Introduction

Chromaffin cells of the adrenal gland are modified postganglionic sympathetic neurons, innervated by the splanchnic nerve, that control the release of catecholamines to the bloodstream. In humans, chromaffin cells have an adrenergic phenotype [34] and they are the primary source for adrenaline production and release. In the human adrenal gland, there is a juxtaposition of medulla and cortex (Fig. 1). This differs from other species such as cow, rat, or mouse, in which the separation between medulla and cortex is preserved. The cortex secretes high concentrations of glucocorticoids to the medulla, which activate the expression of the enzyme phenylethanolamine N-methyltransferase (PNMT) [51], and also the α7 subunit gene [9]. Transcriptional activators of the PNMT gene (Egr-1, AP2, Ps 1, and MAZ) also contribute to the adrenergic phenotype of the human chromaffin cell [50]. Following an acute sympathetic response to stress, high plasma levels of noradrenaline and adrenaline provided by the sympathetic nervous system and adrenaline secreted by the medulla increase the metabolic rate of the body to produce energy and adapt the cardiovascular system and musculature to defense or escape. Whenever sympathetic activity increases, the splanchnic nerve releases acetylcholine (ACh) that will bind to nicotinic acetylcholine receptors (nAChRs) and muscarinic receptors in the chromaffin cells of the adrenal gland.

Fig. 1
figure 1

Photographs of human adrenal glands obtained from organ donors. a Two whole human adrenal glands in which the surrounding fat has been removed. b Human adrenal gland sectioned. Note that under the capsule the cortex and medulla are intermingled and there is no separation between them

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nAChRs are ligand-gated cationic channels that mediate fast synaptic transmission. There are 16 different nAChR subunits, α1–7, α9, α10, β1-β4, δ, and ε in adult mammals and γ in embryonic muscle, that assemble into pentamers to form a variety of nAChR subtypes. As the α3β4* (asterisk indicates the possible presence of additional subunits) nAChR subtype is classically considered the ganglionic subtype, chromaffin cells are expected to mainly express this receptor as well. The initial studies on nAChRs in chromaffin cells were performed in bovine and rat species, and showed that this was indeed the case. However, controversy exits regarding the precise subunit composition of nAChRs in bovine and rat chromaffin cells. Bovine chromaffin cells express α3β4* nAChRs but it is unknown whether they also express subtypes with β2 subunits [7]. In rat chromaffin cells, a previous report suggested the presence of α3β4 nAChRs as well as a subtype(s) with β2 subunits [14]. The functional role of α7 nAChR subunits in chromaffin cells is less clear. The α7 gene was cloned [18] and cell surface expression of α7 nAChRs was determined by binding experiments using the α7 and α9* nAChR antagonist α-bungarotoxin (α-Bgtx) [38, 49] and by antibody detection [16]. In addition, partial or full α-Bgtx block of nAChR agonist-elicited currents, calcium signals, or secretion in bovine chromaffin or PC12 cells [5, 13, 27] has also been shown. However, the catecholamine release process [25, 26, 45, 47] was found to be insensitive to α-Bgtx. Evidence for the presence of α7 nAChRs has also been provided in rat chromaffin cells through α7 mRNA detection [11, 14, 32, 41], and in PC12 cells by affinity purification and immunoblotting of α-Bgtx binding receptors [15]. In rat chromaffin cells, however, inward currents elicited by nicotine pulses were insensitive to α-Bgtx and low doses of methyllycaconitine (MLA), demonstrating lack of functional α7 receptors [14].

Given the relevance of nAChRs to the release of catecholamines by adrenal gland chromaffin cells and sympathetic neurons, and the number of drugs acting on nAChRs that are clinically used, it was of interest to characterize these receptors in native human cells. Our first study on human chromaffin cell nAChRs was published in 2007 [33]. It showed that the achievement of stable electrophysiological recordings of nAChRs and nAChR-evoked exocytosis in the perforated-patch mode of the patch-clamp technique was possible in chromaffin cells obtained from the adrenal glands of organ donors. Afterwards, we performed a detailed characterization of nAChR subtypes in human chromaffin cells using α-conotoxins (α-CTxs). We also investigated the contribution of these receptors to the exocytosis that would lead to neurotransmitter release, and the effect of some drugs of particular relevance for nicotine addition on the membrane potential.

nAChRs in human chromaffin cells of adrenal glands obtained from organ donors: characterization, contribution to exocytosis and pharmacology

Chromaffin cells were collected from adrenal glands of organ donors with “encephalic” or “cardiac” death. In the “encephalic death,” donor brain has suffered an irreversible loss of function. In these donors, the heart continued to beat, kept “alive” by means of vasoactive drugs and mechanical ventilation that guaranteed the perfusion of organs. In the “cardiac death” condition, the donor heart has stopped beating due to cardiac arrest, and the lack of blood circulation rapidly provoked the death of the brain. To be considered viable, other organs must be perfused through cannulation of the femoral vessels within 2 h of cardiac arrest. Donors are transferred to the hospital with mechanical ventilation, cardiocompression, and medication to assess donation. In both cases, donors are considered to be dead since resuscitation is not possible. After obtaining the permits of the family and a judge, organs to be transplanted and adrenals were removed from the donor. Adrenal glands were then placed into a preservation liquid and transported to our laboratory, where we performed the isolation and culture of the chromaffin cells [21, 33]. Experiments to record nAChR currents were started 24–48 h after platting the cells to allow recovery from enzyme digestion [2].

Characterization of nAChRs in human chromaffin cells

We first sought to address the subunit composition of nAChRs in these cells, with special attention paid to the α7 receptor, which is crucial in mediating rapid synaptic transmission [1, 17, 48, 53]. α7 mRNA detection in human medulla was previously shown by Mousavi and colleagues in 2001 [32]. Thus, the probability that this receptor subtype was expressed in human chromaffin cells was high. We found that the nAChR antagonists α-Bgtx (1 μM) and MLA (10 nM) blocked the nicotinic currents elicited by ACh by 6 ± 1.7 and 7 ± 1.6%, in an irreversible and reversible manner, respectively. Choline (10 mM) pulses induced a biphasic current with an initial α7 component (5.5 ± 0.4 ms rise time, 8.5 ± 0.4 ms time constant decay time), which was blocked by α-Bgtx or MLA, followed by a slower non-α7 component. The α7 nAChR specific agonist PNU-282987 also elicited rapidly activated currents (7.1 ± 0.4 ms for 3 μM and 5.5 ± 0.4 ms for 30 μM) that were also rapidly inactivated (10 ± 0.9 ms for 3 μM, and 9.8 ± 1.8 ms for 30 μM). α7 nAChR positive allosteric modulators, such as 5-hydroxyindole (1 mM) and PNU-120596 (10 μM), potentiated nAChR currents that could be blocked by α-Bgtx. α7 nAChR currents could be clearly recorded in all human chromaffin cells tested [35]. It is interesting to note that in bovine adrenal gland slices, α-BgTx sensitive receptors are restricted to medullary areas adjacent to the adrenal cortex and are colocalized with PNMT. Also, α7 nAChR transcripts are localized exclusively in adrenergic cells [12]. The expression of PNMT [51] and the α7 nAChR subunit gene [9] are activated by glucocorticoids. These findings may explain why α7 nAChR currents could be recorded in human chromaffin cells in which 99% of cells are adrenergic, while in other laboratories, their presence could not be clearly established in non-human species with more of a mixture of adrenergic and non-adrenergic cells.

To characterize non-α7 nAChR subtypes expressed in chromaffin cells, α-Ctxs were used. These toxins are peptides isolated from the venom of marine cone snails and have been useful in developing peptide analogs that selectively target specific nAChR subtypes. The use of these peptides together with molecular biology tools allowed us to conclude that the predominant heteromeric nAChR subtype expressed by human chromaffin cells is α3β4* with a minor population of β2* nAChRs [21]. This conclusion was achieved by means of the use of the following peptides: LvIA(N9R,V10A) that targets human α3β2, α6/α3β2β3, and β3α6β2α4β2 nAChRs heterologusly expressed in Xenopus oocytes with IC50 values of 3.3, 13.5, and 11.4 nM, respectively [21]; α-CTx BuIA(T5A,P6O) that targets α3β4 and α6/α3β4 nAChRs heterologusly expressed in Xenopus oocytes with IC50 values of 166 and 7.4 nM, respectively [21]; and α-CTx PeIA(A7V,S90H,V10A,N11R,E14A, an α6β2 and α6β4 nAChR antagonist that targets α6M211L,cytα3β4, α6/α3β2β3, and β3α6β2α4β2 receptors heterologously expressed in Xenopus oocytes with IC50 values of 1.6, 3.8, and 6.3 nM, respectively [21]; and α-CTx ArIB(V11L,V16D), that targets α7 human native receptors [24], whereas human non-α7 nAChRs heterologously expressed in Xenopus oocytes are insensitive to this toxin [21].

nAChR currents elicited by ACh pulses in human chromaffin cells were blocked by 7 ± 2% with 100 nM LvIA(N9R,V10A), showing a minor contribution of β2 subunits in the nAChR composition. Currents were blocked by 98 ± 0.3% with 1 μM α-CTx BuIA(T5A,P6O) in the same cells, treated with 100 nM α-CTx ArIB(V11L,V16D) to block α7 nAChRs. In addition, α-CTx PeIA(A7V,S90H,V10A,N11R,E14A) only inhibited nicotinic currents in human chromaffin cells at concentrations of 100 nM or higher suggesting that there were few α6*-containing nAChRs present in these cells [21].

We performed molecular biology to confirm electrophysiological data. We assessed human adrenal gland tissue for the expression of nAChR subunit mRNAs using both end-point and quantitative real-time PCR (qPCR) methodologies. mRNAs for multiple nAChR subunits including α2, α3, α4, α5, α6, α7, α10, β2, and β4 subunits were detected. However, transcripts for α3, α7, and β4 subunits were found to be the most abundant subunits present. Transcripts for α5 and β2 were somewhat less abundant while those for α2, α6, and α10 were nearly absent. Transcripts for α4, α9, and β3 were detected infrequently. Internal controls were also performed by comparing the expression levels of α2, α3, α4, α5, α6, α7, α10, β2, and β4 subunits in adrenal medullary tissue to human brain. These experiments indicated that in adrenal gland, transcripts for α3 were more abundant compared to α6 whereas in human brain, α6 were more abundant than α3. We reassessed these results by performing the experiments on adrenal chromaffin cells isolated and cultured to avoid contamination of other cells. qPCR experiments in isolated chromaffin cells confirmed results obtained in adrenal medulla [21].

Contribution to exocytosis of nAChRs in human chromaffin cells

We recorded the plasma membrane capacitance increment in the voltage-clamp mode of the patch-clamp technique as an index of exocytosis to investigate the contribution of nAChR currents to the exocytotic process. However, ACh evokes action potentials that depolarize the cell and activate voltage-dependent calcium channels (VDCC). Therefore, to evaluate the overall exocytosis evoked by ACh, it is necessary to apply a protocol that allows recording of capacitance increments elicited under non-voltage-clamped conditions. To achieve this condition, we developed a “triple-step” protocol to measure plasma membrane capacitance increments due to changes in the membrane potential elicited by the nicotinic agonist. In this way, the plasma membrane capacitance that can be only measured under the whole-cell configuration is recorded before and after a pulse of ACh applied in the current-clamp configuration. By performing this protocol, we found that the contribution to the exocytosis of calcium entry through the nAChR ionophore may be of similar magnitude to that achieved by calcium entry through VDCC due to depolarization [33]. Using this protocol, we have been able to observe that α7 nAChR currents did not evoke exocytosis by themselves, but the depolarization provoked by these currents was able to elicit exocytosis [35]. On the other hand, current flowing through the α3β4* nAChR ionophore is able to elicit exocytosis by itself or by evoking depolarization [33, 36].

Use of α-Ctxs: some considerations

The fact that α-CTxs selective for rat α6*-containing nAChRs expressed in heterologous systems inhibited human chromaffin cell nAChRs with similar IC50 values led us to initially conclude that the predominant nAChR expressed in human chromaffin cells was the α6β4* subtype [36]. In this previous study, we used the mutant analog of α-Ctx MII, the α-Ctx MII[H9A,L15A] (α-Ctx MII[H9A,L15A]) which primarily targets rat α6* nAChRs with respect to rat α3 nAChRs expressed heterologouly in Xenopus oocytes [30] and exhibited a lower IC50 for α6β2* (2.4 nM) with respect to α6β4* nAChRs (269 nM). The IC50 value obtained for this toxin in human chromaffin cells was 217.8 nM [36], similar to the data previously reported for rat α6β4* nAChRs expressed in Xenopus oocytes. For that reason, the conclusion was that an α6β4* nAChRs was the predominant nAChR subtype expressed in these cells.

Later on some reports showed evidences on the differential effect of α-Ctxs on rat versus human nAChRs expressed in heterologous systems [3, 52], which was further confirmed in our lab [21]. In addition, we found some inconsistencies between α-Ctx MII[H9A, L15A] IC50 values for inhibition of human adrenal chromaffin cell nAChRs (217.8 nM) [36] and human α6/α3β4* nAChRs heterologously expressed in Xenopus oocytes (13.3 nM) [19]. Thus, species differences are a key factor to be considered when characterizing nAChR subtypes.

The effects of α-Ctxs on human nAChRs expressed in Xenopus oocytes and chromaffin cells are summarized in Table 1. A comparison between the effects of α-Ctxs on rat and human α3β4 and α6β4 subtypes is summarized in Table 2.

Table 1 IC50 values for inhibition of human nAChRs expressed in Xenopus oocytes and adrenal chromaffin cell nAChRs. Values in parentheses are 95% confidence intervals
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Table 2 Comparison of α-Ctx IC50 values for inhibition of rat versus human nAChRs expressed in Xenopus oocytes (taken from Hone et al. [22] with some modifications)
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Pharmacology of nAChRs in human chromaffin cells

Tobacco smoking is the major cause of cardiovascular morbidity and mortality, and therefore, quitting smoking is crucial, especially for patients with some cardiovascular disease. However, some drugs used in the cessation smoking therapy are central nervous system nAChR agonists and may be also acting on ganglionic α3β4 nAChRs. This is the case of varenicline, a drug clinically used for the treatment of nicotine addiction. Varenicline activates heterologously expressed nAChRs of the α4β2 [31, 39], α6β2 [8], α3β4 [6, 31, 40, 43, 46], and α7 subtypes [31].

A case-report of varenicline-triggered pheochromocytoma crisis in a smoking subject suggested that varenicline might have activity towards adrenal gland chromaffin cell nAChRs [23]. In addition, some evidences regarding possible cardiovascular adverse effects of varenicline have been reported [37, 42]. However, information concerning the activity of varenicline on native human α3β4 nAChRs was not available. Thus, we evaluated the action of varenicline alone and in the presence of nicotine on nAChRs in human chromaffin cells, as well as on the excitability of these cells.

We performed patch-clamp experiments under the current-clamp configuration to evaluate the effects of varenicline on the plasma membrane excitability elicited by native α3β4* nAChRs in human chromaffin cells and compared them to those of nicotine. Varenicline and nicotine activated α3β4* nAChRs with EC50 values of 1.8 (1.2–2.7) μM and 19.4 (11.1–33.9) μM, respectively. Perfusion of therapeutically relevant doses of varenicline (50 or 100 nM) showed very little effect on action potential firing evoked by 10 ms ACh in the current-clamp mode. However, perfusion of 250 nM varenicline increased the number of action potentials fired by 436 ± 150% compared to control conditions. In contrast, nicotine showed no effect on action potential firing at any of the concentrations tested (50, 100, 250, and 500 nM). However, the presence of nicotine may potentiate the effects of varenicline. In smoking cessation therapy with varenicline, smokers should establish a date to stop smoking and treatment with varenicline should start 1 to 2 weeks before this date. In addition, nicotine replacement therapy is sometimes combined with varenicline to improve quit rates [10]. To examine this, we tested 50 nM nicotine together with 100 nM varenicline and obtained an increase of the action potential firing by 290 ± 104%. These results demonstrate that therapeutic concentrations of varenicline alone are unlikely to alter the adrenal chromaffin cell’s behavior and response to ACh, but in combination with nicotine, varenicline increases action potential firing, which may lead to an increase in neurotransmitter release [21].