Unmasking the functions of the chromaffin cell α₇ nicotinic receptor by using short pulses of acetylcholine and selective blockers

Abstract

Methyllycaconitine (MLA), α-conotoxin ImI, and α-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 μM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC₅₀s of 100 nM for MLA and 300 nM for α-conotoxin ImI and α-bungarotoxin. MLA (100 nM), α-conotoxin ImI (1 μM), and α-bungarotoxin (1 μM) halved the entry of ⁴⁵Ca²⁺ stimulated by 5-s pulses of 300 μM ACh applied to incubated cells. These supramaximal concentrations of α₇ nicotinic receptor blockers depressed by 30% (MLA), 25% (α-bungarotoxin), and 50% (α-conotoxin ImI) the inward current generated by 1-s pulses of 100 μM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain α₇ neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, α-conotoxin ImI, and α-bungarotoxin with IC₅₀s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through α₃β₄ nAChR was unaffected by α-conotoxin ImI and α-bungarotoxin, and weakly blocked by MLA (IC₅₀ = 1 μM). The functions of controlling the electrical activity, the entry of Ca²⁺, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to α₃β₄ nAChR; the present results constitute the first evidence to support a prominent role of α₇ nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

In 1977, Wilson and Kirshner (1) first reported the presence of α-bungarotoxin-binding sites in bovine adrenal medullary chromaffin cells. Eighteen years later, a bovine chromaffin cell α₇ subunit of the neuronal nicotinic receptor subtype for acetylcholine (nAChR) was cloned; injection in Xenopus oocytes of its cRNA produced α-bungarotoxin-binding sites and functional acetylcholine (ACh) currents that were blocked by α-bungarotoxin (2). α₃ and β₄ subunits of nAChR have been also cloned and characterized in bovine chromaffin cells (3). Thus, the question arises as to why different nAChR are expressed in bovine chromaffin cells and what their functions might be. Early attempts to block with α-bungarotoxin (a selective α₇ receptor blocker), the catecholamine secretory responses generated by various nicotinic receptor agonists produced negative results (1, 4–6) or provided some conflicting data (7). Thus, the actual view is that α₃β₄ receptors control the catecholamine secretory response in chromaffin cells and that α₇ receptors play no role in the regulation of such function. By using new protocols (short ACh pulses) and pharmacological tools, i.e., the novel selective α₇ nAChR blockers α-conotoxin ImI (8, 9) and methyllycaconitine (MLA) (10), we obtained evidence that α₇ receptors do have a role in the generation of inward nAChR currents (I_ACh), as well as in the triggering of Ca²⁺ entry and catecholamine release in response to ACh stimulation of bovine chromaffin cells. In addition, to strengthen the functional data obtained in chromaffin cells, we present here additional experiments that corroborate the high selectivity of α-conotoxin ImI and methyllycaconitine on the neuronal nAChR of the α₇-subtype and their poor blockade of α₃β₄-subtype of receptors expressed in Xenopus oocytes.

MATERIALS AND METHODS

Isolation of Bovine Chromaffin Cells.

Bovine chromaffin cells were isolated as described by Moro et al. (11). Cells were suspended in DMEM supplemented with 5% fetal calf serum, 10 μM cytosine arabinoside, 10 μM fluorodeoxyuridine, 50 units ml⁻¹ penicillin, and 50 μg ml⁻¹ streptomycin. For secretion experiments, 5 × 10⁶ cells were plated in 10-cm diameter Petri dishes, and for ⁴⁵Ca uptake experiments, 2 × 10⁵ cells per dish were plated in 96-well dishes. For patch-clamp experiments, cells were plated on 1-cm diameter glass coverslips at a density of 50 × 10³ cells ml⁻¹. Cells were kept in an incubator at 37°C with 5% CO₂ in a water-saturated atmosphere. Media were renewed after 24 h and every 2–3 days.

Measurement of Catecholamine Release from Bovine Chromaffin Cells.

Cells were collected by gentle rubbing, centrifuged, resuspended in 200 μl of Krebs-Hepes (composition in mM: NaCl, 144/KCl, 5.9/MgCl₂, 1.2/CaCl₂, 2/Hepes10/glucose, 11) pH 7.4 at room temperature (22 ± 2°C), and introduced in a microchamber with glass wool. Then, the cells were continuously superfused with Krebs-Hepes at a rate of 0.5 ml min⁻¹; the liquid flowing from the perfusion chamber reached an electrochemical detector model Metrohn 641VA under the amperometric mode, which monitored on-line the amount of catecholamines secreted (12). Catecholamine secretion was stimulated by perfusing for 5 s every 6 min, a Krebs-Hepes solution containing 100 μM ACh. Known concentrations of adrenaline were used as external standards. Secretion was quantified by measuring the peaks (and in some experiments also the areas) of the amperometric signals in nanoamperes.

Measurement of ⁴⁵Ca²⁺ Uptake.

Cells were washed once with Krebs-Hepes and then the toxins were incubated for 30 min at 37°C. Ca²⁺ uptake was induced by adding ⁴⁵Ca²⁺ (9 μCi ml⁻¹) dissolved in Krebs-Hepes containing 300 μM ACh for 10 s; basal uptake was performed in normal Krebs-Hepes. The reaction was stopped by adding once an ice-cold Krebs-Hepes solution containing 10 mM La³⁺ and OCa²⁺; excess extracellularly bound ⁴⁵Ca²⁺ was washed five times with a Ca²⁺-free Krebs-Hepes containing 10 mM LaCl₃ and 2 mM EGTA, at 15-s intervals. At the end, 100 μl of 10% trichloroacetic acid was added and the cells were scrapped off with a pipette tip and introduced in a vial; scintillation liquid was added and radioactivity was counted in a Packard scintillation counter Model L1500.

Electrophysiological Recordings of nAChR Currents in Chromaffin Cells.

Currents through the nAChR channels were recorded by using the whole-cell configuration of the patch-clamp technique (13). Briefly, nicotinic currents were elicited by fast application of 100 μM ACh through a multibarrel pipette controlled by miniature solenoid valves and placed within ≈100 μm of the cell. The flow rate (0.5–1 ml min⁻¹) was regulated by gravity to achieve a complete replacement of cell surroundings in 50–100 ms. External superfusion medium was (in mM): NaCl, 137; KCl, 5; MgCl₂, 1; CaCl₂, 2; glucose, 11; and Hepes-NaOH, 10; pH, 7.4. Soft glass patch-clamp electrodes were filled with (in mM): NaCl, 10; CsCl, 100; tetraethylammonium cloride, 20; MgATP, 5; EGTA, 14; GTP.Na, 0.3; and Hepes-KOH, 20; pH 7.2. The holding potential was −80 mV, and electrode resistance ranged from 2 to 5 MΩ.

Expression of α₃β₄ and α₇ nAChR in Oocytes and Measurements of nAChR Channel Currents.

The plasmids pPCA48E, pZPC13, and pHIP306 containing the entire coding regions of rat nAChR α₃, β₄, and α₇ genes were linearized by using the restriction enzymes EcoRI, XhoI, and SmaI, respectively. Capped mRNAs were synthesized in vitro from the corresponding plasmids by using SP6 (α₃), T3 (β₄), or T7 (α₇) RNA polymerases and a mCAP RNA-capping Kit (Stratagene). Preparation of oocytes and microinjection of RNAs were done according to Miledi et al. (14) with some modifications (15). Oocytes were injected with 50 nl (50 ng) of α₇ cRNA or 50 nl (25:25 ng) of a combination of α₃ and β₄ cRNAs by using a nanoject automatic injector (Drummond Scientific, Broomall, PA). Oocytes were defolliculated with collagenase (type I, 200 units mg⁻¹ at a final concentration of 0.5 mg ml⁻¹). Electrophysiological recordings were made 2–5 days after RNA injection.

Inward currents through nAChR were recorded at room temperature (22 ± 2°C) in Ringer’s solution containing (in mM): NaCl, 115; KCl, 2; CaCl₂, 1.8; and Hepes, 5; buffered to pH 7.4 with NaOH. To block endogenous muscarinic receptors, atropine (1 μM) was used in all the solutions employed for the experiments expressing the combination α₃β₄ subunits. Membrane currents were recorded with a two-electrode voltage clamp amplifier (OC-725-B Warner Instrument Corporation, Hamden, CT) by using microelectrodes with resistance of 0.5–5 MΩ made from borosilicate glass (GC100TF-15, Clark Electromedical Instruments, Pangbourne, UK) and filled with KCl (3 M). The holding potential in all experiments was −60 mV. Single oocytes were held in a chamber with a volume of 0.3 ml and constantly superfused with Ringer’s solution by gravity (4 ml min⁻¹). Toxins were superfused during variable periods of time (ranging from 3 to 20 min) until a maximal and stable effect was observed in some cases, or the absence of any effect was confirmed in others. Voltage protocols, ACh pulses and data acquisition were controlled by using a Digidata 1200 interface and clampex software (Axon Instruments, Foster City, CA). The IC₅₀ of the toxins on ACh-induced currents were estimated through nonlinear regression analysis, by graphpad Software (San Diego, CA) for a PC computer.

Materials and Solutions.

Acetylcholine was purchased from Sigma and ⁴⁵Ca²⁺ with a specific activity 10–40 mCi mg⁻¹, from Amersham. α-Conotoxin ImI was obtained as described by Myers et al. (16) and McIntosh et al. (8). α-Bungarotoxin was purchased from Research Biochemicals (Natick, MA) and Sigma; methyllycaconitine was also from Research Biochemicals. Toxins were dissolved in distilled water at the concentration of 10⁻³ M, aliquoted, and maintained at −20°C until use. Before each experiment, they were dissolved in the saline solutions used in each experiment and diluted to the desired final concentration.

Statistics.

Differences between means of group data fitting a normal distribution were assessed by using the Student’s t test. A P value equal or <0.05 was taken as the limit of significance.

RESULTS

Blockade of ACh-Induced Secretory Responses by Selective Antagonists of α₇ nAChR.

To study the effects of toxins on catecholamine release responses, chromaffin cells were continuously superfused with Krebs-Hepes solution, and after an initial equilibration period (≈10 min), they were stimulated at 6-min intervals with short ACh pulses (100 μM, 5 s). The initial secretory response (345 ± 39 nA in 19 different cell batches) declined by 30% after eight ACh pulses (control curve in Fig. 1B). A typical experimental protocol to study the effects of a given toxin on secretion appears in Fig. 1A, where original secretory spikes are shown; the toxin (one single concentration in each cell batch) was applied during 12 min (6 min before an ACh pulse and 6 min before the subsequent pulse). Fig. 1A shows an example of the blockade of secretion induced by 1 μM α-conotoxin ImI as well as the recovery of the ACh responses after the toxin washout. Fig. 1B shows averaged results for three selective α₇ nAChR blockers. α-Bungarotoxin blocked the response by 44% after 6 min and by 63% after 12 min; α-conotoxin ImI (1 μM) caused 70% inhibition at 6 and 12 min. Methyllycaconitine (0.1 μM) caused 70% inhibition of secretion in 6 min. The effects of α-conotoxin ImI and methyllycaconitine reversed quickly upon washout, whereas those of α-bungarotoxin did not. Concentration-response curves for three of the toxins are shown in Fig. 1C; approximate IC₅₀s were 300 nM for α-conotoxin ImI and α-bungarotoxin, and 100 nM for MLA.

Figure 1.

Blockade and recovery of ACh-induced secretory responses after superfusion of chromaffin cells with MLA (0.1 μM), α-bungarotoxin (BGTx; 1 μM), and α-conotoxin ImI (ImI; 1 μM). The blockers were given during the time period indicated on the bottom horizontal bar in A and B (12 min). In each individual experiment, cells were stimulated 10 times at 6 min intervals with ACh pulses (100 μM, 5 s) (see example of A using α-conotoxin ImI as a blocker). The first secretory pulse was discarded and the secretory peak of the second pulse was normalized to 100% (initial, B). The rest of the peaks were expressed as % of the initial secretion peak. Data are means ± SE of 6–11 experiments (B). (C) Concentration-response curves for BGTx, α-conotoxin ImI, and MLA for blockade of ACh-evoked secretion (protocols as in A); the value of secretion after 12 min of toxin superfusion was taken to calculate the % blockade of secretion. A separate batch of cells was used for each blocker concentration. Data are means ± SE of 6–8 experiments performed with different batches of cell cultures.

Inhibition by α₇ Blockers of ACh-Induced ⁴⁵Ca²⁺ Uptake.

The physiological catecholamine release response triggered by ACh in the intact adrenal gland implies the previous entry into the cytosol of extracellular Ca²⁺ (17) through voltage-dependent Ca²⁺ channels (18). So, it was expected that the blockade of secretion by α-toxins would parallel the inhibition of Ca²⁺ entry. Chromaffin cells incubated in Krebs-Hepes solution for 10 s took up 70 ± 5.7 cpm as ⁴⁵Ca²⁺ (n = 18 wells). A pulse of 300 μM ACh for 10 s was necessary to get a near 4-fold increase of ⁴⁵Ca²⁺ uptake (277 ± 14 cpm in 18 wells from different cell batches).

Toxins (0.1 μM) were kept in contact with the cells for 30 min before applying the ACh pulse. α-Bungarotoxin blocked the ⁴⁵Ca²⁺ uptake by 44 ± 3.7%, α-conotoxin ImI by 45.6 ± 3.1%, and MLA by 42 ± 4.2% (Fig. 2). Blockade of ⁴⁵Ca²⁺ entry could be due to a direct action of the toxins on Ca²⁺ channels, which are activated secondarily to ACh stimulation of nAChR, causing cell depolarization. However, none of the toxins inhibited the ⁴⁵Ca²⁺ uptake induced by high K⁺ (70 mM, 10 s), thus precluding a direct effect on Ca²⁺ channels (data not shown). ⁴⁵Ca²⁺ influx into chromaffin cells stimulated with ACh may occur via nicotinic receptors directly and/or via voltage-dependent Ca²⁺ channels. The present experiments do not distinguish between these possibilities.

Figure 2.

Effects of α-conotoxins ImI (ImI), α-bungarotoxin (BGTx), and MLA on ⁴⁵Ca²⁺ entry into chromaffin cells stimulated with ACh. Cells were incubated for 30 min with the compounds at the concentration of 100 nM. Data are means ± SE of the number of wells shown in parentheses on top of each bar, from at least four different batches of cells. ***P < 0.001, compared with controls.

Effects of α-Toxins on Inward Currents Through Native Nicotinic Receptors in Chromaffin Cells.

If the α₇ blockers inhibited Ca²⁺ entry and secretion stimulated by nicotinic receptors, then they also should affect the ACh-induced currents through native nAChR of chromaffin cells. The application of 1-s pulses of ACh (100 μM) to a voltage-clamped (holding potential −80 mV) chromaffin cell, at 30 s intervals, generated inward currents (I_ACh); the current amplitude was reproducible during the application of at least 18 ACh pulses (Fig. 3A). In 98 cells, the average peak current amounted to 1,736 ± 80 pA.

Figure 3.

Effects of α-conotoxin ImI (ImI; B), α-bungarotoxin (BGTx; C), and MLA (C) on inward currents induced by ACh pulses (100 μM, 1 s, given at 30-s intervals), in voltage-clamped chromaffin cells (holding potential −80 mV). (A) Reproducibility of inward currents upon repeated application of ACh pulses. (B) Time course of I_ACh before, during, and after adding ImI (1 μM; top horizontal bars). (Insets) Two original inward current traces generated by ACh pulses before or in the presence of the blocker. (C) Time course of the blocking effects of both MLA (0.1 μM) and BGTx (1 μM). These results were reproduced in 4–9 cells for each blocker.

The time courses of the effects of α₇ blockers on I_ACh are shown in Fig. 3 B and C. After breaking into the cell with the patch pipette, pulses of ACh (1 s, 100 μM) were applied at 30-s intervals. Once the I_ACh peaks stabilized, 1 μM α-conotoxin ImI was applied as shown on the top horizontal bars (Fig. 3B). The inhibition with each concentration was achieved in a single step and was maximum after 30 s of superfusion with the toxin. Note also that the blockade was reversible and that the current recovered its initial size after 3 min of toxin washout. The addition of the toxin for a second time produced similar effects. The Inset shows original I_ACh traces in the absence (control) and in the presence of 1 μM toxin; the kinetics of I_ACh was not apparently modified by α-conotoxin ImI. Fig. 3C shows similar time course experiments for I_ACh blockade induced by MLA (0.1 μM) and by α-bungarotoxin (1 μM). MLA caused a fast blockade of I_ACh (≈65%) that quickly reversed after washout of the compound. The subsequent addition of α-bungarotoxin caused ≈40% blockade of I_ACh that reversed slowly and partially upon washout.

Effects of α₇ Blockers on ACh Currents in Xenopus Oocytes Expressing the α₃β₄ and α₇ Subtypes of nAChR.

The selectivity of the three α₇ blockers used above was studied in Xenopus oocytes injected with RNAs coding for α₇ and α₃β₄ receptors. Fig. 4A shows the kinetics of I_ACh in oocytes expressing α₇ and α₃β₄ receptors. The application of long pulses of ACh (100 μM, 20 s) generated inward currents that in the case of α₇ receptors inactivated promptly with a τ of 1.5 s. In contrast, the current generated by α₃β₄ receptors suffered a very tiny inactivation (only ≈5–10% of current loss was observed at the end of the 20 s ACh pulse). The size of peak currents also differed between α₇-injected oocytes (80 ± 9 nA, n = 25 oocytes) and α₃β4 oocytes (3.4 ± 0.73 μA; n = 20 oocytes), 3–4 days after injecting the mRNAs.

Figure 4.

Kinetics and blockade by α-toxins of I_ACh, in Xenopus oocytes injected with cRNAs for α₇ or α₃β₄ nAChR. Oocytes maintained at a holding potential of −60 mV, were stimulated with pulses of ACh (100 μM) as shown by the top horizontal bars. (A) Two original I_ACh traces (normalized currents) obtained by stimulation with ACh (20 s) in one oocyte expressing α₇ receptors and in another expressing α₃β₄ receptors. (B) Effect of α-conotoxin ImI on I_ACh generated by 1 s pulses of ACh (100 μM) applied to oocytes expressing α₇ receptors. (C and D) Concentration-response curves of α-toxins to block I_ACh in oocytes expressing α₇ nAChR (C) or α₃β₄ receptors (D). Protocols to estimate the blockade exerted by each toxin on I_ACh were as those described in B. Repeated pulses of ACh (1s, 100 μM) generated inward currents that were normalized to 100% (before adding each toxin concentration). The blockade exerted by each toxin concentration was expressed as percentage of the initial current. Data are means ± SE of 5–10 oocytes for each blocker.

The effects of α-toxins on I_ACh were tested in oocytes stimulated with 1-s pulses of 100 μM ACh, applied at 1-min intervals. With this protocol, I_ACh was quite reproducible during several minutes; so, it was possible to test the blocking effects of a given compound as well as the recovery of I_ACh after washing out such compound. Fig. 4B shows that 1 μM α-conotoxin ImI completely suppressed the α₇ I_ACh, which recovered quickly after 5-min washout. α-Bungarotoxin (0.1 μM) suppressed I_ACh through α₇ receptors, and the current did not recover 10 min after the toxin washout (not shown).

Quantitative data on toxin blockade of I_ACh in α₇- and α₃β₄-injected oocytes are presented in Figs. 4 C and D, respectively. α-Conotoxin ImI blocked α₇ currents with an IC₅₀ of 100 nM; α₃β₄ currents were unaffected even by 3 μM of this toxin. α-Bungarotoxin blocked α₇ currents with an IC₅₀ of 1.6 nM and had no effects on α₃β₄ currents at concentrations up to 3 μM. Finally, methyllycaconitine blocked α₇ currents with an IC₅₀ of 0.1 nM and α₃β₄ currents with an IC₅₀ of 1 μM.

DISCUSSION

In this study, we used three blockers with high selectivity for α₇ nAChR to explore their effects on native nicotinic receptor currents as well as on Ca²⁺ uptake and catecholamine release triggered by stimulation of chromaffin cells with short pulses of ACh. Their claimed selectivity (8, 10) was corroborated in our hands, since the brain homomeric α₇ nicotinic receptor currents in oocytes were potently blocked by α-bungarotoxin, α-conotoxin ImI, and methyllycaconitine; α-bungarotoxin and α-conotoxin ImI did not affect the I_ACh through heteromeric α₃β₄ receptors whereas MLA blocked it with a potency four orders of magnitude lower than that exhibited for α₇ receptors (Fig. 4). The IC₅₀ values for MLA and α-bungarotoxin blockade of α₇ receptors expressed in oocytes were slightly higher than those obtained by other authors (9, 19); this could be explained on the basis of the shorter exposure periods used in our experiments or the lower superfusion rates used. Because the bovine adrenal chromaffin cells express α₇ receptors with high homology to brain receptors (2), we used these specific blockers as tools to investigate whether such receptors had a functional role in chromaffin cells.

Our chromaffin cell cultures are enriched in epinephrine-containing cells (11), where α₇ receptors seem to be preferentially expressed (20). We cannot exclude the expression of native heteromeric receptors formed by the combination of α₇ and α₃β₄ subunits, as the case is in oocytes injected with α₃β₄ and β₃ subunits (21). However, it is likely that the whole-cell native currents through nAChR, generated by 1-s pulses of ACh, are mainly the result of the simultaneous activation of a mixed population of α₇ and α₃β₄ receptors. This is supported by the fact that the three α₇ receptor blockers used in this study inhibited only partially the ACh-stimulated inward currents (Fig. 3), the entry of ⁴⁵Ca²⁺ (Fig. 2), and the release of catecholamines (Fig. 1), in chromaffin cells, at concentrations that inhibited fully the ACh-induced current in oocytes expressing α₇ nAChR (Fig. 4).

The reasons for the failure to show a functional role for chromaffin cell α₇ nAChR in earlier studies (1, 4–6, 22, 25), might rest in the use of long periods of stimulation (minutes) of chromaffin cells. As shown for brain receptors (26; and Fig. 4A), the bovine chromaffin cell α₇ receptor desensitizes in <1 s when expressed in oocytes (2). If the native α₇ receptor had similar kinetics in bovine chromaffin cells, exposure of these cells to nAChR agonists during several minutes in those earlier studies, would necessarily mask any participation of α₇ receptors in the fast component of the secretory response. As seen in Fig. 4A, α₃β₄ receptors expressed in oocytes did not desensitize even after 20 s of ACh application; it seems therefore that under the experimental conditions used in earlier reports (minutes of ACh stimulation) the observed secretory responses will be due to α₃β₄ receptor stimulation. But this by no means excludes that under more physiological conditions as those used here (1–5 s stimulation with ACh), α₇ receptors do certainly participate in the triggering and regulation of fast Ca²⁺ entry and catecholamine release.

Finding a function for α₇ nAChR in brain tissues have proven to be also elusive. Earlier experiments showing specific and saturable binding sites for α-bungarotoxin could not find that the toxin affected some functions in several synapses (27–32). However, more recent studies have provided data implicating α₇ receptors in some neural functions. For instance, α₇ receptors have been suggested to enhance fast excitatory transmission via a presynaptic mechanism (33), the increase of cytosolic Ca²⁺ (34, 35), the guidance of nerve fibers (36), the inhibition of neurite extension (37), the expression of neurotrophic growth factor in rat hippocampus (38), the death of motoneurons (39), the modulation of cell proliferation in lung tumor cells (40, 41), and the generation of postsynaptic neuronal responses (42, 43).

With the same blockers used in the present study, Codignola et al. (40) also found that α-conotoxin ImI inhibits nicotine-evoked hormone secretion in human small cell lung carcinoma, and Vijayaraghavan et al. (34) found that α-bungarotoxin and methyllycaconitine inhibit the increase of [Ca²⁺]_i induced by 2- to 4-s pulses of nicotine in chicken ciliary ganglion neurons. Interestingly, the latter authors found that the inhibition was higher, the lower the concentration of nicotine used. Thus, it seems that depending on the conditions of stimulation (duration of the stimulus, concentration of agonist used), the functions associated to the activation of α₇ receptors can be unmasked or not. This might explain the extensive controversy that has surrounded the numerous attempts to find a function for α₇ receptors. In this context, we believe that our present results clearly establish a functional role in the control of Ca²⁺ entry and secretion triggered by the physiological cholinergic neurotransmitter in the adrenal medullary chromaffin cells. Why two (or more) nAChR subtypes (α₇, α₃β₄) are required to control the secretory process is still a mystery. It may be related to the need to secrete differentially norepinephrine or epinephrine under particular stressful conditions. Or it may be related to the generation of a fast but transient secretory response (α₇ are fast desensitizing receptors) or a more sustained catecholamine release response (α₃β₄ are slow desensitizing receptors).

ABBREVIATIONS

MLA

methyllycaconitine

ACh

acetylcholine

nAChR

neuronal nicotinic receptor for acetylcholine

ImI

α-conotoxin ImI