RESUMO
This review summarizes our research on nicotinic acetylcholine receptors in human chromaffin cells. Limited research has been conducted in this field on human tissue, primarily due to the difficulties associated with obtaining human cells. Receptor subtypes were characterized here using molecular biology and electrophysiological patch-clamp techniques. However, the most significant aspect of this study refers to the cross-talk between the two main subtypes identified in these cells, the α7- and α3ß4* subtypes, aiming to avoid their desensitization. The article also reviews other aspects, including the regulation of their expression, function or physical interaction by choline, Ca2+, and tyrosine and serine/threonine phosphatases. Additionally, the influence of sex on their expression is also discussed.
Assuntos
Células Cromafins , Receptores Nicotínicos , Humanos , Receptores Nicotínicos/metabolismo , Colina/metabolismo , Fenômenos Eletrofisiológicos , Células Cromafins/metabolismoRESUMO
We have recently reported that α7 and α3ß4 nicotinic acetylcholine receptor (nAChR) subtypes are expressed in human chromaffin cells in the plasma membrane where they colocalize and physically interact. The present study was designed to evaluate whether those receptor subtypes also colocalize at the central nervous system to mutually interact, and whether their expression and colocalization are regulated by phosphorylation/dephosphorylation processes, as they are in human chromaffin cells. We have here found that in isolated and maintained in culture mouse hippocampal neurons, nAChR expression and colocalization of α7, but not α3ß4, nAChR subtypes decreased by tyrosine (Tyr)- and serine/threonine (Ser/Thr)-phosphatase inhibition. However, Tyr-kinase inhibition or protein-phosphatase 2A (PP2A) activation increased α3ß4 nAChR expression, diminishing receptor subtypes colocalization. Furthermore, colocalization is not recovered if the inhibitors of Tyr-phosphatase and kinases, or the inhibitor of Ser/Thr-phosphatases and the activator of PP2A are applied together. Therefore, regulation of α7 and α3ß4 nAChR subtypes expression by Tyr- and Ser/Thr kinases and phosphatases exhibit differential mechanisms in mouse hippocampal neurons. Colocalization of nAChR subtypes, however, is altered by any maneuver that affects these kinases or phosphatases, which might have consequences in the functional activity of nAChR subtypes.
Assuntos
Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa7 , Camundongos , Animais , Humanos , Fosforilação , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Tirosina/metabolismo , Receptores Nicotínicos/metabolismo , Neurônios/metabolismo , Hipocampo/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3ß4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3ß4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3ß4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3ß4 subtype is dependent on Ca2+ In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+ In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3ß4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3ß4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3ß4 current increase. Because choline is an α3ß4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.
Assuntos
Células Cromafins/metabolismo , Receptor Cross-Talk/fisiologia , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cardiovascular side effects of varenicline and a case report of a hypertensive crisis in a varenicline-prescribed patient with pheochromocytoma have been reported. The goal of the present study was to determine whether such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in human chromaffin cells of the adrenal gland. In this study, we performed electrophysiological plasma membrane capacitance and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors. Cells were stimulated with short pulses (10 ms) of acetylcholine (ACh; 300 µM) applied at 0.2 Hz, in order to closer mimic the physiological situation at the splanchnic nerve-chromaffin cell synapse. In addition, rat chromaffin cells were used to compare the effects obtained in cells from a more readily available species. Varenicline increased the exocytosis of secretory vesicles in human and rat chromaffin cells in the presence of nicotine, effects that were not due to an increase of plasma membrane capacitance or currents triggered by the nicotinic agonists alone. These results should be considered in nicotine addiction therapies when varenicline is used.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Vareniclina/farmacologia , Acetilcolina/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Animais , Bovinos , Células Cromafins/metabolismo , Humanos , RatosRESUMO
Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, ß2, and ß4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3ß2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3ß4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3ß4 nAChRs alone or α3ß4 together with the α3ß2ß4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3ß2ß4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.
Assuntos
Medula Suprarrenal/metabolismo , Células Cromafins/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/biossíntese , Animais , Células Cultivadas , Conotoxinas/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/biossínteseRESUMO
High catecolamine plasma levels because of sympathetic nervous system over-activity contribute to cirrhosis progression. The aim of this study was to investigate whether chromaffin cells of the adrenal gland might potentiate the deleterious effect exerted by this over-activity. Electrophysiological patch-clamp and amperometric experiments with carbon-fibre electrodes were conducted in single chromaffin cells of control and CCl4 -induced cirrhotic rats. The spontaneous action potential firing frequency was increased in chromaffin cells of cirrhotic rats with respect to control rats. The exocytosis evoked by that firing was also increased. However, exocytosis elicited by ACh did not vary between control and cirrhotic rats. Exocytosis triggered by depolarizing pulses was also unchanged. Amperometric recordings confirmed the lack of increased catecholamine charge released in cirrhosis after ACh or depolarization stimuli. However, the amperometric spikes exhibited faster kinetics of release. The overall Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), or in particular through Cav1 channels, did not vary between chromaffin cells of control and cirrhotic rats. The inhibition of VDCC by methionine-enkephaline or ATP was not either altered, but it was increased by adrenaline in cells of cirrhotic rats. When a cocktail composed by the three neurotransmitters was tested in order to approach a situation closer to the physiological condition, the inhibition of VDCC was similar between both types of cells. In summary, chromaffin cells of the adrenal gland might contribute to exacerbate the sympathetic nervous system over-activity in cirrhosis because of an increased exocytosis elicited by an enhanced spontaneous electrical activity.
Assuntos
Potenciais de Ação/fisiologia , Células Cromafins/metabolismo , Exocitose/fisiologia , Cirrose Hepática/metabolismo , Animais , Canais de Cálcio/metabolismo , Tetracloreto de Carbono/toxicidade , Catecolaminas/metabolismo , Progressão da Doença , Cirrose Hepática/induzido quimicamente , Masculino , Ratos , Ratos WistarRESUMO
During the last 10 years, we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for recording of α7 nAChR currents, something that had previously not been achieved in non-human species. The use of α-conotoxins allowed us to characterize non-α7 nAChR subtypes and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3ß4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxß2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage-dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on catecholamine release in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.
Assuntos
Células Cromafins/metabolismo , Conotoxinas/farmacologia , Neurotoxinas/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Potenciais de Ação , Sinalização do Cálcio , Células Cromafins/efeitos dos fármacos , Células Cromafins/fisiologia , Humanos , Receptores Nicotínicos/químicaRESUMO
Varenicline is a nicotinic acetylcholine receptor (nAChR) agonist used to treat nicotine addiction, but a live debate persists concerning its mechanism of action in reducing nicotine consumption. Although initially reported as α4ß2 selective, varenicline was subsequently shown to activate other nAChR subtypes implicated in nicotine addiction including α3ß4. However, it remains unclear whether activation of α3ß4 nAChRs by therapeutically relevant concentrations of varenicline is sufficient to affect the behavior of cells that express this subtype. We used patch-clamp electrophysiology to assess the effects of varenicline on native α3ß4* nAChRs (asterisk denotes the possible presence of other subunits) expressed in human adrenal chromaffin cells and compared its effects to those of nicotine. Varenicline and nicotine activated α3ß4* nAChRs with EC50 values of 1.8 (1.2-2.7) µM and 19.4 (11.1-33.9) µM, respectively. Stimulation of adrenal chromaffin cells with 10 ms pulses of 300 µM acetylcholine (ACh) in current-clamp mode evoked sodium channel-dependent action potentials (APs). Under these conditions, perfusion of 50 or 100 nM varenicline showed very little effect on AP firing compared to control conditions (ACh stimulation alone), but at higher concentrations (250 nM) varenicline increased the number of APs fired up to 436 ± 150%. These results demonstrate that therapeutic concentrations of varenicline are unlikely to alter AP firing in chromaffin cells. In contrast, nicotine showed no effect on AP firing at any of the concentrations tested (50, 100, 250, and 500 nM). However, perfusion of 50 nM nicotine simultaneously with 100 nM varenicline increased AP firing by 290 ± 104% indicating that exposure to varenicline and nicotine concurrently may alter cellular behavior such as excitability and neurotransmitter release.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Córtex Suprarrenal/efeitos dos fármacos , Células Cromafins/efeitos dos fármacos , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Vareniclina/administração & dosagem , Potenciais de Ação/fisiologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/fisiologia , Adulto , Idoso , Animais , Células Cromafins/fisiologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Xenopus laevisRESUMO
The present study was performed to evaluate the Cav1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Cav1.2 and Cav1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Cav1.2 and Cav1.3 subtypes, but not Cav1.1 or Cav1.4. Electrophysiological experiments were conducted to investigate the contribution of Cav1 channels to the exocytotic process and cell excitability. Cav1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3µM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (-34mV), which elicits 10.4mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Cav1 channels in exocytosis and cell excitability.
Assuntos
Potenciais de Ação , Caveolina 1/metabolismo , Células Cromafins/citologia , Células Cromafins/metabolismo , Exocitose , Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Células Cromafins/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Humanos , Isradipino/farmacologia , Nifedipino/farmacologiaRESUMO
Ligands that selectively inhibit human α3ß2 and α6ß2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3ß4 and α6ß4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3ß2 than α3ß4 and 165-fold more potent on human α6/α3ß2ß3 than α6/α3ß4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with ß2 ligand-binding sites. In contrast, the ß4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained ß4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3ß4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, ß2, and ß4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.
Assuntos
Medula Suprarrenal/metabolismo , Células Cromafins/metabolismo , Conotoxinas/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Humanos , Técnicas de Patch-Clamp , Isoformas de Proteínas , Ratos , Receptores Nicotínicos/classificação , Xenopus laevisRESUMO
Nicotinic acetylcholine receptors (nAChRs) that contain α6 and ß4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6ß4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3ß4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6ß4* receptors. PCR was used to show the presence of transcripts for α6 and ß4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6ß4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and ß2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6ß4* nAChRs.
Assuntos
Células Cromafins/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Conotoxinas/genética , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Potenciais Evocados/efeitos dos fármacos , Haplorrinos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Receptores Nicotínicos/genéticaRESUMO
Chromaffin cells have been widely used to study neurosecretion since they exhibit similar calcium dependence of several exocytotic steps as synaptic terminals do, but having the enormous advantage of being neither as small or fast as neurons, nor as slow as endocrine cells. In the present study, secretion associated to experimental measurements of the exocytotic dynamics in human chromaffin cells of the adrenal gland was simulated by using a model that combines stochastic and deterministic approaches for short and longer depolarizing pulses, respectively. Experimental data were recorded from human chromaffin cells, obtained from healthy organ donors, using the perforated patch configuration of the patch-clamp technique. We have found that in human chromaffin cells, secretion would be mainly managed by small pools of non-equally fusion competent vesicles, slowly refilled over time. Fast secretion evoked by brief pulses can be predicted only when 75% of one of these pools (the "ready releasable pool" of vesicles, abbreviated as RRP) are co-localized to Ca²âº channels, indicating an immediately releasable pool in the range reported for isolated cells of bovine and rat (Álvarez and Marengo, J Neurochem 116:155-163, 2011). The need for spatial correlation and close proximity of vesicles to Ca²âº channels suggests that in human chromaffin cells there is a tight control of those releasable vesicles available for fast secretion.
Assuntos
Células Cromafins/fisiologia , Exocitose/fisiologia , Modelos Biológicos , Dinâmica não Linear , Glândulas Suprarrenais/citologia , Biofísica , Cálcio/metabolismo , Células Cultivadas , Estimulação Elétrica , Humanos , Processos EstocásticosRESUMO
The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Exocitose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ácido gama-Aminobutírico/análogos & derivados , Glândulas Suprarrenais/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Bovinos , Humanos , Isoleucina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Neurotransmissores/metabolismo , Pregabalina , Ácido gama-Aminobutírico/farmacologiaRESUMO
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 300µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4ms rise time) and inactivated component (8.5 ± 0.4ms 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 (1mM) and PNU-120596 (10µM), 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.
Assuntos
Células Cromafins/fisiologia , Exocitose/fisiologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Aconitina/análogos & derivados , Aconitina/farmacologia , Adolescente , Adulto , Idoso , Bungarotoxinas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/fisiologia , Adulto Jovem , Receptor Nicotínico de Acetilcolina alfa7RESUMO
In the present study, we have electrophysiologically characterized native nicotinic acetylcholine receptors (nAChRs) in human chromaffin cells of the adrenal gland as well as their contribution to the exocytotic process. α-Conotoxin AuIB blocked by 14 ± 1% the acetylcholine (ACh)-induced nicotinic current. α-Conotoxin MII (α-Ctx MII) exhibited an almost full blockade of the nicotinic current at nanomolar concentrations (IC(50)=21.6 nM). The α6*-preferring α-Ctx MII mutant analogs, α-Ctx MII[H9A,L15A] and α-Ctx MII[S4A,E11A,L15A], blocked nAChR currents with an IC(50) of 217.8 and 33 nM, respectively. These data reveal that nAChRs in these cells include the α6* subtype. The washout of the blockade exerted by α-conotoxin BuIA (α-Ctx BuIA; 1 µM) on ACh-evoked currents was slight and slow, arguing in favor of the presence of a ß4 subunit in the nAChR composition. Exocytosis was almost fully blocked by 1 µM α-Ctx MII, its mutant analogs, or α-Ctx BuIA. Finally, the fluorescent analog Alexa Fluor 546-BuIA showed distinct staining in these cells. Our results reveal that α6ß4* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under stressful situations.
Assuntos
Medula Suprarrenal/fisiologia , Células Cromafins/fisiologia , Exocitose/fisiologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Células Cromafins/efeitos dos fármacos , Conotoxinas/farmacologia , Exocitose/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Humanos , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Compostos de Quinolínio/farmacologiaRESUMO
This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo L/fisiologia , Células Cromafins/fisiologia , Exocitose/fisiologia , Animais , Células Cultivadas , Células Cromafins/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal gland. Most existing knowledge of these cells has been the outcome of extensive research performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the physiology of this neuroendocrine cell in humans has been gained. This review summarizes the main findings reported in human chromaffin cells under physiological or disease conditions and discusses the clinical implications of these results.
Assuntos
Células Cromafins/fisiologia , Células Cromafins/transplante , Doença , Medula Suprarrenal/citologia , Medula Suprarrenal/embriologia , Medula Suprarrenal/transplante , Grânulos Cromafim/metabolismo , HumanosRESUMO
In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca2+ channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca2+ channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca2+ channels. R-type Ca2+ channels, blocked by 77% with 20 muM Cd2+ and not affected by 50 muM Ni2+, were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca2+ channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca2+ channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca2+ channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/fisiologia , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Dopamina beta-Hidroxilase/metabolismo , Eletrofisiologia , Humanos , Imuno-Histoquímica , Técnicas de Patch-Clamp , Feniletanolamina N-Metiltransferase/metabolismoRESUMO
The whole-cell secretory response evoked by acetylcholine (ACh) in human chromaffin cells was examined using a new protocol based on quickly switching from the voltage-clamp to the current-clamp (CC) configuration of the patch-clamp technique. Our experiments revealed that Ca(2+) entry through the nicotinic receptor at hyperpolarized membrane potentials contributed as much to the exocytosis (100.4 +/- 27.3 fF) evoked by 200 ms pulses of ACh, as Ca(2+) flux through voltage-dependent Ca(2+) channels at depolarized membrane potentials. The nicotinic current triggered a depolarization event with a peak at +49.3 mV and a 'plateau' phase that ended at -23.9 mV, which was blocked by 10 mumol/L mecamylamine. When a long ACh stimulus (15 s) was applied, the nicotinic current at the end of the pulse reached a value of 15.45 +/- 3.6 pA, but the membrane potential depolarization still remained at the 'plateau' stage until withdrawal of the agonist. Perfusion with 200 mumol/L Cd(2+) during the 15 s ACh pulse completely abolished the plasma membrane depolarization at the end of the pulse, indicating that Ca(2+) entry through Ca(2+) channels contributed to the membrane potential depolarization provoked by prolonged ACh pulses. These findings also reflect that voltage-dependent Ca(2+) channels were recruited by the small current flowing through the desensitized nicotinic receptor to maintain the depolarization. Finally, muscarinic receptor activation triggered a delayed exocytotic process after prolonged ACh stimulation, dependent on Ca(2+) mobilization from the endoplasmic reticulum. In summary, we show here that nicotinic and muscarinic receptors contribute to the exocytosis of neurotransmitters in human chromaffin cells, and that the nicotinic receptor plays a key role in several stages of the stimulus-secretion coupling process in these cells.
Assuntos
Medula Suprarrenal/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Neurotransmissores/metabolismo , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/fisiologia , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Medula Suprarrenal/citologia , Adulto , Idoso , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
This study was designed to examine the kinetics of neurotransmitter release using the carbon fiber amperometric technique on cells in slices of mouse adrenal glands superfused with bicarbonate phosphate buffer-based solutions. The exocytotic amperometric response evoked by electrical stimulation was significantly faster than that produced after exogenous application of ACh or K+. Splanchnic nerve-evoked neurotransmitter release was blocked by hexamethonium, indicating the involvement of ACh nicotinic receptors. We discuss the implications of our data for understanding the mechanisms underlying the vesicle fusion process.