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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2012 Feb;165(4):908–921. doi: 10.1111/j.1476-5381.2011.01596.x

Pharmacological characterization of native α7 nicotinic ACh receptors and their contribution to depolarization-elicited exocytosis in human chromaffin cells

Alberto Pérez-Alvarez 1, Alicia Hernández-Vivanco 1, Sergio Alonso y Gregorio 2, Angel Tabernero 2, J Michael McIntosh 3, Almudena Albillos 1
PMCID: PMC3312488  PMID: 21790533

Abstract

BACKGROUND AND PURPOSE

Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells.

EXPERIMENTAL APPROACH

To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors.

KEY RESULTS

The nicotinic current provoked by 300 µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1 µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10 nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10 mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4 ms rise time) and inactivated component (8.5 ± 0.4 ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1 mM) and PNU-120596 (10 µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists.

CONCLUSIONS AND IMPLICATIONS

Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.

Keywords: nicotinic receptor, α7, patch clamp, capacitance, human chromaffin cell

Introduction

We previously showed that nicotinic acetylcholine receptors (nAChRs; nomenclature follows Alexander et al., 2011) in human chromaffin cells control several stages of the stimulus-secretion coupling process that occurs when these cells are activated with short or long pulses of ACh (Pérez-Alvarez and Albillos, 2007). Also, human chromaffin cells mainly exhibit an ‘adrenergic phenotype’ (Pérez-Alvarez et al., 2008), i.e., they release more adrenaline than noradrenaline, confirming previous data (Takiyyuddin et al., 1994). Thus, nAChRs play an essential role in the fast and more sustained catecholamine secretion that takes place under stress situations in humans.

In relation to the fast response elicited by short pulses of ACh, nAChRs in human chromaffin cells play a prominent role in exocytosis according to the following evidence from our laboratory (Pérez-Alvarez and Albillos, 2007): (i) 200 ms pulses of ACh elicited a nicotinic current, fully abolished by mecamylamine, that contributed to overall exocytosis at hyperpolarized membrane potentials; this contribution was as large as that of voltage-dependent Ca2+ channels (VDCC) at depolarized potentials; and (ii) this ACh-evoked nicotinic current triggered plasma membrane depolarization, recruiting VDCC and eliciting exocytosis.

From a methodological point of view, the ACh secretory response elicited by Ca2+ entry through the nicotinic receptor ionophore could be quantified by capacitance measurements at high resolution using the voltage clamp configuration of the ‘patch-clamp’ technique, which allows the recording of secretory vesicle fusion with the plasma membrane (Neher and Marty, 1982). However, the need to fix the voltage to perform capacitance recordings precludes the recruitment of Na+ and Ca2+ channels by ACh to evoke action potentials, the subsequent depolarization, activation of VDCC and exocytosis. In order to overcome this limitation, we designed a ‘triple-step protocol’ (Pérez-Alvarez and Albillos, 2007). This method allows the measurement of ACh-elicited exocytosis by recording plasma membrane capacitance (Cm) in the voltage clamp configuration of the patch-clamp technique before (step 1) and after (step 3) the depolarization elicited by the agonist takes place, which must be recorded in the current clamp configuration (step 2).

In this context, we sought to address the subtypes of nAChRs involved in the exocytotic process triggered by short ACh pulses in human chromaffin cells, with special attention paid to the α7 receptor, which is crucial in mediating rapid transmission (Zhang et al., 1996; Ullian et al., 1997; Alkondon et al., 1998; Frazier et al., 1998), especially in periods of high-frequency stimulation (Chang and Berg, 1999; Bibevski et al., 2000). This receptor generates a quickly inactivated current that is readily blocked by α-bungarotoxin (α-Bgtx) or methyllycaconitine (MLA) (Alkondon et al., 1992; Bertrand et al., 1992; Zorumski et al., 1992). α7 receptor currents raise intracellular Ca2+ (Vijayaraghavan et al., 1992; Zhang et al., 1994) due to the high Ca2+ permeability of α7 nAChRs (Bertrand et al., 1993; Séguéla et al., 1993; Delbono et al., 1997), depolarize the plasma membrane when located postsynaptically (Zhang et al., 1996; Frazier et al., 1998; Chang and Berg, 1999) and presynaptically modulate neurotransmitter release (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998; Li et al., 1998).

In chromaffin cells from other species where nAChRs have been investigated, there is controversy over the nAChR subunit sensitivity to α-Bgtx, the homomeric or heteromeric distribution of α7 nAChRs and their functionality. In bovine chromaffin cells, expression of α7 nAChRs has been determined by specific α-Bgtx binding (Wilson and Kirshner, 1977; Quik et al., 1987), cloning (García-Guzmán et al., 1995) and antibody detection (El-Hajj et al., 2007). However, in these cells nAChRs have also been found to be insensitive to α-Bgtx (Free et al., 2002) as is the catecholamine release process (Kumakura et al., 1980; Trifaró and Lee, 1980; Kilpatrick et al., 1981; Tachikawa et al., 2001). These findings contrast with those of other researchers who report partial or full α-Bgtx blockade of currents and secretion in bovine chromaffin or PC12 cells (Blumenthal et al., 1997; López et al., 1998; Fuentealba et al., 2004). A recent study by Del Barrio et al. (2011) shows that minimal cytosolic Ca2+ concentrations transients, induced by low concentrations of the selective α7 nAChR agonist PNU-282987 and their corresponding elicited exocytosis, were markedly increased by PNU-120596, and these responses were blocked by α-Bgtx. Evidence for the presence of α7 nAChRs has been also provided in human (Mousavi et al., 2001) and rat (Rust et al., 1994; Mousavi et al., 2001; Colomer et al., 2010) chromaffin cells through α7 mRNA detection, and in PC12 cells by affinity purification and immunoblotting of α-Bgtx receptors (Drisdel and Green, 2000).

The present study was designed to explore the presence and function of α7 nAChRs in primary cultures of human chromaffin cells. Here, we show pure α7 nAChR currents, recorded for the first time in chromaffin cells. Our data indicate that α7 nAChRs are functionally expressed and contribute to exocytosis of secretory vesicles in human chromaffin cells.

Methods

Cell cultures

The study protocol was approved by the Ethics Committees of the Hospital Ramón y Cajal, Hospital Universitario La Paz and Universidad Autónoma de Madrid. After obtaining informed consent from the donors' relatives, adrenal glands were collected from nine adult organ donors (both sexes; age range: 16–78 years) who had died of cerebral haemorrhage. After confirmation of brain death, adequate organ perfusion was ensured and adrenal glands were obtained during surgical procedures to remove kidneys for transplantation. After removal, glands were maintained at 4°C in sterile Locke's solution and taken to the laboratory within 4 hours. The inherent difficulties in obtaining human adrenal glands limited the number of experiments that could be performed. The method used for isolation and culture of human chromaffin cells has been previously described (Pérez-Alvarez and Albillos, 2007). Experiments were started 48 h after plating to allow recovery of the nicotinic receptor after collagenase treatment (Almazán et al., 1984).

Electrophysiological recordings

Perforated patch recordings were made in the whole-cell configuration of the patch-clamp technique. The external solution used to record nicotinic currents was (in mM): 2 CaCl2, 145 NaCl, 5.5 KCl, 1 MgCl2, 10 HEPES and 10 glucose; the pH was adjusted to 7.4 with NaOH. Intracellular solution composition was (in mM): 145 K-glutamate, 8 NaCl, 1 MgCl2, 10 HEPES and 0.5 amphotericin B (Sigma-Aldrich, Madrid, Spain); the pH was adjusted to 7.2 with KOH.

An amphotericin B stock solution was prepared daily at a concentration of 50 mg/mL dimethyl sulphoxide (DMSO) and kept protected from light. The final concentration of amphotericin B was prepared by ultrasonicating 10 µL of stock amphotericin B in 1 mL of internal solution in the dark. Pipettes were tip-dipped in amphotericin-free solution for several seconds and back-filled with freshly mixed intracellular amphotericin solution.

The perfusion system for drug application consisted of a multibarrelled polyethylene pipette positioned close to the cell under study. The exchange time of solutions of this system calculated with open-tip experiments (Zhang et al., 1994) was 10 ms. The agonist was always delivered from the same tube. Antagonists or modulators were perfused between pulses (5 min), and this flow was only interrupted during agonist perfusion (200 ms). The level of the bath fluid was continuously controlled by a custom-designed fibre optics system coupled to a pump used to aspirate excess fluid.

Pipettes of 2–3 MΩ resistance were pulled from borosilicate glass capillary tubes, partially coated with wax and fire polished. After seal formation and perforation, only recordings in which the access resistance of the pipette and the leak current were lower than 20 MΩ and 20 pA, respectively, were accepted. The holding potential (Vh) was −80 mV in all cases except in Figure 5 (Vh = −60 mV). Series resistance was 80% compensated. A four-pole Bessel filter set to 2.9 kHz was used. Currents were sampled at 12 kHz.

Figure 5.

Figure 5

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Characterization of α7 currents and the corresponding exocytosis evoked by the α7 nAChR agonist PNU-282987. Original recordings of currents and membrane capacitances elicited by the selective α7 agonist PNU-282987 (3 µM) and choline in the same cell (A). MLA abolished the α7 component activated by both agonists. PNU-282987 at 30 µM elicited an α7 current of similar magnitude to 3 µM and no exocytosis (B). In (C), summary results of the current and charge elicited by pulses of PNU-282987 (3 µM and 30 µM), normalized to the choline response in the same cell. ***P < 0.001, significantly different from choline alone; paired Student's t-test (n = 6).

Simultaneous electrophysiological current and plasma Cm measurements (as an index of exocytosis) were undertaken using an EPC-10 amplifier and PULSE software (HEKA Elektronik, Lambrecht, Germany) running on a PC computer. Increments in Cm accurately measure the fusion of vesicles to the plasma membrane that takes place during the exocytotic event (Neher and Marty, 1982; Albillos et al., 1997; Neher, 1998; 2006) and have been previously employed to record secretory responses induced by nicotinic agonists (Mollard et al., 1995; Pérez-Alvarez and Albillos, 2007).

Capacitance changes were estimated by the Lindau–Neher (Lindau and Neher, 1988; Gillis, 1995) technique implemented in the ‘Sine + DC’ feature of the ‘PULSE’ lock-in software. A 1 kHz, 70 mV peak-to-peak amplitude sinewave was applied at the holding potential. Plasma Cm was sampled at 1 kHz. The ‘triple-step’ protocol employed in this study is a slight modification of the method reported previously (Pérez-Alvarez and Albillos, 2007) to measure agonist-induced exocytosis by means of Cm changes. It combines the voltage clamp and the current clamp configurations of the patch-clamp technique. Initial plasma Cm is recorded in the voltage clamp configuration (step 1), then by switching to the current clamp configuration, the agonist is able to depolarize the plasma membrane (step 2), and finally by returning to the voltage clamp mode, the increase produced in plasma Cm can be recorded, reflecting the overall exocytosis evoked by the agonist (step 3). For analysis of Cm changes, the maximum Cm value in step 3 was taken after at least 100 ms from the start of the step to avoid capacitance artefacts (Horrigan and Bookman, 1994) and compared with the basal value before stimulation.

Analysis of data was conducted on a PC using IGOR Pro software (Wavemetrics, Lake Oswego, OR, USA). Changes in seal conductance (Gs) after the application of agonists or α7 nAChR potentiators, and in membrane conductance (Gm) after the application of ACh were observed. Given that large conductance changes affect capacitance measurements, these were always determined after the Gs and Gm changes had ceased (Pérez-Alvarez and Albillos, 2007). The fast component charge of the choline-evoked current was calculated by determining the integral from the baseline of the recording until 10 ms after the peak current.

Data analysis

Unless otherwise stated, data are given as the mean ± SEM. Paired or unpaired Student's t-tests were used to compare data.

Materials

PNU-120596 was purchased from TOCRIS Bioscience (Bristol, UK). ACh, choline, PNU-282987, 5-HI, α-Bgtx and MLA were from Sigma (Madrid, Spain). All drugs were dissolved in water, except PNU-120596, PNU-282987 and 5-hydroxyindole (5-HI) that were dissolved in DMSO. The concentrations of DMSO used to dissolve these drugs were 0.03% for 5-HI, 0.1% for PNU-120596, 0.01% for 3 µM PNU-282987 and 0.1% for 30 µM PNU-282987. Stock aliquots were stored at −20°C. Choline was always used at 10 mM, and ACh at 300 µM.

Results

α7 nAChR antagonists partially block currents elicited by ACh but not exocytosis

Using the voltage clamp mode in the perforated patch configuration of the patch-clamp technique, the presence of functional α7 nAChRs in the plasma membrane was investigated using ACh as stimulus (300 µM, 200 ms pulses applied every 2 min). We chose this concentration of ACh because it was sufficient to evoke substantial currents, without the large rebound produced using 1 mM ACh (Pérez-Alvarez and Albillos, 2007). In addition, 300 µM is the concentration considered to be attained by ACh at the neuromuscular junction (Kuffler and Yoshikami, 1975; Corringer et al., 2000; Changeux and Edelstein, 2005). After obtaining stable responses, α-Bgtx (1 µM) or MLA (1 nM or 10 nM) were perfused to rapidly and selectively block α7 nAChRs. The nicotinic peak currents elicited by ACh (2.6 ± 0.3 nA, n = 10) were blocked by 6.0 ± 1.7% using α-Bgtx (n = 5) and by 7.1 ± 1.6% using 10 nM MLA (n = 5). Blockade was irreversible after wash out of α-Bgtx (Figure 1A and B) but not MLA (Figure 1D and E). Exocytosis, measured as a plasma Cm increase, was unaffected by the antagonists (n = 5) (Figure 1C and F).

Figure 1.

Figure 1

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α-Bgtx and MLA blockade of ACh-evoked currents and exocytosis. 200 ms pulses of 300 µM ACh were applied every 2 min. Time course of blockade exerted by α-Bgtx 1 µM (A) or MLA 1–10 nM (D) on the ACh-elicited currents; perfusion of blocker was followed by washout (w/o\) to test reversibility. Bar diagrams represent means ± SEM and show the extent and reversibility of blockade by α-Bgtx (n = 5) (B) or MLA (n = 5) (E). Exocytotic responses as measured by Cm were unaffected by α-Bgtx (C) or MLA (F). Normalized data were obtained as the ratio of the agonist response in the presence of the antagonist, with respect to control conditions. *P < 0.05, significantly different from control; paired Student's t-test.

α7 nAChR-positive allosteric modulators (PAMs) potentiate ACh-elicited current and exocytosis

As the magnitude of the α7 current was small, an additional strategy was used to confirm the presence of α7 nAChRs. PAMs such as PNU-120596 (Hurst et al., 2005; Gronlien et al., 2007) or 5-hydroxyindole (5-HI) (Zwart et al., 2002) can increase the current through homomeric α7 nAChRs. We tested these compounds in human chromaffin cells to assess their effects on the nicotinic currents elicited by ACh.

Perfusion with PNU-120596 (10 µM) potentiated the nicotinic current charge (1.7 ± 0.2 nC to 3.4 ± 0.9 nC) and the corresponding ACh-elicited exocytosis (116.2 ± 69 fF to 414 ± 233 fF) (n = 4) but did not increase the peak current (Fig 2). This effect was reversible after wash out (Figure 2A and B), and fully and irreversibly abolished after α-Bgtx perfusion in the presence of PNU-120596 (Supporting Information Figures S1A and B).

Figure 2.

Figure 2

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Effect of α7 nAChR PAMs on ACh-evoked currents and exocytosis. 200 ms pulses of 300 µM ACh were applied every 5 min. Original recordings of currents (A) and Cm (B) evoked by ACh in the presence or absence of PNU-120596 10 µM. Perfusion and washout (w/o) of 5-HI (1 mM) (C). Bar diagram comparing the effects of PNU-120596 (n = 4) and 5-HI (n = 5) on the ACh current, charge and exocytosis (D). Normalized data were obtained as the ratio of the agonist response in the presence of the modulator, with respect to control conditions. *P < 0.05, **P < 0.01, significantly different from control; paired Student's t-test.

5-HI, at a concentration of 1 mM, blocked the peak current and the current charge elicited by ACh by 37 ± 6% and 64 ± 3% (Figure 2C) (n = 5), without significantly affecting exocytosis. This could be explained by the blockade exerted on non-α7 nAChRs by 5-HI, documented by other authors (Zwart et al., 2002). A summary of the effects of these compounds on the peak current, charge and exocytosis is provided in Figure 2D.

The isolated α7 current component of the ACh response does not evoke exocytosis

To dissect the α7 current component of the ACh response, we assessed the effect of α-conotoxin (α-Ctx) MII[S4A,E11A,L15A], which has been shown to primarily target α6* nAChRs (Azam et al., 2008), the main nAChR subtype expressed in human chromaffin cells (Albillos et al., 2011). Perfusion with the toxin at 500 nM concentration showed a clear blockade of the peak current and exocytotic responses evoked by ACh (Figure 3A). The percentage of peak current remaining (∼5%, n = 5) was consistent with the α7 contribution to the total current. Nevertheless, the slow kinetics of current inactivation and the exocytotic response observed in the presence of the toxin argued against a total blockade of non-α7 nAChRs. Application of higher concentrations (1–2 µM) unveiled a fast nicotinic current that was irreversibly blocked upon perfusion with α-Bgtx 1 µM (n = 5) (Figure 3B). The overall non-α7-current blockade was also reflected in the total abolition of the secretory response.

Figure 3.

Figure 3

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Characterization of the α7 current component and the corresponding exocytosis elicited by ACh. Panel A (left) shows the time course of blockade exerted by α-Ctx MII[S4A,E11A,L15A] (500 nM) on ACh-induced currents. The insets show original recordings of ACh before and after perfusion with the toxin. On the right, the original traces of the exocytotic responses elicited by ACh before and after perfusion with α-Ctx MII[S4A,E11A,L15A] are displayed. Panel B shows the time course of blockade exerted by α-Ctx MII[S4A,E11A,L15A] at a higher concentration (2 µM) on ACh-induced currents. α-Bgtx perfusion exerted an additional and irreversible blockade on the α-Ctx MII[S4A,E11A,L15A]-resistant current (n = 5). The insets show original recordings of ACh before and after perfusion with the toxins. On the right, the original traces of the exocytotic responses elicited by ACh before and after perfusion with α-Ctx MII[S4A,E11A,L15A] are shown.

Choline and PNU-282987 evoke α7 nicotinic currents that do not trigger exocytosis in human chromaffin cells

We used choline (10 mM), an α7 nAChR agonist (Papke et al., 1996; Alkondon et al., 1997; 1999; Frazier et al., 1998), to characterize the α7 current and to assess the effects of selective α7 nAChR antagonists or PAMs. Choline binds with millimolar affinity to α7 nAChRs (Papke et al., 1996; Alkondon et al., 1997). It was used at 10 mM (EC100 on α7 nAChRs, Alkondon et al., 1997; Fuentealba et al., 2004) to ensure activation of α7 nAChRs.

Cell stimulation with choline 10 mM (200 ms) evoked a biphasic current with two clearly defined components. The first component was rapidly activated and inactivated. The second current component exhibited a slow activated stage with no inactivation (Figure 4A). The peak current amounted to 130 ± 16 pA (n = 31). However, the Ca2+ entry elicited by choline could not trigger exocytosis of secretory vesicles (Figure 4A, inset). In response to perfusion with the α7 receptor antagonist α-Bgtx, only the quickly activated and inactivated component was blocked (98 ± 1%, n = 6), and this component did not recover after toxin washout (Figure 4B). When tested in different cells, the α7 receptor antagonist MLA also blocked the initial current component elicited by choline by 97 ± 2% (n = 8). MLA washout resulted in recovery of the fast component of the nicotinic current (Figure 4C). A summary of the effects of α7 receptor antagonist blockade and washout is provided in Figure 4D.

Figure 4.

Figure 4

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Characterization of α7 currents and the corresponding exocytosis evoked by the α7 nAChR agonist choline. Choline pulses (10 mM, bar represents 200 ms of drug application) elicit a biphasic current response (A) with a fast (α7) and slow component (non-α7). There is no sizeable exocytotic response to this stimulus (see inset for the Cm recording). Original recordings showing selective blockade of the α7 component by α-Bgtx 1 µM (B) or MLA 10 nM (C). In (D), summary results of the block and washout(w/o) of the α7 component, exerted by α-Bgtx (n = 6) and MLA (n = 8). Normalized data were obtained as the ratio of the agonist response in the presence of the antagonist, with respect to control conditions. ***P < 0.001, significantly different from control; paired Student's t-test. In (E), original recordings of the 10 nM MLA blockade and wash out (w/o) of the α7 currents elicited by choline after perfusion of 500 nM α-Ctx MII[S4A,E11A,L15A].

Choline is a partial non-α7 agonist (Papke et al., 1996; Fuentealba et al., 2004). The α7 component of the choline response could be isolated by applying α-Ctx MII[S4A, E11A, L15A] at a concentration of 500 nM and this component was fully and reversibly blocked with 10 nM MLA (n = 6) (Figure 4E). These data show that the initial component of the biphasic current elicited by choline corresponds to activation of α7 nAChRs.

In addition, we evaluated the effect of PNU-282987, a synthetic selective α7 nAChR agonist. This compound evoked a peak current of 42 ± 9.8 pA at 3 µM and of 57.7 ± 9.2 pA at 30 µM (n = 6). Choline was applied to the same cells as PNU-282987 to compare their responses (Figure 5A and B). The PNU-282987 peak current amounted to 37 ± 5% (at 3 µM) and 42 ± 6% (at 30 µM) of the peak current response shown by the choline α7 component (Figure 5C). MLA (10 nM) fully abolished the nicotinic current elicited by PNU-282987 as well as the initial peak current evoked by choline in the same cells, confirming the α7 nature of the PNU-282987 response (Figure 5A).

The α7 current kinetics elicited by choline and PNU-282987 were analysed in order to be compared with previous data reported in other cell systems (Supporting Information Figure S2). In the case of choline, the time measured for 10–90% activation (t10–90) was 5.5 ± 0.4 ms. The inactivation phase could be fitted to a single exponential curve, giving a time constant of inactivation (τinactiv) of 8.5 ± 0.4 ms (n = 31). The kinetic study of the currents yielded by PNU-282987 indicated quickly activated and inactivated stages for both concentrations, with t10–90 = 7.1 ± 0.4 ms and τinactiv = 10.0 ± 0.9 ms for 3 µM, and t10–90 = 5.5 ± 0.4 ms and τinactiv = 9.8 ± 1.8 ms for 30 µM (n = 6).

α7 nAChR PAMs potentiate choline- and PNU-282987- elicited responses

When the agonist used was choline, 5-HI (1 mM) evoked a large increase in the initial peak current (α7 component as previously described). The potentiation achieved by 5-HI was 3.5-fold in the cell shown in Figure 6A. On average, the peak current elicited by choline was augmented (132 ± 43 pA to 426 ± 139 pA) (n = 5) (Figure 6B). This increase was abolished, together with the initial peak current elicited by choline, after perfusion with MLA (10 nM) and was fully restored after washout. Potentiation of α7 peak current by 5-HI, however, did not cause overall current charge (α7 and non-α7) potentiation. This may be explained by the blocking effects of 5-HI on the non-α7 component of the choline-elicited current (Figure 6A and B).

Figure 6.

Figure 6

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Potentiation of the α7 nAChR current and exocytosis by PAMs. Original recordings of potentiation and blockade of choline currents by 5-HI 1 mM and 5-HI+MLA (A). In (B), summary results of the effects on choline responses exerted by 5-HI (n = 5) or 5-HI+MLA. Normalized data were obtained as the ratio of the agonist response in the presence of the modulator, with respect to control conditions. #P < 0.05, **P < 0.005, ***P < 0.001, significantly different from control; paired Student's t-test.The potentiation by 10 µM PNU-120596 of choline-elicited currents is shown in panel C along with washout (w/o). The corresponding exocytotic response was also selectively potentiated (D). In (E), summary results of the effect on choline responses exerted by PNU-120596 (n = 6) or PNU-120596 plus α−Bgtx. Normalized data were obtained as the ratio of the agonist response in the presence of the modulator, with respect to control conditions. #P < 0.05, *P < 0.01, ***P < 0.001, significantly different from control; paired Student's t-test. Currents (F) and exocytosis (G) elicited by 3 µM PNU-282987 were also strongly potentiated by PNU-120596. In (H), summary results of the effect of PNU-120596 on the 3 µM PNU-282987 responses. Normalized data were obtained as the ratio of the agonist response in the presence of the antagonist or modulator respect to control conditions. #P < 0.05, significantly different from control (PNU-282987 alone); paired Student's t-test. (n = 5).

PNU-120596 caused a marked potentiation of the choline response, which was reversible after washout. The choline-evoked peak current was increased to 1.5 ± 0.4 nA and the current charge to 5.7 ± 2.2 nC (n = 6) (Figure 6C–E). Exocytosis was also largely potentiated although a numerical value for the potentiation from a null value could not be calculated. Current, charge and exocytosis increments were irreversibly abolished after application of α-Bgtx (1 µM) (Supporting Information Figure S3).

When the agonist used was PNU-282987 (3 µM), currents and exocytosis were also increased by PNU-120596. Peak currents were increased to 2.4 ± 0.2 nA and the current charge to 12.5 ± 2.9 nC (n = 5) (Figure 6F–H).

The kinetics of the fast current component were preserved in the 5-HI-potentiated choline-elicited currents (t10–90 = 7.2 ± 0.7 ms, τinactiv = 10.1 ± 0.6 ms), in good agreement with the kinetic properties of type I PAMs, which do not influence inactivation (Gronlien et al., 2007). However, in the presence of PNU-120596, the current elicited by choline showed significantly slower kinetics (t10–90 = 776.2 ± 33.3 ms, τinactiv = 2.32 ± 0.2 s) consistent with the actions of a type II PAM (Gronlien et al., 2007). When the agonist was PNU-282987, PNU-120596 potentiated current kinetics were also significantly slower as compared with controls (t10–90 = 890.7 ± 113.8 ms, τinactiv = 4.8 ± 0.6 s) (n = 5) (Supporting Information Figure S4).

α7 activation triggers exocytosis by depolarizing the cell membrane

Although Ca2+ entry through the ionophore associated with the α7 nAChR yielded no secretory response, we next tried to determine whether this receptor could contribute to the exocytosis by means of plasma membrane depolarization and subsequent recruitment of Ca2+ channels, as reported for nAChRs in human chromaffin cells (Pérez-Alvarez and Albillos, 2007). This goal was achieved by investigating the exocytotic response elicited by the selective stimulation of the α7 nAChRs, and consequent evoked depolarization, by choline in the presence of 500 nM α-Ctx MII[S4A, E11A, L15A] or PNU-282987. Exocytosis elicited by agonist depolarization was measured using the ‘triple-step’ protocol. The exocytotic response elicited by 10 mM choline after perfusion with 500 nM α-Ctx MII[S4A, E11A, L15A] or 30 µM PNU-282987 was 26 ± 4 fF (n = 5) and 50 ± 13 fF (n = 7), respectively (Figure 7A and B), calculated as the difference between the Cm values of steps 3 and 1.

Figure 7.

Figure 7

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Contribution of α7 nAChRs to depolarization-elicited exocytosis revealed by their selective potentiation and activation. A modification of the ‘triple-step’ protocol described by Pérez-Alvarez and Albillos, (2007) was used to measure the exocytotic responses evoked by the selective activation of α7 nAChRs by choline plus α-Ctx-MII[S4A, E11A, L15A] 500 nM (A) or PNU-282987 30 µM (B), applied in the current clamp (CC) configuration to induce depolarization. In the first step, the basal Cm was recorded by applying a sine wave in the voltage clamp (VC) mode. Step 2 was performed under the current clamp configuration to allow the agonist to depolarize the plasma membrane and evoke exocytosis, which was measured in step 3 by recording Cm in the voltage clamp configuration. Vh was −60 mV, as a potential close to the resting membrane potential of human chromaffin cells.

Finally, PAMs are being developed to improve cognitive disorders (Hajós and Rogers, 2010). In this sense, it would be relevant to analyse the effect of these drugs on the exocytotic process that would lead to catecholamine release in human chromaffin cells. PNU-120596 (1 µM) was tested on the agonist depolarization (ACh or choline)-elicited exocytosis, which was measured using the ‘triple-step’ protocol. The depolarization elicited by 300 µM ACh generated a secretory response of 61.7 ± 7 fF (n = 4), which was potentiated by 1 µM PNU-120596. The depolarization duration also largely increased in these cells (Supporting Information Figure S5A). When 10 mM choline was used as agonist, secretion was 40 ± 13 fF (n = 5). In the cell shown in Supporting Information Figure S5B, the choline secretory response was potentiated by PNU-120596, from 15 fF to 914 fF, and the duration of the depolarization from 0.9 s to 28.8 s.

Discussion

Although the presence of mRNA for α7 nAChRs in human chromaffin cells has been previously reported (Mousavi et al., 2001), our results constitute the first electrophysiological recordings and characterization of functional α7 nAChRs in primary cultures of human chromaffin cells and provide evidence for their contribution to depolarization-elicited exocytosis in these cells. ACh-evoked currents attributable to α7 nAChRs have also been described in the peripheral nervous system (Zhang et al., 1994; Cuevas and Berg, 1998) and CNS (Zorumski et al., 1992; Alkondon and Albuquerque, 1993) of other mammals.

Choline behaves as an agonist of α7 nAChRs (Papke et al., 1996; Alkondon et al., 1997; 1999; Frazier et al., 1998), and its dual effect has been reported of potentiating or inhibiting α4β4 nAChRs (Zwart and Vijverberg, 2000), as well as activating α3β4 nAChRs expressed in Xenopus oocytes (Fuentealba et al., 2004). This could explain the biphasic current, showing an initial peak and a second plateau stage, elicited by this agonist in the present study. This initial peak current was triggered through activation of rapidly activated and inactivated α7 nAChRs, as it was blocked by the α7 nAChR antagonists MLA or α-Bgtx and was potentiated by the α7 nAChR PAMs 5-HI or PNU-120596. The α7 nAChR current could be isolated using the selective α6* receptor blocker α-Ctx MII[S4A, E11A,L15A] (Azam et al., 2008), and further abolished by MLA. Rapid inactivation of this initial α7 current peak in the presence of choline (8.5 ± 0.4 ms) was of the same order of magnitude as the ACh-elicited current reported in native systems, such as cultured postnatal rat hippocampal neurons (8 ± 2 ms) (Zorumski et al., 1992), or choline-elicited responses in tuberomammillary histamine neurons of the posterior hypothalamus (9.65 ± 1.1 ms) (Uteshev et al., 2002). The second stage of the choline-evoked current was produced by activation of non-α7 nAChRs, as indicated by the blockade of these nAChRs by α-Ctx MII[S4A, E11A,L15A].

α-Bgtx and MLA have also been shown to block α9* nAChRs (Elgoyhen et al., 1994; 2001; Verbitsky et al., 2000). However, the irreversible nature of the α-Bgtx blockade observed here, the fast activation and desensitization kinetics of the currents involved, and the selective action of the modulators on α7 nAChRs (Zwart et al., 2002; Hurst et al., 2005) make it unlikely that the nAChR subtypes sensitive to α-Bgtx and MLA reported here are of the α9* subtype.

In human adult chromaffin cells, mRNAs for α7 and β2 receptor subunits have been detected (Mousavi et al., 2001). Given the confirmation by Liu et al. (2009) of earlier data (Khiroug et al., 2002) supporting the presence of a functional α7β2 receptor in rat brain, it could be that such a receptor is expressed in human chromaffin cells. However, two lines of evidence refute this possibility: first, the α7 current observed in our study exhibits fast activation and inactivation kinetics, comparable to those shown by homomeric receptors (Zorumski et al., 1992) yet contrary to the slow current kinetics reported for α7β2 heteromeric nAChRs (Khiroug et al., 2002; Liu et al., 2009); and second, the lack of an effect of 3 µM dihydro-β-erythroidine (DHβE; data not shown) on human α7 nAChRs here reported, a concentration well above the IC50 of 0.17 µM described for the highly sensitive α7β2 receptor (Liu et al., 2009).

Evidence for functional α7 nAChRs has been reported in bovine chromaffin cells as the blockade by α7 nAChR antagonists of the current or secretory response elicited by long pulses of agonists (1–5 s) (López et al., 1998), or as the cytosolic Ca2+ transient or secretion induced by low concentrations of selective α7 nAChR agonists perfused for 10–30 s in the presence of PNU-120596 (Del Barrio et al., 2011). However, in this latter study, direct recording of the α7 nAChR Ca2+ signal using α7 nAChR agonists was not possible, probably because of the long stimuli and static system of drug application used. Indeed, it has been reported that rapid drug application is crucial to record fast activated and inactivated α7 nAChR currents (Vijayaraghavan et al., 1992; Zhang et al., 1994). Neither have functional α7 nAChRs been found in rat chromaffin cells (Di Angelantonio et al., 2003), most likely because 100 µM nicotine mainly activates non-α7 nAChRs as shown in bovine chromaffin cells (Del Barrio et al., 2011). In effect, the present results are the first description of the recording of isolated native α7 nAChR currents in chromaffin cells using short α7 nAChR agonist stimuli and a rapid agonist perfusion system. Thus, it seems that the fast activation and inactivation kinetics of α7 nAChR currents dictate a need for high resolution techniques and fast agonist perfusion systems to record these currents.

Interestingly, autoradiography of bovine adrenal gland slices with α-Bgtx indicates that α7 nAChRs are restricted to medullary areas adjacent to the adrenal cortex and co-localize with the enzyme PNMT, which methylates noradrenaline to adrenaline and thus confers the ability to release adrenaline on chromaffin cells (the ‘adrenergic phenotype’) (Criado et al., 1997). In addition, glucocorticoid-activated PNMT expression increases with stress (Wurtman and Axelrod, 1965; Kvetnansky et al., 2006; Tai et al., 2007). Accordingly, because human chromaffin cells mainly display an adrenergic phenotype (99% of cells) (Pérez-Alvarez et al., 2008), it is plausible that α7 nAChR expression also increases with stress to produce the faster neurotransmitter response necessary. However, in a recent study by Colomer et al., (2010) in rat chromaffin cells, no changes in the expression levels of α7 nAChR transcripts were observed.

Here we also show that α7 nAChRs contribute to exocytosis when plasma membrane depolarization is evoked by specific α7 nAChR agonists (i.e. choline after perfusion with α-Ctx MII[S4A, E11A, L15A] or PNU-282987). In bovine chromaffin cells, α7 nAChRs were shown to contribute to exocytosis using α7 nAChR antagonists to block ACh-evoked catecholamine release from a population of cells (López et al., 1998). Also, the α7 agonist PNU-282987, in the presence of the allosteric potentiator PNU-120596, stimulated catecholamine release in a population of bovine cells (Del Barrio et al., 2011). In the present study, through the use of a high-resolution patch-clamp technique and brief exposure to agonists, we were able to record the exocytotic responses produced to α7 nAChR agonists. In other cell systems, α7 nAChRs have been found to presynaptically modulate (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998; Li et al., 1998) or influence (Coggan et al., 1997) neurotransmitter release. Postsynaptically, they are known to generate depolarizing currents (Frazier et al., 1998) but no evidence of a role in exocytosis has been reported.

There is also mounting evidence for a role of choline as a modulator of synaptic cholinergic neurotransmission at the splachnic nerve chromaffin cell synapse (Holz and Senter, 1981) acting through α7 nAChRs (Fuentealba et al., 2004). However, the incomplete blockade exerted by 100 nM of α-Bgtx and MLA on the choline-elicited secretory response has cast doubts on the nature of the nAChR subtype involved in such choline effects (Fuentealba et al., 2004). Here we show that choline activated α7 nAChRs and, thus, could have a functional role in regulating cholinergic synaptic activity by acting on these receptors.

Finally, our data could also have therapeutic implications. α7 nAChRs are considered pharmacological targets to treat several diseases, and α7 nAChR PAMs are being investigated as candidates for improving cognitive deficits and neurodegeneration (Hurst et al., 2005; Dunlop et al., 2009; Dinklo et al., 2011). Our data show that exocytosis is greatly enhanced in the presence of PNU-120596, suggesting that treatment with PAMs might have some effect on peripheral catecholamine secretion via actions on chromaffin cells. This effect would need to be considered as these medications are developed. In addition, our study validates the use of human chromaffin cells as a model to investigate the functional roles of α7 nAChRs.

Acknowledgments

The authors thank the donors of the human adrenal glands and their relatives. We also thank Dr Agustín Albillos and the transplant teams of the Hospital Ramón y Cajal and Hospital Universitario La Paz for their excellent coordination in supplying the human adrenal glands. AHV holds a fellowship award from the Universidad Autónoma de Madrid. This work was supported by a grant from the Ministerio de Ciencia y Tecnología No. BFU2008-01382/BFI awarded to AA, and NIH Grants MH53631 and GM48677 to JMM.

Glossary

5-HI

5-hydroxyindole

α-Bgtx

α-bungarotoxin

α-Ctx

MII[S4A, E11A, L15A], α-conotoxin MII[S4A, E11A, L15A]

Cm

membrane capacitance

DHβE

dihydro-β-erythroidine

Gm

membrane conductance

Gs

seal conductance

MLA

methyllycaconitine

nAChR

nicotinic acetylcholine receptor

PAM

positive allosteric modulator

VDCC

voltage-dependent Ca2+ channels

Conflicts of interest

None declared.

Supporting Information

Figure S1 Blockade of PNU-120596 potentiationof ACh-elicited currents and exocytosis by α-Bgtx 1 μM areshown in panels A and B, respectively.

bph0165-0908-SD1.tif (140.1KB, tif)

Figure S2 Kinetics of α7 currents.Current activation and inactivation kinetics elicited by choline orPNU-282987 3 and 30 μM were analysed and compared. Activationwas determined as the time between 10 and 90% activation(t10–90), while inactivation was fitted to a singleexponential curve (dashed line is the fitted curve).

bph0165-0908-SD2.tif (172.2KB, tif)

Figure S3 Blockade of PNU-120596 potentiationof choline-elicited currents and exocitosis by α-Bgtx 1μM.

bph0165-0908-SD3.tif (201.8KB, tif)

Figure S4 Kinetics of potentiated α7currents. The current activation and inactivation kinetics elicitedby choline 10 mM in the presence of 5-HI 1 mM or PNU-120596 10μM, or PNU-282987 plus PNU-120596, were analysed and compared.Activation was determined as the time between 10 and 90% activation(t10–90), while inactivation was fitted to a singleexponential curve (dashed line is the fitted curve).

bph0165-0908-SD4.tif (176.4KB, tif)

Figure S5 Contribution of α7 nAChRs todepolarization-elicited secretion revealed by their selectivepotentiation. Perfusion of PNU-120596 1 μM increases thedepolarizing and secretory responses following nAChR activation byACh (a) or choline (b).

bph0165-0908-SD5.tif (148.2KB, tif)

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