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1.
Pharmacol Res ; 140: 56-66, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29894763

RESUMO

Adrenal chromaffin cells comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the appropriate stress response. Deletion of the serotonin (5-HT) transporter (SERT) gene in mice (SERT-/- mice) or pharmacological block of SERT function in rodents and humans augments this sympathoadrenal stress response (epinephrine secretion). The prevailing assumption is that loss of CNS SERT alters central drive to the peripheral sympathetic nervous system. Adrenal chromaffin cells also prominently express SERT where it might coordinate accumulation of 5-HT for reuse in the autocrine control of stress-evoked catecholamine secretion. To help test this hypothesis, we have generated a novel mouse model with selective excision of SERT in the peripheral sympathetic nervous system (SERTΔTH), generated by crossing floxed SERT mice with tyrosine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was abolished in the adrenal gland but not perturbed in the CNS of SERTΔTH mice. SERT-mediated [3H] 5-HT uptake was unaltered in midbrain, hindbrain, and spinal cord synaptosomes, confirming transporter function was intact in the CNS. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in SERTΔTH mice, contrasting with the depleted 5-HT content in SERT-/- mice. Selective SERT excision reduced adrenal gland 5-HT content by ≈ 50% in SERTΔTH mice but had no effect on adrenal catecholamine content. This novel model confirms that SERT expressed in adrenal chromaffin cells is essential for maintaining wild-type levels of 5-HT and provides a powerful tool to help dissect the role of SERT in the sympathetic stress response.


Assuntos
Glândulas Suprarrenais/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Antidepressivos , Feminino , Masculino , Mesencéfalo/metabolismo , Camundongos Transgênicos , Modelos Animais , Rombencéfalo/metabolismo , Serotonina/metabolismo , Medula Espinal/metabolismo , Tirosina 3-Mono-Oxigenase
2.
J Biol Chem ; 292(29): 12165-12177, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28515322

RESUMO

Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gßγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gßγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gßγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gßγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gßγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gßγ for SNARE-binding sites in lipid environments. Mutant Gßγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gßγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gßγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gß and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gßγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gßγ-SNARE interaction and show that the target of Gßγ, downstream of VGCC, is the membrane-embedded SNARE complex.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Bicamadas Lipídicas , Modelos Moleculares , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Sintaxina 1/metabolismo , Animais , Ligação Competitiva , Sinalização do Cálcio , Bovinos , Linhagem Celular , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Humanos , Lipossomos , Fusão de Membrana , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sintaxina 1/química
3.
J Neurochem ; 143(2): 171-182, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28815595

RESUMO

Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 µM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.


Assuntos
Medula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/fisiologia , Receptores sigma/fisiologia , Medula Suprarrenal/citologia , Medula Suprarrenal/efeitos dos fármacos , Animais , Anisóis/farmacologia , Catecolaminas/antagonistas & inibidores , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Propilaminas/farmacologia , Receptores sigma/agonistas , Receptor Sigma-1
4.
Biochim Biophys Acta ; 1828(7): 1629-43, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23063655

RESUMO

Voltage gated calcium channels (Ca²âº channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of Ca(V)2 (N- and P/Q-type) Ca²âº-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein ßγ dimer (Gßγ) to the α1 subunit of Ca(V)2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gßγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. This article is part of a Special Issue entitled: Calcium channels.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos
5.
Vitam Horm ; 124: 39-78, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38408804

RESUMO

The adrenal glands are key components of the mammalian endocrine system, helping maintain physiological homeostasis and the coordinated response to stress. Each adrenal gland has two morphologically and functionally distinct regions, the outer cortex and inner medulla. The cortex is organized into three concentric zones which secrete steroid hormones, including aldosterone and cortisol. Neural crest-derived chromaffin cells in the medulla are innervated by preganglionic sympathetic neurons and secrete catecholamines (epinephrine, norepinephrine) and neuropeptides into the bloodstream, thereby functioning as the neuroendocrine arm of the sympathetic nervous system. In this article we review serotonin (5-HT) and the serotonin transporter (SERT; SLC6A4) in the adrenal gland. In the adrenal cortex, 5-HT, primarily sourced from resident mast cells, acts as a paracrine signal to stimulate aldosterone and cortisol secretion through 5-HT4/5-HT7 receptors. Medullary chromaffin cells contain a small amount of 5-HT due to SERT-mediated uptake and express 5-HT1A receptors which inhibit secretion. The atypical mechanism of the 5-HT1A receptors and interaction with SERT fine tune this autocrine pathway to control stress-evoked catecholamine secretion. Receptor-independent signaling by SERT/intracellular 5-HT modulates the amount and kinetics of transmitter release from single vesicle fusion events. SERT might also influence stress-evoked upregulation of tyrosine hydroxylase transcription. Transient signaling via 5-HT3 receptors during embryonic development can limit the number of chromaffin cells found in the mature adrenal gland. Together, this emerging evidence suggests that the adrenal medulla is a peripheral hub for serotonergic control of the sympathoadrenal stress response.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina , Serotonina , Animais , Humanos , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Aldosterona/metabolismo , Hidrocortisona , Glândulas Suprarrenais , Mamíferos
6.
J Neuroendocrinol ; 35(11): e13255, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-36970756

RESUMO

Adrenomedullary chromaffin cells respond to splanchnic (sympathetic) nerve stimulation by releasing stress hormones into the circulation. The signal for hormone secretion is encoded in the neurotransmitters - especially acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP) - that are released into the splanchnic-chromaffin cell synapse. However, functional differences in the effects of ACh and PACAP on the chromaffin cell secretory response are not well defined. Here, selective agonists of PACAP receptors or nicotinic and muscarinic acetylcholine receptors were applied to chromaffin cells. The major differences in the effects of these agents were not on exocytosis, per se, but rather on the steps upstream of exocytosis. In almost every respect, the properties of individual fusion events triggered by PACAP and cholinergic agonists were similar. On the other hand, the properties of the Ca2+ transients evoked by PACAP differed in several ways from those evoked by muscarinic and nicotinic receptor stimulation. A defining feature of the PACAP-stimulated secretory pathway was its dependence on signaling through exchange protein directly activated by cAMP (Epac) and PLCε. However, the absence of PLCε did not disrupt Ca2+ transients evoked by cholinergic agonists. Accordingly, inhibition of Epac activity did not disrupt secretion triggered by acetylcholine or specific agonists of muscarinic and nicotinic receptors. Thus, PACAP and acetylcholine stimulate chromaffin cell secretion via separate and independent pathways. This feature of stimulus-secretion coupling may be important for sustaining hormone release from the adrenal medulla under conditions associated with the sympathetic stress response.


Assuntos
Células Cromafins , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Acetilcolina/metabolismo , Catecolaminas/metabolismo , Catecolaminas/farmacologia , Agonistas Colinérgicos/metabolismo , Agonistas Colinérgicos/farmacologia , Células Cromafins/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hormônios , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Camundongos , Receptores Colinérgicos/metabolismo
7.
J Gen Physiol ; 155(2)2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36538657

RESUMO

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.


Assuntos
Células Cromafins , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Acetilcolina/farmacologia , Acetilcolina/metabolismo , Cálcio/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo
8.
Anesthesiology ; 116(5): 1013-24, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22417967

RESUMO

BACKGROUND: Gabapentin is most commonly prescribed for chronic pain, but acute perioperative effects, including preemptive analgesia and hemodynamic stabilization, have been reported. Adrenal chromaffin cells are a widely used model to investigate neurosecretion, and adrenal catecholamines play important physiologic roles and contribute to the acute stress response. However, the effects of gabapentin on adrenal catecholamine release have never been tested. METHODS: Primary cultures of bovine adrenal chromaffin cells were treated with gabapentin or vehicle for 18-24 h. The authors quantified catecholamine secretion from dishes of cells using high-performance liquid chromatography and resolved exocytosis of individual secretory vesicles from single cells using carbon fiber amperometry. Voltage-gated calcium channel currents were recorded using patch clamp electrophysiology and intracellular [Ca2+] using fluorescent imaging. RESULTS: Gabapentin produced statistically significant reductions in catecholamine secretion evoked by cholinergic agonists (24 ± 3%, n = 12) or KCl (16 ± 4%, n = 8) (mean ± SEM) but did not inhibit Ca2+ entry or calcium channel currents. Amperometry (n = 51 cells) revealed that gabapentin inhibited the number of vesicles released upon stimulation, with no change in quantal size or kinetics of these unitary events. CONCLUSIONS: The authors show Ca2+ entry was not inhibited by gabapentin but was less effective at triggering vesicle fusion. The work also demonstrates that chromaffin cells are a useful model for additional investigation of the cellular mechanism(s) by which gabapentin controls neurosecretion. In addition, it identifies altered adrenal catecholamine release as a potential contributor to some of the beneficial perioperative effects of gabapentin.


Assuntos
Glândulas Suprarrenais/metabolismo , Aminas/farmacologia , Catecolaminas/antagonistas & inibidores , Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Ácidos Cicloexanocarboxílicos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido gama-Aminobutírico/farmacologia , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Gabapentina , Hemodinâmica/fisiologia , Técnicas In Vitro , Técnicas de Patch-Clamp , Cloreto de Potássio/antagonistas & inibidores , Cloreto de Potássio/farmacologia , Vesículas Secretórias/efeitos dos fármacos
10.
Mol Pharmacol ; 79(6): 987-96, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21383044

RESUMO

Prostaglandin (PG) E(2) controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1-EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE(2) in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE(2) might modulate chromaffin cell function. PGE(2) did not alter resting intracellular [Ca(2+)] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit Ca(V)2 voltage-gated Ca(2+) channel currents (I(Ca)). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gßγ subunit-mediated mechanism common to other G(i/o)-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of I(Ca). Finally, changes in membrane capacitance showed that Ca(2+)-dependent exocytosis was reduced in parallel with I(Ca). To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE(2).


Assuntos
Canais de Cálcio/fisiologia , Células Cromafins/metabolismo , Exocitose/fisiologia , Receptores de Prostaglandina/fisiologia , Animais , Sequência de Bases , Primers do DNA , Dinoprostona/farmacologia , Masculino , Camundongos , RNA Mensageiro/genética , Receptores de Prostaglandina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Cell Mol Neurobiol ; 30(8): 1201-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21061161

RESUMO

Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the "fight-or-flight" response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca2+ entry through voltage-gated Ca2+ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein ßγ heterodimers (Gbc) bind to and inhibit Ca2+ channels. Here I review the mechanisms by which GPCRs inhibit Ca2+ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca2+ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gßγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.


Assuntos
Glândulas Suprarrenais/metabolismo , Canais de Cálcio/metabolismo , Catecolaminas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Glândulas Suprarrenais/citologia , Animais , Células Cromafins/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos
12.
Sci Rep ; 10(1): 1300, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992767

RESUMO

The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.


Assuntos
Potenciais de Ação , Receptores de Superfície Celular/deficiência , Células Receptoras Sensoriais/metabolismo , Animais , Biomarcadores , Linhagem Celular , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Técnicas de Patch-Clamp , Células Receptoras Sensoriais/ultraestrutura
13.
J Neurosci ; 26(51): 13373-83, 2006 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-17182788

RESUMO

Precise regulation of N-type (Ca(V)2.2) voltage-gated calcium channels (Ca-channels) controls many cellular functions including neurotransmitter and hormone release. One important mechanism that inhibits Ca2+ entry involves binding of G-protein betagamma subunits (Gbetagamma) to the Ca-channels. This shifts the Ca-channels from "willing" to "reluctant" gating states and slows activation. Voltage-dependent reversal of the inhibition (facilitation) is thought to reflect transient dissociation of Gbetagamma from the Ca-channels and can occur during high-frequency bursts of action potential-like waveforms (APW). Inactivation of Ca-channels will also limit Ca2+ entry, but it remains unclear whether G-proteins can modulate inactivation. In part this is because of the complex nature of inactivation, and because facilitation of Ca-channel currents (I(Ca)) masks the extent and kinetics of inactivation during typical stimulation protocols. We used low-frequency trains of APW to activate I(Ca). This more closely mimics physiological stimuli and circumvents the problem of facilitation which does not occur at < or = 5 Hz. Activation of endogenous G-proteins reduced both Ca2+-dependent, and voltage-dependent inactivation of recombinant I(Ca) in human embryonic kidney 293 cells. This was mimicked by expression of wild-type Gbetagamma, but not by a point mutant of Gbetagamma with reduced affinity for Ca-channels. A similar decrease in the inactivation of I(Ca) was produced by P2Y receptors in adrenal chromaffin cells. Overall, our data identify and characterize a novel effect of G-proteins on I(Ca), and could have important implications for understanding how G-protein-coupled receptors control Ca2+ entry and Ca2+-dependent events such as neurotransmitter and hormone release.


Assuntos
Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo N/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Potenciais de Ação/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/fisiologia , Sinalização do Cálcio/fisiologia , Bovinos , Linhagem Celular , Células Cromafins/metabolismo , Células Cromafins/fisiologia , Proteínas de Ligação ao GTP/farmacologia , Humanos
14.
ACS Chem Neurosci ; 8(5): 943-954, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28406285

RESUMO

Serotonin (5-HT) is an important neurotransmitter in the central nervous system where it modulates circuits involved in mood, cognition, movement, arousal, and autonomic function. The 5-HT transporter (SERT; SLC6A4) is a key regulator of 5-HT signaling, and genetic variations in SERT are associated with various disorders including depression, anxiety, and autism. This review focuses on the role of SERT in the sympathetic nervous system. Autonomic/sympathetic dysfunction is evident in patients with depression, anxiety, and other diseases linked to serotonergic signaling. Experimentally, loss of SERT function (SERT knockout mice or chronic pharmacological block) has been reported to augment the sympathetic stress response. Alterations to serotonergic signaling in the CNS and thus central drive to the peripheral sympathetic nervous system are presumed to underlie this augmentation. Although less widely recognized, SERT is robustly expressed in chromaffin cells of the adrenal medulla, the neuroendocrine arm of the sympathetic nervous system. Adrenal chromaffin cells do not synthesize 5-HT but accumulate small amounts by SERT-mediated uptake. Recent evidence demonstrated that 5-HT1A receptors inhibit catecholamine secretion from adrenal chromaffin cells via an atypical mechanism that does not involve modulation of cellular excitability or voltage-gated Ca2+ channels. This raises the possibility that the adrenal medulla is a previously unrecognized peripheral hub for serotonergic control of the sympathetic stress response. As a framework for future investigation, a model is proposed in which stress-evoked adrenal catecholamine secretion is fine-tuned by SERT-modulated autocrine 5-HT signaling.


Assuntos
Medula Suprarrenal/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Estresse Fisiológico/fisiologia , Sistema Nervoso Simpático/metabolismo , Animais , Nível de Alerta/fisiologia , Células Cromafins/metabolismo , Humanos , Transmissão Sináptica/fisiologia
15.
Neuropharmacology ; 110(Pt A): 438-448, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27544824

RESUMO

Adrenal chromaffin cells (ACCs), the neuroendocrine arm of the sympathetic nervous system, secrete catecholamines to mediate the physiological response to stress. Although ACCs do not synthesize 5-HT, they express the serotonin transporter (SERT). Genetic variations in SERT are linked to several CNS disorders but the role(s) of SERT/5-HT in ACCs has remained unclear. Adrenal glands from wild-type mice contained 5-HT at ≈ 750 fold lower abundance than adrenaline, and in SERT(-/-) mice this was reduced by ≈80% with no change in catecholamines. Carbon fibre amperometry showed that SERT modulated the ability of 5-HT1A receptors to inhibit exocytosis. 5-HT reduced the number of amperometric spikes (vesicular fusion events) evoked by KCl in SERT(-/-) cells and wild-type cells treated with escitalopram, a SERT antagonist. The 5-HT1A receptor antagonist WAY100635 blocked the inhibition by 5-HT which was mimicked by the 5-HT1A agonist 8-OH-DPAT but not the 5-HT1B agonist CP93129. There was no effect on voltage-gated Ca(2+) channels, K(+) channels, or intracellular [Ca(2+)] handling, showing the 5-HT receptors recruit an atypical inhibitory mechanism. Spike charge and kinetics were not altered by 5-HT receptors but were reduced in SERT(-/-) cells compared to wild-type cells. Our data reveal a novel role for SERT and suggest that adrenal chromaffin cells might be a previously unrecognized hub for serotonergic control of the sympathetic stress response.


Assuntos
Glândulas Suprarrenais/metabolismo , Células Cromafins/metabolismo , Receptores de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Cátions Bivalentes/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Serotonina/metabolismo , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
16.
PLoS One ; 10(7): e0134117, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26222492

RESUMO

CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel ß-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-ß-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Ativação Enzimática/efeitos dos fármacos , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Cinética , Técnicas de Patch-Clamp , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tionucleotídeos/metabolismo
17.
Metabolism ; 52(5): 528-34, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12759879

RESUMO

Genetic variation in the gene for a cytosolic cysteine protease, calpain-10, increases the susceptibility to type 2 diabetes apparently by altering levels of gene expression. In view of the importance of altered beta-cell function in the pathophysiology of type 2 diabetes, the present study was undertaken to define the effects on insulin secretion of exposing pancreatic islets to calpain inhibitors for 48 hours. Exposure of mouse islets to calpain inhibitors (ALLN, ALLM, E-64-d, MDL 18270, and PD147631) of different structure and mechanism of action for 48 hours reversibly suppresses glucose-induced insulin secretion by 40% to 80%. Exposure of islets to inhibitors of other proteases, ie, cathepsin B and proteasome, did not affect insulin secretion. The 48-hour incubation with calpain inhibitors also attenuates insulin secretory responses to the mitochondrial fuel alpha-ketoisocaproate (KIC). The same incubation also suppresses glucose metabolism and intracellular calcium ([Ca(2+)](i)) responses to glucose or KIC in islets. In summary, long-term inhibition of islet calpain activity attenuates insulin secretion possibly by limiting the rate of glucose metabolism. A reduction of calpain activity in islet could contribute to the development of beta-cell failure in type 2 diabetes thereby providing a link between genetic susceptibility to diabetes and the pathophysiologic manifestations of the disease.


Assuntos
Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Ilhotas Pancreáticas/metabolismo , Leucina/análogos & derivados , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Separação Celular , Dipeptídeos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , NADP/metabolismo , Oxirredução
18.
PLoS One ; 9(10): e109203, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25275439

RESUMO

Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca)) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca). We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.


Assuntos
Butanóis/farmacologia , Canais de Cálcio/efeitos dos fármacos , Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Ativação do Canal Iônico , Animais , Butanóis/química , Cálcio/metabolismo , Bovinos , Células Cromafins/metabolismo , Isomerismo
19.
Lab Chip ; 13(23): 4663-73, 2013 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-24126415

RESUMO

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.


Assuntos
Catecolaminas/análise , Células Cromafins/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas Eletroquímicas , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Cálcio/metabolismo , Carbacol/farmacologia , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Dimetilpolisiloxanos , Eletrodos , Cinética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Platina/química , Cloreto de Potássio/farmacologia , Silício/química , Estimulação Química
20.
Biosens Bioelectron ; 34(1): 30-6, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22398270

RESUMO

Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 µm×300 µm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 µM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrode's solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 µM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Catecolaminas/análise , Técnicas Eletroquímicas/métodos , Microfluídica , Técnicas Biossensoriais/instrumentação , Células Cromafins/química , Desenho de Equipamento , Exocitose , Humanos , Irídio/química , Luminescência , Microeletrodos , Trombina/análise
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