Ca2+ permeability of nicotinic acetylcholine receptors from rat dorsal root ganglion neurones
1 Istituto Pasteur Fondazione Cenci-Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza Biologia e Medicina Molecolare, Università di Roma ‘La Sapienza’, P.le Aldo Moro 5, I-00185 Roma, Italy
2 Fondazione Santa Lucia, Via Ardeatina, Roma, Italy
Abstract
Ca2+ entry through neuronal nicotinic ACh receptors (nAChRs) modulates many biological processes in nervous tissue. In order to study the functional role of nAChRs in peripheral sensory signalling, we measured their Ca2+ permeability in rat dorsal root ganglion (DRG) neurones, and analysed the effects of nAChR-mediated Ca2+ influx on the function of the vanilloid receptor TRPV1. The fractional Ca2+ current (Pf, i.e. the percentage of current carried by Ca2+ ions) flowing through nAChR channels was measured by Ca2+ imaging fluorescence microscopy in combination with the patch-clamp technique. Functional nAChRs were expressed in a subset of adult DRG neurones (about 24% of the cells), typically with small to medium size as measured by their capacitance (40 ± 3 pF). In most cells, ACh evoked slowly desensitizing currents, insensitive to methyllycaconitine (MLA, 10 nM), a potent antagonist of homomeric nAChRs. Fast decaying currents, probably mediated by 7*-nAChRs (i.e. native 7-containing nAChRs), were observed in 15% of ACh-responsive cells, in which slowly decaying currents, mediated by heteromeric nAChRs, were simultaneously present. The nAChRs of adult DRG neurones exhibited a Pf value of 2.2 ± 0.6% in the presence of MLA and 1.9 ± 0.6% (P > 0.1) in the absence of MLA, indicating that homomeric MLA-sensitive nAChRs do not contribute to Ca2+ entry into adult DRG neurones. Conversely, 10% of neonatal DRG neurones showed ACh-evoked currents completely blocked by MLA. In these neurones, nAChRs showed a larger Pf value (9.5 ± 1.5%), indicating the expression of bona fide 7*-nAChRs. Finally, we report that Ca2+ influx through nAChRs in adult DRG neurones negatively modulated the TRPV1-mediated responses, representing a possible mechanism underlying the analgesic properties of nicotinic agonists on sensory neurones.
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Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric cation-selective ligand-gated channels. To date, 12 neuronal nicotinic subunits have been cloned in vertebrates (2–10 and 2–4) that are widely expressed in the nervous system (Dani, 2001) and in non-neuronal cells, such as glial and endothelial cells, keratinocytes, macrophages and lymphocytes (Sharma & Vijayaraghavan, 2002; Wang et al. 2003). It is generally thought that some neuronal nAChRs are located at presynaptic sites, where they modulate the neurotransmitter release by controlling the intracellular free Ca2+ concentration ([Ca2+]i) via the influx of Ca2+ through their channels (Wonnacott, 1997; MacDermott et al. 1999).
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A major difficulty in studying the Ca2+ permeability of native nAChRs is represented by the fact that the subunit composition of nAChRs (largely undefined in native preparations) determines the selectivity of the ion channel, as shown by studies on heterologously expressed nAChRs, with known subunit composition. A large body of findings supports the division of neuronal nAChRs into two classes (see for review Fucile, 2004). The first class is that of homopentameric nAChRs composed of the subunits 7–9, sensitive to -bungarotoxin (-BTX), which show a high Ca2+ permeability. The second class groups heteropentameric, -BTX-insensitive nAChRs (non--BTX nAChRs), formed by both (out of 2–6) and (out of 2–4) subunits, showing lower Ca2+ permeability.
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An extensive analysis of the Ca2+ permeability of native nAChRs is still lacking, mainly because neurones express multiple nAChR subunits, yielding receptors of unknown composition. The paucity of selective agonists and/or antagonists has not helped the problem of identifying the native nAChRs. Our study starts to repair this deficiency by examining the Ca2+ permeability of nAChRs in dorsal root ganglion (DRG) neurones, which express several nicotinic subunits comprising 3, 4, 5, 7, 9 and 10 (Genzen et al. 2001; Khan et al. 2003; Haberberger et al. 2004). In particular, our aim is to correlate different classes of DRG nAChRs with different Ca2+ permeability values.
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DRG neurones convey sensory information from the periphery to the CNS, using neuropeptides and amino acids as neurotransmitters (Salt & Hill, 1983). nAChRs may be involved in the flow of information, as nicotine and nicotinic agonists induce analgesic effects, acting both at central and peripheral sites (Flores, 2000; Jain, 2004). Moreover, variations of [Ca2+]i alter the DRG nociceptive responses through the modulation of the vanilloid receptor TRPV1 (transient receptor potential channel, vanilloid subfamily 1; Caterina et al. 1997; Koplas et al. 1997; Guenther et al. 1999). The Ca2+ permeability of the nAChRs expressed in DRG neurones may thus be physiologically relevant to sensory transduction.
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We measured the fractional Ca2+ current (Pf, the current carried by Ca2+ ions) of methyllycaconitine (MLA)-sensitive and -insensitive nAChRs in DRG neurones, finding values comparable to those of nAChRs expressed in heterologous systems. We also analysed the effects of the nAChR-mediated Ca2+ influx in DRG neurones on the function of the capsaicin-activated TRPV1 receptors, showing that it might contribute to the modulation of peripheral sensory processing.
Methods
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Animals
All DRG preparations were obtained from Wistar rats. Animals were used in accordance with the European Directive 86/609/EEC requirements and with the rules of the Ethical Committee of the Medical Faculty of the University of Rome ‘La Sapienza’. All rats were anaesthetized with halothane and killed by cervical dislocation.
Preparation of acutely dissociated DRG neurones
Acutely dissociated DRG neurones were prepared according to methods reported in Hu & Li (1997), with minor modifications. Thoracic and lumbar ganglia were excised and placed into phosphate-buffered saline (PBS) without calcium or magnesium. After removing the roots and surrounding connective tissue, we minced and incubated the DRGs with an enzymatic mix containing trypsin (0.5 mg ml–1), collagenase (1.0 mg ml–1), and DNase (0.1 mg ml–1) at 37°C for 25 min. Enzymatic digestion was stopped by substituting the enzymatic mix with Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS), penicillin and streptomycin. After enzymatic treatment, the cells were drawn gently up and down in a 1 ml plastic pipette, centrifuged at 233 g (1150 r.p.m.) at room temperature for 4 min, and the supernatant removed. Cells were re-suspended with an adequate amount of DMEM, plus 10% FBS, penicillin and streptomycin, and nerve growth factor (NGF, 0.25 mg ml–1). Dissociated DRG neurones were plated on poly L-lysine- (0.01 mg ml–1) and laminin- (0.04 mg ml–1) coated Petri dishes and cultured under an atmosphere containing 5% CO2. Cells were used within 1 day of plating.
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Electrophysiology
Whole-cell currents were recorded at 25°C from cells voltage clamped at –70 mV (unless otherwise specified) using borosilicate glass patch pipettes having a tip resistance of 3–5 M. Membrane currents were filtered at 2 kHz upon acquisition with an Axopatch 200B amplifier (Axon Instruments, USA) and analysed using pCLAMP8 software (Axon). During recordings, cells were continuously superfused using a gravity-driven perfusion system consisting of independent tubes for normal and drug-containing external solutions. The terminals of the tubes were positioned 50–100 μm away from the patched cell and connected, on the other side, to a fast exchanger system (RSC-100, Bio-Logic, France).
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Determination of Ca2+ transients
The methods for Ca2+ determinations are fully reported in Fucile et al. (2000). Cells were incubated at 37°C with the membrane-permeant fluorescent dye fura-2 AM (4 μM) for 45 min in DMEM. Fluorescence determinations were made using a conventional system driven by Axon Imaging Workbench software (Axon). All optical parameters and digital camera settings were maintained throughout this study to avoid non-homogeneous data. Recordings of fluorescence signals and membrane currents were synchronized, and images acquired and stored on a PC and analysed off-line. The changes in intracellular Ca2+ were expressed as F/F, the ratio of time-resolved fluorescence variations over the basal fluorescence at a single excitation wavelength (380 nm), to increase the temporal resolution of the determinations, when measuring the fractional Ca2+ current Pf. Increases of [Ca2+]i due to the activation of nicotinic or muscarinic receptors could be easily recognized on the basis of the response kinetics. Little delay (<< 100 ms) and fast rise times were typical features of nicotinic Ca2+ transients, while cells showing delayed muscarinic Ca2+ transients were discarded from subsequent analysis.
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Whole-cell patch-clamp recordings were performed on cells expressing nAChRs, identified as those generating a fluorescence transient in response to a brief nicotine application. Electrodes were filled with intracellular solution (see below) containing cell-impermeant fura-2 (250 μM, Molecular Probes). Determinations were carried out after the basal fluorescence had reached a stable value. Cells displaying high basal F340/F380 ratio values (> 2 in our conditions) and/or low basal F380 values (< 100 a.u.) were discarded. In order to evaluate Pf, the F/Q ratio between the fluorescence increase (F) and total charge that had entered the cell at each fluorescence acquisition time (Q) was defined as:
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For each cell, we used the F/Q points that immediately after the onset of the nicotine-induced response exhibited a linear relationship, indicating that the Ca2+-buffering capability of fura-2 was not saturated. The F/Q ratio value was then measured as the slope of the linear regression best fitting the F–Q plot. Finally, the Pf was determined by normalizing the ratio obtained in standard medium (F/QS) to the calibration ratio, measured when Ca2+ ions were the only permeant ionic species (F/QCa):
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In some experiments, the changes in intracellular Ca2+ were expressed as variations of [Ca2+]i calculated, as previously described (Grynkiewicz et al. 1985; Grassi et al. 1993), using the equation:
where R is the ratio between the cell fluorescence images acquired at two excitation wavelengths (340 and 380 nm, emission 510 nm), Rmin= 0.4, Rmax= 8.9, and Keff= 1.55 μM. Keff=KD, where KD is the FuRA-2 dissociation constant and is an experimentally determined constant.
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Solutions and chemicals
Cells were superfused with a standard external medium containing (mM): NaCl, 140; KCl, 2.8; CaCl2, 2; MgCl2, 2; Hepes–NaOH, 10; glucose, 10; pH 7.3. The Ca2+-free solution was obtained by omitting CaCl2 in the solution. The standard intracellular solution for whole-cell recording contained (mM): CsCl, 140; Hepes–CsOH, 10; Mg2ATP3, 2; and EGTA, 0.5; pH 7.3. F/Q calibrations were performed in a medium (calibration medium) containing only Ca2+ as permeant ions (mM): NMDG, 142; CaCl2, 2; and Hepes–Ca(OH)2, 10; pH 7.3; or NMDG, 130; CaCl2, 10; and Hepes–Ca(OH)2, 10; pH 7.3. For all the measurements of Pf, patch-clamp electrodes were filled with an internal solution containing (mM): NMDG, 140; Hepes–HCl, 10; fura-2, 0.25; thapsigargin, 0.001, pH 7.3. Agonists/antagonists of nAChRs and TRPV1 receptors (acetylcholine, nicotine, choline, MLA, 5-hydroxyindole (5OH-indole), dihydro-erithroidine (DHE) and capsaicin) were diluted with the appropriate extracellular solution to the final concentration. All chemicals were purchased from Sigma (USA), except for fura-2 and fura-2 AM which were from Molecular Probes (USA).
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Data analysis and statistics
Data are given as means ±S.E.M., and statistical significance tested using ANOVA (P < 0.05). Dose–response curves were constructed by fitting the values obtained at different concentrations, after normalization. The non-linear fitting routine of Sigma Plot software (Jandel Scientific, CA, USA) was used to fit the data to the Hill equation: I= 1/(1 + (EC50/[A])nH), where I is the normalized current amplitude induced by the agonist at concentration [A], nH is the Hill coefficient and EC50 the concentration at which a half-maximum response was induced. The F/Q ratio values used in the Pf determinations were obtained as linear regressions of the data, using Sigma Plot software. The fitting routine of Clampfit 8 (Axon) was used to fit the current decay to a single or double exponential function.
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Results
Characterization of nicotinic currents in adult DRG neurones
To distinguish the cells with nicotine-evoked Ca2+ transients, adult rat DRG neurones loaded with the cell-permeant form of fura-2 were exposed to brief pulses of nicotine (100 μM). Fluorescence transients were detected in 38 out of 158 cells examined (24%), which had a small to medium diameter (data not shown). We subsequently performed whole-cell patch clamp recordings on these nAChR-expressing cells, which had a capacitance of 40 ± 3 pF (n= 38). ACh (100 μM) elicited a slowly desensitizing inward current, with mean amplitude of –1.2 ± 0.2 nA and mean decay time constant () of 0.78 ± 0.05 s (Fig. 1A). In five of these cells, nicotine (100 μM) evoked inward currents with similar mean amplitude (–1.3 ± 0.6 nA; n= 5), but a significantly smaller (0.39 ± 0.03 s; P < 0.05; Fig. 1A). The relatively slow current decay suggests that ACh-evoked responses were mainly due to the activation of heteromeric nAChRs, composed of both and subunits, as confirmed by their insensitivity to MLA (10 nM; Fig. 1B).
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A, samples of ACh- and nicotine-evoked whole-cell currents in two different voltage-clamped dorsal root ganglion (DRG) cells. Horizontal bars represent the drug applications at indicated concentrations. Superimposed traces represent single exponential functions best fitting the data, with values of 0.57 s (left) and 0.42 s (right). B, whole-cell currents elicited by ACh (100 μM) in another DRG neurone in the absence or presence of methyllycaconitine (MLA; 10 nM), as indicated. Superimposed traces represent single exponential functions best fitting the data, with a value of 0.77 s for both left and right traces. C, whole-cell currents elicited by the indicated agonists and upon 5OH-indole treatment in a neurone different from those in A and B.
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Since the 7 subunit is expressed in DRG neurones, we further investigated the presence of 7*-nAChRs by examining the whole-cell currents evoked, in the same set of cells, by choline, which is a full agonist for 7*-nAChR, but a partial agonist of 34*-nAChRs (Alkondon et al. 1997). In all the ACh-responsive cells tested, choline (10 mM) elicited slow inward currents, with amplitude smaller than the responses evoked by ACh or nicotine (–130 ± 30 pA; n= 38). In 6 out of these 38 cells (capacitance 57 ± 7 pF) choline also elicited a fast-decaying component, with a of 0.03 ± 0.01 s, followed by the slower component (Fig. 1C), suggesting the expression of functional nAChRs of different subunit composition in some DRG neurones. This coexpression was confirmed by using 5-hydroxyindole (5OH-indole; 1 mM), a drug known to potentiate 7*-nAChRs while inhibiting heteromeric nAChRs (Zwart et al. 2002; Fucile et al. 2004). In cells exhibiting biphasic responses to choline, 5OH-indole potentiated the fast component (9.0 ± 1.5 fold, n= 3), while reducing the slow component of the ACh-evoked response (Fig. 1C).
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The apparent affinity for ACh and the voltage dependence of the slowly decaying ACh-evoked currents were also characterized. The ACh dose–response curve was best fitted to a Hill equation (not shown), yielding an EC50 value of 115 μM, compatible with low affinity heteromeric nAChRs such as 3*-nAChRs (Brioni et al. 1997). The current–voltage curve showed a clear current rectification (not shown), a common feature of neuronal nAChRs. All these data show that adult DRG neurones express bona fide heteromeric receptors, along with the rare expression of 7*-nAChRs.
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Pf determinations in adult DRG neurones
The brief application of either ACh or nicotine (2–4 s) to DRG neurones loaded with fura-2 via the patch pipette caused the elevation of [Ca2+]i in all the cells examined (e.g. Fig. 2A). The Pf of heteromeric nAChRs expressed in adult DRG neurones was measured in the presence of MLA (10 nM; Fig. 2B) to block any homomeric 7*- or 9*-nAChRs. In standard medium, the F/QS ratio had a mean value of 0.09 ± 0.02 nC–1 (n= 12; Fig. 2B and D). The calibration F/QCa value was obtained by measuring, in calibration medium, the Ca2+ current activated by a 0 mV depolarization step, and the related fluorescence variation, yielding a mean value of 4.1 ± 0.4 nC–1 (n= 10; Fig. 2C and D). Using this value to normalize F/QS gave, for MLA-insensitive nAChRs in adult DRG neurones, a Pf value of 2.2 ± 0.6% (Fig. 2D). This value is similar to that previously measured in heterologous expression systems for mammalian 34- and 42-nAChRs (Lax et al. 2002).
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A, samples of Ca2+ transients elicited by either ACh or nicotine at indicated concentrations on two different adult DRG neurones. B, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by ACh in an adult DRG neurone equilibrated in standard medium in the presence of MLA, as indicated. Current traces and fluorescence signals are aligned and share the same time scale. Note the decrease of F/F indicating the rise of [Ca2+]i. C, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by depolarization (from –70 mV to 0 mV, as indicated) in a DRG neurone equilibrated in a medium containing only Ca2+ as permeant ion. D, linear relationships between F/F and Q obtained from the cells shown in B and C. The Pf value, calculated by normalizing the slope obtained in B (standard medium) to the slope obtained in C (Ca2+ medium), is 2.5%.
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When the same measurements were repeated using ACh in the absence of MLA, the Pf value (1.9 ± 0.6%; n= 10) was not significantly different (P > 0.1) from that measured in the presence of MLA, confirming that in adult DRG neurones nicotinic responses are primarily due to the activation of heteromeric, MLA-insensitive nAChRs, while homomeric nAChRs are expressed in a minority of cells.
Ca2+ permeability of 7*-nAChRs expressed by neonatal DRG neurones
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The measurement of the Ca2+ permeability of the 7*-nAChRs in adult DRG neurones is impaired by the fact that this nAChR subtype is invariably coexpressed with other nAChR populations. While it is relatively easy to block 7*-nAChRs using the potent antagonist MLA, it is more difficult to selectively block the MLA-insensitive heteromeric nAChRs in order to focus on the 7*-nAChRs. To overcome this problem, we performed experiments on neonatal DRG neurones, since 12 out of 124 (9.7%) ACh-responsive neurones (capacitance 19 ± 2 pF) showed a pure MLA-sensitive response (Fig. 3A and B). Since DRG cells express 7 as well as 9 subunits, both capable of forming MLA-sensitive homomeric nAChRs, we compared the whole-cell current responses to ACh (500 μM) and nicotine (500 μM), which is an agonist of 7*-nAChRs but an antagonist of 9*-nAChRs (Verbitsky et al. 2000). In neurones with a pure MLA-sensitive current, nicotine and ACh evoked whole-cell currents of similar amplitude (–70 ± 30 pA and –70 ± 30 pA, respectively, n= 5) but different decay constant (= 110 ± 15 ms, and = 41 ± 5 ms, n= 5; Fig. 3A), indicating that 7*-nAChRs represent the main, if not the unique, population of functional nAChRs in MLA-sensitive neonatal DRG neurones. Thus, we measured the Pf of 7*-nAChRs in MLA-sensitive neonatal DRG neurones using nicotine (200 μM) in the presence of 5OH-indole (1 mM; Fig. 3B), a compound that increases the 7*-mediated currents without modifying the nAChR Ca2+ permeability (Fucile et al. 2003). Under these conditions, we found a F/QS ratio of 0.39 ± 0.02 nC–1 (Fig. 3C and D; n= 4). Normalizing this value to the same calibration F/QCa value used for adult DRG neurones, we obtained a Pf of 9.5 ± 1.5%, similar to the value measured for the rat 7-nAChRs in a heterologous expression system (Fucile et al. 2003).
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A, whole-cell currents elicited by ACh or nicotine at indicated concentration in a single cell, with or without MLA (as indicated). B, whole-cell currents elicited by nicotine in a single neonatal DRG neurone, blocked by MLA, and enhanced by 5OH-indole, at indicated concentrations. C, fluorescence variation recorded simultaneously with the bottom whole-cell current shown in B, in the presence of 5OH-indole (1 mM). D, linear relationship between F/F (i.e. Ca2+-dependent fluorescence variation) and Q (total electric charge) obtained from the recording shown in B and C. Resulting F/Q ratio and Pf values: 0.341 nC–1 and 8.4%, respectively.
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Modulation of the TRPV1 function by Ca2+ entry through DRG nAChRs
In order to determine whether the Ca2+ entry through nAChRs modulates the sensory function of DRG neurones, we analysed the changes induced by nicotinic stimulation in the responses mediated through TRPV1, which is physiologically activated by mild heat and acidification, or, pharmacologically, by capsaicin (for review see Caterina & Julius, 2001). We chose this receptor because it is well known that TRPV1 function, in particular its desensitization, is modulated by [Ca2+]i variations (Koplas et al. 1997; Guenther et al. 1999). It is, however, unknown whether Ca2+ influx through nAChRs affects the nociceptive TRPV1 response.
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This issue was investigated in adult DRG neurones responsive to both nicotine and capsaicin. These cells were identified using the Ca2+ imaging technique. Under our conditions, 185 out 347 DRG neurones (53 ± 5%; 69 optical fields, 6 different experiments) exhibited detectable Ca2+ transients when challenged with capsaicin (0.5 μM) for 1–4 s. About 25% of these cells were also sensitive to nicotine (see above), and had a capacitance of 38 ± 4 pF (n= 33).
Since the amplitude of capsaicin-induced currents rapidly runs down in the presence of standard external Ca2+, the TRPV1 function was monitored in a Ca2+-free medium. The whole-cell currents were elicited by repetitive applications of capsaicin (0.5 μM; duration 0.5 s, every 20 s; Fig. 4A). Ca2+ entry through nAChRs was achieved by a prolonged nicotine application (500 μM for 15 s) in the presence of standard Ca2+ medium (Fig. 4A). This stimulation accelerated the decay of capsaicin-induced currents (Fig. 4B), with the time course shown in Fig. 4C, and reduced their amplitudes (Fig. 4D). The capsaicin-induced currents were not significantly altered by a comparable (15 s) application of nicotine in Ca2+-free medium (n= 5), or by superfusion of the cell with Ca2+-containing medium in the absence of nicotine (n= 5; Fig. 4E). These data indicate that the reported effects on TRPV1-evoked currents were due to Ca2+ entry through nAChRs.
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A, simultaneous recordings of whole-cell currents (bottom) and of [Ca2+]i changes (top) in a single DRG neurone. Capsaicin pulses (0.5 μM, 0.5 s) were applied every 20 s in a Ca2+-free medium (as indicated). Note the prolonged nicotine application (500 μM, 15 s) in the presence of normal external Ca2+, inducing inward current (grey line) and Ca2+ entry, and modifying the subsequent capsaicin-evoked currents. B, capsaicin-induced currents recorded before and after nicotine application. Current amplitudes were normalized to allow a better comparison of desensitization kinetics. Superimposed lines represent single exponential functions best fitting the data, with values of 3.70 s (before nicotine) and 2.69 s (after nicotine). Asterisks indicate the corresponding currents in A and B. C, time course of normalized of capsaicin-induced currents before and after a 15 s nicotine application (horizontal line; mean of the current preceding nicotine application, 3.0 ± 1.0 s). Note absence of recovery after nicotine washout. D, time course of normalized amplitudes of capsaicin-induced currents before and after nicotine application (mean amplitude of the current preceding nicotine application, 480 ± 70 pA). Note again absence of current recovery. E, effects of different treatments on of capsaicin-evoked currents. Same protocol as A. Values were obtained averaging the normalized from the first five currents following the treatment. *P < 0.05.
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We also tested the effects of the chronic exposure of DRG neurones to a low concentration of nicotine (200 nM). For this purpose, we analysed the capsaicin-induced [Ca2+]i variations after a 16–24 h treatment of DRG neurones with nicotine. No significant difference in the number of responsive cells was found between control (56 ± 8%, n= 178), nicotine-treated neurones (55 ± 12%, n= 122), and neurones treated with nicotine in the presence of nicotinic antagonists (10 μM DHE and 10 nM MLA; 43 ± 7%, n= 47). This finding indicates that the chronic treatment did not select any specific subsets of DRG neurones. Upon chronic nicotine treatment, the mean amplitude of the capsaicin-induced Ca2+ transients significantly decreased to 64% of the control value, and this effect was antagonized by nAChR blockers (Fig. 5A and B). The rise-time of the Ca2+ transients was significantly shorter in the treated cells (77% of control; Fig. 5A and C), consistent with the accelerated decay of the capsaicin-induced currents observed upon acute nicotinic stimulation. Again, the nicotinic antagonists blocked this action.
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A, typical Ca2+ transients induced by a 1 s pulse of 1 μM capsaicin in a control DRG neurone (black line) and in a DRG neurone incubated for 18 h with 200 nM nicotine (grey line). B, histogram of the mean amplitude of capsaicin-induced Ca2+ transients upon indicated treatments. [Ca2+]i=[Ca2+]i,peak–[Ca2+]i,basal. C, histogram of rise time of capsaicin-induced Ca2+ transients upon indicated treatments. *P < 0.05.
Altogether, these findings indicate that both the acute and chronic activation of nAChRs affect the subsequent TRPV1-mediated responses similarly, reducing their amplitude and accelerating their desensitization.
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Discussion
In this study we measured the Ca2+ permeability through nAChRs in a native cell system, the rat DRG neurone, and we report that both heteromeric and homomeric 7*-nAChRs exhibit Pf values similar to those determined for cloned receptors in heterologous expression systems (Fucile, 2004). We also demonstrate that TRPV1 is negatively modulated by Ca2+ entry through nAChRs, indicating a new way of investigating the peripheral nociceptive mechanisms.
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Functional nAChRs expressed by DRG neurones
Sensory neurones, including nociceptors, express a wide variety of functionally active nAChR subtypes (Genzen et al. 2001; Khan et al. 2003; Genzen & McGehee, 2003; Haberberger et al. 2004). We report that nicotinic stimulation activates currents with differential functional and pharmacological properties, probably mediated by different nAChR subtypes, in acutely dissociated adult DRG neurones. The ACh-responsive DRG cells exhibited a capacitance of 40 pF, suggesting their classification as small- to medium-sized nociceptive cells (see Rau et al. 2004). Specifically, the nAChR subunits expressed by DRG neurones, identified using many experimental approaches, include all cloned neuronal subunits to date, 2–10 and 2–4 (Genzen et al. 2001; Lips et al. 2002; Lang et al. 2003). The most represented nAChR subtype generates slowly desensitizing current, partially agonized by choline, insensitive to MLA, and with a relatively low apparent affinity for ACh. All these features suggest the major involvement of 34*-nAChRs (see also Alkondon et al. 1997; Khan et al. 2003; Rau et al. 2004), rather than 42*-nAChRs exhibiting a higher apparent affinity for ACh (Brioni et al. 1997). In the same cells, a low expression of nAChRs generating fast-decaying MLA-sensitive currents, namely 7*- and/or 9*-nAChRs (Brioni et al. 1997; Verbitsky et al. 2000) was also found. We report instead that, in neonatal DRG neurones, currents fully blocked by MLA, probably mediated only through 7*-nAChRs, are more frequently detected, in agreement with previous studies (Genzen et al. 2001). These neonatal DRG neurones exhibited lower capacitance than adult ACh-responsive cells, consistent with the finding that 7 is expressed as unique nicotinic subunits only in small-diameter C-fibre nociceptors (Rau et al. 2004).
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DRG neurones are the only neuronal cell type shown, to date, to express the 9 and 10 nAChR subunits (Lips et al. 2002; Haberberger et al. 2004) able to form highly Ca2+-permeable channels (Katz et al. 2000; Weisstaub et al. 2002). We could not find any functional expression of 9*-nAChRs in isolated DRG neurones using pharmacological tools. Since no significant difference exists between maximal current amplitudes elicited by either ACh (agonist) or nicotine (9 receptor antagonist; Verbitsky et al. 2000), we conclude that the detected MLA-sensitive currents are probably mediated through 7*- rather than 9*-AChRs in DRG neurones, in agreement with the finding that isolated DRG neurones after 1 day in culture are devoid of 9*-nAChRs (Haberberger et al. 2004).
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Ca2+ permeability of nAChRs in DRG neurones
In this study, we report Pf values from DRG native nerve cells that compare with those measured in heterologous systems (Fig. 6; Fucile, 2004). Specifically, the Pf value of human 34-nAChRs previously determined in a heterologous cell expression system (2.7%; Lax et al. 2002) is similar to the value reported here from adult rat DRG neurones in the presence of MLA (2.2%). Furthermore, in adult DRG neurones the Pf value obtained activating all nAChR subtypes is not significantly different from the Pf value of MLA-insensitive nAChRs, suggesting that 34*-nAChRs represent the main nAChR population. Yet, the Pf value of 7*-nAChRs determined in neonatal DRG neurones (9.5%) matches that of heterologously expressed rat and human 7-nAChRs (8.8% and 11.4%, respectively; Lax et al. 2002; Fucile et al. 2003). These data suggest that native and heterologously expressed nAChRs, sharing similar functional and pharmacological features, also display similar Ca2+ permeability.
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Histogram of Pf values of heterologously expressed nAChRs (filled columns) and nAChRs expressed in native DRG neurones (open columns), as indicated. h, human; r, rat. For the Pf values of h 34- and h 42-nAChRs (expressed in HEK293 cells) see Lax et al. (2002). For the Pf values of h and r 7-nAChRs (expressed in GH4C1 cells) see Fucile et al. (2003). ‘Mixed’ indicates Pf values obtained by activating all nAChR subtypes present in the examined adult DRG neurones. The Pf of 7*-nAChRs was measured in neonatal DRG neurones.
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Before our study, the Pf value of native nAChRs expressed by neurones was measured by Rogers & Dani, 1995; Rogers et al. 1997), for -bungarotoxin-insensitive nAChRs in adult rat superior cervical ganglion cells. These authors found a value of 4.7 ± 0.3% using a less negative holding potential and a higher extracellular Ca2+ concentration, compared to our conditions (–50 mV versus–70 mV and 2.5 mMversus 2.0 mM, respectively). These different experimental settings reduce the difference between Pf values. However, it is likely that the populations of heteromeric nAChRs present in DRG and superior cervical ganglion neurones are not homogeneous.
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Functional consequences of nAChR activation in DRG neurones
Many studies report the ability of nicotinic agonists to modulate the peripheral algesic reactivity, but the actual physiological role of nAChR expression in primary sensory neurones is still under question. It is likely that DRG nAChRs contribute to the chemoreceptor complexity required to detect a broad spectrum of algesic molecules, the nicotinic stimulation activating nociceptors in vivo and in vitro (Jancso, 1961; Sucher et al. 1990; Liu et al. 1993; Steen & Reeh, 1993; Bernardini et al. 2001; Genzen & McGehee, 2003), and several cell types from DRG-innervated tissues producing ACh (Wessler et al. 1999). Another functional role probably played by DRG nAChRs is the modulation of the neurotransmitter release from the central endings of DRG neurones, since spinal cord interneurones are proved to produce ACh and to presynaptically contact the terminals of DRG afferents in the dorsal horn (Ribeiro-da-Silva & Cuello, 1990).
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Our findings reported here suggest that DRG nAChRs may play an additional role in sensory modulation. It is well known that the activation of cholinergic nicotinic pathways elicits anti-nociceptive effects (for reviews see: Flores, 2000; Jain, 2004). Although several CNS regions express nAChRs capable of producing anti-nociception (Christensen & Smith, 1990; Iwamoto, 1991; Iwamoto & Marion, 1993; Damaj et al. 1998), part of the nAChR-mediated analgesia is believed to be due to a peripheral action (Caggiula et al. 1995; Rueter et al. 2003). We show that acute stimulation of DRG nAChRs produces Ca2+ entry and a subsequent modification of the capsaicin-induced TRPV1-mediated currents, with faster desensitization and smaller amplitude. These effects are probably due to Ca2+ entry through nAChRs, being absent in Ca2+-free medium and not explained by a direct interaction with TRPV1 receptor–channels such as that exerted by nicotine or other nicotinic agonists on vanilloid receptors in trigeminal ganglia neurones (Liu et al. 2004). Interestingly, we report a reduced Ca2+ response to capsaicin after a prolonged incubation with doses of nicotine as low as those detected in smokers' extracellular fluids (Dani & Heinemann, 1996), likely to produce a long-lasting stimulation of extrasynaptic nAChRs.
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Capsaicin-activated TRPV1-mediated currents recorded in DRG neurones exhibit both acute desensitization (the inactivation of the current during a prolonged application of capsaicin) and tachyphylaxis (the diminution of the maximal current amplitude during repetitive deliveries of the same capsaicin concentrations). Both phenomena have been demonstrated to be Ca2+ dependent (Cholewinski et al. 1993; Caterina et al. 1997; Koplas et al. 1997) and we have shown here to be caused by nicotine. Considered together, our data indicate that the activation of DRG nAChRs can decrease the sensitivity of TRPV1 channels to subsequent sensory stimuli. Such a negative nicotinic modulation of TRPV1, if proved in vivo, could be physiologically important, representing a protective mechanism against the hyperexcitation and Ca2+ overloading of DRG cells. However, it is likely that the activation of DRG nAChRs modulates the sensory signalling differently depending on (i) the expressed nAChR subtypes, (ii) their localization, and (iii) the timing of activation compared to other sensory stimuli. Thus, further studies should be done to address these questions.
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2 Fondazione Santa Lucia, Via Ardeatina, Roma, Italy
Abstract
Ca2+ entry through neuronal nicotinic ACh receptors (nAChRs) modulates many biological processes in nervous tissue. In order to study the functional role of nAChRs in peripheral sensory signalling, we measured their Ca2+ permeability in rat dorsal root ganglion (DRG) neurones, and analysed the effects of nAChR-mediated Ca2+ influx on the function of the vanilloid receptor TRPV1. The fractional Ca2+ current (Pf, i.e. the percentage of current carried by Ca2+ ions) flowing through nAChR channels was measured by Ca2+ imaging fluorescence microscopy in combination with the patch-clamp technique. Functional nAChRs were expressed in a subset of adult DRG neurones (about 24% of the cells), typically with small to medium size as measured by their capacitance (40 ± 3 pF). In most cells, ACh evoked slowly desensitizing currents, insensitive to methyllycaconitine (MLA, 10 nM), a potent antagonist of homomeric nAChRs. Fast decaying currents, probably mediated by 7*-nAChRs (i.e. native 7-containing nAChRs), were observed in 15% of ACh-responsive cells, in which slowly decaying currents, mediated by heteromeric nAChRs, were simultaneously present. The nAChRs of adult DRG neurones exhibited a Pf value of 2.2 ± 0.6% in the presence of MLA and 1.9 ± 0.6% (P > 0.1) in the absence of MLA, indicating that homomeric MLA-sensitive nAChRs do not contribute to Ca2+ entry into adult DRG neurones. Conversely, 10% of neonatal DRG neurones showed ACh-evoked currents completely blocked by MLA. In these neurones, nAChRs showed a larger Pf value (9.5 ± 1.5%), indicating the expression of bona fide 7*-nAChRs. Finally, we report that Ca2+ influx through nAChRs in adult DRG neurones negatively modulated the TRPV1-mediated responses, representing a possible mechanism underlying the analgesic properties of nicotinic agonists on sensory neurones.
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Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric cation-selective ligand-gated channels. To date, 12 neuronal nicotinic subunits have been cloned in vertebrates (2–10 and 2–4) that are widely expressed in the nervous system (Dani, 2001) and in non-neuronal cells, such as glial and endothelial cells, keratinocytes, macrophages and lymphocytes (Sharma & Vijayaraghavan, 2002; Wang et al. 2003). It is generally thought that some neuronal nAChRs are located at presynaptic sites, where they modulate the neurotransmitter release by controlling the intracellular free Ca2+ concentration ([Ca2+]i) via the influx of Ca2+ through their channels (Wonnacott, 1997; MacDermott et al. 1999).
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A major difficulty in studying the Ca2+ permeability of native nAChRs is represented by the fact that the subunit composition of nAChRs (largely undefined in native preparations) determines the selectivity of the ion channel, as shown by studies on heterologously expressed nAChRs, with known subunit composition. A large body of findings supports the division of neuronal nAChRs into two classes (see for review Fucile, 2004). The first class is that of homopentameric nAChRs composed of the subunits 7–9, sensitive to -bungarotoxin (-BTX), which show a high Ca2+ permeability. The second class groups heteropentameric, -BTX-insensitive nAChRs (non--BTX nAChRs), formed by both (out of 2–6) and (out of 2–4) subunits, showing lower Ca2+ permeability.
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An extensive analysis of the Ca2+ permeability of native nAChRs is still lacking, mainly because neurones express multiple nAChR subunits, yielding receptors of unknown composition. The paucity of selective agonists and/or antagonists has not helped the problem of identifying the native nAChRs. Our study starts to repair this deficiency by examining the Ca2+ permeability of nAChRs in dorsal root ganglion (DRG) neurones, which express several nicotinic subunits comprising 3, 4, 5, 7, 9 and 10 (Genzen et al. 2001; Khan et al. 2003; Haberberger et al. 2004). In particular, our aim is to correlate different classes of DRG nAChRs with different Ca2+ permeability values.
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DRG neurones convey sensory information from the periphery to the CNS, using neuropeptides and amino acids as neurotransmitters (Salt & Hill, 1983). nAChRs may be involved in the flow of information, as nicotine and nicotinic agonists induce analgesic effects, acting both at central and peripheral sites (Flores, 2000; Jain, 2004). Moreover, variations of [Ca2+]i alter the DRG nociceptive responses through the modulation of the vanilloid receptor TRPV1 (transient receptor potential channel, vanilloid subfamily 1; Caterina et al. 1997; Koplas et al. 1997; Guenther et al. 1999). The Ca2+ permeability of the nAChRs expressed in DRG neurones may thus be physiologically relevant to sensory transduction.
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We measured the fractional Ca2+ current (Pf, the current carried by Ca2+ ions) of methyllycaconitine (MLA)-sensitive and -insensitive nAChRs in DRG neurones, finding values comparable to those of nAChRs expressed in heterologous systems. We also analysed the effects of the nAChR-mediated Ca2+ influx in DRG neurones on the function of the capsaicin-activated TRPV1 receptors, showing that it might contribute to the modulation of peripheral sensory processing.
Methods
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Animals
All DRG preparations were obtained from Wistar rats. Animals were used in accordance with the European Directive 86/609/EEC requirements and with the rules of the Ethical Committee of the Medical Faculty of the University of Rome ‘La Sapienza’. All rats were anaesthetized with halothane and killed by cervical dislocation.
Preparation of acutely dissociated DRG neurones
Acutely dissociated DRG neurones were prepared according to methods reported in Hu & Li (1997), with minor modifications. Thoracic and lumbar ganglia were excised and placed into phosphate-buffered saline (PBS) without calcium or magnesium. After removing the roots and surrounding connective tissue, we minced and incubated the DRGs with an enzymatic mix containing trypsin (0.5 mg ml–1), collagenase (1.0 mg ml–1), and DNase (0.1 mg ml–1) at 37°C for 25 min. Enzymatic digestion was stopped by substituting the enzymatic mix with Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS), penicillin and streptomycin. After enzymatic treatment, the cells were drawn gently up and down in a 1 ml plastic pipette, centrifuged at 233 g (1150 r.p.m.) at room temperature for 4 min, and the supernatant removed. Cells were re-suspended with an adequate amount of DMEM, plus 10% FBS, penicillin and streptomycin, and nerve growth factor (NGF, 0.25 mg ml–1). Dissociated DRG neurones were plated on poly L-lysine- (0.01 mg ml–1) and laminin- (0.04 mg ml–1) coated Petri dishes and cultured under an atmosphere containing 5% CO2. Cells were used within 1 day of plating.
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Electrophysiology
Whole-cell currents were recorded at 25°C from cells voltage clamped at –70 mV (unless otherwise specified) using borosilicate glass patch pipettes having a tip resistance of 3–5 M. Membrane currents were filtered at 2 kHz upon acquisition with an Axopatch 200B amplifier (Axon Instruments, USA) and analysed using pCLAMP8 software (Axon). During recordings, cells were continuously superfused using a gravity-driven perfusion system consisting of independent tubes for normal and drug-containing external solutions. The terminals of the tubes were positioned 50–100 μm away from the patched cell and connected, on the other side, to a fast exchanger system (RSC-100, Bio-Logic, France).
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Determination of Ca2+ transients
The methods for Ca2+ determinations are fully reported in Fucile et al. (2000). Cells were incubated at 37°C with the membrane-permeant fluorescent dye fura-2 AM (4 μM) for 45 min in DMEM. Fluorescence determinations were made using a conventional system driven by Axon Imaging Workbench software (Axon). All optical parameters and digital camera settings were maintained throughout this study to avoid non-homogeneous data. Recordings of fluorescence signals and membrane currents were synchronized, and images acquired and stored on a PC and analysed off-line. The changes in intracellular Ca2+ were expressed as F/F, the ratio of time-resolved fluorescence variations over the basal fluorescence at a single excitation wavelength (380 nm), to increase the temporal resolution of the determinations, when measuring the fractional Ca2+ current Pf. Increases of [Ca2+]i due to the activation of nicotinic or muscarinic receptors could be easily recognized on the basis of the response kinetics. Little delay (<< 100 ms) and fast rise times were typical features of nicotinic Ca2+ transients, while cells showing delayed muscarinic Ca2+ transients were discarded from subsequent analysis.
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Whole-cell patch-clamp recordings were performed on cells expressing nAChRs, identified as those generating a fluorescence transient in response to a brief nicotine application. Electrodes were filled with intracellular solution (see below) containing cell-impermeant fura-2 (250 μM, Molecular Probes). Determinations were carried out after the basal fluorescence had reached a stable value. Cells displaying high basal F340/F380 ratio values (> 2 in our conditions) and/or low basal F380 values (< 100 a.u.) were discarded. In order to evaluate Pf, the F/Q ratio between the fluorescence increase (F) and total charge that had entered the cell at each fluorescence acquisition time (Q) was defined as:
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For each cell, we used the F/Q points that immediately after the onset of the nicotine-induced response exhibited a linear relationship, indicating that the Ca2+-buffering capability of fura-2 was not saturated. The F/Q ratio value was then measured as the slope of the linear regression best fitting the F–Q plot. Finally, the Pf was determined by normalizing the ratio obtained in standard medium (F/QS) to the calibration ratio, measured when Ca2+ ions were the only permeant ionic species (F/QCa):
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In some experiments, the changes in intracellular Ca2+ were expressed as variations of [Ca2+]i calculated, as previously described (Grynkiewicz et al. 1985; Grassi et al. 1993), using the equation:
where R is the ratio between the cell fluorescence images acquired at two excitation wavelengths (340 and 380 nm, emission 510 nm), Rmin= 0.4, Rmax= 8.9, and Keff= 1.55 μM. Keff=KD, where KD is the FuRA-2 dissociation constant and is an experimentally determined constant.
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Solutions and chemicals
Cells were superfused with a standard external medium containing (mM): NaCl, 140; KCl, 2.8; CaCl2, 2; MgCl2, 2; Hepes–NaOH, 10; glucose, 10; pH 7.3. The Ca2+-free solution was obtained by omitting CaCl2 in the solution. The standard intracellular solution for whole-cell recording contained (mM): CsCl, 140; Hepes–CsOH, 10; Mg2ATP3, 2; and EGTA, 0.5; pH 7.3. F/Q calibrations were performed in a medium (calibration medium) containing only Ca2+ as permeant ions (mM): NMDG, 142; CaCl2, 2; and Hepes–Ca(OH)2, 10; pH 7.3; or NMDG, 130; CaCl2, 10; and Hepes–Ca(OH)2, 10; pH 7.3. For all the measurements of Pf, patch-clamp electrodes were filled with an internal solution containing (mM): NMDG, 140; Hepes–HCl, 10; fura-2, 0.25; thapsigargin, 0.001, pH 7.3. Agonists/antagonists of nAChRs and TRPV1 receptors (acetylcholine, nicotine, choline, MLA, 5-hydroxyindole (5OH-indole), dihydro-erithroidine (DHE) and capsaicin) were diluted with the appropriate extracellular solution to the final concentration. All chemicals were purchased from Sigma (USA), except for fura-2 and fura-2 AM which were from Molecular Probes (USA).
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Data analysis and statistics
Data are given as means ±S.E.M., and statistical significance tested using ANOVA (P < 0.05). Dose–response curves were constructed by fitting the values obtained at different concentrations, after normalization. The non-linear fitting routine of Sigma Plot software (Jandel Scientific, CA, USA) was used to fit the data to the Hill equation: I= 1/(1 + (EC50/[A])nH), where I is the normalized current amplitude induced by the agonist at concentration [A], nH is the Hill coefficient and EC50 the concentration at which a half-maximum response was induced. The F/Q ratio values used in the Pf determinations were obtained as linear regressions of the data, using Sigma Plot software. The fitting routine of Clampfit 8 (Axon) was used to fit the current decay to a single or double exponential function.
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Results
Characterization of nicotinic currents in adult DRG neurones
To distinguish the cells with nicotine-evoked Ca2+ transients, adult rat DRG neurones loaded with the cell-permeant form of fura-2 were exposed to brief pulses of nicotine (100 μM). Fluorescence transients were detected in 38 out of 158 cells examined (24%), which had a small to medium diameter (data not shown). We subsequently performed whole-cell patch clamp recordings on these nAChR-expressing cells, which had a capacitance of 40 ± 3 pF (n= 38). ACh (100 μM) elicited a slowly desensitizing inward current, with mean amplitude of –1.2 ± 0.2 nA and mean decay time constant () of 0.78 ± 0.05 s (Fig. 1A). In five of these cells, nicotine (100 μM) evoked inward currents with similar mean amplitude (–1.3 ± 0.6 nA; n= 5), but a significantly smaller (0.39 ± 0.03 s; P < 0.05; Fig. 1A). The relatively slow current decay suggests that ACh-evoked responses were mainly due to the activation of heteromeric nAChRs, composed of both and subunits, as confirmed by their insensitivity to MLA (10 nM; Fig. 1B).
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A, samples of ACh- and nicotine-evoked whole-cell currents in two different voltage-clamped dorsal root ganglion (DRG) cells. Horizontal bars represent the drug applications at indicated concentrations. Superimposed traces represent single exponential functions best fitting the data, with values of 0.57 s (left) and 0.42 s (right). B, whole-cell currents elicited by ACh (100 μM) in another DRG neurone in the absence or presence of methyllycaconitine (MLA; 10 nM), as indicated. Superimposed traces represent single exponential functions best fitting the data, with a value of 0.77 s for both left and right traces. C, whole-cell currents elicited by the indicated agonists and upon 5OH-indole treatment in a neurone different from those in A and B.
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Since the 7 subunit is expressed in DRG neurones, we further investigated the presence of 7*-nAChRs by examining the whole-cell currents evoked, in the same set of cells, by choline, which is a full agonist for 7*-nAChR, but a partial agonist of 34*-nAChRs (Alkondon et al. 1997). In all the ACh-responsive cells tested, choline (10 mM) elicited slow inward currents, with amplitude smaller than the responses evoked by ACh or nicotine (–130 ± 30 pA; n= 38). In 6 out of these 38 cells (capacitance 57 ± 7 pF) choline also elicited a fast-decaying component, with a of 0.03 ± 0.01 s, followed by the slower component (Fig. 1C), suggesting the expression of functional nAChRs of different subunit composition in some DRG neurones. This coexpression was confirmed by using 5-hydroxyindole (5OH-indole; 1 mM), a drug known to potentiate 7*-nAChRs while inhibiting heteromeric nAChRs (Zwart et al. 2002; Fucile et al. 2004). In cells exhibiting biphasic responses to choline, 5OH-indole potentiated the fast component (9.0 ± 1.5 fold, n= 3), while reducing the slow component of the ACh-evoked response (Fig. 1C).
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The apparent affinity for ACh and the voltage dependence of the slowly decaying ACh-evoked currents were also characterized. The ACh dose–response curve was best fitted to a Hill equation (not shown), yielding an EC50 value of 115 μM, compatible with low affinity heteromeric nAChRs such as 3*-nAChRs (Brioni et al. 1997). The current–voltage curve showed a clear current rectification (not shown), a common feature of neuronal nAChRs. All these data show that adult DRG neurones express bona fide heteromeric receptors, along with the rare expression of 7*-nAChRs.
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Pf determinations in adult DRG neurones
The brief application of either ACh or nicotine (2–4 s) to DRG neurones loaded with fura-2 via the patch pipette caused the elevation of [Ca2+]i in all the cells examined (e.g. Fig. 2A). The Pf of heteromeric nAChRs expressed in adult DRG neurones was measured in the presence of MLA (10 nM; Fig. 2B) to block any homomeric 7*- or 9*-nAChRs. In standard medium, the F/QS ratio had a mean value of 0.09 ± 0.02 nC–1 (n= 12; Fig. 2B and D). The calibration F/QCa value was obtained by measuring, in calibration medium, the Ca2+ current activated by a 0 mV depolarization step, and the related fluorescence variation, yielding a mean value of 4.1 ± 0.4 nC–1 (n= 10; Fig. 2C and D). Using this value to normalize F/QS gave, for MLA-insensitive nAChRs in adult DRG neurones, a Pf value of 2.2 ± 0.6% (Fig. 2D). This value is similar to that previously measured in heterologous expression systems for mammalian 34- and 42-nAChRs (Lax et al. 2002).
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A, samples of Ca2+ transients elicited by either ACh or nicotine at indicated concentrations on two different adult DRG neurones. B, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by ACh in an adult DRG neurone equilibrated in standard medium in the presence of MLA, as indicated. Current traces and fluorescence signals are aligned and share the same time scale. Note the decrease of F/F indicating the rise of [Ca2+]i. C, simultaneous recordings of the whole-cell current (top) and fluorescent transient (bottom) elicited by depolarization (from –70 mV to 0 mV, as indicated) in a DRG neurone equilibrated in a medium containing only Ca2+ as permeant ion. D, linear relationships between F/F and Q obtained from the cells shown in B and C. The Pf value, calculated by normalizing the slope obtained in B (standard medium) to the slope obtained in C (Ca2+ medium), is 2.5%.
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When the same measurements were repeated using ACh in the absence of MLA, the Pf value (1.9 ± 0.6%; n= 10) was not significantly different (P > 0.1) from that measured in the presence of MLA, confirming that in adult DRG neurones nicotinic responses are primarily due to the activation of heteromeric, MLA-insensitive nAChRs, while homomeric nAChRs are expressed in a minority of cells.
Ca2+ permeability of 7*-nAChRs expressed by neonatal DRG neurones
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The measurement of the Ca2+ permeability of the 7*-nAChRs in adult DRG neurones is impaired by the fact that this nAChR subtype is invariably coexpressed with other nAChR populations. While it is relatively easy to block 7*-nAChRs using the potent antagonist MLA, it is more difficult to selectively block the MLA-insensitive heteromeric nAChRs in order to focus on the 7*-nAChRs. To overcome this problem, we performed experiments on neonatal DRG neurones, since 12 out of 124 (9.7%) ACh-responsive neurones (capacitance 19 ± 2 pF) showed a pure MLA-sensitive response (Fig. 3A and B). Since DRG cells express 7 as well as 9 subunits, both capable of forming MLA-sensitive homomeric nAChRs, we compared the whole-cell current responses to ACh (500 μM) and nicotine (500 μM), which is an agonist of 7*-nAChRs but an antagonist of 9*-nAChRs (Verbitsky et al. 2000). In neurones with a pure MLA-sensitive current, nicotine and ACh evoked whole-cell currents of similar amplitude (–70 ± 30 pA and –70 ± 30 pA, respectively, n= 5) but different decay constant (= 110 ± 15 ms, and = 41 ± 5 ms, n= 5; Fig. 3A), indicating that 7*-nAChRs represent the main, if not the unique, population of functional nAChRs in MLA-sensitive neonatal DRG neurones. Thus, we measured the Pf of 7*-nAChRs in MLA-sensitive neonatal DRG neurones using nicotine (200 μM) in the presence of 5OH-indole (1 mM; Fig. 3B), a compound that increases the 7*-mediated currents without modifying the nAChR Ca2+ permeability (Fucile et al. 2003). Under these conditions, we found a F/QS ratio of 0.39 ± 0.02 nC–1 (Fig. 3C and D; n= 4). Normalizing this value to the same calibration F/QCa value used for adult DRG neurones, we obtained a Pf of 9.5 ± 1.5%, similar to the value measured for the rat 7-nAChRs in a heterologous expression system (Fucile et al. 2003).
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A, whole-cell currents elicited by ACh or nicotine at indicated concentration in a single cell, with or without MLA (as indicated). B, whole-cell currents elicited by nicotine in a single neonatal DRG neurone, blocked by MLA, and enhanced by 5OH-indole, at indicated concentrations. C, fluorescence variation recorded simultaneously with the bottom whole-cell current shown in B, in the presence of 5OH-indole (1 mM). D, linear relationship between F/F (i.e. Ca2+-dependent fluorescence variation) and Q (total electric charge) obtained from the recording shown in B and C. Resulting F/Q ratio and Pf values: 0.341 nC–1 and 8.4%, respectively.
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Modulation of the TRPV1 function by Ca2+ entry through DRG nAChRs
In order to determine whether the Ca2+ entry through nAChRs modulates the sensory function of DRG neurones, we analysed the changes induced by nicotinic stimulation in the responses mediated through TRPV1, which is physiologically activated by mild heat and acidification, or, pharmacologically, by capsaicin (for review see Caterina & Julius, 2001). We chose this receptor because it is well known that TRPV1 function, in particular its desensitization, is modulated by [Ca2+]i variations (Koplas et al. 1997; Guenther et al. 1999). It is, however, unknown whether Ca2+ influx through nAChRs affects the nociceptive TRPV1 response.
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This issue was investigated in adult DRG neurones responsive to both nicotine and capsaicin. These cells were identified using the Ca2+ imaging technique. Under our conditions, 185 out 347 DRG neurones (53 ± 5%; 69 optical fields, 6 different experiments) exhibited detectable Ca2+ transients when challenged with capsaicin (0.5 μM) for 1–4 s. About 25% of these cells were also sensitive to nicotine (see above), and had a capacitance of 38 ± 4 pF (n= 33).
Since the amplitude of capsaicin-induced currents rapidly runs down in the presence of standard external Ca2+, the TRPV1 function was monitored in a Ca2+-free medium. The whole-cell currents were elicited by repetitive applications of capsaicin (0.5 μM; duration 0.5 s, every 20 s; Fig. 4A). Ca2+ entry through nAChRs was achieved by a prolonged nicotine application (500 μM for 15 s) in the presence of standard Ca2+ medium (Fig. 4A). This stimulation accelerated the decay of capsaicin-induced currents (Fig. 4B), with the time course shown in Fig. 4C, and reduced their amplitudes (Fig. 4D). The capsaicin-induced currents were not significantly altered by a comparable (15 s) application of nicotine in Ca2+-free medium (n= 5), or by superfusion of the cell with Ca2+-containing medium in the absence of nicotine (n= 5; Fig. 4E). These data indicate that the reported effects on TRPV1-evoked currents were due to Ca2+ entry through nAChRs.
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A, simultaneous recordings of whole-cell currents (bottom) and of [Ca2+]i changes (top) in a single DRG neurone. Capsaicin pulses (0.5 μM, 0.5 s) were applied every 20 s in a Ca2+-free medium (as indicated). Note the prolonged nicotine application (500 μM, 15 s) in the presence of normal external Ca2+, inducing inward current (grey line) and Ca2+ entry, and modifying the subsequent capsaicin-evoked currents. B, capsaicin-induced currents recorded before and after nicotine application. Current amplitudes were normalized to allow a better comparison of desensitization kinetics. Superimposed lines represent single exponential functions best fitting the data, with values of 3.70 s (before nicotine) and 2.69 s (after nicotine). Asterisks indicate the corresponding currents in A and B. C, time course of normalized of capsaicin-induced currents before and after a 15 s nicotine application (horizontal line; mean of the current preceding nicotine application, 3.0 ± 1.0 s). Note absence of recovery after nicotine washout. D, time course of normalized amplitudes of capsaicin-induced currents before and after nicotine application (mean amplitude of the current preceding nicotine application, 480 ± 70 pA). Note again absence of current recovery. E, effects of different treatments on of capsaicin-evoked currents. Same protocol as A. Values were obtained averaging the normalized from the first five currents following the treatment. *P < 0.05.
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We also tested the effects of the chronic exposure of DRG neurones to a low concentration of nicotine (200 nM). For this purpose, we analysed the capsaicin-induced [Ca2+]i variations after a 16–24 h treatment of DRG neurones with nicotine. No significant difference in the number of responsive cells was found between control (56 ± 8%, n= 178), nicotine-treated neurones (55 ± 12%, n= 122), and neurones treated with nicotine in the presence of nicotinic antagonists (10 μM DHE and 10 nM MLA; 43 ± 7%, n= 47). This finding indicates that the chronic treatment did not select any specific subsets of DRG neurones. Upon chronic nicotine treatment, the mean amplitude of the capsaicin-induced Ca2+ transients significantly decreased to 64% of the control value, and this effect was antagonized by nAChR blockers (Fig. 5A and B). The rise-time of the Ca2+ transients was significantly shorter in the treated cells (77% of control; Fig. 5A and C), consistent with the accelerated decay of the capsaicin-induced currents observed upon acute nicotinic stimulation. Again, the nicotinic antagonists blocked this action.
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A, typical Ca2+ transients induced by a 1 s pulse of 1 μM capsaicin in a control DRG neurone (black line) and in a DRG neurone incubated for 18 h with 200 nM nicotine (grey line). B, histogram of the mean amplitude of capsaicin-induced Ca2+ transients upon indicated treatments. [Ca2+]i=[Ca2+]i,peak–[Ca2+]i,basal. C, histogram of rise time of capsaicin-induced Ca2+ transients upon indicated treatments. *P < 0.05.
Altogether, these findings indicate that both the acute and chronic activation of nAChRs affect the subsequent TRPV1-mediated responses similarly, reducing their amplitude and accelerating their desensitization.
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Discussion
In this study we measured the Ca2+ permeability through nAChRs in a native cell system, the rat DRG neurone, and we report that both heteromeric and homomeric 7*-nAChRs exhibit Pf values similar to those determined for cloned receptors in heterologous expression systems (Fucile, 2004). We also demonstrate that TRPV1 is negatively modulated by Ca2+ entry through nAChRs, indicating a new way of investigating the peripheral nociceptive mechanisms.
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Functional nAChRs expressed by DRG neurones
Sensory neurones, including nociceptors, express a wide variety of functionally active nAChR subtypes (Genzen et al. 2001; Khan et al. 2003; Genzen & McGehee, 2003; Haberberger et al. 2004). We report that nicotinic stimulation activates currents with differential functional and pharmacological properties, probably mediated by different nAChR subtypes, in acutely dissociated adult DRG neurones. The ACh-responsive DRG cells exhibited a capacitance of 40 pF, suggesting their classification as small- to medium-sized nociceptive cells (see Rau et al. 2004). Specifically, the nAChR subunits expressed by DRG neurones, identified using many experimental approaches, include all cloned neuronal subunits to date, 2–10 and 2–4 (Genzen et al. 2001; Lips et al. 2002; Lang et al. 2003). The most represented nAChR subtype generates slowly desensitizing current, partially agonized by choline, insensitive to MLA, and with a relatively low apparent affinity for ACh. All these features suggest the major involvement of 34*-nAChRs (see also Alkondon et al. 1997; Khan et al. 2003; Rau et al. 2004), rather than 42*-nAChRs exhibiting a higher apparent affinity for ACh (Brioni et al. 1997). In the same cells, a low expression of nAChRs generating fast-decaying MLA-sensitive currents, namely 7*- and/or 9*-nAChRs (Brioni et al. 1997; Verbitsky et al. 2000) was also found. We report instead that, in neonatal DRG neurones, currents fully blocked by MLA, probably mediated only through 7*-nAChRs, are more frequently detected, in agreement with previous studies (Genzen et al. 2001). These neonatal DRG neurones exhibited lower capacitance than adult ACh-responsive cells, consistent with the finding that 7 is expressed as unique nicotinic subunits only in small-diameter C-fibre nociceptors (Rau et al. 2004).
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DRG neurones are the only neuronal cell type shown, to date, to express the 9 and 10 nAChR subunits (Lips et al. 2002; Haberberger et al. 2004) able to form highly Ca2+-permeable channels (Katz et al. 2000; Weisstaub et al. 2002). We could not find any functional expression of 9*-nAChRs in isolated DRG neurones using pharmacological tools. Since no significant difference exists between maximal current amplitudes elicited by either ACh (agonist) or nicotine (9 receptor antagonist; Verbitsky et al. 2000), we conclude that the detected MLA-sensitive currents are probably mediated through 7*- rather than 9*-AChRs in DRG neurones, in agreement with the finding that isolated DRG neurones after 1 day in culture are devoid of 9*-nAChRs (Haberberger et al. 2004).
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Ca2+ permeability of nAChRs in DRG neurones
In this study, we report Pf values from DRG native nerve cells that compare with those measured in heterologous systems (Fig. 6; Fucile, 2004). Specifically, the Pf value of human 34-nAChRs previously determined in a heterologous cell expression system (2.7%; Lax et al. 2002) is similar to the value reported here from adult rat DRG neurones in the presence of MLA (2.2%). Furthermore, in adult DRG neurones the Pf value obtained activating all nAChR subtypes is not significantly different from the Pf value of MLA-insensitive nAChRs, suggesting that 34*-nAChRs represent the main nAChR population. Yet, the Pf value of 7*-nAChRs determined in neonatal DRG neurones (9.5%) matches that of heterologously expressed rat and human 7-nAChRs (8.8% and 11.4%, respectively; Lax et al. 2002; Fucile et al. 2003). These data suggest that native and heterologously expressed nAChRs, sharing similar functional and pharmacological features, also display similar Ca2+ permeability.
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Histogram of Pf values of heterologously expressed nAChRs (filled columns) and nAChRs expressed in native DRG neurones (open columns), as indicated. h, human; r, rat. For the Pf values of h 34- and h 42-nAChRs (expressed in HEK293 cells) see Lax et al. (2002). For the Pf values of h and r 7-nAChRs (expressed in GH4C1 cells) see Fucile et al. (2003). ‘Mixed’ indicates Pf values obtained by activating all nAChR subtypes present in the examined adult DRG neurones. The Pf of 7*-nAChRs was measured in neonatal DRG neurones.
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Before our study, the Pf value of native nAChRs expressed by neurones was measured by Rogers & Dani, 1995; Rogers et al. 1997), for -bungarotoxin-insensitive nAChRs in adult rat superior cervical ganglion cells. These authors found a value of 4.7 ± 0.3% using a less negative holding potential and a higher extracellular Ca2+ concentration, compared to our conditions (–50 mV versus–70 mV and 2.5 mMversus 2.0 mM, respectively). These different experimental settings reduce the difference between Pf values. However, it is likely that the populations of heteromeric nAChRs present in DRG and superior cervical ganglion neurones are not homogeneous.
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Functional consequences of nAChR activation in DRG neurones
Many studies report the ability of nicotinic agonists to modulate the peripheral algesic reactivity, but the actual physiological role of nAChR expression in primary sensory neurones is still under question. It is likely that DRG nAChRs contribute to the chemoreceptor complexity required to detect a broad spectrum of algesic molecules, the nicotinic stimulation activating nociceptors in vivo and in vitro (Jancso, 1961; Sucher et al. 1990; Liu et al. 1993; Steen & Reeh, 1993; Bernardini et al. 2001; Genzen & McGehee, 2003), and several cell types from DRG-innervated tissues producing ACh (Wessler et al. 1999). Another functional role probably played by DRG nAChRs is the modulation of the neurotransmitter release from the central endings of DRG neurones, since spinal cord interneurones are proved to produce ACh and to presynaptically contact the terminals of DRG afferents in the dorsal horn (Ribeiro-da-Silva & Cuello, 1990).
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Our findings reported here suggest that DRG nAChRs may play an additional role in sensory modulation. It is well known that the activation of cholinergic nicotinic pathways elicits anti-nociceptive effects (for reviews see: Flores, 2000; Jain, 2004). Although several CNS regions express nAChRs capable of producing anti-nociception (Christensen & Smith, 1990; Iwamoto, 1991; Iwamoto & Marion, 1993; Damaj et al. 1998), part of the nAChR-mediated analgesia is believed to be due to a peripheral action (Caggiula et al. 1995; Rueter et al. 2003). We show that acute stimulation of DRG nAChRs produces Ca2+ entry and a subsequent modification of the capsaicin-induced TRPV1-mediated currents, with faster desensitization and smaller amplitude. These effects are probably due to Ca2+ entry through nAChRs, being absent in Ca2+-free medium and not explained by a direct interaction with TRPV1 receptor–channels such as that exerted by nicotine or other nicotinic agonists on vanilloid receptors in trigeminal ganglia neurones (Liu et al. 2004). Interestingly, we report a reduced Ca2+ response to capsaicin after a prolonged incubation with doses of nicotine as low as those detected in smokers' extracellular fluids (Dani & Heinemann, 1996), likely to produce a long-lasting stimulation of extrasynaptic nAChRs.
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Capsaicin-activated TRPV1-mediated currents recorded in DRG neurones exhibit both acute desensitization (the inactivation of the current during a prolonged application of capsaicin) and tachyphylaxis (the diminution of the maximal current amplitude during repetitive deliveries of the same capsaicin concentrations). Both phenomena have been demonstrated to be Ca2+ dependent (Cholewinski et al. 1993; Caterina et al. 1997; Koplas et al. 1997) and we have shown here to be caused by nicotine. Considered together, our data indicate that the activation of DRG nAChRs can decrease the sensitivity of TRPV1 channels to subsequent sensory stimuli. Such a negative nicotinic modulation of TRPV1, if proved in vivo, could be physiologically important, representing a protective mechanism against the hyperexcitation and Ca2+ overloading of DRG cells. However, it is likely that the activation of DRG nAChRs modulates the sensory signalling differently depending on (i) the expressed nAChR subtypes, (ii) their localization, and (iii) the timing of activation compared to other sensory stimuli. Thus, further studies should be done to address these questions.
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