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This corrects the article DOI: 10.1038/ncomms11947.
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Vascular endothelial transient potential (TRP) channels, located mostly on the plasma membrane of cells, are critical in regulatory and pathophysiological circumstances. The objective of this chapter is to describe several well-established approaches, ranging from function to molecular assays, to investigate the mechanistic role of TRP channels in vascular endothelial cells. We show experimental procedures and representative figures on the following methods: (1) Isolation and culture of vascular endothelial cells, (2) examination of electrophysiological activity of TRP channel by patch-clamping with whole-cell configuration and its function in vascular tone and blood flow by isometric tension and isobaric diameter measurements, and Laser Doppler flowmetry, (3) detection of TRP channel-mediated intracellular Ca2+ imaging by using fluorescent microscopy, and (4) determination of TRP channel interaction by coimmunoprecipitation, double immunofluorescence staining and Förster resonance energy transfer (FRET) detection.
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Células Endoteliales/fisiología , Potenciales de la Membrana , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Aorta/citología , Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ratas , Ratas Sprague-DawleyRESUMEN
Blood pressure is maintained within a normal physiological range by a sophisticated regulatory mechanism. Baroreceptors serve as a frontline sensor to detect the change in blood pressure. Nerve signals are then sent to the cardiovascular control centre in the brain in order to stimulate baroreflex responses. Here, we identify TRPC5 channels as a mechanical sensor in aortic baroreceptors. In Trpc5 knockout mice, the pressure-induced action potential firings in the afferent nerve and the baroreflex-mediated heart rate reduction are attenuated. Telemetric measurements of blood pressure demonstrate that Trpc5 knockout mice display severe daily blood pressure fluctuation. Our results suggest that TRPC5 channels represent a key pressure transducer in the baroreceptors and play an important role in maintaining blood pressure stability. Because baroreceptor dysfunction contributes to a variety of cardiovascular diseases including hypertension, heart failure and myocardial infarction, our findings may have important future clinical implications.
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Aorta/fisiología , Presión Sanguínea/fisiología , Presorreceptores/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Frecuencia Cardíaca/fisiología , Activación del Canal Iónico , Masculino , Mecanotransducción Celular , Ratones Noqueados , Neuronas/metabolismo , Concentración Osmolar , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²âº ([Ca²âº](i)) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²âº](i) rise in iPSC-ECs from normal individuals but a sustained [Ca²âº](i) elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca²âº](i) rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²âº](i) rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²âº](i) elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.
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Calcio/metabolismo , Citosol/metabolismo , Regulación de la Expresión Génica/fisiología , Células Madre Pluripotentes/metabolismo , Progeria/metabolismo , Canales Catiónicos TRPV/metabolismo , Humanos , Immunoblotting , Microscopía Confocal , Presión Osmótica/fisiología , Progeria/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVES: Early life psychological stress is an essential factor contributing to the development of irritable bowel syndrome (IBS), with corticotrophin releasing factor (CRF) having been implicated in this common gastrointestinal disorder. The aim of our study is to examine the effect of neonatal maternal separation (NMS), an early life stress model, on the brain CRF expression following visceral pain induced by colorectal distension (CRD) stimuli in male rats. METHODS: Male neonatal Sprague-Dawley rats were subjected to 3-hr daily maternal separation on postnatal day 2-21, with unseparated normal (N) rats serving as controls. Electromyogram signals (EMG) in response to phasic CRD were measured. The results demonstrated an increased pain response and EMG magnitudes in NMS rats as compared to N rats in response to CRD stimulation. The mRNA and protein expressions of CRF in hippocampus, cortex and thalamus of NMS and N group following the CRD stress were determined by real-time quantitative PCR and western-blotting studies respectively. RESULTS: There was an increased mRNA and protein level of CRF in thalamus of NMS rats but no apparent change in CRF expression in hippocampus and cortex of both groups. Furthermore, an increased expression of CRF type 1 receptor (CRF-R1) was observed in the thalamus of NMS rats. CONCLUSION: These results suggested an up-regulation of thalamus CRF-R1 is associated with visceral hyperalgesia in the rat model of NMS.
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Dolor Abdominal/metabolismo , Hiperalgesia/metabolismo , Privación Materna , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Estrés Psicológico/metabolismo , Tálamo/metabolismo , Dolor Abdominal/fisiopatología , Dolor Abdominal/psicología , Animales , Animales Recién Nacidos/metabolismo , Animales Recién Nacidos/psicología , Corteza Cerebral/metabolismo , Colon/fisiopatología , Dilatación Patológica/fisiopatología , Electromiografía , Hipocampo/metabolismo , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Umbral del Dolor/psicología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/genética , Recto/fisiopatología , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: The employment of well characterized test samples prepared from authenticated, high quality medicinal plant materials is key to reproducible herbal research. The present study aims to demonstrate a quality assurance program covering the acquisition, botanical validation, chemical standardization and good manufacturing practices (GMP) production of IBS-20, a 20-herb Chinese herbal formula under study as a potential agent for the treatment of irritable bowel syndrome. METHODS: Purity and contaminant tests for the presence of toxic metals, pesticide residues, mycotoxins and microorganisms were performed. Qualitative chemical fingerprint analysis and quantitation of marker compounds of the herbs, as well as that of the IBS-20 formula was carried out with high-performance liquid chromatography (HPLC). Extraction and manufacture of the 20-herb formula were carried out under GMP. Chemical standardization was performed with liquid chromatography-mass spectrometry (LC-MS) analysis. Stability of the formula was monitored with HPLC in real time. RESULTS: Quality component herbs, purchased from a GMP supplier were botanically and chemically authenticated and quantitative HPLC profiles (fingerprints) of each component herb and of the composite formula were established. An aqueous extract of the mixture of the 20 herbs was prepared and formulated into IBS-20, which was chemically standardized by LC-MS, with 20 chemical compounds serving as reference markers. The stability of the formula was monitored and shown to be stable at room temperature. CONCLUSION: A quality assurance program has been developed for the preparation of a standardized 20-herb formulation for use in the clinical studies for the treatment of irritable bowel syndrome (IBS). The procedures developed in the present study will serve as a protocol for other poly-herbal Chinese medicine studies.
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Sleep apnea associated with chronic intermittent hypoxia (IH) impairs hippocampal functions but the pathogenic mechanisms involving dysfunction of nitric oxide (NO) and ionic channels remain unclear. We examined the hypothesis that hippocampal NO deficit impairs the activity of large conductance calcium-activated potassium (BK) channels in rats with chronic IH, mimicking conditions in patients with sleep apnea. A patch-clamp study was performed on hippocampal CA1 neurons acutely dissociated from IH and control rats. The levels of endogenous NO and intracellular calcium in the CA1 region of the hippocampal slices were measured respectively by electrochemical microsensors and spectrofluorometry. We found that the open probability of BK channels remarkably decreased in the CA1 pyramidal neurons in a time-dependent manner with the IH treatment, without changes in the unitary conductance and reversal potential. NO donors, SNP or DETA/NO, significantly restored the activity of BK channels in the IH neurons, which was prevented by blockade of S-nitrosylation with NEM or MTSES but not by inhibition of the cGMP pathway with ODQ or 8-bromo-cGMP. Endogenous NO levels were substantially lowered in the IH hippocampus during resting and hypoxia. Also, the level of protein expression of neuronal NO synthase was markedly lessened in the IH neurons with decreased intracellular calcium response to hypoxia. Collectively, the results suggest that the IH-induced NO deficit mediated by a down-regulation of the expression of neuronal NO synthase plays a causative role in the impaired activity of BK channels, which could account for the hippocampal injury in patients with sleep apnea.
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Hipocampo/metabolismo , Hipoxia/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Óxido Nítrico/fisiología , Animales , Calcio/metabolismo , Enfermedad Crónica , Coenzimas/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/análisis , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Hypoxia regulates the local expression of angiotensin-generating system in the rat carotid body and the me-tabolite angiotensin IV (Ang IV) may be involved in the modulation of carotid body function. We tested the hypothesis that Ang IV-binding angiotensin AT(4) receptors play a role in the adaptive change of the carotid body in hypoxia. The expression and localization of Ang IV-binding sites and AT(4) receptors in the rat carotid bodies were studied with histochemistry. Specific fluorescein-labeled Ang IV binding sites and positive staining of AT(4) immunoreactivity were mainly found in lobules in the carotid body. Double-labeling study showed the AT(4) receptor was localized in glomus cells containing tyrosine hydroxylase, suggesting the expression in the chemosensitive cells. Intriguingly, the Ang IV-binding and AT(4) immunoreactivity were more intense in the carotid body of chronically hypoxic (CH) rats (breathing 10% oxygen for 4 weeks) than the normoxic (Nx) control. Also, the protein level of AT(4) receptor was doubled in the CH comparing with the Nx group, supporting an upregulation of the expression in hypoxia. To examine if Ang IV induces intracellular Ca(2+) response in the carotid body, cytosolic calcium ([Ca(2+)](i)) was measured by spectrofluorimetry in fura-2-loaded glomus cells dissociated from CH and Nx carotid bodies. Exogenous Ang IV elevated [Ca(2+)](i) in the glomus cells and the Ang IV response was significantly greater in the CH than the Nx group. Hence, hypoxia induces an upregulation of the expression of AT(4) receptors in the glomus cells of the carotid body with an increase in the Ang IV-induced [Ca(2+)]i elevation. This may be an additional pathway enhancing the Ang II action for the activation of chemoreflex in the hypoxic response during chronic hypoxia.
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Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.
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Isquemia Encefálica/metabolismo , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Hipoxia de la Célula , Hipocampo/metabolismo , Neuronas/metabolismo , Óxido Nítrico/farmacología , Animales , Arginina/farmacología , Isquemia Encefálica/patología , Células Cultivadas , GMP Cíclico/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Fura-2 , Hipocampo/citología , NG-Nitroarginina Metil Éster/farmacología , Neuronas/citología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Oxígeno/metabolismo , Ratas , Ratas Sprague-Dawley , S-Nitrosotioles/metabolismoRESUMEN
Melatonin modulates the carotid chemoreceptor response to chemical stimuli, and chronic hypoxia changes circadian activities and carotid body function. The purpose of this study was to test the hypothesis that chronic hypoxia alters the function and expression of melatonin receptors in the rat carotid body. Effects of melatonin on the carotid responses to hypercapnic acidosis and to hypoxia were determined by spectrofluorometric measurement of cytosolic calcium ([Ca(2+)](i)) in fura-2-loaded type-I (glomus) cells dissociated from carotid bodies obtained from normoxic (Nx) or chronically hypoxic (CH) rats breathing 10% oxygen for 4 wk. In the Nx control, melatonin concentration dependently attenuated the peak [Ca(2+)](i) response to hypercapnic acidosis, whereas it augmented the [Ca(2+)](i) response to cyanide or deoxygenated buffer. Yet, melatonin enhanced the peak [Ca(2+)](i) responses to hypercapnic acidosis or hypoxia in the CH glomus cells. An agonist of melatonin receptors, iodomelatonin also elevated the hypercapnic or hypoxic responses in the CH groups. The melatonin-induced changes in the [Ca(2+)](i) responses were abolished by pretreatment with nonselective mt(1)/MT(2) antagonist, luzindole, and by MT(2) antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest a functional modulation of melatonin receptors in the glomus cells in chronic hypoxia. To evaluate the level of expression of the melatonin receptors, in situ hybridization study with antisense mt(1) and MT(2) receptor mRNA oligonucleotide probes was performed on the Nx and CH carotid bodies. There were significant increases in the expression of mt(1) and MT(2) receptors in the CH comparing with the Nx group. Taken together, our results suggest an upregulation of the carotid expression of melatonin receptors by chronic hypoxia, which modulates the carotid response to melatonin for the circadian influence on breathing.
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Cuerpo Carotídeo/metabolismo , Hipoxia/metabolismo , Receptores de Melatonina/metabolismo , Animales , Calcio/metabolismo , Cuerpo Carotídeo/patología , Enfermedad Crónica , Hipercapnia/metabolismo , Hipoxia/patología , Ratas , Receptores de Melatonina/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
Melatonin attenuates carotid chemoreceptor response to hypercapnic acidosis and may contribute to the effect of circadian rhythms on the chemoreflex. The purpose of this study was to test the hypothesis that melatonin modulates rat carotid chemoreceptor response to hypoxia. To examine the effect of melatonin on the hypoxic response of the chemosensitive cells, cytosolic calcium ([Ca2+]i) was measured by spectrofluorometry in fura-2-loaded type-I (glomus) cells dissociated from rat carotid bodies. Melatonin (0.01-10 nm) did not change the resting Ca2+]i level of the glomus cells but it concentration-dependently increased peak Ca2+]i response to cyanide or deoxygenated buffer. An agonist of melatonin receptors, iodomelatonin also enhanced the Ca2+]i response to hypoxia. The melatonin-induced enhancement of the Ca2+]i response was abolished by pretreatment with nonselective mt1/MT2 antagonist, luzindole, and by MT2 antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest that melatonin receptors in the glomus cells mediate the effect of melatonin on the chemoreceptor response to hypoxia. In addition, melatonin increased the carotid afferent response to hypoxia in unitary activities recorded from the sinus nerve in isolated carotid bodies superfused with bicarbonate-buffer saline. Furthermore, plethysmographic measurement of ventilatory activities in unanesthetized rats revealed that melatonin (1 mg/kg, i.p.) increased the ventilatory response to hypoxia. Hence, the circadian rhythm of melatonin in arterial blood can modulate the carotid chemoreceptor response to hypoxia. This modulation may be a physiological mechanism involved in the day-light differences in ventilatory activities.
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Cuerpo Carotídeo/efectos de los fármacos , Cuerpo Carotídeo/fisiopatología , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/fisiopatología , Hipoxia/etiología , Hipoxia/fisiopatología , Melatonina/análogos & derivados , Melatonina/farmacología , Animales , Calcio/metabolismo , Ritmo Circadiano/fisiología , Técnicas In Vitro , Melatonina/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Melatonina/efectos de los fármacos , Receptores de Melatonina/fisiología , Respiración/efectos de los fármacos , Tetrahidronaftalenos/farmacología , Triptaminas/farmacologíaRESUMEN
Respiratory activity is under circadian modulation and the physiological mechanisms may involve the pineal secretory product, melatonin, and the carotid chemoreceptor. We hypothesized that melatonin modulates the carotid chemoreceptor response to hypercapnic acidosis. To determine whether the effect of melatonin on the chemoreceptor response to hypercapnic acidosis is mediated by melatonin receptors in the chemosensitive cells, cytosolic calcium ([Ca2+]i) was measured by spectrofluorometry in fura-2-loaded glomus cells dissociated from rat carotid bodies. Melatonin (0.01-10 nm) per se did not change the [Ca2+]i levels of the glomus cells but it concentration-dependently attenuated the peak [Ca2+]i response to hypercapnic acidosis in the glomus cells. In addition, the [Ca2+]i response was attenuated by 2-iodomelatonin, an agonist of melatonin receptors. The melatonin-induced attenuation of the [Ca2+]i response to hypercapnic acidosis was abolished by pretreatment with an non-selective mt1/MT2 antagonist, luzindole, and by MT2 antagonists, 4-phenyl-2-propionamidotetraline or DH97. In situ hybridization study with antisense mt1 and MT2 receptor mRNA oligonucleotide probes showed an expression of mt1 and MT2 receptors in the rat carotid body. Also, melatonin attenuated the carotid afferent response to hypercapnic acidosis in single- or pauci-fibers recorded from the sinus nerve in isolated carotid bodies superfused with bicarbonate-buffer saline. Results suggest that an activation of the melatonin receptors expressed in the glomus cells of the rat carotid body reduces the chemoreceptor response to hypercapnic acidosis. This modulation may play a physiological role in the influence of the circadian rhythms on the chemoreflex.
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Acidosis Respiratoria/tratamiento farmacológico , Cuerpo Carotídeo/efectos de los fármacos , Células Quimiorreceptoras/efectos de los fármacos , Hipercapnia/tratamiento farmacológico , Melatonina/análogos & derivados , Melatonina/farmacología , Acidosis Respiratoria/metabolismo , Animales , Calcio/metabolismo , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/metabolismo , Electrofisiología/métodos , Fura-2/metabolismo , Hipercapnia/metabolismo , Melatonina/antagonistas & inhibidores , Melatonina/fisiología , Ratas , Ratas Sprague-Dawley , Receptor de Melatonina MT1/antagonistas & inhibidores , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/antagonistas & inhibidores , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Receptores de Melatonina/antagonistas & inhibidores , Receptores de Melatonina/efectos de los fármacos , Tetrahidronaftalenos/farmacología , Triptaminas/farmacologíaRESUMEN
Endothelin-1 (ET-1) excites carotid body (CB) chemoreceptors and induces mitosis of the chemoreceptors in chronic hypoxia. The aim of the present study was to examine the hypothesis that up-regulation of both ETA receptor and endogenous ET-1 expression in CB chemoreceptors enhances the response of intracellular Ca2+ to ET-1 following adaptation to chronic hypoxia (10% inspired O2 for 3-4 weeks). Cytosolic free [Ca2+] ([Ca2+]i) in type-I (glomus) cells freshly dissociated from rat CBs was measured by spectrofluorometry. Application of exogenous ET-1 (1-100 nM) concentration-dependently elevated [Ca2+]i in the glomus cells. This response to ET-1 (100 nM) was 49% greater in the chronically hypoxic (CH) group. The ET-1 response was abolished completely by the ETA receptor antagonist BQ610 (1 microM), but not by the ETB antagonist BQ788 (1 microM). The transient [Ca2+]i elevation induced by caffeine (30 mM) in the normoxic group was similar to that in the CH group, suggesting no differences in the intracellular Ca2+ stores. In situ hybridization with a digoxigenin-labelled antisense ETA receptor mRNA oligonucleotide probe revealed very intense and ubiquitous specific expression of ETA receptors in the lobules of glomus cells in the CH group, whereas staining in normoxic controls was light. Immunohistochemical studies revealed intense cytoplasmic staining for ET-1-immunoreactivity in most of the cell clusters in glomera in the CBs of CH rats but was faint in normoxic CBs. These findings indicate increased expression of both the ETA receptor and ET-1 in CB chemoreceptors during chronic hypoxia. Taken together, our results suggest that the [Ca2+]i response to ET-1 in rat CB chemoreceptors is augmented by up-regulation of ETA receptors and ET-1 expression. The enhancement of the paracrine/autocrine effect of ET-1 on the chemoreceptors is consistent with an excitatory and mitogenic role of the ET-1 and ETA receptor in the CB during chronic hypoxia.