Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling
© Wang et al; licensee BioMed Central Ltd. 2013
Received: 21 December 2012
Accepted: 22 February 2013
Published: 23 February 2013
Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.
Honokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding.
It is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.
KeywordsHonokiol Calcium signaling Store operated calcium entry (SOCE) Inositol trisphosphate (IP3) Muscarinic acetylcholine receptor Phospholipase Cβ
Honokiol is a small, cell-permeable phenolic compound derived from the bark of magnolia trees that is present in Asian herbal teas. Honokiol has a unique array of antiangiogenic, antitumor and anti-inflammatory actions .
In the central and peripheral nervous systems, honokiol has a neuroprotective effect, as well as effects on certain signal transduction pathways. Honokiol is an effective scavenger of reactive oxygen species , which may be an important factor in its anti-inflammatory and neuroprotective actions [3–5]. Honokiol also has pronounced anti-nociceptive effects, notably the nociception associated with glutamate receptor activation [6, 7]. Honokiol inhibits muscarinic receptor- and depolarization-mediated contraction of isolated porcine trachea, guinea pig ileum and rat uterus, apparently by blocking the influx of extracellular calcium [8–10]. Thus, common aspects of honokiol action in excitable tissues are an antioxidative action and depression of glutamate-receptor and calcium mediated signaling. It is possible that these are interrelated factors.
The present research was undertaken to determine the effects of honokiol on calcium signaling underlying transmission mediated by muscarinic receptors expressed in Chinese hamster ovary (CHO) cells, particularly receptor-mediated release from the endoplasmic reticulum through activated IP3 receptors and the subsequent store-operated calcium entry (SOCE) that is induced by depletion of calcium from the endoplasmic reticulum . We report that honokiol is a potent inhibitor of SOCE, with lesser effects on resting calcium concentrations and calcium release from the endoplasmic reticulum.
Cell culture and cytotoxicity measurements
CHO cells stably transfected with a clone of the human M3 muscarinic acetylcholine receptor (cDNA Resource Center, http://www.cdna.org) were cultured in DME/F12 media containing 10% FBS and penicillin/streptomycin (100 IU/100 μg/ml) at 37° in a 5% CO2 humidified atmosphere. Cell viability was determined using an MTS reduction assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Promega, Madison). Cells (≈ 7,000) were seeded into each well of a 96-well plate and incubated for 16 h. The cells were exposed to honokiol for 24 h before cell viability was determined.
Muscarinic receptor binding
[3H]N-methylscopolamine ([3H]MS; PerkinElmer), a non-selective muscarinic antagonist, was used to label muscarinic receptors from rat brain. Membranes from the neocortex were homogenized in 50 mmol/l Tris–HCl (pH 7.4), and an aliquot (15-30 μg protein) was incubated in the presence of [3H]MS and 50 mmol/l Tris–HCl (pH 7.4) for 60 minutes at room temperature in a final volume of 1 ml. Binding was determined by filtration of the suspension through glass fiber filters (1 μm pore size), followed by measurement of the radioactivity trapped on the filters by liquid scintillation counting. Non-specific binding was determined in the presence of 10 μmol/l atropine.
[3H]MS binding parameters (receptor concentration [Bmax] and dissociation constant [KD]) were determined by nonlinear regression (DeltaGraph) using a model for a single population of independent binding sites. Agonist binding was determined in competition studies using carbamylcholine and 0.2 nmol/l [3H]MS.
Measurement of intracellular calcium
Cytosolic calcium concentration ([Ca2+]i) was measured in a ratiometric assay using the calcium-sensitive fluorescent dye, fura-2 (Molecular Probes). Cells (≈120,000) were seeded onto 29 mm glass bottom dishes and incubated for 16 h. The cells were then incubated with fura-2 AM for 60 min at room temperature in a basal salt solution (BSS) comprised of 130 mmol/l NaCl, 5.4 mmol/l KCl, 5.5 mmol/l glucose, 2 mmol/l CaCl2, 1 mmol/l MgCl2, and 20 mmol/l HEPES, at pH 7.4. The cells were then washed twice with BSS, and the incubation continued for an additional 30 min before measuring calcium levels. Honokiol was present during the last 10 min of the final incubation period.
[Ca2+]i. was determined at room temperature using an InCyt Basic IM™ Fluorescence Imaging System (Intracellular Imaging, Inc., Cincinnati, OH). The imaging system was calibrated for calcium concentration using standard calcium solutions of known concentration (Invitrogen #C3722), according to manufacturer protocol. The ratio of fluorescence intensities measured at 512 nm after excitation at 340 and 380 nm is a reliable indicator of [Ca2+]i..
Signals from 17-25 cells were measured in each plate. Cells in which the resting [Ca2+]i. was not maintained at a constant level (typically 1-2 cells/plate) were excluded from the analysis, as were cells that completely failed to respond to a muscarinic agonist (typically < 1 cell/plate). To measure responses in the absence of extracellular calcium, the cells were switched to calcium-free BSS 3 min before imaging.
Parameters (calcium concentrations, dissociation constants, receptor concentrations, IC50’s) were compared as the means from 3-6 independent experiments (with 3 replicates in each binding measurement protocol and 17-23 replicates in each calcium measurement protocol) using Student’s t-test. Values from experiments with multiple independent variables were compared by ANOVA and Tukey’s test using GraphPad Prism software. Significant differences were indicated by P values of < 0.05.
Honokiol inhibition of muscarinic signaling
The resting cytosolic calcium concentration ([Ca2+]i) of CHO-M3 cells in BSS in this series of experiments was 32.7 ± 9.8 nmol/l (mean ± S.D.; N = 5). Stimulation with the muscarinic agonist carbamylcholine (10 μmol/l) led to a rapid and sustained increase in cytosolic calcium to a concentration that ranged from 500 to 900 nmol/l (Figure 1A). The threshold concentration for elicitation of a response to carbamylcholine was approximately 0.2 μmol/l. Responses to carbamylcholine were completely blocked by a 10 min pretreatment with 10 μmol/l atropine, and CHO cells not transfected with a muscarinic receptor gene did not respond to carbamylcholine. Addition of EDTA (5 mmol/l) to the external medium caused a rapid decrease in [Ca2+]i, indicating that the sustained elevation of [Ca2+]i following muscarinic receptor activation was dependent upon the entry of extracellular calcium (Figure 1A).
The initial change in [Ca2+]i largely reflects calcium release from the endoplasmic reticulum, while the sustained plateau phase almost exclusively reflects calcium entry from the extracellular medium [12, 13]. Initial calcium release from the ER is mediated by IP3 receptor ionophores, while the plateau phase involves “capacitive” calcium entry through Orai channels in the plasma membrane. STIM1 proteins span the ER membrane and possess a domain that senses calcium depletion. Calcium depletion induces an interaction between STIM1 and Orai, thereby activating the influx of extracellular calcium [13, 14]. This influx of extracellular calcium in response to depletion of calcium in the endoplasmic reticulum is referred to as store-operated calcium entry (SOCE).
Honokiol blocked sustained calcium entry (i.e., SOCE) at concentrations at which it did not affect initial calcium release (i.e., calcium release from the ER (Figure 1B). For example, 10 μmol/l honokiol completely inhibited SOCE without significantly depressing the initial rise in [Ca2+]i. At higher concentrations (20 and 30 μmol/l), honokiol blocked both calcium responses.
Reversibility of honokiol effects on muscarinic signaling
Influence of honokiol on ligand binding to muscarinic receptors
Muscarinic signaling in CHO cells
The influence of honokiol on muscarinic signaling was investigated in CHO cells expressing the human M3 muscarinic receptor. In wild-type CHO cells, we cannot detect acetylcholinesterase activity, muscarinic receptor binding, or muscarinic signaling as reflected in either calcium mobilization or alteration of cAMP synthesis in response to carbamylcholine. However, CHO cells express components of both the phospholipase Cβ  and adenylate cyclase  signaling pathways, and CHO cells transfected with transgenes for the different muscarinic receptor subtypes respond to muscarinic agonists in pharmacologically and physiologically appropriate fashions: Activation of human M1, M3 or M5 receptors expressed in CHO cells leads to the production of IP3, release of calcium from the ER, activation of SOCE, and modulation of the expression of genes under control of the AP-1 or NFAT response elements  (AL Martin et al., unpublished observations). Activation of human M2 and M4 receptors expressed in CHO cells leads to the inhibition of forskolin-stimulated cAMP formation and alteration of the expression of genes under the control of the cAMP response element (CRE)  (AL Martin et al., unpublished observations).
M3 receptors activate Gαq transducer proteins that stimulate phospholipase Cβ (PLCβ) . PLCβ hydrolyzes phosphatidylinositol 4.5-bisphosphate, releasing both diacylglycerol and inositol trisphosphate (IP3). IP3 binds to the IP3 receptor/ionophore on the endoplasmic reticulum (ER) to release calcium into the cytosol. Sensors in STIM1 proteins that span ER membranes are sensitive to the depletion of ER calcium. This leads to a close apposition of ER and plasma membranes, allowing STIM1 proteins to interact with Orai channel proteins in the plasma membrane, thereby stimulating the entry of extracellular calcium. This is called capacitive calcium entry or store-operated calcium entry (SOCE) [11, 13, 18].
Honokiol disruption of muscarinic signaling
Calcium release from the ER through the IP3 receptor ionophore was visualized as the initial release of calcium in response to carbamylcholine. This release was not affected by honokiol in either the presence or absence of extracellular calcium (Figures 1 & 2). Honokiol had variable effects on calcium release from the ER induced by thapsigargin. In multiple instances, honokiol did not affect thapsigargin-induced release at concentrations that produced strong inhibition of SOCE. However, at higher concentrations, an inhibition of both thapsigargin-induced release and SOCE was observed. The precise relationship between the thapsigargin-induced release, ER calcium concentration, and SOCE gating requires additional clarification.
SOCE was visualized by depleting calcium from the ER by stimulating the muscarinic-IP3 receptor pathway or by inhibiting SERCA pumps in the absence of extracellular calcium. When [Ca2+]i returned to its baseline level (3-5 min), calcium was added to the extracellular medium (final concentration = 2 mmol/l). Calcium immediately entered the cell via the SOCE channels that had opened in response to depletion of ER calcium. In both paradigms, honokiol displayed a strong inhibition of extracellular calcium entry through SOCE channels.
Honokiol’s effect on SOCE was selective insofar as honokiol inhibited SOCE at lower concentrations than it affected ER calcium release induced by either IP3 receptor activation or thapsigargin inhibition of SERCA. Honokiol’s effect was rapid and partially reversible (at concentrations below 30 μmol/l), and was observed at concentrations and under conditions in which honokiol was not cytotoxic and did not interfere with ligand binding to the muscarinic receptor. Previous work has documented honokiol inhibition of muscarinic receptor- and depolarization-mediated contraction of isolated porcine trachea, guinea pig ileum and rat uterus, apparently by blocking the influx of extracellular calcium [8–10]. The present work suggests that an inhibition of SOCE is the mechanism underlying these effects on autonomic signaling.
The threshold for honokiol inhibitory effects in vitro was ≈ 3 μM. This is consistent with the concentration dependence of in vitro antiinflammatory, antithrombosis, signaling and antioxidant effects of honokiol. For example, Alexeev et al.  noted magnonol and honokiol effects on GABAergic transmission in the 1-30 μM range, Tian et al. used 150 μM honkiol to enhance cell death of cultured cancer cells , Nagalingam et al. found that honokiol concentrations of 5 μM honokiol were required to inhibit the clonogenicity of cultured breast cancer cells by activation of AMP-activated protein kinase , and Fukuyama et al., demonstrated a neurtorphic acivity (neurtie outgrowth) of 0.1 – 10 μM honokiol on cultured rat cortical neurons . Moreover, Lu et al. used 1 -100 μM honokiol to inhibit responses of uteri to cholinergic agonists  and Ko et al used 0.1 -100 μM honokiol to inhibited cholinergic responses of tracheal muscle .
In earlier work we demonstrated that oxidative stress (e.g., by exposure to metal oxide nanoparticles) also has a selective inhibitory effect on SOCE . Honokiol, however, has antioxidant properties and is cytoprotective in the face of multiple insults, including oxidative stress. This raises the possibility that honokiol actions reflect a direct interaction with the ion channel (Orai) itself, rather than an indirect effect on the redox state of the cell. This would be consistent with honokiol’s antioxidant activity, lack of cytotoxicity, and rapid and partially reversible inhibition of SOCE. Another possibility that remains to be evaluated is whether honokiol disrupts Orai – Stim1 interactions. It is interesting that honokiol, unlike most pharmacologically exploited ion channel blockers, is not a hydrophobic amine.
The effect of honokiol on resting [Ca2+]i. was biphasic. The decrease in [Ca2+]i. at honokiol concentrations below 10 μmol/l might reflect the role of Orai channels as one of the transport mechanisms that determine cellular calcium concentration . It is also possible that honokiol alters the resting calcium content aof the endoplasmic reticulum, which might reflect changes in active sequestration mechanisms (SERCA) or ER channel conductances. Increases in resting [Ca2+]i. are associated with incipient cytotoxicity. Honokiol recently was reported to promote calcium release associated with mitochondrial dysfunction in human chondrosarcoma cells . However, even at high and ultimately cytotoxic concentrations of honokiol, virtually all of the cells remained capable of maintaining [Ca2+]i at constant levels over the time course of these acute experiments.
Honokiol had a potent inhibitory effect on store operated calcium entry (SOCE) stimulated by activation of the M3 muscarinic receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated release, or disruption of M3 receptor binding. It is likely that inhibition of SOCE accounts for honokiol disruption of parasympathetic motor functions, and may contribute to certain of its beneficial pharmacological properties.
Co-Corresponding author: Ming-Huan Chan, Ph.D. Institute of Neuroscience, Research Ctr for Mind, Brian & Learning, National, Chengchi University, No. 64, Sec 2, ZhiNan Rd. Taipei, Taiwan.
Basal salt solution
Intracellular calcium concentration
Chinese hamster ovary
Sarco/endoplasmic reticulum calcium-ATPase
Store operated calcium entry
The authors thank Dr. Chimpiao (Natinal Dong Hwa University, Hualien Taiwan) for providing the honokiol used in this work. This research was supported by the Missouri S&T cDNA Resource Center (http://www.cdna.org).
- Fried LE, Arbiser JL: Honokiol, a multifunctional antiangiogenic and antitumor agent. Antioxid Redox Signal. 2009, 11 (5): 1139-1148. 10.1089/ars.2009.2440.PubMed CentralView ArticlePubMedGoogle Scholar
- Dikalov S, Losik T, Arbiser JL: Honokiol is a potent scavenger of superoxide and peroxyl radicals. Biochem Pharmacol. 2008, 76 (5): 589-596. 10.1016/j.bcp.2008.06.012.PubMed CentralView ArticlePubMedGoogle Scholar
- Chao LK, Liao PC, Ho CL, Wang EI, Chuang CC, Chiu HW, Hung LB, Hua KF: Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein kinase, and the NF-kappaB pathway to reduce LPS-induced TNFalpha and NO expression. J Agric Food Chem. 2010, 58 (6): 3472-3478. 10.1021/jf904207m.View ArticlePubMedGoogle Scholar
- Lee J, Jung E, Park J, Jung K, Lee S, Hong S, Park E, Kim J, Park S, Park D: Anti-inflammatory effects of magnolol and honokiol are mediated through inhibition of the downstream pathway of MEKK-1 in NF-kappaB activation signaling. Planta Med. 2005, 71 (4): 338-343. 10.1055/s-2005-864100.View ArticlePubMedGoogle Scholar
- Lee SY, Cho JY: Inhibitory effects of honokiol on LPS and PMA-induced cellular responses of macrophages and monocytes. BMB Rep. 2009, 42 (9): 574-579. 10.5483/BMBRep.2009.42.9.574.View ArticlePubMedGoogle Scholar
- Lin YR, Chen HH, Ko CH, Chan MH: Effects of honokiol and magnolol on acute and inflammatory pain models in mice. Life Sci. 2007, 81 (13): 1071-1078. 10.1016/j.lfs.2007.08.014.View ArticlePubMedGoogle Scholar
- Lin YR, Chen HH, Lin YC, Ko CH, Chan MH: Antinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory pain. J Biomed Sci. 2009, 16: 94-10.1186/1423-0127-16-94.PubMed CentralView ArticlePubMedGoogle Scholar
- Chan SS, Zhao M, Lao L, Fong HH, Che CT: Magnolol and honokiol account for the anti-spasmodic effect of magnolia officinalis in isolated guinea pig ileum. Planta Med. 2008, 74 (4): 381-384. 10.1055/s-2008-1034320.View ArticlePubMedGoogle Scholar
- Ko CH, Chen HH, Lin YR, Chan MH: Inhibition of smooth muscle contraction by magnolol and honokiol in porcine trachea. Planta Med. 2003, 69 (6): 532-536.View ArticlePubMedGoogle Scholar
- Lu YC, Chen HH, Ko CH, Lin YR, Chan MH: The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus. Naunyn Schmiedebergs Arch Pharmacol. 2003, 368 (4): 262-269. 10.1007/s00210-003-0802-8.View ArticlePubMedGoogle Scholar
- Putney JW: Capacitative calcium entry: from concept to molecules. Immunol Rev. 2009, 231 (1): 10-22. 10.1111/j.1600-065X.2009.00810.x.View ArticlePubMedGoogle Scholar
- Smyth JT, Hwang SY, Tomita T, Dehaven WI, Mercer JC, Putney JW: Activation and regulation of store-operated calcium entry. J Cell Mol Med. 2010, 14 (10): 2337-2349. 10.1111/j.1582-4934.2010.01168.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Putney JW: Pharmacology of store-operated calcium channels. Mol Interv. 2010, 10 (4): 209-218. 10.1124/mi.10.4.4.PubMed CentralView ArticlePubMedGoogle Scholar
- Bird GS, DeHaven WI, Smyth JT, Putney JW: Methods for studying store-operated calcium entry. Methods. 2008, 46 (3): 204-212. 10.1016/j.ymeth.2008.09.009.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang HJ, Growcock AC, Tang TH, O’Hara J, Huang YW, Aronstam RS: Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway. Toxicol In Vitro. 2010, 24 (7): 1953-1961. 10.1016/j.tiv.2010.08.005.View ArticlePubMedGoogle Scholar
- Varga EV, Stropova D, Rubenzik M, Wang M, Landsman RS, Roeske WR, Yamamura HI: Identification of adenylyl cyclase isoenzymes in CHO and B82 cells. Eur J Pharmacol. 1998, 348 (2–3): R1-R2.View ArticlePubMedGoogle Scholar
- Aronstam RS, Patil P: Muscarinic receptors: autonomic neurons. Encyclopedia of neuroscience. Edited by: Squire LR. 2009, Oxford: Academic, 1141-1149. 4View ArticleGoogle Scholar
- Smyth JT, Dehaven WI, Bird GS, Putney JW: Ca2 + -Store-dependent and -independent reversal of stim1 localization and function. J Cell Sci. 2008, 121 (Pt 6): 762-772.PubMed CentralView ArticlePubMedGoogle Scholar
- Alexeev M, Grosenbaugh DK, Mott DD, Fisher JL: The natural products magnolol and honokiol are positive allosteric modulators of both synaptic and extra-synaptic GABA(A) receptors. Neuropharmacology. 2012, 62 (8): 2507-2514. 10.1016/j.neuropharm.2012.03.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Tian W, Deng Y, Li L, He H, Sun J, Xu D: Honokiol synergizes chemotherapy drugs in multidrug resistant breast cancer cells via enhanced apoptosis and additional programmed necrotic death. Int J Oncol. 2013, 42 (2): 721-732.PubMedGoogle Scholar
- Nagalingam A, Arbiser JL, Bonner MY, Saxena NK, Sharma D: Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis. Breast Cancer Res: BCR. 2012, 14 (1): R35-10.1186/bcr3128.PubMed CentralView ArticlePubMedGoogle Scholar
- Fukuyama Y, Nakade K, Minoshima Y, Yokoyama R, Zhai H, Mitsumoto Y: Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons. Bioorganic Med Chem Lett. 2002, 12 (8): 1163-1166. 10.1016/S0960-894X(02)00112-9.View ArticleGoogle Scholar
- Ermak G, Davies KJ: Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol. 2002, 38 (10): 713-721. 10.1016/S0161-5890(01)00108-0.View ArticlePubMedGoogle Scholar
- Chen YJ, Wu CL, Liu JF, Fong YC, Hsu SF, Li TM, Su YC, Liu SH, Tang CH: Honokiol induces cell apoptosis in human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress. Cancer Lett. 2010, 291 (1): 20-30. 10.1016/j.canlet.2009.08.032.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.