RESUMEN
Studies of inflammatory bowel disease (IBD) have been inconclusive in relating microbiota with distribution of inflammation. We report microbiota, host transcriptomics, epigenomics and genetics from matched inflamed and non-inflamed colonic mucosa [50 Crohn's disease (CD); 80 ulcerative colitis (UC); 31 controls]. Changes in community-wide and within-patient microbiota are linked with inflammation, but we find no evidence for a distinct microbial diagnostic signature, probably due to heterogeneous host-microbe interactions, and show only marginal microbiota associations with habitual diet. Epithelial DNA methylation improves disease classification and is associated with both inflammation and microbiota composition. Microbiota sub-groups are driven by dominant Enterbacteriaceae and Bacteroides species, representative strains of which are pro-inflammatory in vitro, are also associated with immune-related epigenetic markers. In conclusion, inflamed and non-inflamed colonic segments in both CD and UC differ in microbiota composition and epigenetic profiles.
Asunto(s)
Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Epigénesis Genética/inmunología , Microbioma Gastrointestinal/inmunología , Interacciones Microbiota-Huesped/inmunología , Adulto , Anciano , Bacteroides/genética , Bacteroides/inmunología , Bacteroides/aislamiento & purificación , Biopsia , Células CACO-2 , Estudios de Casos y Controles , Estudios de Cohortes , Colitis Ulcerosa/genética , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/diagnóstico por imagen , Colon/inmunología , Colon/microbiología , Colon/patología , Colonoscopía , Enfermedad de Crohn/genética , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , ADN Bacteriano/aislamiento & purificación , Enterobacteriaceae/genética , Enterobacteriaceae/inmunología , Enterobacteriaceae/aislamiento & purificación , Epigenómica , Femenino , Microbioma Gastrointestinal/genética , Interacciones Microbiota-Huesped/genética , Humanos , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S/genética , RNA-Seq , Adulto JovenRESUMEN
Research on arterial chemoreceptors, particularly on the carotid body, has been fruitful in the last fifty years, to which this review is addressed. The functional anatomy of the organ appears to be well established. The biophysical bases by which glomus cells transduce chemical changes in the milieu intérieur (hypoxia, hypercapnia, acidosis) into electrical and biochemical changes in glomus cells have received much attention. Physical changes (in temperature, flow and osmolarity) are also detected by the carotid body. Electrical coupling between glomus cells themselves appears as very extensive. Sustentacular cells classically considered as ensheathing glia for glomus cells and nerve endings now appear to behave as stem cells precursors for glomus cells under chronic hypoxic conditions. Many papers have been devoted to transmitters released from glomus cells (acetylcholine, dopamine, ATP) and well as to their effects upon chemosensory nerve activity. Chemosensory neurons have been explored from generation of action potentials at peripheral nerve endings, passing to properties of perikarya at petrosal ganglia and finally at characterization of synaptic transmission at solitary tract nuclei. There is abundant literature on ventilatory and cardiovascular reflexes elicited from arterial chemoreceptors. The transient effects of sudden and brief withdrawal of chemosensory discharges by hyperoxia also provide clues on the role played by carotid bodies in the homeostasis of full organisms.
Asunto(s)
Cuerpo Carotídeo/fisiología , Animales , Cuerpo Carotídeo/efectos de los fármacos , Humanos , Reflejo/efectos de los fármacos , Reflejo/fisiología , Estimulación QuímicaAsunto(s)
Cuerpo Carotídeo/fisiología , Células Quimiorreceptoras/fisiología , Acetilcolina/metabolismo , Adenosina/farmacología , Animales , Cuerpo Carotídeo/efectos de los fármacos , Gatos , Células Quimiorreceptoras/efectos de los fármacos , Dopamina/metabolismo , Técnicas In Vitro , Neurotransmisores/metabolismo , Óxido Nítrico/fisiología , Norepinefrina/metabolismoRESUMEN
The carotid body is a major arterial chemoreceptor that senses low O2 tension, high CO2 tension and low pH in the arterial blood. It is generally believed that neurotransmitters, including acetylcholine (ACh), participate in the genesis of afferent neural output from the carotid body and modulate the function of chemoreceptor cells (glomus cells). Previous pharmacological studies suggest that M1 and M2 muscarinic ACh receptors (mAChRs) are involved in these processes. This study was designed to demonstrate the presence and localization of M1 and M2 mAChRs in the carotid body and in the petrosal ganglion of the cat. Since DNA sequences of the cat M1 and M2 mAChRs were not known, we first determined partial DNA sequences. These sequences and deduced amino acid sequences highly resembled those of human and the rat. Subsequent reverse transcription-polymerase chain reaction (RT-PCR)analysis has demonstrated that mRNAs for M1 and M2 mAChRs are present in the carotid body and the petrosal ganglion of the cat. Immunohistochemistry has indicated that the localization of these receptors appears different. Immunoreactivity for M1 mAChR was strong in nerves in the carotid body. Nerve endings positively stained for M1 mAChR appear to innervate glomus cells. Weak staining for M1 mAChRs was seen in glomus cells. On the other hand, M2 receptor protein seems to be present in glomus cells but not on nerve endings. One third of the neurons in the petrosal ganglion showed immunoreactivity for M1 mAChR. Many neurons and nerve fibers in the petrosal ganglion expressed M2 mAChR immunoreactivity. The results were consistent with previous pharmacological studies. Thus, activation of M1 mAChRs on afferent nerve endings may be linked to the increase in neural output during hypoxia. Further, M1 and M2 mAChRs on glomus cells modulate the release of neurotransmitters.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Carotídeo/metabolismo , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Gatos , ADN Complementario/metabolismo , Femenino , Ganglios Sensoriales/citología , Ganglios Sensoriales/metabolismo , Nervio Glosofaríngeo/citología , Nervio Glosofaríngeo/metabolismo , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Ratas , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genética , Células Receptoras Sensoriales/citología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transmisión Sináptica/fisiologíaAsunto(s)
Cuerpo Carotídeo/metabolismo , Dopamina/metabolismo , Hipoxia/metabolismo , Norepinefrina/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Cuerpo Carotídeo/efectos de los fármacos , Gatos , Diaminas/farmacología , Antagonistas Muscarínicos/farmacología , Pirenzepina/farmacología , Receptor Muscarínico M1 , Receptor Muscarínico M2RESUMEN
We investigated if neuronal nicotinic acetylcholine receptors (nAChRs) are localized in chemoreceptor afferent neurons in the cat petrosal ganglion (PG) and if acetylcholine (ACh) excites chemoreceptor afferent neurons. Immunocytochemistry revealed that a majority of PG neurons expressed alpha 4 and/or alpha 7 subunits of neuronal nAChRs, and a part of them were tyrosine hydroxylase positive. Excitability of cultured PG neurons was studied with patch clamp techniques (whole cell configuration). ACh and nicotine evoked both inward and outward currents. The inward current was partially blocked by removal of extracellular calcium and by antagonists for alpha 4 beta 2 (dihydro-beta-erythroidine) or alpha 7 nAChRs (methyllycaconitine). Outward current was blocked by 4-aminopyridine (4-AP) and sometimes by atropine. ACh-induced membrane potential changes were well correlated with ACh-induced currents. Depolarization and hyperpolarization occurred in response to ACh. Occasionally depolarization was followed by a train of action potentials. The results suggest that heterogeneous neuronal nAChRs are widely distributed in both chemoreceptor and other PG neurons. In some neurons nAChRs may be functionally coupled with outward K+ channels. Further studies are required to determine whether chemoreceptor neurons have a distinct distribution pattern of nAChRs and K+ channels.
Asunto(s)
Acetilcolina/farmacología , Ganglios Sensoriales/efectos de los fármacos , Neuronas/efectos de los fármacos , 4-Aminopiridina/farmacología , Animales , Gatos , Células Cultivadas , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/metabolismo , Ganglios Sensoriales/citología , Ganglios Sensoriales/metabolismo , Inmunohistoquímica , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Nicotina/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismoRESUMEN
Previous pharmacological, immunocytochemical, electrophysiological, and microfluorometric studies have suggested that acetylcholine (ACh) is a critically important excitatory transmitter in the chemotransduction of hypoxia by the cat carotid body (CB). With the use of HPLC this study shows that the in vitro cat CB releases ACh under normoxic conditions; this release is increased when the CB is challenged with hypoxia. The preliminary observation that greater amounts of ACh are liberated in the presence of gallamine and AFDX116 suggests the presence of functioning M2 muscarinic receptors on the glomus cells of the CB.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Carotídeo/metabolismo , Hipoxia/fisiopatología , Animales , Atropina/farmacología , Cuerpo Carotídeo/efectos de los fármacos , Gatos , Trietyoduro de Galamina/farmacología , Técnicas In Vitro , Mecamilamina/farmacología , Parasimpatolíticos/farmacología , Pirenzepina/análogos & derivados , Pirenzepina/farmacología , Receptor Muscarínico M2 , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiologíaRESUMEN
Current modelling of carotid body (CB) chemotransduction postulates an essential role for neurotransmitters, including dopamine (DA). Catecholamines (CA) released from incubated/superfused cat CBs has often been reported to diminish rapidly over the course of the exposure. The purpose of the first set of experiments was to determine the effects of including L-dihydroxyphenylalanine (L-DOPA), the immediate precursor to DA, in the incubation medium. CBs were removed from deeply anesthetized cats, cleaned of connective tissue, and placed in separate incubation tubes containing Krebs Ringer Bicarbonate solution (KRB) at 37 degrees C. One tube contained 40 microM L-DOPA. Both tubes were bubbled for 2 hr with a normoxic gas mixture (21% O2/6% CO2). This was followed immediately by a 30-minute exposure to a hypoxic gas mixture (4% O2/5% CO2). The mean amounts of DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and norepinephrine (NE) released during 30 min exposures were always greater when L-DOPA was present. The use of gas mixture like the above normoxic gas mixture in incubation studies has often been considered quasi-hypoxic. Hence, in a second set of experiments we tested the effect of high oxygen mixture (95% O2/5% CO2). All other features of these experiments were the same as the above. The high oxygen environment correlated with lower DA release suggesting a reduced excitation/inhibition. The subsequent exposure to hypoxia, however, provoked a much larger release of DA and NE. The data demonstrate the substantial effect of oxygen on the release of CAs and the apparent need of a DA precursor like L-DOPA to allow detection of the changes in CA release from the CBs upon exposure to a hypoxic stimulus.
Asunto(s)
Cuerpo Carotídeo/efectos de los fármacos , Cuerpo Carotídeo/metabolismo , Catecolaminas/metabolismo , Levodopa/farmacología , Oxígeno/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Gatos , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/fisiología , Dopamina/metabolismo , Femenino , Ácido Homovanílico/metabolismo , Técnicas In Vitro , Levodopa/metabolismo , Masculino , Norepinefrina/metabolismoRESUMEN
Oxygen can be said to be the most fundamentally necessary substrate for life. In those organisms having a cardiopulmonary system for delivering it in blood to the tissues the carotid body functions as the principal detector of decreases in arterial oxygen. Such a decrease stimulates an increase in neural output from the carotid body to the nucleus tractus solitarii, and this can precipitate a wide array of systemic reflex responses. The neural mechanisms involved in the genesis of increased signal from the carotid body remain unclear. But a current model of carotid body chemotransduction postulates that transmitter-laden glomus cells initiate the neural activity by being depolarized by hypoxemia and releasing an excitatory transmitter which binds to postsynaptic receptors of the adjacent sensory afferent fibers as well as to presynaptic glomus cell autoreceptors. This Frontiers Review evaluates anew the data supporting the hypothesis that acetylcholine (ACh) is an (the) essential excitatory transmitter in this process by examining ACh's fulfillment of criteria required to establish a substance as a synaptic transmitter. All eight criteria are fulfilled in the case of ACh. Indeed, additional data further support the Cholinergic Hypothesis.
Asunto(s)
Acetilcolina/fisiología , Cuerpo Carotídeo/fisiología , Células Quimiorreceptoras/fisiología , Oxígeno/sangre , Animales , Historia del Siglo XX , Modelos Neurológicos , Oxígeno/historiaRESUMEN
Hypoxia, hypercapnia and acidosis stimulate the carotid body (CB) sending increased neural activity via a branch of the glossopharyngeal nerve to nucleus tractus solitarius; this precipitates an impressive array of cardiopulmonary, endocrine and renal reflex responses. However, the cellular mechanisms by which these stimuli generate the increased CB neural output are only poorly understood. Central to the understanding of these mechanisms is the determination of which agents are released within the CB in response to hypoxia, and serve as the stimulating transmitter(s) for chemosensory nerve endings. Acetylcholine (ACh) has been proposed as such an agent from the outset, but this proposal has been, and remains, controversial. The present study tests two hypotheses: (1) The CB releases ACh under normoxic/normocapnic conditions; and (2) The amount released increases during hypoxia and other conditions known to increase neural output from the CB. These hypotheses were tested in 12 experiments in which both CBs were removed from the anesthetized cat and incubated at 37 degrees C in a physiological salt solution while the solution was bubbled with four different concentrations of oxygen and carbon dioxide. The incubation medium was exchanged at 10 min intervals for 30 min (three periods of incubation). The medium was analyzed with high performance liquid chromatography-electrochemical detection for ACh content. Normoxic/normocapnic conditions (21% O2/6% CO2) produced a total of 0.639 +/- 0.106 pmol/150 microl (mean +/- S.E.M.; n = 12). All stimulating conditions produced larger total outputs: 4% O2/2% CO2 produced 1.773 +/- 0.46 pmol/150 microl; 0% O2/5% CO2, 0.868 +/- 0.13 pmol/150 microl; 4% O2/10% CO2, 1.077 +/- 0.21 pmol/150 microl. These three amounts were significantly greater than the normoxic/normocapnic condition, but indistinguishable among themselves. Further, the amount of ACh released did not diminish over the 30 min of stimulation. These data support the concept that during hypoxia ACh functions as a stimulating transmitter in the CB, and are consistent with the earlier reports of cholinergic enzymes and receptors found in the CB.
Asunto(s)
Acetilcolina/metabolismo , Cuerpo Carotídeo/fisiología , Neuronas/fisiología , Acidosis/fisiopatología , Animales , Cuerpo Carotídeo/fisiopatología , Gatos , Colina/metabolismo , Femenino , Nervio Glosofaríngeo/fisiología , Nervio Glosofaríngeo/fisiopatología , Hipoxia Encefálica/fisiopatología , Técnicas In Vitro , Masculino , Núcleo Solitario/fisiología , Núcleo Solitario/fisiopatología , Factores de TiempoRESUMEN
With immunocytochemical techniques using a monoclonal antibody for alpha7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that alpha7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed alpha7 subunits. These data support a role for acetylcholine in carotid body neurotransmission.
Asunto(s)
Cuerpo Carotídeo/química , Terminaciones Nerviosas/química , Receptores Nicotínicos/análisis , Vías Aferentes/química , Animales , Gatos , Inmunohistoquímica , Transmisión Sináptica/fisiología , Tirosina 3-Monooxigenasa/análisisRESUMEN
Acetylcholine increases intracellular calcium of arterial chemoreceptor cells of adult cats. J. Neurophysiol. 78: 2388-2395, 1997. Several neurotransmitters have been reported to play important roles in the chemoreception of the carotid body. Among them acetylcholine (ACh) appears to be involved in excitatory processes in the cat carotid body. As one of the steps to elucidate possible roles of ACh in carotid body chemoreception in the cat, we examined the effect of ACh on intracellular calcium concentration ([Ca2+]i) of cultured carotid body cells. The carotid body from adult cats was dissociated and cultured for up to 2 wk. [Ca2+]i was measured from clusters of cells with a microfluorometric technique using Indo-1 AM. Experiments were performed at 37 degrees C, and cells were continuously superfused with modified Krebs solutions equilibrated with 5% CO2-16% O2-79% N2. ACh (100 mu M) caused a marked increase in [Ca2+]i in approximately 70% of clusters, and the responses to 1-300 mu M of ACh were concentration dependent. The magnitude and kinetics of the ACh response were mimicked by the application of nicotine, whereas muscarinic agonists, pilocarpine, and muscarine failed to evoke a similar response. ACh-induced increase in [Ca2+]i was dependent on extracellular Ca2+: it was greatly reduced or completely abolished by a transient removal of extracellular Ca2+. The response was consistently but only partially reduced by caffeine (5 mM) or nifedipine (10 mu M). The effect of mecamylamine (100 mu M) was inhibitory but small. Moreover, the increase in [Ca2+]i in response to ACh was also observed in some clusters that did not respond to high K (100 mM) Krebs. These results suggest that ACh increases [Ca2+]i of cultured carotid body cells by activating neuronal nicotinic ACh receptors, leading to Ca2+ influx via nicotinic channels. In addition, other pathways such as Ca2+ influx through L-type calcium channels, perhaps secondary to membrane depolarization, and Ca2+ release from intracellular stores may participate in increasing [Ca2+]i in response to ACh. Muscarinic receptors appear to play only a small role, if any.
Asunto(s)
Acetilcolina/farmacología , Calcio/metabolismo , Cuerpo Carotídeo/fisiología , Células Quimiorreceptoras/fisiología , Animales , Cuerpo Carotídeo/citología , Gatos , Células Cultivadas , Células Quimiorreceptoras/citología , Células Quimiorreceptoras/efectos de los fármacos , Colorantes Fluorescentes , Indoles , Cinética , Mecamilamina/farmacología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Nifedipino/farmacología , Pilocarpina/farmacología , Potasio/farmacologíaRESUMEN
From the 1930s into the 1970s, the role of acetylcholine (ACh) in the carotid body's chemotransduction of hypoxia was debated. Since the late 1970s, the issue has been pursued only intermittently or not at all. The purpose of this study was to test again with a new preparation the hypothesis that ACh is an excitatory neurotransmitter in the cat carotid body's chemotransduction of hypoxia. We tested the effect of the specific nicotinic blocker mecamylamine and the muscarinic blocker of all five muscarinic receptors, atropine. We further tested the effects of M1 and M2 muscarinic-receptor blockers. The carotid body region was selectively perfused with hypoxic Krebs-Ringer bicarbonate (KRB) solutions that were blocker free or contained varying doses of the blockers. Both mecamylamine and atropine reduced the response to hypoxic KRB in a dose-related manner. The M2 muscarinic-receptor blockers gallamine and AFDX 116 increased the response to hypoxic KRB, whereas the M1 muscarinic-receptor blocker pirenzepine reduced the response to hypoxic KRB. These data are consistent with an excitatory role for ACh in the carotid body chemotransduction of hypoxia in the cat.
Asunto(s)
Acetilcolina/fisiología , Atropina/farmacología , Cuerpo Carotídeo/efectos de los fármacos , Células Quimiorreceptoras/efectos de los fármacos , Hipoxia/fisiopatología , Mecamilamina/farmacología , Animales , GatosRESUMEN
The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.
Asunto(s)
Respiración/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
The purpose of this study was to examine the role of afferent input in the reflex modulation of renal sympathetic nerve activity (SNA) in response to apnea. Apneas of 20-, 40-, and 60-s duration were induced in the anesthetized, paralyzed cat (n = 7) ventilated with either room air or 100% oxygen. While receiving room air, there were increases (p < 0.005) in renal SNA of 34.5 +/- 4.2%, 53.3 +/- 6.4%, and 59.9 +/- 7.2% of maximum during the 20-, 40-, and 60-s apneas, respectively. There were corresponding increases (p < 0.025) in mean arterial pressure (Pa) of 9 +/- 3, 30 +/- 9, and 45 +/- 12 mm Hg during the 20-, 40-, and 60-s apneas while receiving room air, respectively. The effect of 100% oxygen was to reduce (p < 0.0001) the renal SNA response to apnea, at a matched level of PaCO2, by at least 80%, and to eliminate any increase in Pa. During the first breath of the postapneic period, there was a partial inhibition of renal SNA. During the second and third breaths of the postapneic period, there was a marked fall in renal SNA that was associated with a precipitous decline in directly recorded carotid chemoreceptor activity (n = 2). The magnitude of the fall in renal SNA after apnea was related to the degree of postapneic hypertension. We conclude that hypoxic chemoreceptor stimulation is the predominant factor generating the renal SNA response to apnea, with modulating inputs from thoracic afferents and arterial baroreceptors likely contributing to the marked inhibition of renal SNA immediately after the apnea.