RESUMEN
KEY POINTS: Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co-cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization-activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih . ABSTRACT: Adenosine and dopamine (DA) act as neurotransmitters or neuromodulators at the carotid body (CB) chemosensory synapse, but their mechanisms of action are not fully understood. Using a functional co-culture model of rat CB chemoreceptor (type I) cell clusters and juxtaposed afferent petrosal neurons (PNs), we tested the hypothesis that adenosine and DA act postsynaptically to modulate a hyperpolarization-activated, cyclic nucleotide-gated (HCN) cation current (Ih ). In whole-cell recordings from hypoxia-responsive PNs, cAMP mimetics enhanced Ih whereas the HCN blocker ZD7288 (2 µm) reversibly inhibited Ih . Adenosine caused a potentiation of Ih (EC50 â¼ 35 nm) that was sensitive to the A2a blocker SCH58261 (5 nm), and an â¼16 mV depolarizing shift in V½ for voltage dependence of Ih activation. By contrast, DA (10 µm) caused an inhibition of Ih that was sensitive to the D2 blocker sulpiride (1-10 µm), and an â¼11 mV hyperpolarizing shift in V½ . Sulpiride potentiated Ih in neurons adjacent to, but not distant from, type I cell clusters. DA also decreased PN action potential frequency whereas adenosine had the opposite effect. During simultaneous paired recordings, SCH58261 inhibited both the presynaptic hypoxia-induced receptor potential in type I cells and the postsynaptic PN response. By contrast, SCH58261 inhibited only the postsynaptic PN response induced by isohydric hypercapnia. Confocal immunofluorescence confirmed the localization of HCN4 subunits in tyrosine hydroxylase-positive chemoafferent neurons in tissue sections of rat petrosal ganglia. These data suggest that adenosine and DA, acting through A2a and D2 receptors respectively, regulate PN excitability via their opposing actions on Ih .
Asunto(s)
Adenosina/farmacología , Dopamina/farmacología , Neuronas/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Cocultivo , Ganglio Geniculado/citología , Ganglio Geniculado/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neuronas/fisiología , Canales de Potasio/metabolismo , Ratas WistarRESUMEN
The carotid body (CB) chemoreflex maintains blood Po2 and Pco2/H+ homeostasis and displays sensory plasticity during exposure to chronic hypoxia. Purinergic signaling via P1 and P2 receptors plays a pivotal role in shaping the afferent discharge at the sensory synapse containing catecholaminergic chemoreceptor (type I) cells, glial-like type II cells, and sensory (petrosal) nerve endings. However, little is known about the family of ectonucleotidases that control synaptic nucleotide levels. Using quantitative PCR (qPCR), we first compared expression levels of ectonucleoside triphosphate diphosphohydrolases (NTPDases1,2,3,5,6) and ecto-5'-nucleotidase (E5'Nt/CD73) mRNAs in juvenile rat CB vs. brain, petrosal ganglia, sympathetic (superior cervical) ganglia, and a sympathoadrenal chromaffin (MAH) cell line. In whole CB extracts, qPCR revealed a high relative expression of surface-located members NTPDase1,2 and E5'Nt/CD73, compared with low NTPDase3 expression. Immunofluorescence staining of CB sections or dissociated CB cultures localized NTPDase2,3 and E5'Nt/CD73 protein to the periphery of type I clusters, and in association with sensory nerve fibers and/or isolated type II cells. Interestingly, in CBs obtained from rats reared under chronic hypobaric hypoxia (~60 kPa, equivalent to 4,300 m) for 5-7 days, in addition to the expected upregulation of tyrosine hydroxylase and VEGF mRNAs, there was a significant upregulation of NTPDase3 and E5'Nt/CD73 mRNA, but a downregulation of NTPDase1 and NTPDase2 relative to normoxic controls. We conclude that NTPDase1,2,3 and E5'Nt/CD73 are the predominant surface-located ectonucleotidases in the rat CB and suggest that their differential regulation during chronic hypoxia may contribute to CB plasticity via control of synaptic ATP, ADP, and adenosine pools.
Asunto(s)
5'-Nucleotidasa/metabolismo , Encéfalo/enzimología , Cuerpo Carotídeo/enzimología , Regulación Enzimológica de la Expresión Génica , Hipoxia/metabolismo , Plasticidad Neuronal , Nervios Periféricos/enzimología , Animales , Enfermedad Crónica , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
Signal processing in the carotid body (CB) is initiated at receptor glomus (or type I) cells which depolarize and release the excitatory neurotransmitter ATP during chemoexcitation by hypoxia and acid hypercapnia. Glomus cell clusters (GCs) occur in intimate association with glia-like type II cells which express purinergic P2Y2 receptors (P2Y2Rs) but their function is unclear. Here we immunolocalize the gap junction-like protein channel pannexin-1 (Panx-1) in type II cells and show Panx-1 mRNA expression in the rat CB. As expected, type II cell activation within or near isolated GCs by P2Y2R agonists, ATP and UTP (100 µm), induced a rise in intracellular [Ca(2+)]. Moreover in perforated-patch whole cell recordings from type II cells, these agonists caused a prolonged depolarization and a concentration-dependent, delayed opening of non-selective ion channels that was prevented by Panx-1 blockers, carbenoxolone (5 µm) and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS; 10 µm). Because Panx-1 channels serve as conduits for ATP release, we hypothesized that paracrine, type II cell P2Y2R activation leads to ATP-induced ATP release. In proof-of-principle experiments we used co-cultured chemoafferent petrosal neurones (PNs), which express P2X2/3 purinoceptors, as sensitive biosensors of ATP released from type II cells. In several cases, UTP activation of type II cells within or near GCs led to depolarization or increased firing in nearby PNs, and the effect was reversibly abolished by the selective P2X2/3 receptor blocker, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 µm). We propose that CB type II cells may function as ATP amplifiers during chemotransduction via paracrine activation of P2Y2Rs and Panx-1 channels.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/metabolismo , Conexinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Conexinas/antagonistas & inhibidores , Conexinas/genética , Fenómenos Electrofisiológicos , Expresión Génica , Potenciales de la Membrana , Modelos Neurológicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neurotransmisores/metabolismo , Comunicación Paracrina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transmisión SinápticaRESUMEN
The carotid body (CB) is a peripheral chemoreceptor organ that initiates compensatory reflex responses so as to maintain gas homeostasis. Stimuli such as low oxygen (hypoxia) and high CO(2)/H(+) (acid hypercapnia) cause an increase in 'afferent' sensory discharge that is relayed via the carotid sinus nerve (CSN) to the brainstem, resulting in corrective changes in ventilation. A parallel autonomic pathway has been recognized for >40 years as the source of 'efferent' inhibition of the CB sensory discharge and, more recently, nitric oxide (NO) has been identified as the key mediator. This review will examine our current understanding of the role of nNOS-positive autonomic neurons, embedded in 'paraganglia' within the glossopharyngeal (GPN) and CSN nerves, in mediating efferent CB chemoreceptor inhibition. We highlight recent data linking the actions of hypoxia, ACh and ATP to NO synthesis/release from GPN neurons. Finally, we consider the novel hypothesis that pannexin-1 channels present in GPN neurons may play a role in NO signaling during hypoxia.
Asunto(s)
Cuerpo Carotídeo/fisiología , Óxido Nítrico/fisiología , Animales , Células Quimiorreceptoras/fisiología , Fenómenos Electrofisiológicos , Nervio Glosofaríngeo/citología , Nervio Glosofaríngeo/fisiología , Humanos , Modelos Neurológicos , Neuronas/fisiología , Neurotransmisores/fisiologíaRESUMEN
Aortic bodies (ABs) are putative peripheral arterial chemoreceptors, distributed near the aortic arch. Though presumed to be analogous to the well-studied carotid bodies (CBs), their anatomical organization, innervation, and function are poorly understood. By using multilabel confocal immunofluorescence, we investigated the cellular organization, innervation, and neurochemistry of ABs in whole mounts of juvenile rat vagus and recurrent laryngeal (V-RL) nerves and in dissociated cell culture. Clusters of tyrosine hydroxylase-immunoreactive (TH-IR) glomus cells were routinely identified within these nerves. Unlike the CB, many neuronal cell bodies and processes, identified by peripherin (PR) and neurofilament/growth-associated protein (NF70/GAP-43) immunoreactivity, were closely associated with AB glomus clusters, especially near the V-RL bifurcation. Some neuronal cell bodies were immunopositive for P2X2 and P2X3 purinoceptor subunits, which were also found in nerve terminals surrounding glomus cells. Immunoreactivity against the vesicular acetylcholine transporter (VAChT) was detected in local neurons, glomus cells, and apposed nerve terminals. Few neurons were immunopositive for TH or neuronal nitric oxide synthase. A similar pattern of purinoceptor immunoreactivity was observed in tissue sections of adult rat V-RL nerves, except that glomus cells were weakly P2X3-IR. Dissociated monolayer cultures of juvenile rat V-RL nerves yielded TH-IR glomus clusters in intimate association with PR- or NF70/GAP-43-IR neurons and their processes, and glial fibrillary acidic protein-IR type II (sustentacular) cells. Cocultures survived for several days, wherein neurons expressed voltage-activated ionic currents and generated action potentials. Thus, this coculture model is attractive for investigating the role of glomus cells and local neurons in AB function.
Asunto(s)
Cuerpos Aórticos/citología , Células Quimiorreceptoras/citología , Técnica del Anticuerpo Fluorescente/métodos , Microscopía Confocal/métodos , Neuronas/citología , Animales , Cuerpos Aórticos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Células Quimiorreceptoras/metabolismo , Nervios Laríngeos/citología , Nervios Laríngeos/metabolismo , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Nervio Vago/citología , Nervio Vago/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
GABA is expressed in carotid body (CB) chemoreceptor type I cells and has previously been reported to modulate sensory transmission via presynaptic GABA(B) receptors. Because low doses of clinically important GABA(A) receptor (GABA(A)R) agonists, e.g. benzodiazepines, have been reported to depress afferent CB responses to hypoxia, we investigated the potential contribution of GABA(A)R in co-cultures of rat type I cells and sensory petrosal neurones (PNs). During gramicidin perforated-patch recordings (to preserve intracellular Cl-), GABA and/or the GABA(A) agonist muscimol (50 microm) induced a bicuculline-sensitive membrane depolarization in isolated PNs. GABA-induced whole-cell currents reversed at approximately -38 mV and had an EC50 of approximately 10 microm (Hill coefficient = approximately 1) at -60 mV. During simultaneous PN and type I cell recordings at functional chemosensory units in co-culture, bicuculline reversibly potentiated the PN, but not type I cell, depolarizing response to hypoxia. Application of the CB excitatory neurotransmitter ATP (1 microm) over the soma of functional PN induced a spike discharge that was markedly suppressed during co-application with GABA (2 microm), even though GABA alone was excitatory. RT-PCR analysis detected expression of GABAergic markers including mRNA for alpha1, alpha2, beta2, gamma2S, gamma2L and gamma3 GABA(A)R subunits in petrosal ganglia extracts. Also, CB extracts contained mRNAs for GABA biosynthetic markers, i.e. glutamate decarboxylase (GAD) isoforms GAD 67A,E, and GABA transporter isoforms GAT 2,3 and BGT-1. In CB sections, sensory nerve endings apposed to type I cells were immunopositive for the GABA(A)R beta subunit. These data suggest that GABA, released from the CB during hypoxia, inhibits sensory discharge postsynaptically via a shunting mechanism involving GABA(A) receptors.
Asunto(s)
Cuerpo Carotídeo/citología , Células Quimiorreceptoras/fisiología , Receptores de GABA-A/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adenosina Trifosfato/farmacología , Animales , Cuerpo Carotídeo/metabolismo , Hipoxia de la Célula/fisiología , Células Cultivadas , Células Quimiorreceptoras/efectos de los fármacos , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Agonistas de Receptores GABA-B , Antagonistas de Receptores de GABA-B , Ganglios/citología , Ganglios/metabolismo , Expresión Génica/efectos de los fármacos , Glutamato Descarboxilasa/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Wistar , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-B/fisiología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
In mammals, ventilation is peripherally controlled by the carotid body (CB), which receives afferent innervation from the petrosal ganglion and efferent innervation from neurons located along the glossopharyngeal nerve (GPN). GPN neurons give rise to the "efferent inhibitory" pathway via a plexus of neuronal nitric oxide (NO) synthase-positive fibers, believed to be responsible for CB chemoreceptor inhibition via NO release. Although NO is elevated during natural CB stimulation by hypoxia, the underlying mechanisms are unclear. We hypothesized that ATP, released by rat CB chemoreceptors (type 1 cells) and/or red blood cells during hypoxia, may directly activate GPN neurons and contribute to NO-mediated inhibition. Using combined electrophysiological, molecular, and confocal immunofluorescence techniques, we detected the expression of multiple P2X receptors in GPN neurons. These receptors involve at least four different purinergic subunits: P2X2 [and the splice variant P2X2(b)], P2X3, P2X4, and P2X7. Using a novel coculture preparation of CB type I cell clusters and GPN neurons, we tested the role of P2X signaling on CB function. In cocultures, fast application of ATP, or its synthetic analog 2',3'-O-(4 benzoylbenzoyl)-ATP, caused type I cell hyperpolarization that was prevented in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium. These data suggest that ATP released during hypoxic stress from CB chemoreceptors (and/or red blood cells) will cause GPN neuron depolarization mediated by multiple P2X receptors. Activation of this pathway will lead to calcium influx and efferent inhibition of CB chemoreceptors via NO synthesis and consequent release.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Nervio Glosofaríngeo/metabolismo , Inhibición Neural/fisiología , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Animales Recién Nacidos , Cuerpo Carotídeo/citología , Células Cultivadas , Células Quimiorreceptoras/fisiología , Vías Eferentes/citología , Vías Eferentes/efectos de los fármacos , Vías Eferentes/metabolismo , Depuradores de Radicales Libres/farmacología , Nervio Glosofaríngeo/citología , Hipoxia/metabolismo , Hipoxia/fisiopatología , Inhibición Neural/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Subunidades de Proteína/agonistas , Subunidades de Proteína/metabolismo , Agonistas del Receptor Purinérgico P2 , Ratas , Ratas Wistar , Receptores Purinérgicos P2X , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaAsunto(s)
Cuerpo Carotídeo/metabolismo , Receptores Presinapticos/metabolismo , Vías Aferentes/metabolismo , Animales , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/efectos de los fármacos , Células Cultivadas , Células Quimiorreceptoras/metabolismo , Retroalimentación , Hipoxia/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Ratas , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de GABA-B/metabolismo , Receptores Presinapticos/efectos de los fármacos , Serotonina/farmacologíaRESUMEN
Background K+ channels exert control over neuronal excitability by regulating resting potential and input resistance. Here, we show that GABAB receptor-mediated activation of a background K+ conductance modulates transmission at rat carotid body chemosensory synapses in vitro. Carotid body chemoreceptor (type I) cells expressed GABAB(1) and GABAB(2) subunits as well as endogenous GABA. The GABAB receptor agonist baclofen activated an anandamide- and Ba2+-sensitive TASK-1-like background K+ conductance in chemoreceptor cell clusters, but was without effect on voltage-gated Ca2+ channels. Hydroxysaclofen (50 microM), 5-aminovaleric acid (100 microM) and CGP 55845 (100 nM), selective GABAB receptor blockers, potentiated the hypoxia-induced receptor potential; this effect was abolished by pre-treatment with pertussis toxin (PTX; 500 ng ml-1), an inhibitor of Gi, or by H-89 (50 microM), a selective inhibitor of protein kinase A. The protein kinase C inhibitor chelerythrine chloride (100 microM) was without effect on this potentiation. GABAB receptor blockers also caused depolarisation of type I cells in clusters, and enhanced spike discharge in spontaneously firing cells. In functional co-cultures of type I clusters and petrosal sensory neurones, GABAB receptor blockers potentiated hypoxia-induced postsynaptic chemosensory responses mediated by the fast-acting transmitters ACh and ATP. Thus GABAB receptor-mediated activation of TASK-1 or a related channel provides a presynaptic autoregulatory feedback mechanism that modulates fast synaptic transmission in the rat carotid body.
Asunto(s)
Autorreceptores/fisiología , Cuerpo Carotídeo/fisiología , Retroalimentación Fisiológica/fisiología , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/fisiología , Receptores de GABA-B/fisiología , Receptores Presinapticos/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Autorreceptores/efectos de los fármacos , Baclofeno/farmacología , Canales de Calcio/metabolismo , Cuerpo Carotídeo/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electrofisiología , Retroalimentación Fisiológica/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-B , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Potasio/farmacología , Potasio/fisiología , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores de GABA-B/genética , Receptores Presinapticos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/fisiologíaRESUMEN
The regulation of K(+) channels by O(2) levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K(+) channels in the immortalised v-myc, adrenal-derived HNK1(+) (MAH) cell line. MAH cells possess a K(+) conductance that is sensitive to Cd(2+), iberiotoxin and apamin, and which is inhibited by ~24 % when exposed to a hypoxic perfusate (O(2) tension 20 mmHg). This conductance was attributed to high-conductance Ca(2+)-activated K(+) (BK) and small-conductance Ca(2+)-activated K(+) (SK) channels, which are major contributors to the O(2)-sensitive K(+) conductance in adrenomedullary chromaffin cells. Under low [Ca(2+)](i) conditions that prevented activation of Ca(2+)-dependent K(+) conductances, a rapidly activating and slowly inactivating K(+) conductance, sensitive to both TEA and 4-aminopyridine (4-AP), but insensitive to 100 nM charybdotoxin (CTX), was identified. This current was also reduced (by ~25 %) when exposed to hypoxia. The hypoxia-sensitive component of this current was greatly attenuated by 10 mM 4-AP, but was only slightly reduced by 10 mM TEA. This suggests the presence of delayed-rectifier O(2)-sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 alpha-subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K(+) channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O(2)-sensitive K(+) channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O(2) sensing in adrenomedullary chromaffin cells.