RESUMEN
Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.
Asunto(s)
Asparagina/metabolismo , Canales Epiteliales de Sodio/metabolismo , Matriz Extracelular/metabolismo , Dominios Proteicos/genética , Animales , Asparagina/química , Modelos Animales de Enfermedad , Células Endoteliales , Endotelio Vascular/citología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Canales Epiteliales de Sodio/química , Canales Epiteliales de Sodio/genética , Femenino , Glicosilación , Células HEK293 , Humanos , Hipertensión/etiología , Hipertensión/patología , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Oocitos , Técnicas de Placa-Clamp , Mutación Puntual , Polisacáridos/química , Estrés Mecánico , Xenopus laevisRESUMEN
PURPOSE OF REVIEW: The ability of endothelial cells to sense mechanical force, and shear stress in particular, is crucial for normal vascular function. This relies on an intact endothelial glycocalyx that facilitates the production of nitric oxide (NO). An emerging arterial shear stress sensor is the epithelial Na+ channel (ENaC). This review highlights existing and new evidence for the interdependent activity of the glycocalyx and ENaC and its implications for vascular function. RECENT FINDINGS: New evidence suggests that the glycocalyx and ENaC are physically connected and that this is important for shear stress sensing. The connection relies on N-glycans attached to glycosylated asparagines of α-ENaC. Removal of specific N-glycans reduced ENaC's shear stress response. Similar effects were observed following degradation of the glycocalyx. Endothelial specific viral transduction of α-ENaC increased blood pressure (â¼40âmmHg). This increase was attenuated in animals transduced with an α-ENaC version lacking N-glycans. SUMMARY: These observations indicate that ENaC is connected to the glycocalyx and their activity is interdependent to facilitate arterial shear stress sensation. Future research focusing on how N-glycans mediate this interaction can provide new insights for the understanding of vascular function in health and disease.
Asunto(s)
Canales Epiteliales de Sodio , Glicocálix , Animales , Células Endoteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Glicocálix/metabolismo , Humanos , Polisacáridos/metabolismo , Sodio/metabolismo , Estrés MecánicoRESUMEN
Vascular epithelial sodium channels (ENaCs) made up of canonical α, ß, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans.
Asunto(s)
Vasos Sanguíneos/metabolismo , Canales Epiteliales de Sodio/metabolismo , Presión Sanguínea , Vasos Sanguíneos/fisiología , Canales Epiteliales de Sodio/genética , Humanos , MutaciónRESUMEN
BACKGROUND: Acetylcholine (ACh) plays a crucial role in the function of the heart. Recent evidence suggests that cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that comprises of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and type-2 muscarinic ACh receptors (M2AChR) to synthesize, release, degrade ACh as well as for ACh to transduce a signal. NNCS is linked to cardiac cell survival, angiogenesis and glucose metabolism. Impairment of these functions are hallmarks of diabetic heart disease (DHD). The role of the NNCS in DHD is unknown. The aim of this study was to examine the effect of diabetes on cardiac NNCS and determine if activation of cardiac NNCS is beneficial to the diabetic heart. METHODS: Ventricular samples from type-2 diabetic humans and db/db mice were used to measure the expression pattern of NNCS components (ChAT, CHT1, VAChT, AChE and M2AChR) and glucose transporter-4 (GLUT-4) by western blot analysis. To determine the function of the cardiac NNCS in the diabetic heart, a db/db mouse model with cardiac-specific overexpression of ChAT gene was generated (db/db-ChAT-tg). Animals were followed up serially and samples collected at different time points for molecular and histological analysis of cardiac NNCS components and prosurvival and proangiogenic signaling pathways. RESULTS: Immunoblot analysis revealed alterations in the components of cardiac NNCS and GLUT-4 in the type-2 diabetic human and db/db mouse hearts. Interestingly, the dysregulation of cardiac NNCS was followed by the downregulation of GLUT-4 in the db/db mouse heart. Db/db-ChAT-tg mice exhibited preserved cardiac and vascular function in comparison to db/db mice. The improved function was associated with increased cardiac ACh and glucose content, sustained angiogenesis and reduced fibrosis. These beneficial effects were associated with upregulation of the PI3K/Akt/HIF1α signaling pathway, and increased expression of its downstream targets-GLUT-4 and VEGF-A. CONCLUSION: We provide the first evidence for dysregulation of the cardiac NNCS in DHD. Increased cardiac ACh is beneficial and a potential new therapeutic strategy to prevent or delay the development of DHD.
Asunto(s)
Acetilcolina/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Cardiomiopatías Diabéticas/prevención & control , Glucosa/metabolismo , Ventrículos Cardíacos/metabolismo , Acetilcolinesterasa/metabolismo , Anciano , Animales , Estudios de Casos y Controles , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatías Diabéticas/etiología , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Muscarínico M2/metabolismo , Simportadores/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC-which is less responsive to shear force (SF)-increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with ß and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC's SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Matriz Extracelular/metabolismo , Mecanotransducción Celular , Oocitos/fisiología , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Canales Epiteliales de Sodio/química , Canales Epiteliales de Sodio/genética , Glicosilación , Humanos , Mutación , Oocitos/citología , Homología de Secuencia , Xenopus laevisRESUMEN
Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.
Asunto(s)
Núcleo Basal de Meynert/metabolismo , Oxitocina/metabolismo , Vasopresinas/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Núcleo Basal de Meynert/patología , Femenino , Hipotálamo/metabolismo , Lactancia/metabolismo , Lactancia/fisiología , Eyección Láctea/efectos de los fármacos , Neuronas/metabolismo , Oxitocina/farmacología , Núcleo Hipotalámico Paraventricular/metabolismo , Embarazo , Ratas , Núcleo Supraóptico/metabolismo , Vasopresinas/farmacologíaRESUMEN
The autonomic influences on the heart have a ying-yang nature, albeit oversimplified, the interplay between the sympathetic and parasympathetic system (known as the cholinergic system) is often complex and remain poorly understood. Recently, the heart has been recognized to consist of neuronal and non-neuronal cholinergic system (NNCS). The existence of cardiac NNCS has been confirmed by the presence of cholinergic markers in the cardiomyocytes, which are crucial for synthesis (choline acetyltransferase, ChAT), storage (vesicular acetylcholine transporter, VAChT), reuptake of choline for synthesis (high-affinity choline transporter, CHT1) and degradation (acetylcholinesterase, AChE) of acetylcholine (ACh). The non-neuronal ACh released from cardiomyocytes is believed to locally regulate some of the key physiological functions of the heart, such as regulation of heart rate, offsetting hypertrophic signals, maintenance of action potential propagation as well as modulation of cardiac energy metabolism via the muscarinic ACh receptor in an auto/paracrine manner. Apart from this, several studies have also provided evidence for the beneficial role of ACh released from cardiomyocytes against cardiovascular diseases such as sympathetic hyperactivity-induced cardiac remodeling and dysfunction as well as myocardial infarction, confirming the important role of NNCS in disease prevention. In this review, we aim to provide a fundamental overview of cardiac NNCS, and information about its physiological role, regulatory factors as well as its cardioprotective effects. Finally, we propose the different approaches to target cardiac NNCS as an adjunctive treatment to specifically address the withdrawal of neuronal cholinergic system in cardiovascular disease such as heart failure.
Asunto(s)
Miocitos Cardíacos/metabolismo , Sistema Colinérgico no Neuronal/fisiología , Acetilcolina/metabolismo , Animales , Enfermedades Cardiovasculares/metabolismo , Humanos , Sistema Colinérgico no Neuronal/genéticaRESUMEN
A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.
Asunto(s)
Arterias Carótidas/metabolismo , Canales Epiteliales de Sodio/metabolismo , Mecanotransducción Celular , Arterias Mesentéricas/metabolismo , Estrés Fisiológico , Vasoconstricción , Vasodilatación , Animales , Presión Arterial , Arterias Carótidas/efectos de los fármacos , Bloqueadores del Canal de Sodio Epitelial/farmacología , Canales Epiteliales de Sodio/efectos de los fármacos , Técnicas In Vitro , Masculino , Mecanotransducción Celular/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Flujo Sanguíneo Regional , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Gadolinium-based-contrast-agents (GBCAs) are used for magnetic-resonance-imaging and associated with renal and cardiovascular adverse reactions caused by released Gd3+ ions. Gd3+ is also a modulator of mechano-gated ion channels, including the epithelial Na+ channel (ENaC) that is expressed in kidney epithelium and the vasculature. ENaC is important for salt-/water homeostasis and blood pressure regulation and a likely target of released Gd3+ from GBCAs causing the above-mentioned adverse reactions. Therefore this study examined the effect of Gd3+ and GBCAs on ENaC's activity. METHODS: Human αßγENaC was expressed in Xenopus laevis oocytes and exposed to Gd3+, linear (Gd-DTPA, Magnevist) or cyclic (Dotarem) GBCAs. Transmembrane ion-currents (IM) were recorded by the two-electrode-voltage-clamp technique and Gd3+-release by Gd-DTPA was confirmed by inductively coupled plasma-mass spectrometry. RESULTS: Gd3+ exerts biphasic effects on ENaC's activity: ≤0.3mmol/l decreased IM which was preventable by DEPC (modifies histidines). Strikingly Gd3+≥0.4mmol/l increased IM and this effect was prevented by cysteine-modifying MTSEA. Linear Gd-DTPA and Magnevist mimicked the effect of ≤0.3mmol/l Gd3+, whereas the chelator DTPA showed no effect. Gd3+ and Gd-DTPA increased the IC50 for amiloride, but did not affect ENaC's self-inhibition. Interestingly, cyclic Gd-DOTA (Dotarem) increased IM to a similar extent as its chelator DOTA, suggesting that the chelator rather than released Gd3+ is responsible for this effect. CONCLUSION: These results confirm Gd3+-release from linear Gd-DTPA and indicate that the released Gd3+ amount is sufficient to interfere with ENaC's activity to provide putative explanations for GBCA-related adverse effects.
Asunto(s)
Medios de Contraste/efectos adversos , Canales Epiteliales de Sodio/efectos de los fármacos , Gadolinio DTPA/efectos adversos , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Gadolinio/efectos adversos , Gadolinio DTPA/farmacocinética , Humanos , Xenopus laevisRESUMEN
IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.
Asunto(s)
Adenosina Trifosfato/farmacología , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Acetilcolina/farmacología , Adenosina Trifosfato/análogos & derivados , Animales , Western Blotting , Células Cultivadas , Colina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Lipopolisacáridos/química , Potenciales de la Membrana/efectos de los fármacos , Monocitos/metabolismo , Nicotina/farmacología , Fosforilcolina/química , Interferencia de ARN , Ratas , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937 , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Chemosensory cells in the mucosal surface of the respiratory tract ("brush cells") use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.
Asunto(s)
Acetilcolina/metabolismo , Células Quimiorreceptoras/metabolismo , Uretra/citología , Uretra/metabolismo , Vejiga Urinaria/fisiología , Animales , Células Quimiorreceptoras/citología , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Microvellosidades/fisiología , Comunicación Paracrina/fisiología , Técnicas de Placa-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiología , Gusto/fisiología , Lengua/citología , Lengua/inervación , Lengua/fisiología , Uretra/inervación , Vejiga Urinaria/inervación , Urodinámica/fisiología , Urotelio/citología , Urotelio/metabolismoRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells.
Asunto(s)
Tamaño de la Célula , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Oocitos/fisiología , Estrés Mecánico , 1-Metil-3-Isobutilxantina/farmacología , Animales , Cloruros/metabolismo , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/fisiología , Femenino , Glicina/análogos & derivados , Glicina/farmacología , Humanos , Hidrazinas/farmacología , Presión Hidrostática , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/fisiología , Potenciales de la Membrana/efectos de los fármacos , Mutación , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Resistencia al Corte , Xenopus laevisRESUMEN
Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.
Asunto(s)
Conexinas/metabolismo , Epitelio/metabolismo , Presión Hidrostática , Canales KATP/metabolismo , Pulmón/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Femenino , Xenopus laevisRESUMEN
[This retracts the article DOI: 10.1016/j.heliyon.2023.e17434.].
RESUMEN
Aims: Type 1 diabetes mellitus (T1DM) is associated with increased risk of cardiovascular disease (CVD) and mortality. The underlying mechanisms for T1DM-induced heart disease still remains unclear. In this study, we aimed to investigate the effects of cardiac non-neuronal cholinergic system (cNNCS) activation on T1DM-induced cardiac remodelling. Methods: T1DM was induced in C57Bl6 mice using low-dose streptozotocin. Western blot analysis was used to measure the expression of cNNCS components at different time points (4, 8, 12, and 16 weeks after T1DM induction). To assess the potential benefits of cNNCS activation, T1DM was induced in mice with cardiomyocyte-specific overexpression of choline acetyltransferase (ChAT), the enzyme required for acetylcholine (Ac) synthesis. We evaluated the effects of ChAT overexpression on cNNCS components, vascular and cardiac remodelling, and cardiac function. Key findings: Western blot analysis revealed dysregulation of cNNCS components in hearts of T1DM mice. Intracardiac ACh levels were also reduced in T1DM. Activation of ChAT significantly increased intracardiac ACh levels and prevented diabetes-induced dysregulation of cNNCS components. This was associated with preserved microvessel density, reduced apoptosis and fibrosis, and improved cardiac function. Significance: Our study suggests that cNNCS dysregulation may contribute to T1DM-induced cardiac remodelling, and that increasing ACh levels may be a potential therapeutic strategy to prevent or delay T1DM-induced heart disease.
RESUMEN
The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, ß2, and ß4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 µM) induced a dose-dependent increase of the transepithelial short-circuit current (EC(50): 14.6 µM). The nicotine-induced effect (I(NIC)) was attenuated by mecamylamine (25 µM, apical) and methyllycaconitine (1 µM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 µM) revealed apical and basolateral location of the receptors. I(NIC) was not affected by the sodium channel inhibitor amiloride (10 µM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172 (20 µM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM, apical), the Na(+)/K(+)/2Cl(-) cotransporter inhibitor bumetanide (200 µM, basolateral), the potassium channel inhibitor Ba(2+) (5 mM, basolateral), and 4.4'-diisothiocyanatostilbene-2.2'-disulfonate (100 µM, apical), indicating a contribution of Ca(2+)-activated chloride channels and potassium channels. Removal of extracellular Na(+) (apical) or Ca(2+) (apical) did not influence I(NIC) but reduced the DMPP effect. Experiments with the Ca(2+)-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP(3)) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 µM, apical) decreased I(NIC), indicating a nicotine-mediated increase of intracellular Ca(2+) and cAMP levels involving the IP(3) signaling pathway. These findings indicate the activity of Ca(2+)-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl(-) and K(+) transport in tracheal epithelium.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Epitelio/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Potasio/metabolismo , Receptores Nicotínicos/metabolismo , Tráquea/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Epitelio/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Nicotina/farmacología , Receptores Nicotínicos/genética , Transducción de Señal/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Tráquea/efectos de los fármacosRESUMEN
The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, ß, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of ß- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αßγ-channels.
Asunto(s)
Endocitosis/fisiología , Canales Epiteliales de Sodio/metabolismo , Neuronas/metabolismo , Anciano , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Corteza Cerebral/citología , Clonación Molecular , Dinaminas/antagonistas & inhibidores , Femenino , Células HEK293 , Humanos , Hidrazonas/farmacología , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp/métodos , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/metabolismo , Transporte de Proteínas/fisiología , Xenopus laevisRESUMEN
P2X(4) receptors are involved in mechanotransduction processes, but it is unknown whether or not P2X(4) receptors form mechanosensitive ion channels. This study questioned, whether laminar shear stress (LSS) can modulate P2X(4) receptor activity. Mouse P2X(4) receptor was cloned and heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage-clamp experiments the application of ATP (100µM) produced a transient inward current that was decreased by about 50% upon a second ATP application, corresponding to the desensitization behavior of P2X(4) receptors. In P2X(4) expressing oocytes LSS (shear forces of ~5.1dynes/cm(2)) did not produce any effect. However, LSS modulated the response of P2X(4) to ATP. With LSS (~5.1dynes/cm(2)) the desensitization of the current due to the second ATP application was diminished. Ivermectin (IVM), a compound which stabilizes the open state of P2X(4) receptors, mimicked the effect of LSS (~5.1dynes/cm(2)), since there was no additional effect of LSS after pre-incubation with IVM detected. This indicates that LSS like IVM stabilizes the open state of the receptor, although the particular mechanism remains unknown. These data demonstrate that LSS modulates the activity of P2X(4) receptors by eliminating the desensitization of the receptors in response to ATP probably by stabilizing the open state of the channel.
Asunto(s)
Receptores Purinérgicos P2X4/metabolismo , Estrés Fisiológico , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.
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Bloqueadores del Canal de Sodio Epitelial , Ajo/química , Exudados de Plantas/farmacología , Compuestos de Sulfhidrilo/química , Ácidos Sulfínicos/farmacología , Animales , Cisteína/farmacología , Disulfuros , Ditiotreitol/farmacología , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Oocitos/fisiología , Técnicas de Placa-Clamp , Exudados de Plantas/química , Ácidos Sulfínicos/química , Ácidos Sulfínicos/aislamiento & purificación , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/fisiologíaRESUMEN
Renal water reabsorption increases in pregnancy and lactation to expand maternal blood volume to cope with the cardiovascular demands of the developing fetus and new-born baby. Vasopressin (antidiuretic hormone) promotes renal water reabsorption and its secretion is principally stimulated by body fluid osmolality. Hence, lowered osmolality normally decreases vasopressin secretion. However, despite water retention profoundly reducing osmolality in pregnancy and lactation, vasopressin levels are maintained to drive blood volume expansion. Despite its importance for successful reproduction, the cellular mechanisms that maintain vasopressin secretion in the face of decreased osmolality during pregnancy and lactation are unknown. Vasopressin is secreted by neurons that are intrinsically osmosensitive through expression of N-terminal truncated-transient receptor potential vanilloid-1 channel, ΔN-TRPV1, which is mechanically activated by osmotically-induced cell shrinkage to increase vasopressin neuron activity. Vasopressin neurons also express TRPV4 but the role of TRPV4 in vasopressin neuron function is not well characterised. Here, we summarise our novel evidence showing that TRPV4 forms functional channels with ΔN-TRPV1 that have a greater single-channel conductance compared to channels with ΔN-TRPV1 alone. We propose that upregulation of TRPV4 heteromerisation with ΔN-TRPV1 might maintain vasopressin secretion in pregnancy and lactation to expand blood volume for successful reproduction.