Human Muscarinic Acetylcholine Receptors Are a Target of the Marine

Chem. Res. Toxicol. 2010, 23, 1753–1761

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Human Muscarinic Acetylcholine Receptors Are a Target of the Marine Toxin 13-Desmethyl C Spirolide Carolina B. Wandscheer, Natalia Vilarinõ, Begonã Espinã, M. Carmen Louzao, and Luis M. Botana* Departamento de Farmacologıá, Facultad de Veterinaria, UniVersidad de Santiago de Compostela, Campus UniVersitario, 27002 Lugo, Spain ReceiVed June 23, 2010

Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxication has been related to their presence in shellfish yet, the possible toxicological consequences to human health are actually unknown. The elucidation of the spirolide mechanism/s of action would help to estimate the threat to human consumers. Previous toxicological studies in mice suggested the involvement of acetylcholine receptors. In this work, the effects of the 13-desmethyl C spirolide on the activity and the expression of muscarinic acetylcholine receptors (mAChR) were analyzed using a human neuroblastoma cell model. The 13-desmethyl C spirolide inhibited the acetylcholine-induced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The 13-desmethyl C spirolide also reduced binding of the mAChR specific antagonist [3H]QNB to neuroblastoma cells. The effect of the 13-desmethyl C spirolide persisted after toxin removal and was inhibited by protection of the primary binding site with high concentrations of atropine suggesting an interaction of the spirolide with the orthologous binding site of mAChR. Moreover, the toxin induced a change in the characteristics of the membrane-associated M3 mAChRs, although it did not alter the total levels of M3 mAChR protein. The 13-desmethyl C spirolide targets mAChRs causing a reduction of function, a decrease of specific antagonist binding to mAChRs, and alteration of membrane-bound receptors that might have important toxicological implications. 1. Introduction The cyclic imine 13-desmethyl C spirolide (Figure 1) belongs to a group of marine toxins with a macrocyclic structure that was designated as spirolide due to the presence of a spiro ring (1, 2). This group of toxins was discovered in the early 1990s in Canada during routine testing for lipophilic marine toxins due to their fast-acting toxicity and the neurological symptoms observed in mouse bioassays (3, 4). The spirolides are produced by phytoplankton (the only species identified until now capable of producing spirolides are Alexandrium ostenfeldii and Alexandrium peruVianun 5, 6) and can reach human consumers by accumulation in filter-feeding mollusks. Spirolide-toxic, algal blooms have been described mainly in America and Europe (3, 7-12), but their toxicity to humans is still unknown. The existing toxicological data have been obtained in laboratory animals and indicate that the intraperitoneal toxicity of the 13desmethyl C spirolide is higher than its oral toxicity. A recently estimated LD50 of 5-8 µgkg-1 for pure 13-desmethyl C spirolide by intraperitoneal injection (13) suggests a higher toxicity than that initially published from a mixture of spirolides, whose main component was 13-desmethyl C spirolide (LD50 of 40 µgkg-1) (3). The oral LD50 when pure 13-desmethyl C spirolide is administered by gavage has an estimated value of approximately 150 µgkg-1 (13). The mechanism of action of these toxins is still not fully elucidated. In vivo toxicological studies demonstrated that the toxin induces neurological symptoms including stiffening and arching of the tail toward the head, tremors progressing to spasm of the hind limbs, respiratory distress, * To whom correspondence should be addressed. Tel/Fax: +34 982252242. E-mail: [email protected].

Figure 1. Chemical structure of the 13-desmethyl C spirolide.

tremors of the whole body, and respiratory arrest (4). Moreover, neuronal damage in mice and an increase of the mRNA levels of muscarinic acetylcholine receptors (mAChR1) (1) and nicotinic acetylcholine receptors (nAChR) in rats were observed after i.p. administration of lethal doses of spirolides (4). Very recently, the spirolides have been demonstrated to bind to nAChRs with high affinity and behave as nAChR antagonists (14-16). However, there is no direct evidence of mAChRs being targeted by spirolides. Muscarinic AChRs are G protein-coupled receptors (GPCR) that can signal through cAMP generation or through an increase 1 Abbreviations: BSA, bovine serum albumin; GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; nAChR, nicotinic acetylcholine receptor; NMS, N-methylscopolamine; PBS, phosphate buffered saline; QNB, quinuclidinyl benzilate; TBS-T, Tris buffered saline-Tween 20; SPX, spirolide.

10.1021/tx100210a  2010 American Chemical Society Published on Web 10/18/2010

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of the cytosolic calcium concentration ([Ca2+]i) depending on the associated heterotrimeric G protein (17, 18). M1, M3, and M5 receptors are usually coupled to Gq/11 proteins and trigger a calcium signal following PLC activation. The stimulation of M2 and M4 mAChRs, which are preferentially coupled to Gi/o proteins, induces a reduction of cAMP levels through inhibition of adenylyl cyclase. Muscarinic AChRs are implicated in a multitude of functions of the central and peripheral nervous systems and the parasympathetic innervated organs. Besides, this “classic” muscarinic signaling, the processes of receptor internalization and up- or down-regulation are an important part of mAChR regulation. Actually, receptor occupation by agonists and antagonists has been shown to induce the internalization of the receptor (19, 20). Down-regulation of total receptor levels is often triggered by persistent stimulation with agonists, while receptor antagonists have been demonstrated to induce upregulation of the total receptor (18). The aim of this study was to elucidate the pharmacologic effect of the 13-desmethyl C spirolide on human mAChRs. In order to do that, we chose a cell model known to have an AChelicited calcium signal which had been previously shown to be dependent on mAChR activation, the neuroblastoma BE(2)-M17 cell line (21). Receptor binding experiments and functional assays based on the measurement of the calcium signal were used to explore the effect of this toxin on calcium signalinglinked mAChRs. Additionally, the effect of the toxin on the levels of the receptor protein was also analyzed.

2. Experimental Procedures 2.1. Reagents. Eagle’s minimum essential medium (EMEM), Ham’s F12, glutamine, and nonessential amino acids were purchased from Biochrom AG (Berlin, Germany). Fetal bovine serum (FBS), gentamycin, Tris base, Trypsin-EDTA solution, mercaptoethanol, atropine sulfate, epibatidine dihydrochloride, acetylcholine chloride, amphotericine B, bovine serum albumin (BSA), and antiβ-tubulin were purchased from Sigma (St. Louis, MO). Thapsigargin and ionomycin free acid were purchased from Alexis Corporation (Laüfelfingen, Switzerland). Fura-2 acetoxymethyl ester was purchased from Molecular Probes (Leiden, Netherlands). See Blue prestained standard, Novex 10% Tris-Glycine gels, and TrisGlycine SDS sample buffer were from Invitrogen (Carlsbad, CA). The polyvinylidene fluoride (PVDF) membrane (Immobilon P membrane) was from Millipore (Billerica, MA). Alamar Blue was from Biosource International (Camarillo, CA). Antimuscarinic acetylcholine receptor M1 (H-120) and M3 (H-210) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the anti-Na+/K+ ATPase alpha 1 subunit antibody was from Abcam (Cambridge, UK). Amersham ECL Western Blotting Detection Reagent and [3H]-quinuclidinyl benzilate ([3H]QNB) were purchased from GE Healthcare (Buckinghamshire, UK). Super Signal West Femto and Pico Chemiluminescent Substrates, antirabbit HRP, and antimouse HRP antibodies were from Thermo Scientific Pierce (Rockford, USA). The culture flasks and plates were purchased from Nunc (Roskilde, Denmark). The scintillation vials were from Beckman Coulter (Brea, CA). The scintillation fluid, Ultima Gold, and [3H]N-methylscopolamine ([3H]NMS) were purchased from PerkinElmer (Walthom, MA). 13-Desmethyl C spirolide was from Cifga Laboratorio (Lugo, Spain). Phosphatebuffered saline (PBS) composition was 137 mM NaCl, 8.2 mM Na2HPO4, 3.2 mM KCl, and 1.5 mM KH2PO4, pH 7.4. TBS-T (Tris buffered saline-Tween 20) was 50 mM Tris base, 150 mM NaCl, and 0.05% Tween 20. The extracellular medium used during intracellular Ca2+ concentration measurements was 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM glucose, 1.2 mM MgCl2, 0.44 mM KH2PO4, and 4.2 mM NaHCO3, pH 7.4. 2.2. Cell Line Culture. Human neuroblastoma BE(2)-M17 cell line (European Collection of Cell Cultures) was cultured in EMEM/

Wandscheer et al. Ham’s F12 (1:1) supplemented with 2 mM glutamine, 1% nonessential amino acids, 10% FBS, 50 mg/L gentamycin, and 50 µg/L amphotericine B. For imaging assays, neuroblastoma cells were grown on glass coverslips at a density of 3-4 × 104 cells/well and used after 5-7 days. Cell cultures were kept at 37 °C in 5% CO2. Cell subcultures were transferred with trypsin/EDTA. 2.3. [Ca2+]i Measurements. For [Ca2+]i measurements, the neuroblastoma cells, previously seeded on coverslips, were loaded for 10 min with the [Ca2+] sensitive dye fura-2 acetoxymethyl ester (0.5 µM) at 37 °C in darkness in a final volume of 1 mL of the experimental standard solution containing 0.1% BSA. After dye loading, the neuroblastoma cells were washed three times with experimental extracellular solution and incubated with 13-desmethyl C spirolide in culture medium for varying periods of time as indicated. After the preincubation time with toxin, the neuroblastoma cells were washed two times with fresh buffer. These coverslips were placed into a thermostatted chamber at 37 °C, and neuroblastoma cell images were registered with a Nikon Diaphot microscope equipped with epifluorescence optics (Nikon 40x immersion fluor objective) and a Lambda 10-2 Sutter Instrument. Fluorescent images were collected at dual excitation wavelengths of 340 and 380 nm, and an emission wavelength of 530 nm. The calculation of [Ca2+]i was carried out by using the method of Grynkiewicz et al. (22). 2.4. Cytotoxicity Assays. The cytotoxicity of 13-desmethyl C spirolide was assessed by measuring cell metabolic activity in control and toxin-exposed cell cultures using the fluorescent probe Alamar blue as described in ref 23. Alamar blue fluorescence was measured at 30 min, 1, 12, and 24 h. 2.5. Western Blot Analysis. Neuroblastoma cells were incubated with 13-desmethyl C spirolide for different periods of time. After two washes with PBS, the cells were harvested by scraping, centrifuged, lysed in sample buffer with 2.5% β-mercaptoethanol, and boiled for 5 min. For the analysis of membrane-associated mAChRs, ProteoExtract Transmembrane Protein Extraction Kit (Novagen, Darmstadt, Germany) was used following the manufacturer’s instructions. Reagent B was used for the separation of the membrane fraction. The membrane extracts were diluted with 2× the sample buffer with 5% β-mercaptoethanol and heated at 60 °C for 10 min. The cellular proteins were separated by electrophoresis and transferred to a PVDF membrane. After blocking with 2% powdered nonfat milk, the membrane was incubated with antimAChR M1 or M3 antibodies, washed, and incubated with an antirabbit HRP antibody. Finally, the membrane was washed again with TBS-T and exposed to the Super Signal West Femto Chemiluminescent Substrate. The images were acquired by a Diversity Imaging System (Syngene, Cambridge, UK), and band density was quantified with the Gene Tools 3.08 software (Syngene, Cambridge, UK). The very same membranes were reblotted with an anti-β-tubulin antibody or an anti-Na+/K+ ATPase antibody as a control for lane loading in whole cell lysate blots and transmembrane protein blots, respectively. 2.6. Interaction with mAChR. The interaction with mAChRs was studied by inhibition of [3H]QNB binding. Neuroblastoma cells were passaged 24 h before the experiment, and 0.1 × 106 cells were seeded in 24 well plates. The cells were incubated for 1 h at 37 °C and 5% CO2 with several concentrations of 13-desmethyl C spirolide. Nonspecific binding was determined in the presence of 100 µM atropine in every experiment. [3H]QNB or [3H]NMS was subsequently added at a final concentration of 50 nM or 2 nM, respectively, and the samples were incubated for 1 h at room temperature. The concentration of atropine and 13-desmethyl C spirolide was maintained in each well for the whole duration of the experiment unless stated otherwise. Finally, the wells were quickly washed twice with PBS, and 400 µL of 0.1 M NaOH was added. A volume of 380 µL from each well was transferred into scintillation vials, and 2 mL of scintillation fluid was added to each vial. Radioactivity was detected in a Beckman counter LS6000TA (Fullerton, CA). The results were expressed as specific binding in counts per minute (cpm). Specific binding was determined as the difference between total cpm for each condition and cpm in the

Spirolides Target Muscarinic Receptors

Figure 2. Characterization of the acetylcholine-induced [Ca2+]i response in human neuroblastoma BE(2)-M17 cells. (A) Neuroblastoma cells were incubated for 10 min in the presence or absence of 100 µM atropine and then stimulated with 100 µM ACh (n ) 3, mean ( SEM). The concentration of atropine was maintained constant through the whole duration of the experiment. (B) [Ca2+]i in neuroblastoma cells stimulated with epibatidine (n ) 3, mean ( SEM). The arrows indicate agonist addition.

presence of atropine. For most mathematical approaches used in curve fitting, the concentration of the free ligand is assumed to be equal to the concentration that is added. The radioactivity of the culture medium with a concentration of 50 nM [3H]QNB did not change after a 1 h incubation with the cells, suggesting that the concentration of free [3H]QNB after binding can be considered equal to the concentration added. 2.7. Statistical Analysis. Statistical significance of differences between treatments was determined by using ANOVA followed by Tukey’s multiple comparison post-test, except when there were only two groups of data in which case a paired t-test was used. Data were expressed as the mean ( standard error of the mean (SEM) of separate experiments. The difference between treatments were considered significant at p < 0.05.

3. Results 3.1. Effect of 13-Desmethyl C Spirolide on the AChElicited Calcium Signal. The effect of 13-desmethyl C spirolide on mAChR was studied using the ACh-elicited [Ca2+]i response in BE(2)-M17 neuroblastoma cells as a read out. The AChdependent response in this neublastoma cell line was characterized using the mAChR antagonist atropine and the nAChR agonist epibatidine. The [Ca2+]i response induced by 100 µM ACh was completely inhibited by the presence of 100 µM atropine (Figure 2A). Epibatidine did not elicit a [Ca2+]i response in BE(2)-M17 neuroblastoma cells at concentrations of 0.1 mM (Figure 2B) and 1 mM. These results demonstrate that the ACh-

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elicited [Ca2+]i response in neuroblastoma cells is due to the stimulation of mAChRs, without the involvement of nAChRs. A 1 h incubation with the cyclic imine 13-desmethyl C spirolide inhibited the [Ca2+]i response induced by the addition of 100 µM ACh to the extracellular medium (Figure 3A). The addition of 13-desmethyl C spirolide to the extracellular medium did not cause any change of the [Ca2+]i in neuroblastoma cells (Figure 3B). The dose-dependency of the 13-desmethyl C spirolide-inhibitory effect was evaluated by the quantification of the [Ca2+]i rise from the baseline before the addition of ACh to the peak of the [Ca2+]i response. Figure 3C shows that an exposure of neuroblastoma cells to concentrations of the spirolide ranging from 20 to 500 nM increasingly inhibited the ACh-induced [Ca2+]i rise. The IC50 was calculated using a four parameter logistic equation and had an estimated value of 59.5 nM (SE of log IC50 ) 0.08). A concentration of 100 nM was selected for further experiments. In order to characterize the inhibition of mAChRs by 13desmethyl C spirolide, dose-response curves to ACh were obtained in the presence and absence of 100 nM 13-desmethyl C spirolide. As expected, the [Ca2+]i rise induced by ACh stimulation was reduced in cells preincubated with 13-desmethyl C spirolide (Figure 3D). Actually, the 13-desmethyl C spirolide reduced the maximum response to ACh (Figure 3D) suggesting that the 13-desmethyl C spirolide might be an irreversible competitive inhibitor of mAChRs. The inhibitory effect of the 13-desmethyl C spirolide was dependent on the time of exposure to the toxin. An exposure of 10 min to 100 nM 13-desmethyl C spirolide was not enough to achieve the full inhibitory effect of this toxin concentration, which was reached after 30 min or 1 h of incubation in the presence of 13-desmethyl C spirolide (Figure 3E). An exposure of 2 min to the spirolide was also tested and had no effect on the response to ACh (data not shown). Moreover, the inhibitory effect of the spirolide on the ACh-induced calcium signal persisted during a relatively short period of time when the toxin was removed from the external medium before stimulation. Even a time lag of 30 min between the removal of the toxin and the stimulation did not affect the spirolide inhibitory effect (data not shown). The inhibitory effect of 100 nM 13-desmethyl C spirolide on the ACh-induced calcium signal was significantly reduced when the spirolide was incubated simultaneously with 100 µM atropine (Figure 3F), suggesting a competition of atropine and 13-desmethyl C spirolide for the same binding site. However, this binding site seems to be responsible only for a fraction of the 13-desmethyl C spirolide inhibitory action. The [Ca2+]i signal induced by thapsigargin (an inhibitor of the intracellular Ca2+ pump) in a Ca2+-containing medium was not affected by the presence of the 13-desmethyl C spirolide. The [Ca2+]i signal elicited by ACh in a Ca2+-free medium was completely inhibited by prestimulation with thapsigargin, indicating that the intracellular Ca2+ stores emptied by ACh are thapsigargin-dependent and that therefore the Ca2+ entry mechanisms elicited by both compounds are probably related (data not shown). 3.2. Binding of 13-Desmethyl C Spirolide to mAChR. In order to elucidate if the effect of the 13-desmethyl C spirolide on the mAChR-dependent response occurred at the receptor level, the binding of the mAChR antagonist [3H]QNB to neuroblastoma cells was quantified in the presence and absence of 13-desmethyl C spirolide. The cells were incubated for 1 h with several concentrations of the toxin and then an equal concentration of [3H]QNB was added to all wells. The spirolide inhibited the specific binding of [3H]QNB to neuroblastoma cells

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Figure 3. Effect of 13-desmethyl C spirolide on [Ca2+]i and on the acetylcholine-induced [Ca2+]i response in human neuroblastoma BE(2)-M17 cells. (A) Neuroblastoma cells were incubated for 1 h in the presence or absence of 100 nM 13-desmethyl C spirolide and then stimulated with 100 µM acetylcholine (n ) 7, mean ( SEM). The arrow indicates ACh addition. (B) [Ca2+]i was recorded during the addition of 500 nM 13-desmethyl C spirolide to the external medium in neuroblastoma cells. The arrow indicates toxin addition (n ) 3, mean ( SEM). (C) [Ca2+]i increase induced by ACh in the presence of several concentrations of 13-desmethyl C spirolide. Neuroblastoma cells were incubated with 20, 50, 80, 100, and 500 nM 13-desmethyl C spirolide for 30 min and then stimulated with 100 µM ACh. The results are expressed as [Ca2+]i increase from the baseline before the addition of the stimulus to the peak after stimulation. The bar shows the response to ACh in the absence of toxin. (n ) 3, mean ( SEM; * statistically different versus 100 µM ACh; and ** statistically different versus low spirolide concentrations plus 100 µM ACh, p < 0.05). (D) Effect of the 13-desmethyl C spirolide on the dose-response curve of ACh-induced [Ca2+]i rise. Neuroblastoma BE(2)-M17 cells were incubated for 60 min in the presence or absence of 100 nM 13-desmethyl C spirolide and then stimulated with 0.1, 5, 100, 500, or 2500 µM ACh. The results are expressed as [Ca2+]i increase from baseline to peak response (n ) 7, mean ( SEM; * statistically different versus matched concentration of ACh, p < 0.05). (E) Kinetics of 13-desmethyl C spirolide-induced inhibition of the ACh-dependent [Ca2+]i rise. Neuroblastoma BE(2)-M17 cells were incubated for 10, 30, and 60 min in the presence of 100 nM 13-desmethyl C spirolide and then stimulated with 100 µM ACh. The ACh control response was monitored in cells incubated for the same periods of time in the absence of toxin. The results are expressed as [Ca2+]i increase from baseline to peak response (n ) 3, mean ( SEM; * statistically different versus 100 µM ACh, p < 0.05; and ** statistically different versus 30 and 60 min time points, p < 0.05). (F) Effect of simultaneous incubation with atropine and 13-desmethyl C spirolide on the peak of the ACh-induced [Ca2+]i rise. The cells were incubated with 100 µM atropine +100 nM 13-desmethyl C spirolide, 100 µM atropine, 100 nM 13-desmethyl C spirolide, or carrier for 1 h, then washed and stimulated with 100 µM ACh (n ) 4, mean ( SEM; * statistically different versus control response, p < 0.05; and ** statistically different versus 13-desmethyl C spirolide treatment, p < 0.05).

(Figure 4A). The biphasic appearance of the inhibition curve suggested two different populations of receptors with different

affinity for the spirolides. Actually, a competitive binding to two-receptor type equation (assuming equal affinity of the


Figure 4. Effect of 13-desmethyl C spirolide on [3H]QNB binding to neuroblastoma BE(2)-M17 cells. (A) Neuroblastoma cells were incubated for 1 h with 0.01, 0.1, 1, 10, 100, and 500 nM 13-desmethyl C spirolide and then incubated for 1 h in the presence of 50 nM [3H]QNB. The concentration of 13-desmethyl C spirolide was maintained constant during the whole duration of the experiment. A control of maximum [3H]QNB binding in the absence of the spirolide (bar on the right panel) was also included in each experiment. A competitive binding to a two-receptor type model was used for curve fitting (n ) 4, mean ( SEM; * statistically different versus 50 nM [3H]QNB). (B) Neuroblastoma cells were incubated for 1 h in the presence or absence of 100 nM 13-desmethyl C spirolide, washed twice to remove the toxin, and incubated in the absence of toxin for an additional hour. Then the cells were incubated for 1 h in the presence of 50 nM [3H]QNB. A control of maximum [3H]QNB binding in the absence of the spirolide and a control of 13-desmethyl C spirolide binding for 1 h followed by incubation with [3H]QNB (no wash) were also included in the experiment (n ) 4, mean ( SEM; * statistically different versus 50 nM [3H]QNB). (C) Neuroblastoma cells were incubated for 1 h with 100 µM atropine and 100 nM 13-desmethyl C spirolide. Then the cells were washed twice to remove both compounds and incubated for 1 h in the presence of 50 nM [3H]QNB. A control of maximum [3H]QNB binding and a control of 13-desmethyl C spirolide binding in the absence of atropine are also shown (n ) 7, mean ( SEM; * statistically different versus 50 nM [3H]QNB control; ** statistically different versus atropine +13-desmethyl C spirolide and atropine alone).


radiolabeled ligand for the two binding sites) fitted the data better than a four-parameter logistic equation based on R2 and residual standard deviation values (GraphPad software, San Diego, CA). The top and bottom of the curve were set constant with the top equal to [3H]QNB binding in the absence of spirolide and the bottom equal to 0. The variables of the equation provided IC50 values of 26 ( 2.9 pM for the first site and 814 ( 20 nM for the second site, with a fraction of the first binding site of 0.39 ( 0.04 (R2 ) 0.9831 and residual standard deviation ) 130.4). The 13-demethyl C spirolide also inhibited [3H]NMS specific binding to the cells, although 500 nM 13-demethyl C spirolide inhibited only 52.7 ( 1.6% of [3H]NMS binding (n ) 3, data not shown), similar to the inhibition of [3H]QNB binding in the same conditions (59.7 ( 0.9%, Figure 4A). The reversibility of the interaction of the 13-desmethyl C spirolide with mAChRs was analyzed by the removal of the toxin from the extracellular medium by 3 washes with fresh incubation medium. The cells were then incubated in the absence of toxin for 10 min, 30 min, and 1 h. Finally, [3H]QNB was added to the cells. The inhibition of [3H]QNB binding by the 13-desmethyl C spirolide remained after the spirolide was removed. Actually, there was no significant difference between [3H]QNB binding to cells treated with the spirolide and to cells treated with the spirolide and washed (Figure 4B). These results did not provide any information about the site of the interaction of the 13-demethyl C spirolide on the mAChR. The protection of the orthologous or primary binding site with high concentrations of a reversible competitive antagonist was used to determine if the 13desmethyl C spirolide binds to this site. The presence of high concentrations of atropine, a mAChR reversible competitive antagonist, during the incubation with the toxin keeps the primary site occupied and precludes binding of the spirolide, provided the 13-desmethyl C spirolide is a competitive antagonist. Therefore, the first step of the experiment was a simultaneous incubation with 100 nM 13-desmethyl C spirolide and 100 µM atropine, followed by thorough washing and an incubation with [3H]QNB. All washes consisted in the removal of the culture medium followed by the addition of fresh medium three times per well, prior to the final addition of medium and [3H]QNB. Controls for [3H]QNB binding, in cells not treated at all or in cells treated with toxin or atropine alone, were also included. The removal of atropine by washing will leave the primary sites free for [3H]QNB binding. Actually, the control for atropine reversibility showed the same [3H]QNB binding signal as [3H]QNB alone (Figure 4C). If the 13-desmethyl C spirolide is a competitive antagonist, [3H]QNB binding after an incubation with atropine and the spirolide and posterior washing should be recovered to control levels. On the contrary, if the 13-desmethyl C spirolide is not a competitive antagonist, the inhibition of [3H]QNB binding should remain after washing. The results showed that [3H]QNB binding levels after a simultaneous incubation with atropine and 13-desmethyl C spirolide was similar to those of the no treatment/[3H]QNB control and the atropine alone control, and statistically different from that of the 13-desmethyl C spirolide alone (Figure 4C), demonstrating that the 13-desmethyl C spirolide is a competitive antagonist of mAChRs. 3.3. Effect of 13-Desmethyl C Spirolide on mAChR Levels. The modulation of M3 mAChR levels by the 13desmethyl C spirolide was analyzed in neuroblastoma cells exposed to the toxin for long periods of time. The presence of the M1 receptor was also tested by Western blot, but no band appeared that could be identified as the M1 receptor. The amount of total M3 mAChRs was not significantly altered by an

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4. Discussion

Figure 5. Effect of 13-desmethyl C spirolide on the level of M3 mAChR protein. Neuroblastoma BE(2)-M17 cells were incubated for 12 h in the presence of 10 or 100 nM 13-desmethyl C spirolide. Control cells were incubated in the same conditions in the absence of toxin. (A) Effect of different concentrations of 13-desmethyl C spirolide on total M3 mAChR levels. Representative Western blot of whole cell lysates for M3 mAChR (upper panel) and the reblot of the same membrane for β-tubulin (lower panel). (B) Effect of 13-desmethyl C spirolide on membrane-associated M3 mAChR levels. Representative Western blot of transmembrane protein extracts for M3 mAChR (upper panel) and the reblot of the same membrane for Na+/K+ ATPase (lower panel). (C) Quantification of the effect of 100 nM 13-desmethyl C spirolide on M3 total protein (n ) 3, mean ( SEM; * statistically different versus control) and (D) on membrane-associated M3 receptors in cells treated for 12 h with the toxin vs control cells (n ) 5, mean ( SEM; * statistically different versus control).

exposure to 10 or 100 nM 13-desmethyl C spirolide for 12 h (Figure 5A and C). Incubations with the toxin for 1, 3, 6, 24, and 48 h did not have an effect either on the total levels of the M3 mAChR (data not shown). On the contrary, when membraneassociated M3 receptors in neuroblastoma cells were analyzed using a transmembrane-protein extraction kit, the amount of receptors extracted by the kit reagent was significantly reduced after 12 h of incubation with 100 nM 13-desmethyl C spirolide versus toxin free cultures (Figure 5B and D). No effect on the membrane-associated M3 receptor was observed at 1 h of incubation time or with 10 nM 13-desmethyl C spirolide. The morphology of neuroblastoma cells was similar to that of the controls in all of the experiments performed during this study (data not shown), and the cytotoxicity experiments demonstrated a 104.11 ( 8.24% viability of cells exposed to 100 nM 13desmethyl C spirolide for 12 h. Similar results were obtained for 10 and 500 nM 13-desmethyl C spirolide and for exposure times of 30 min, 1, and 24 h.

This study demonstrates that the 13-desmethyl C spirolide targets calcium signal-linked mAChRs in the human neuroblastoma cell line BE(2)-M17. The calcium response induced by stimulation with ACh, which is dependent on mAChRs in this cell type, is inhibited by the presence of the 13-desmethyl C spirolide indicating a possible antagonistic effect on receptor activation. This inhibition could be also due to blockage of downstream signaling events, cell death, or changes of plasma membrane-associated receptors. However, regarding downstream signaling, the calcium signal triggered by thapsigargin stimulation was not inhibited by the toxin, and more importantly, the spirolide inhibited binding of the specific antagonist [3H]QNB to mAChRs and this inhibition, as well as the inhibition of the calcium signal, was blocked by atropine, indicating that the action of the 13-desmethyl C spirolide occurs at the receptor level. The reduction of the ACh-induced calcium response and the decrease of [3H]QNB binding cannot be due to cell death because the spirolide does not affect cell viability. Internalization of mAChRs has been widely reported, and it might as well be responsible for the reduction of the mAChRrelated calcium response and [3H]QNB binding caused by the 13-desmethyl C spirolide. Actually, kinetic studies of agonistinduced desensitization due to mAChR internalization demonstrate a maximum effect around 15 to 20 min (20, 24, 25). This internalization-related desensitization has been shown to be accompanied by a change in the membrane form of the mAChR reported as a migration to a different sucrose gradient fraction (24). However, the 13-desmethyl C spirolide does not have any agonist activity, and the changes in the characteristics of membrane receptors that are reported in this work were not detectable at times shorter than 12 h of toxin exposure. Although receptor internalization of GPCRs has been mainly related to agonist binding, while antagonists have been generally related to receptor up-regulation, internalization triggered by antagonists or even antibodies has also been reported (26, 27). Receptor internalization might be responsible for the spirolide-induced reduction of the ACh-triggered calcium response; however, the probability of this mechanism of action being the only explanation for the reduction of the calcium response seems very low given the small number of reports on nonagonist-induced internalization and the data supporting toxin binding to the orthosteric binding site of the mAChR. The inhibition of the 13-desmethyl C spirolide effect on [3H]QNB binding by protection of the primary binding site of mAChRs with the reversible competitive antagonist atropine (28) demonstrates that the spirolide binds to the orthosteric binding site of the mAChR. The presence of a high concentration of atropine during the incubation with the 13-desmethyl C spirolide also reduced the inhibitory effect of the toxin on the ACh-triggered calcium signal. However, the toxin-dependent inhibition of the calcium signal was not completely blocked by atropine, suggesting that the toxin may act additionally as an allosteric antagonist of mAChRs. Some GPCR antagonists have been shown to display irreversible noncompetitive antagonism in addition to their wellknown irreversible competitive antagonism (28). Although it had been suggested that the spirolides might target mAChRs based on the symptoms displayed by animals injected intraperitoneally with these toxins and the increase of mAChR mRNA levels in brain tissue in some species (3, 4), direct evidence of mAChR targeting has not been described before. The spirolide displays an apparent “short-term irreversibility” in relatively short periods of time which is supported by the lack of recovery of the response of BE(2)-M17 cells to ACh


and of [3H]QNB binding after toxin removal. Moreover, the 13-desmethyl C spirolide-induced inhibition is time dependent, needing periods of exposure to the toxin longer than 10 min to be fully effective. The mechanism for this “short-term irreversibility” could be related to a high affinity interaction between the receptor and the toxin with extremely low dissociation of the complex, or it could be due to a real irreversible antagonism that implies covalent bonding between the receptor and the toxin, in which case we could be talking about a long-term inhibition. “Short term irreversible” functional inhibition by the 13desmethyl C spirolide due to high affinity interaction has been already reported for nAChRs (16). Theoretically, the carbon at position 3 in the butyrolactone structure (Figure 1) could be an acceptor of 1,4-Michael addition of nucleophilic groups from the amino acids of the receptor protein (29, 30). Although, thiol from cysteines is a likely nucleophilic candidate, other amino acids, such as lysine, tryptophane, and tyrosine have been also shown to react with electrophiles (29). Actually, several tryptophane and tyrosine residues have been shown to participate in orthosteric ligand binding in mAChRs, and some cysteines are located in the proximities of these residues or the orthosteric binding site (31, 32). These amino acids could be available for 1,4-Michael addition, but irreversible binding of the spirolide to the receptor needs to be confirmed experimentally. The interaction of the 13-desmethyl C spirolide with nAChRs, as well as their functional inhibition by this toxin, has been recently reported (14-16). Targeting of nAChRs could explain on its own the neurologic symptoms observed after intraperitoneal administration of the toxin. Our results confirm that the 13-desmethyl C spirolide also targets human mAChRs since our cell model does not express functional nAChRs linked to a calcium signal, and [3H]QNB binding is reduced in the presence of the toxin. Presumably, both muscarinic and nicotinic AChRs could be implicated in the mechanism of toxicity of this marine toxin group. The fact that the spirolides need relatively long periods of incubation to inhibit mAChR-related function in Vitro may be considered an argument against the implication of these receptors in the mechanism of action of these so-called “fast acting toxins”. Although it is true that these toxins can cause death in 2 min, the doses required for this fast toxicity are considerably higher than the LD50 (3, 4, 13). The animals receiving a lethal dose of spirolides die between 3 and 20 min, and the time to death decreases as the dose of toxin increases. For doses close to the LD50, the time to death is 10-15 min (3). In our hands, the mAChR-dependent response was 45% of the control response after 10 min of exposure to the toxin in Vitro. The question now is what degree of impairment of a certain receptor is necessary to elicit a toxic consequence in ViVo. To the extent of our knowledge, this question has not been answered for mAChRs. Additionally, we do not think mAChR antagonism is the only mechanism responsible for spirolide toxic effects; on the contrary, we believe that several mechanisms may be acting in parallel to enhance toxicity. High doses of toxin might be enough to cause a rapid death due to nAChR antagonism, but lower doses could cause death after a longer period of time when mAChR impairment joins to nAChR blockage. The fast and apparently complete recovery after 20 min if the animal does not die would be difficult to relate only to pharmaco/toxicodynamics since the toxicokinetic processes, such as elimination of the toxin from the organism, are extremely relevant to determining the duration of the toxin effect. Moreover, these toxins have been described as fast acting toxins on the basis of the time to death and symptom appearance,


but the toxicity of these compounds may also involve other effects not easily observable during routine bioassay monitoring in rodents. The concentrations of 13-desmethyl C spirolide tested in this work did not completely inhibit the calcium signal elicited by ACh or [3H]QNB binding to the receptor. The lack of complete inhibition in our experimental conditions could be due to the use of a not-high-enough concentration of the spirolide. Unfortunately, the amount of pure spirolides is very limited worldwide, and therefore, we could not increase the amount of toxin used in these experiments. However, there are other possible explanations for this incomplete inhibition. One of them is the presence of mAChR subtypes not sensitive to the toxin. Another explanation would be the externalization to the membrane of unoccupied receptors leaving a subset of toxinfree receptors at any given time due to the relatively slow kinetics of the spirolide-dependent inhibition of the calcium signal. Actually, mAChRs have been described to undergo processes of drug-induced recycling (18). For the incomplete inhibition of [3H]QNB binding, a lack of binding of the spirolide to intracellular receptors which are known to bind [3H]QNB could also be a possible explanation. However, 100 nM 13desmethyl C spirolide inhibited approximately the same percentage of [3H]QNB, which is cell permeable, and [3H]NMS, which is not cell permeable, indicating that the spirolide also binds intracellular receptors. Incomplete inhibition of radioactive antagonist binding even by the same cold (nonradioactive) molecule has been described before for mAChRs (28). In addition to this apparently spirolide-insensitive pool of receptors, the [3H]QNB competition curve also suggests the presence of two populations of mAChRs with very different affinities for the 13-desmethyl C spirolide. The concentrations needed to obtain an inhibition of the calcium signal suggest that either the binding site responsible for the inhibition of the calcium signal is the low-affinity binding site or that there are a number of spare receptors/binding sites of the first kind. Obviously, the presence of two or more mAChR subtypes in these cells and a higher affinity for a particular subtype could easily explain these results. However, the affinity of agonists and antagonists for the mAChRs also depends on the receptor state, and therefore, there are other possible explanations for this results. For example, the affinity of mAChRs for agonists and also certain antagonists has been described to be dependent on guanine nucleotides (33, 34). Since [3H]QNB is cell permeable, the spirolide is also a lipophilic molecule, and our results suggest that it binds intracellular receptors, the pool of internal receptors could have a different affinity for the toxin than external receptors, and explain our results that suggest the presence of two receptor populations. Therefore, two receptor subpopulations of the same mAChR subtype might be also identified as two different binding sites in terms of affinity for the ligand. Although we did not detect M1 receptors by Western blot in this cell model, their presence cannot be ruled out, as well as the presence of other mAChR subtypes, and may explain the biphasic inhibition curve of [3H]QNB binding and/or the lack of complete inhibition of the calcium response to ACh and [3H]QNB binding. The selectivity of the spirolide for the mAChR subtypes bears a special pharmacological interest, and we have already started the study of the spirolide pharmacology related to mAChR subtypes. The spirolides not only inhibit the receptor functionality but also affect the expression of the mAChRs. An increase of mRNA levels of the M1, M4, and M5 mAChRs was reported in rats exposed to lethal doses of spirolides intraperitoneally

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(4). However, the effect on the levels of receptor protein expressed in this species was not studied, and the mAChR mRNA was unaltered in mice (4). The effect of these toxins on the protein expression levels of the human mAChRs is extremely relevant to the evaluation of the health threat derived from human exposure to these toxins. The mAChRs preferentially linked to the calcium signal are the M1, M3, and M5 subtypes (17, 18). Since M1 and M3 are more commonly expressed, they were selected to study the modulation of receptor levels by this toxin. However, the M1 receptor was very poorly expressed or nonexistent in our cell line. In human neuroblastoma BE(2)M17 cells, the levels of total protein of M3 receptors did not change significantly after an exposure to the toxin. We did not study the mRNA levels of these receptors in our cells. However, an increase of mRNA levels that is not translated into a higher level of functional protein would not be relevant from a toxicological or therapeutic point of view. The synthesis of mRNA and the new receptor could be important to maintain a functional population of receptors and therefore for the recovery of a toxic aggression. Protein expression levels might change at higher levels of toxin, but its toxicological and therapeutic relevance would be questionable. Actually, we already tested a concentration that causes an almost complete inhibition of mAChR-related cell function. Interestingly, the amount of M3 mAChRs that could be extracted with a transmembrane-protein extraction kit was reduced by the 13-desmethyl C spirolide 12 h after initial exposure. These results suggest that the properties of the mAChRs have changed after binding to the toxin in a way that their fractionation in a particular membrane protein extraction reagent has been modified. An example of physiological processes that could explain these results would be internalization of toxin-bound M3 receptors, labeling for protein degradation, or the movement to a different signaling compartment in the plasma membrane. Actually, as mentioned before, a shift in the fractionation of the receptor in sucrose gradients has been previously related to receptor internalization (24). In any case, the change of receptor properties at relatively late time points could have important functional and toxicological implications since a delayed alteration of receptors (no effect was observed at 1 h of exposure) could affect the cholinergic neurotransmission system after the subject has recovered from acute toxicity. Actually, the reports to date refer to a complete recovery from acute toxicity of experimental animals exposed to nonlethal doses of spirolides (13). However, some of the possible long-term consequences of spirolide toxicity have probably not been evaluated. For example, it is widely accepted nowadays that mAChRs are implicated in mental functions such as memory and learning (17, 35). In fact, an exposure to mAChR antagonists has been shown to induce cognitive impairment in laboratory animals (36). The exposure to nonlethal doses of spirolides might therefore have some toxicological consequences to humans that have not been considered yet. These consequences could even be irrelevant to healthy subjects but extremely dangerous for individuals with a previous impairment of the cholinergic system, as is the case in Alzheimer’s disease, schizophrenia, Parkinson’s disease, or dementia with Lewy body patients, among others (17, 37-39). Studies of short and longterm internalization as well as long-term effects on receptortriggered responses will be needed to explore the possible longterm toxicity of these group of compounds before in ViVo evaluation of chronic toxicity. This study is the first report on the antagonist effect of the 13-desmethyl C spirolide, a compound of the spirolide group of marine toxins, on human mAChRs. The 13-desmethyl C

Wandscheer et al.

spirolide behaves as a competitive antagonist of mAChRs expressed in human neuroblastoma cells. Interestingly, the spirolide also causes a change of the properties of membraneassociated mAChRs. These effects of the spirolide on mAChRs might have important toxicological consequences to animals and humans that should be considered for the design of future toxicological studies and for risk assessment of human exposure to these marine toxins. Acknowledgment. This work was supported by Ministerio de Ciencia y Tecnologıá, Spain (AGL2007-60946/ALI, SAF200912581 (subprograma NEF), AGL2009-13581-CO2-01, and TRA2009_0189), Xunta de Galicia, Spain (GRC 30/2006 and PGIDT07CSA012261PR, PGDIT 07MMA006261PR, 2009/ XA044 (Consell. Educacioń), and 2008/CP389 (EPITOX, Consell. Innovacioń e Industria, programa IN.CI.TE.)), EU VIth Frame Program (IP FOOD-CT-2004-06988 (BIOCOP) and CRP 030270-2 (SPIES-DETOX)), and EU VIIth Frame Program (211326-CP (CONffIDENCE); STC-CP2008-1-555612 (Atlantox); and 2009-1/117 Pharmatlantic). C.B.W. is the recipient of a predoctoral fellowship from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), of the Ministry of Science and Technology of Brasil.

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Human Muscarinic Acetylcholine Receptors Are a Target of the Marine

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