in the presence of bisabolol. Responses normalized to maximal response under control conditions. EC50 and slope values were determined by fitting the curves from 6 to 8 oocytes to the logistic equation as described in the methods section. Data points obtained before (control) and after 10 min treatment with bisabolol (5 μM) were indicated by filled circles and open circles, respectively. (B) The effects of bisabolol on the specific binding of [125I] -bungarotoxin to oocyte membrane preparations. In the presence and absence of bisabolol, specific binding as a function of the concentration of [125I] -bungarotoxin is presented. Data points for controls and bisabolol (10 μM) are indicated by filled circles, and open circles, respectively. Data points are the means of three independent experiments carried out in triplicate.

Figure 5: The effect of bisabolol on the other members of cys-loop family of ligand-gated ion channels. Data presents mean inhibition of homomerically expressed α7-nicotinic, 5-HT3 and glycine α1 receptors by 10 µM bisabolol. Numbers of cells tested were shown on top each bar.

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Bisabolol inhibits α7-nACh receptors

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Figure 6: Effects of bisabolol on choline-induced ion currents recorded in CA1 area stratum radiatum interneurons of rat hippocampal slices. (A) Recordings of choline-induced currents before (control, left panel), during (10 min of bisabolol), and after (10 min recovery) the bath application of 10 µM bisabolol in hippocampal interneurons. Choline application time was indicated by a solid bar on top of the current traces. Dashed line indicates continuing bath application of bisabolol. (B) Bisabolol inhibits α7-nicotinic acetylcholine receptor function in a concentration-dependent manner. Each data point represents the mean ± S.E.M. of 4 to 5 oocytes. The curve is the best fit of the data to the logistic equation described in the methods section.

Figure 7: Computer modeling of the bisabolol binding to transmembrane regions of nicotinic receptor. (A) Position of bisabolol at lowest energy conformation (-5.7 kcal/mol) between helices of transmembrane domain of the receptor. (B) Bottom view of bisabolol in hydrophobic cavity between transmembrane helices.

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Hartzell C, Putzier I and Arreola J (2005) Calcium-activated chloride channels. Annu Rev Physiol 67:719-758. Hille, B. (2001) Ion Channels of Excitable Membranes, 3rd ed. Sinauer Associates, Sunderland, MA. Hogg, R.C., Raggenbass, M., Bertrand, D. (2003) Nicotinic acetylcholine receptors: from structure to brain function. Rev. Physiol. Biochem. Pharmacol. 147: 1-46. Kabbani N, Nordman JC, Corgiat BA, Veltri DP, Shehu A, Seymour VA, Adams DJ. (2013) Are nicotinic receptors coupled to G proteins? Bioessays.35: 1025-1034. Kamatou GP, Makunga NP, Ramogola WP, Viljoen AM. (2008) South African Salvia species: a review of biological activities and phytochemistry. J Ethnopharmacol. 119:664-672. Lafargue M, Xu L, Carles M, Serve E, Anjum N, Iles KE, Xiong X, Giffard R, Pittet J-F. (2012) Stroke-induced activation of the α7 nicotinic receptor increases Pseudomonas aeruginosa lung injury. FASEB J. 26: 2919-2929. Leite Gde O, Leite LH, Sampaio Rde S, Araruna MK, de Menezes IR, da Costa JG, Campos AR. (2011) (-)-α-Bisabolol attenuates visceral nociception and inflammation in mice. Fitoterapia. 82: 208-211. Leite Gde O, Fernandes CN, de Menezes IR, da Costa JG, Campos AR. (2012)Attenuation of visceral nociception by α-bisabolol in mice: investigation of mechanisms. Org Med Chem Lett.;2:18. Marin M (2012) Calcium signaling in Xenopus oocyte. Adv Exp Med Biol. 740: 1073-1094. Matsunaga, K., Klein, T. W., Friedman, H., and Yamamoto, Y. (2001) Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine. J. Immunol. 167: 6518-6524 McKay DL, Blumberg JB. (2006) A review of the bioactivity and potential health benefits

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of chamomile tea (Matricaria recutita L.). Phytother Res. 20:519-530. Morris, G. M., D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew and A. J. Olson (1998) "Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function." Journal of Computational Chemistry 19: 1639-1662. Morris, G. M., R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell and A. J. Olson (2009). "AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility." J Comput Chem 30: 2785-2791. Nordman JC, Kabbani N. (2012) An interaction between α7 nicotinic receptors and a G-protein pathway complex regulates neurite growth in neural cells. J Cell Sci. 125: 5502-5513. Nordman JC, Kabbani N. (2014) Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3 receptor pathway. FASEB J. 28: 2995-3006. Onaran HO, Costa T. (2009) Allosteric coupling and conformational fluctuations in proteins. Curr Protein Pept Sci. 2009 10:110-115. Oz, M., Melia, M.T., Soldatov, N.M., Abernethy, D.R., Morad, M. (1998) Functional coupling of human L-type Ca2+ channels and angiotensin AT1A receptors coexpressed in Xenopus laevis oocytes: involvement of the carboxyl-terminal Ca2+ sensors. Mol. Pharmacol. 54: 1106-1112. Oz, M., Spivak, C.E., Lupica, C.R., (2004a) The solubilizing detergents, Tween 80 and Triton X-100 non-competitively inhibit alpha 7-nicotinic acetylcholine receptor function in Xenopus oocytes. J. Neurosci. Methods 137: 167-173. Oz, M., Zakharova, I., Dinc, M., Shippenberg, T. (2004b) Cocaine inhibits cromakalim-activated K+ currents in follicle-enclosed Xenopus oocytes. Naunyn Schmiedebergs Arch. Pharmacol. 369: 252-259. Papke RL. (2014) Merging old and new perspectives on nicotinic acetylcholine receptors. Biochem Pharmacol. 89:1-11. Papke RL, Bagdas D, Kulkarni AR, Gould T, AlSharari SD, Thakur GA, Damaj MI. (2014)

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Figure 1

A

ACh ACh

Bisabolol (5 µM)

ACh

0.2 µA 2 sec

B % of control

100 80 60 40 20 0

Bisabolol (5 µM) 0

10

20

Time (min)

30

40

F ig u re 2

A

1 0 0

o f c o n tro l

8 0 6 0 τ1

4 0

= 0 .7 m in

/2

%

2 0 0 0

2

4

6

8

1 0

T im e (m in )

B

%

o f c o n tro l

1 0 0 8 0

IC

6 0

5 0

4 0 2 0 0 1

B i s a b o l o l ( µM )

1 0

= 3 . 1 µM

A

F ig u re 3

o f c o n tro l

1 0 0 8 0 6 0 4 0

(n = 7 )

(n = 6 )

C o n tro l

B A P T A

%

2 0 0

B

0 .0 -1 2 0 -1 0 0 -8 0

-6 0

-4 0

-2 0

- 0 .2 - 0 .4 - 0 .6

c o n tro l B is a b o lo l

- 0 .8 - 1 .0

C

n o rm a liz e d c u rre n t

v o lta g e (m V )

8 0 6 0 4 0 2 0

%

o f c o n tro l

1 0 0

0 -1 2 0 -1 0 0

-8 0

-6 0

-4 0

V o lta g e (m V )

-2 0

Figure 4

% of maximal control

A

100

control bisabolol

80 60 40 20 0

1

10

100

1000

125

B

I-α-Bungarotoxin binding (pmol/mg protein)

concentration of ACh (µM) 2.5 2.0 1.5 1.0 control bisabolol

0.5 0.0

0

1 125

2

3

I-α-Bungarotoxin (nM)

4

Figure 5

Bisabolol (10 µΜ) 100

(n=8)

% inhibition

80 60 40

(n=12)

20 0 (n=8)

-20

α7-nACh

5-HT3

α1−Gly

F ig u re 6

A C h o lin e B i s a b o l o l ( 1 0 µM )

C h o lin e (1 0 m M )

C h o lin e

1 0 0 p A 1 se c

B

1 0 0

IC

5 0

= 4 . 6 µM

6 0 4 0

%

in h ib itio n

8 0

2 0 0 0 .1

1

1 0

B i s a b o l o l ( µM )

1 0 0

Figure 7

B

A

Phe453

carvacrol thymoquinone

vanillin Phe2303 eugenole carvacrol carvone

thymoquinone

C TM1

TM2 carvacrol thymoquinone

TM3 TM4

   

Bisabolol is a naturally occurring monocyclic sesquiterpene alcohol with anti-nociceptive and anti-inflammatory actions. Bisabolol reversibly and concentration dependently inhibits ACh induced α7 receptor mediated currents. The effect of bisabolol was noncompetitive, not dependent on the membrane potential or intracellular Ca2+ levels. Choline-induced currents in rat hippocampal neurons were also inhibited by bisabolol.