Abstract
The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of ∼100 pM to nAChRs in the pineal gland, and the density of these sites is ∼5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at α3β4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous α3β4 nAChRs. Measurements of mRNA indicated the presence of transcripts for α3, β2, and β4 nAChR subunits but not those for α2, α4, α5, α6, α7, or β3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained α3 and β4 subunits associated in one complex. The β2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the α3β4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels composed of α and β subunits. Nine α (α2–α10) and three β (β2–β4) subunits have been identified in vertebrates, and different subunit combinations define specific receptor subtypes. All of these subtypes pass Na+, K+, and Ca2+, but they exhibit distinct biophysical and pharmacological properties. Studies of nAChRs in Xenopus laevis oocytes and transfected mammalian cells have provided valuable information on the biophysical properties, pharmacology, and possible regulation of several different well-defined nAChR subtypes that might play important physiological roles. However, the precise subunit compositions of the subtypes of nAChRs that actually exist in most native tissues are not well-defined. Therefore, identifying the subunit composition of native nAChRs is a crucial step in establishing the physiological roles played by the different receptor subtypes that exist in vivo.
Considerable progress has been made in determining the subunit composition of the predominant receptor subtypes in the rat forebrain, namely the α4β2 subtype (Whiting and Lindstrom, 1987; Flores et al., 1992), which has high affinity for most agonists, and the α7 subtype, which has high affinity for α-bungarotoxin (α-BTX) (Couturier et al., 1990; Schoepfer et al., 1990; Orr-Urtreger et al., 1997). However, other nAChR subtypes are found in various amounts throughout many regions of the central nervous system (Marks et al., 1998; Perry et al., 2002), and some of these less prevalent receptors may play critical roles because of their strategic location (e.g., α6-containing receptors on dopamine axons) (Quik et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004). Moreover, under some conditions in vivo, such as when α4β2 and/or α7 receptors are desensitized or inactivated by exposure to nicotine or during certain disease states that may involve the loss of specific nAChR subtypes, the less prevalent receptors may take on critical roles in mediating cholinergic signals. In addition, α3β2 and α3β4 nAChR subtypes are the predominant nAChRs in autonomic ganglia and thus are critical to the homeostatic functioning of virtually all organ systems in the body.
The pineal gland is part of the photoneuroendocrine system of vertebrates and functions in response to signals from photoreceptor cells in the retina and endogenous oscillators within the suprachiasmatic nucleus to translate light stimuli into neuroendocrine responses. The main role of the pineal gland seems to be to produce and secrete the hormone melatonin, which influences circadian and seasonal biological rhythms in animals. Melatonin production is stimulated when norepinephrine released by sympathetic axons from the superior cervical ganglia activate β-adrenergic receptors in the pineal gland (Axelrod, 1974). More recent studies have demonstrated a cholinergic innervation of the pineal gland from the parasympathetic nervous system (Korf et al., 1996; Larsen et al., 1998; Schafer et al., 1998) and possibly from the medial habenula within the brain (Schafer et al., 1998). This cholinergic innervation seems to play an important role in pineal gland physiology by inhibiting melatonin synthesis via the activation of nAChRs (Stankov et al., 1993; Yamada et al., 1998a).
The pineal expresses mRNA encoding the α3, β2, and β4 nAChR subunits (Wada et al., 1989; Zoli et al., 1995), and nAChR binding sites have been found in mouse and rat pineal gland (Marks et al., 1998; Hernandez et al., 1999; Perry et al., 2002; Dávila-García et al., 2003). Here, we studied the pharmacology of the rat pineal nAChR binding sites and functional responses and used subunit-selective antibodies to determine the subunit composition of the nAChR subtype expressed in the rat pineal gland. Our data indicate that the pineal gland expresses a single subtype of nAChR; it thus provides a simple and convenient model system in which to study a native nAChR of defined subtype.
Materials and Methods
Materials. [3H]Epibatidine ([3H]EB) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). 125I-Epibatidine (125I-EB) used for autoradiography was a kind gift from Dr. John Musachio (National Institute of Mental Health, Bethesda, MD). 125I-α-Bungarotoxin (125I-α-BTX) and [α-32P]ATP were purchased from Amersham Biosciences Inc. (Piscataway, NJ). Tissue culture medium, fetal bovine serum, and antibiotics were purchased from Invitrogen (Carlsbad, CA). DNase1 was purchased from Roche Diagnostics (Indianapolis, IN). Bacterial cell walls containing protein-A (Pansorbin) and protein G (Omnisorb) were purchased from Calbiochem (San Diego, CA). Whatman GF/C filters were obtained from Brandell (Gaithersburg, MD). Other drugs and chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific Co. (Fairlawn, NJ). The α3 nAChR subunit-specific polyclonal antibody was raised in rabbits and has been described previously (Yeh et al., 2001). The β4 nAChR subunit specific polyclonal antibody was a generous gift from Dr. Scott Rogers (University of Utah, Salt Lake City, UT). The β2 nAChR subunit-specific monoclonal antibody (mAb) 270 was produced from hybridoma stocks purchased from American Type Culture Collection (Manassas, VA). This mAb was originally developed and characterized by Whiting and Lindstrom (1987). It is an excellent antibody for immunoprecipitation, but it does not detect rat nAChR β2 subunits in Western blots. Therefore, an nAChR β2 subunit-specific polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used for Western blotting analysis.
Tissues. Frozen pineal glands and cerebral cortex from male Sprague-Dawley rats weighing 225 to 250 g were purchased from Zivic-Miller Laboratories (Portersville, PA) and stored at -80°C until assayed.
Primary Cell Culture. Male Sprague-Dawley rats purchased from Taconic Farms (Germantown, NY) were group-housed with food and water ad libitum in a temperature- and light-controlled room (24°C, lights on from 7:00 AM to 7:00 PM). At 30 to 35 days old (weighing 150 to 200 g), the rats were anesthetized with methoxyflurane inhalation and decapitated. Fresh pineal glands were dissected and then used for primary tissue culture. The isolated pineal glands were washed two times in phosphate-buffered saline and cut into small pieces under a dissecting microscope. The tissue pieces were transferred to a sterile tube containing 5 ml of 0.02% trypsin and 1% DNase in phosphate-buffered saline and incubated with gentle shaking for 10 min at 37°C. The tissues were triturated with a fire-polished pasture pipette and incubated for another 10 min at 37°C. The tissues were then triturated a second time, and 0.5 ml of fetal bovine serum was added to stop the enzymatic reaction. The dissociated cells were washed four times by centrifugation at 800g in culture media containing basal Eagle's medium supplemented with 10% fetal bovine serum, 25 mM glutamine, and 50 μg/ml gentamycin. The dissociated pineal cells were resuspended in fresh culture media and plated onto poly-d-lysine coated coverslips in a 30-mm dish. Cultures were maintained at 37°C with 5% CO2 in a humidified incubator for 1 to 3 days. HEK 293 cells stably expressing the α3β4 nAChR (KXα3β4 cells) and α3β2 nAChR (KXα3β2 cells) were grown and maintained as described previously (Xiao et al., 1998).
Radioligand Binding Assays. nAChR receptor binding sites in membrane homogenates from rat pineal gland and HEK cells expressing α3β4 and α3β2 nAChRs were measured with [3H]EB. Tissues were homogenized with a Polytron homogenizer in 50 mM Tris-HCl, pH 7.4, and the homogenates were centrifuged at 35,000g for 10 min. Membrane pellets were washed twice and then resuspended in fresh buffer. Membrane aliquots (∼35 μg of protein) were incubated with [3H]EB (∼5–3000 pM) for 4 h at 24°C in a volume of 1 ml of Tris-HCl buffer. In competition binding experiments, a series of concentrations of each drug was incubated with ∼500 pM [3H]EB. Bound and free radioligand were separated by vacuum filtration through Whatman GF/C filters pretreated with 0.5% polyethylenimine. The radioactivity bound to the filter was measured by liquid scintillation counting. Nonspecific binding was determined in the presence of 300 μM (-)-nicotine, and specific binding was defined as the difference between total and nonspecific binding. In saturation binding experiments, Bmax and Kd values were determined by nonlinear regression analysis (Prism software; GraphPad Software Inc., San Diego, CA). In competition binding experiments, inhibition curves and the IC50 values were determined by nonlinear regression analysis (GraphPad Prism). The affinities of drugs (Ki values) at nAChRs were calculated from the IC50 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).
Autoradiography. Autoradiography of 125I-EB and 125I-α-BTX binding sites was carried out in 20-μm cryostat-cut sections of the rat brain through the superior colliculus with the pineal gland in place, as described by Perry et al. (2002). Adjacent brain sections were mounted onto slides and incubated with ∼500 pM 125I-EB or ∼5 nM 125I-α-BTX and then rinsed, air-dried, and apposed to autoradiographic film for 1 to 6 days. Nonspecific labeling was determined in the presence of 300 μM nicotine.
Immunoprecipitation. Rat pineal glands were prepared as for a radioligand binding assay, and the membrane pellet was resuspended in fresh buffer and incubated with ∼3 nM [3H]EB for 2 h at room temperature. The tissue was then solubilized by the addition of Triton X-100 at a final concentration of 2% with gentle mixing for 2 h at room temperature. After solubilization, the mixture was centrifuged at 35,000g for 30 min. Aliquots of clear supernatant equivalent to 4 mg of original tissue weight were added to tubes containing either crude antiserum directed at the β4 subunit, affinity-purified antibody directed at the α3 subunit, or an mAb directed at the β2 subunit. The β4 antiserum was tested at dilutions from 1:50 to 1:6 to determine the optimal concentration. The stock α3 and β2 antibodies each contained 1 μg/μl and were tested at dilutions from 2:100 to 15:100 to determine the optimal concentration. Tubes containing normal rabbit serum or an irrelevant monoclonal antibody served as controls. The mixtures were incubated for 4 to 16 h at 4°C with gentle rotation. After this incubation period, 50-μl aliquots of stripped Protein A (Pansorbin) or Protein G (Omnisorb) (Wall et al., 1991) were added to each assay tube and incubated for an additional hour at 4°C with gentle rotation. The [3H]EB-labeled receptor-antibody complex was precipitated by centrifugation at 14,000g for 30 s. The pellets were washed two times with 1 ml of Tris/EDTA buffer, dissolved in 400 μl of 0.1 NaOH and 3% deoxycholate, and counted by liquid scintillation spectroscopy. For the sequential immunoprecipitation assays, the individual clear supernatant from the first immunoprecipitation was collected and immediately added to tubes containing antisera or antibody directed at another nAChR subunit and allowed to incubate for an additional 4 h. The immunoprecipitation procedure with the second antibody was carried out as described above.
Western Blots. Western blot analyses to measure subunit proteins were carried out as described previously (Yeh et al., 2001) using the polyclonal antibodies directed at each of the subunits.
RNA Isolation and RNase Protection Assay. Expressions of mRNAs encoding nAChR subunits were determined as described previously (Xiao et al., 1998) with modifications. In brief, total cellular RNA was isolated using RNA-STAT-60 (Tel-Test Inc., Friendswood, TX). Antisense riboprobes for the α2, α3, α4, α5, α6, α7, β2, β3, and β4 nAChR subunits were generated from DNA templates using T7 RNA polymerase and [α-32P]CTP. The RNase protection assays were carried out using the RPA II kit (Ambion, Austin, TX). Total RNA (20 μg) from the tissue samples was hybridized overnight at 42°C with the subunit riboprobes and a riboprobe for rat GAPDH, which was used as an internal and loading control. Specific activities of [α-32P]CTP used for synthesizing the probes of rat nAChR subunit genes and the probe of GAPDH were 800 and 40 Ci/mmol, respectively. After hybridization, nonprotected fragments were digested with a combination of RNase A and RNase T1 for 30 min at 37°C. The numbers of bases of the full-length probes and the protected fragments of the probes were as follows: α2, 416 and 332; α3, 306 and 230; α4, 496 and 408; α5, 411 and 380; α6, 462 and 396; α7, 450 and 376; β2, 322 and 263; β3, 430 and 394; β4, 252 and 170; and GAPDH, 204 and 135. To avoid overlap of signal bands, three reactions were carried out for each RNA sample separately using three groups of probes: group 1 contained α2, α3, α4, and GAPDH; group 2 contained α5, α6, β4, and GAPDH; and group 3 contained α7, β2, β3, and GAPDH. The protected probe fragments were separated by electrophoresis on a 6% denaturing polyacrylamide gel, and the fragments were visualized using X-ray film or by filmless autoradiographic analysis (PhosphorImager, 445 SI, Amersham Biosciences).
Electrophysiology. Functional responses of nAChRs in rat pineal primary cell culture were measured using the whole-cell configuration of the voltage patch clamp technique. Dissociated rat pinealocytes were plated onto glass cover slips and positioned into a recording chamber (1 ml volume) mounted on the stage of an upright microscope (Axioskop; Carl Zeiss, Jena, Germany) used to visually identify the cells under study. The cells were bathed with an extracellular solution containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 5 mM HEPES and adjusted to 325 mOsM with sucrose, pH adjusted to 7.4 with NaOH. The recording chamber was continuously perfused with the extracellular solution at a rate of 1 ml/min. Ionic currents were monitored with an Axopatch 1-D patch amplifier (Axon Instruments Inc., Union City, CA). Recording patch electrodes were pulled from borosilicate glass capillaries to a resistance of 5 to 7 MΩ when filled with internal electrode solution containing 145 mM potassium gluconate, 5 mM EGTA, 5 mM MgCl2, 10 mM HEPES, 5 mM ATP 0.5 mM GTP, and 10 mM BAPTA, pH adjusted to 7.42 with CsOH. Pinealocytes were distinguished from glial cells visually and selected for whole-cell recording. The size and capacitance were very similar between pinealocytes, and recordings were made from single isolated cells. No differences in responses between cells kept in culture for 1 or 3 days were noted. Drugs were applied using a gravity-fed Y-shaped tubing system positioned within 500 μm of the cell under investigation. Drugs were applied and exchanged rapidly with an onset of <100 ms. For experiments requiring α-BTX incubation, the extracellular solution was exchanged with the same solution with the addition of toxin and allowed to incubate for 2.5 min. Generation of voltage-clamp protocols and acquisition of data were carried out using the pCLAMP software. All experiments were performed at room temperature.
Results
Expressions of nAChR Subunit mRNAs in Rat Pineal Glands. Expressions of mRNAs encoding nine nAChR subunits (α2, α3, α4, α5, α6, α7, β2, β3, and β4) in rat pineal glands were examined with a multiplex RNase protection assay. The mRNA expression pattern detected in the rat pineal gland is shown in Fig. 1. The pineal gland expressed nAChR mRNAs encoding the α3 (lane 1), β4 (lane 2), and β2 (lane 3) subunits only, and the intensity of the bands indicated that the expression levels of the α3 and β4 subunit mRNAs were somewhat higher than that of the β2 subunit mRNA. Although no signals for the mRNA encoding any other nAChR subunits were detected in the pineal gland, strong signals for these other subunits were detected in tissues used as positive controls for the assay (data not shown).
nAChR Binding Sites in the Rat Pineal Gland. Autoradiographic studies of nAChR binding sites in coronal brain sections at the level of the superior colliculus revealed dense 125I-EB binding sites in the pineal gland (Fig. 2A); in contrast, autoradiography of 125I-α-BTX binding in the pineal did not exceed the background level (Fig. 2B). These autoradiographic results combined with the mRNA analysis indicate that the pineal gland expresses one or more heteromeric nAChRs but not α7 receptors or other subtypes that bind 125I-α-BTX. As shown in Fig. 3, saturation binding measurements of [3H]EB binding sites in membrane homogenates demonstrate that the pineal gland expresses a high density of nAChRs (∼300 fmol/mg of protein) that is approximately five times the density found in the rat forebrain (∼60 fmol/mg of protein). The [3H]EB binding curves in the pineal fit a model for a single site, with a Kd of ∼100 pM.
From the nAChR subunit mRNA it expresses, the pineal could contain either an α3β2 or an α3β4 subtype, both subtypes, or a receptor composed of α3 subunits in association with both β2 and β4 subunits (i.e., an α3β2β4 subtype). To begin to determine the characteristics of the nAChR subtype(s) in the pineal gland, the affinities of drugs for the [3H]EB-labeled sites were assessed in binding competition assays and compared with the affinities at defined α3β4 and α3β2 receptors heterologously expressed in HEK 293 cells. The agonists A-85380, cytisine, and nicotine all competed for [3H]EB binding sites in the pineal gland with affinities between ∼24 and 200 nM, whereas the antagonist dihydro-β-erythroidine (DHβE) displayed an affinity of ∼65 μM (Fig. 4A and Table 1). These affinities are much closer to those found at the defined α3β4 receptor than at the α3β2 receptor, especially for A-85380 and DHβE, two drugs that are good discriminators between these subtypes (Fig. 4, B and C; Table 1). In fact, the correlation line between the Ki values for the pineal and the α3β4 subtype was very close to the line of identity (Fig. 4D), suggesting that, as is seen in Table 1, the pharmacology of the pineal nAChR binding sites is very similar to, and probably indistinguishable from, that of the α3β4 subtype.
Functional Responses of nAChRs in the Pineal Gland. The functional responses of the nAChRs in the pineal were examined in whole-cell patch-clamp studies of dissociated pineal cells maintained in culture for ∼3 days. All nicotinic agonists examined activated inward currents in a concentration-dependent manner, with epibatidine being at least 100 times more potent than any other agonist tested and ∼1000 times more potent than acetylcholine (Fig. 5, A and B; Table 2). Most of the agonists examined here, including cytisine, functioned as full agonists compared with acetylcholine, eliciting peak currents of 250 to 300 pA; the exception was DMPP, which seemed to be a partial agonist with approximately half the efficacy of acetylcholine (Fig. 5C). The receptors seemed to desensitize in a manner that was also concentration-dependent, but they recovered their sensitivity within approximately 2 min after removal of agonists (data not shown). At agonist concentrations ≥500 μM, there was usually an obvious loss of receptor function, which probably reflects channel blockade by most of the agonists (data not shown). That could account for the apparently slightly higher efficacy seen with epibatidine, which, because of its high potency, can fully activate the receptors at concentrations that do not significantly block the channel.
As shown in Table 2, the EC50 values for these six agonists to activate whole-cell currents via nAChRs in the pineal gland are very similar to their EC50 values to stimulate 86Rb+ efflux via defined α3β4 receptors heterologously expressed in HEK 293 cells. In fact, as shown in Fig. 6, comparison of the EC50 values for these two very different functional assays carried out in different cell types, one expressing its native receptor and the other expressing a heterologous receptor, reveals that the values are highly correlated (r = 0.99); and, more important, the best fit line for the correlation is again close to the line of identity, indicating that the absolute EC50 values are very similar, as would be expected if both assays were measuring the same receptor.
The potencies of antagonists to block nicotine-activated currents were evaluated by first measuring activation by 30 μM nicotine alone followed at 2-min intervals by measurements after simultaneous application of 30 μM nicotine and decreasing concentrations of mecamylamine, curare, or DHβE in the same cell. Each of these antagonists blocked nicotine-activated currents (Fig. 7A), and the block was concentration-dependent (Fig. 7B). In contrast, α-BTX, which is a highly selective α7 nAChR antagonist, failed to block the nicotine-induced currents in the pineal cells even when the cells were perfused with the toxin during and for 2.5 min before application of nicotine (Fig. 7A).
As shown in Table 3, the IC50 values for the three antagonists to block nicotine-activated whole-cell currents in the pineal are similar to their IC50 values to block 86Rb+ efflux via defined α3β4 receptors heterologously expressed in HEK 293 cells. Moreover, for DHβE, the only effective competitive antagonist examined here, its Ki value calculated from its IC50 to inhibit whole-cell currents in pineal cells was indistinguishable from its Ki value calculated from its IC50 to inhibit 86Rb+ efflux in the heterologously expressed cells (20 versus 22 μM).
Subunit Composition of nAChRs in Pineal Gland. The pharmacological characteristics of the nAChRs in the pineal gland are consistent with an α3β4 receptor subtype, but pharmacology cannot prove subtype, which is defined by subunit composition. Therefore, we carried out immunoprecipitation assays to identify the subunits that comprise the pineal nAChRs. To do this, we used a polyclonal antibody selective for the α3 subunit (Yeh et al., 2001), another directed at the β4 subunit (Flores et al., 1996), and the monoclonal antibody mAb 270, which is selective for the β2 subunit (Whiting and Lindstrom, 1987). As shown in Fig. 8, the polyclonal antibodies directed at the α3 and β4 subunits each immunoprecipitated [3H]EB-labeled nAChRs from detergent-solubilized extracts of pineal tissue in a concentration-dependent manner. In contrast, the β2 subunit-selective mAb 270 did not immunoprecipitate any [3H]EB-labeled receptors from the pineal extracts (Fig. 8). To confirm that mAb 270 was, in fact, capable of immunoprecipitating nAChRs that contained β2 subunits, it was tested in similarly prepared extracts from rat cerebral cortex, where it effectively immunoprecipitated the α4β2 receptor (Fig. 8, inset), which predominates in that tissue (Whiting and Lindstrom, 1987; Flores et al., 1992).
The immunoprecipitation of nAChRs in the pineal extracts only with antibodies directed at α3 and β4 subunits indicates that these two subunits probably comprise the nAChR in the pineal gland. To further test this, sequential immunoprecipitation assays were carried out to determine whether the α3 and β4 subunits are physically associated with each other in the pineal gland. In these studies, [3H]EB-labeled nAChRs in pineal membranes were solubilized and immunoprecipitated with one subunit-specific antibody, and then, after centrifugation, to collect the immunoprecipitated receptors in the pellet, the remaining (“cleared”) supernatant was subjected to a second immunoprecipitation with a second subunit-specific antibody. The degree to which the first antibody decreases immunoprecipitation by the second antibody indicates the degree of association between the subunits (Flores et al., 1992).
The results from these sequential immunoprecipitation studies are shown in Fig. 9. When the pineal extracts were first immunoprecipitated with the α3 subunit-specific antibody, subsequent immunoprecipitations of the supernatant with the antibodies directed at the α3, β4, or β2 subunits yielded essentially no additional immunoprecipitation (Fig. 9A). Nearly identical results were found when the extracts were first immunoprecipitated with the β4 subunit-specific antisera (Fig. 9B). In contrast, the β2 subunit-specific mAb 270 antibody did not immunoprecipitate significant amounts of [3H]EB-labeled receptors from the pineal extracts; thus, after a first exposure to mAb 270, subsequent exposure to the antibodies directed at either the α3 or β4 antisera subunits immunoprecipitated virtually all of the receptors available in the supernatant (Fig. 9C). Western blot analyses of pineal extracts clearly demonstrated the presence of the α3 and β4 subunits, but no signal was detected for the β2 subunit (data not shown).
Discussion
nAChRs mediate the actions of acetylcholine, nicotine, and other nicotinic agonists throughout the central and peripheral nervous systems. Multiple subtypes of these receptors may form from the eight α and three β subunits known to be expressed in mammalian tissues, but because the rules of assembly are not known, the number of theoretically possible heteromeric nAChR subtypes is not yet established. More important, there are very few tissues in which the subunit composition of native nAChRs is known with some certainty. The α7 subtype is identifiable in brain by its rapid kinetics and especially by its sensitivity to α-BTX. In contrast, the many different potential subtypes of heteromeric receptors are not so readily distinguished. The predominant heteromeric nAChR in mammalian brain is the α4β2 subtype (Whiting and Lindstrom, 1987; Flores et al., 1992); however, other subtypes also are found in many areas of brain and spinal cord (Marks et al., 1998; Perry et al., 2002), usually making it difficult to know whether a nicotinic response is mediated by a single nAChR subtype or is a composite response reflecting more than one receptor. Peripheral ganglia seem to contain both α3β2 and α3β4 receptors, possibly incorporating an α5 subunit in some cases (Conroy and Berg, 1995; Flores et al., 1996; Xu et al., 1999); so again, even with their more limited number of nAChR subunits, it is usually not readily apparent which subtype mediates a particular response.
The main finding of this study is that the rat pineal gland expresses the α3β4 subtype of nAChR apparently exclusively. The evidence for this is based on the pharmacology of the receptor and on direct studies of its subunit composition. The pharmacology of both the receptor's binding site and its function correspond quantitatively (Ki and EC50 values) to the defined α3β4 receptor heterologously expressed in HEK 293 cells. Moreover, definitive evidence for the subunit composition of the receptor was derived from the immunoprecipitation studies with subunit-specific antibodies, which indicate clearly that the α3 and β4 subunits in the pineal gland are associated exclusively with each other in the same [3H]EB binding complex. This conclusion is consistent with data from autoradiography studies using 125I-EB in conjunction with drugs that mask certain receptor subtypes (Perry et al., 2002). The pineal gland thus serves as one of the few tissues available for studies of a defined subtype of native heteromeric nAChR.
We measured strong signals for α3 and β4 subunit mRNA, as well as a somewhat weaker mRNA signal for the β2 subunit in the rat pineal gland. These results are consistent with previous mRNA analyses of nicotinic receptor subunits in the rat pineal gland (Wada et al., 1989; Zoli et al., 1995). However, we found no evidence that rat pineal gland nAChRs incorporate β2 subunits; in fact, we found no evidence for the presence of the β2 protein via Western blots. From the sensitivity of our binding and immunoprecipitation assays, we estimate we could have detected β2-containing receptors if they constituted ∼5% or more of the pineal receptors. The presence of the β2 mRNA might reflect a transient nAChR present during an earlier developmental stage or even a role in another protein expressed at low levels.
The main function of the pineal gland is the coupling of central circadian timing systems to effectors by the rhythmic production and release of melatonin. Therefore, pineal function and melatonin have been implicated in reproductive cycles, gonad size (in some species), and sleep-wake cycles (including jet lag). The pineal gland seems to be innervated by both limbs of the autonomic nervous system. Sympathetic axons from the superior cervical ganglia synapse on pinealocytes where they release norepinephrine at β-adrenergic receptors, which leads to an increase in cAMP and activation of melatonin synthesis (Axelrod, 1974). Parasympathetic cholinergic axons to the pineal gland probably originate in the sphenopalantine ganglia (Korf et al., 1996; Larsen et al., 1998). In addition, there is a central cholinergic innervation of the pineal gland from the medial habenula in some species (Schafer et al., 1998). Previous studies have found [3H]EB binding sites in the pineal gland, indicating the presence of nAChRs (Marks et al., 1998; Hernandez et al., 1999; Perry et al., 2002; Dávila-García et al., 2003). Moreover, application of acetylcholine to isolated pinealocytes stimulates membrane depolarization that is coupled to activation of L-type calcium channels, and this action is mimicked by nicotine and blocked by d-tubocurarine (Letz et al., 1997).
Previous studies have found that nAChRs mediate the inhibition of melatonin synthesis in rat pineal explants (Stankov et al., 1993; Yamada et al., 1998a). In an elegant series of studies, Yamada et al. (1996a,b, 1998a,b) established a link between pineal nAChRs, L-type calcium channels, glutamate release, metabotropic glutamate receptors, and inhibition of cAMP in a signaling pathway leading to the inhibition of melatonin synthesis. The signaling pathway begins with activation of the nAChRs, which by depolarizing the pinealocyte membrane activate L-type calcium channels. The increased intracellular Ca2+ levels trigger exocytotic release of glutamate, which activates mGluR3 metabotropic glutamate receptors and leads to decreased cAMP. This results in decreased transcriptional activation of serotonin N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis. Our studies identify the nAChR in pinealocytes that begins this signaling cascade as an α3β4 subtype. This conclusion is consistent with observations from previous reports. For example, our EC50 values for activation of whole-cell currents by nicotine and acetylcholine in dissociated pinealocytes are similar to the values reported for release of glutamate and inhibition of melatonin synthesis (Yamada et al., 1998a). Moreover, both nicotine-activated whole-cell currents (present data) and acetylcholine-induced release of glutamate (Yamada et al., 1998a) in pinealocytes are blocked by d-tubocurarine but not by α-BTX.
A second major finding from our studies reported here is the remarkably close correspondence between the function of α3β4 nAChRs measured by whole-cell patch-clamp analysis in pinealocytes and 86Rb+ efflux in transfected HEK 293 cells that heterologously express these receptors. Thus, the EC50 values for the activation of whole-cell currents in pinealocytes by six nicotinic agonists were nearly indistinguishable from their EC50 values for the activation of 86Rb+efflux measured in HEK 293 cells expressing the α3β4 nAChR subtype. Moreover, cytisine, which is a full agonist at β4-containing receptors but a weak partial agonist at β2-containing receptors (Luetje and Patrick, 1991; Papke and Heinemann, 1994), is a full agonist at the pineal receptor as well as at the heterologously expressed α3β4 receptor (Meyer et al., 2001). Likewise, DMPP seems to be a partial agonist at the pineal receptors, as it is at the heterologously expressed α3β4 receptors (Meyer et al., 2001). In addition to the evidence from the activation of the receptors by agonists, the Ki values for the competitive antagonist DHβE derived from functional studies in pineal cells and the heterologously expressed α3β4 receptors are nearly identical.
Nevertheless, although the pharmacological evidence that the pineal nAChR is an α3β4 subtype is very strong, there are at present no drugs that can conclusively establish the identity of a heteromeric nAChR subtype. Furthermore, very little is known about the pharmacological properties of a possible α3β2β4 receptor (Colquhoun and Patrick, 1997). Therefore, the immunoprecipitation studies with subunit-specific antibodies provide the most definitive evidence for the identity of the pineal receptor subtype. Only the antibodies that recognize the α3 and β4 subunits were effective in immunoprecipitating the nAChR in the pineal gland; moreover, immunoprecipitation with either antibody removed virtually all of the receptors from the remaining supernatant, indicating that the α3 and β4 subunits are physically associated. In contrast, the β2-directed antibody did not immunoprecipitate nAChRs from the pineal extracts, although it efficiently immunoprecipitated receptors from the cerebral cortex, which is known to contain nAChRs with the β2 subunit, namely the α4β2 subtype (Whiting and Lindstrom, 1987; Flores et al., 1992).
In summary, these studies have shown that the rat pineal gland expresses a high density of nAChRs and that this receptor is virtually exclusively an α3β4 subtype. The characteristics of this receptor are consistent with the one that mediates the cholinergic signals that lead to decreased melatonin production and thereby plays an important role in pineal physiology. Thus, the rat pineal gland provides a readily obtainable native tissue with a high concentration of an identified subtype of nAChR. This should allow detailed studies of, for example, the channel properties, regulation, and turnover rate of this receptor in its native cell, as opposed to receptors expressed by transfection into heterologous cell systems. In addition, studies to determine proteins associated with the α3β4 nAChR require a native tissue that expresses a high level of receptor. The pineal gland is such a tissue and should be useful for this purpose.
Acknowledgments
We thank Dr. Scott Rogers for the generous gift of the β4 subunit selective antibody and Brandon C. Cox for assistance with some of the receptor binding assays.
Footnotes
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This research was supported by National Institutes of Health grant DA12976.
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Preliminary reports of this work have been presented previously: Hernandez SC, Musachio JL, Wang Y, Davila-Garia MI, Ebert S, Wolfe BB, and Kellar KJ (1999) The rat pineal gland expresses a nicotinic receptor with the characteristics of the α3β4 subtype.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.104.002345.
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ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; [3H]EB, [3H](±)-epibatidine; 125I-EB, 125I-(±)-epibatidine; EB, (±)-epibatidine; 125I-α-BTX, 125I-α-bungarotoxin; α-BTX, α-bungarotoxin; EB, epibatidine; DHβE, dihydro-β-erythroidine; DMPP, 1,1-dimethly-4-phenylpiperazinium; HEK, human embryonic kidney; mAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; A-85380, 3-[2(S)-azetidinylmethoxy]pyridine dihydrochloride.
- Received May 5, 2004.
- Accepted July 8, 2004.
- The American Society for Pharmacology and Experimental Therapeutics