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1.
J Biol Chem ; 294(46): 17395-17408, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31586031

RESUMEN

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/- cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.


Asunto(s)
Interleucina-6/genética , Canales Iónicos/genética , Miocardio/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Señalización del Calcio/genética , Endopeptidasas/genética , Células Endoteliales/química , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/química , Canales Iónicos/química , Sistema de Señalización de MAP Quinasas/genética , Mecanotransducción Celular/genética , Ratones , Miocardio/química , Fosforilación/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Tioléster Hidrolasas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/química
2.
Br J Pharmacol ; 180(16): 2039-2063, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-36457143

RESUMEN

BACKGROUND AND PURPOSE: The protein PIEZO1 forms mechanically activated, calcium-permeable, non-selective cation channels in numerous cell types from several species. Options for pharmacological modulation are limited and so we modified a small-molecule agonist at PIEZO1 channels (Yoda1) to increase the ability to modulate these channels. EXPERIMENTAL APPROACH: Medicinal chemistry generated Yoda1 analogues that were tested in intracellular calcium and patch-clamp assays on cultured cells exogenously expressing human or mouse PIEZO1 or mouse PIEZO2. Physicochemical assays and wire myography assays on veins from mice with genetic disruption of PIEZO1. KEY RESULTS: A Yoda1 analogue (KC159) containing 4-benzoic acid instead of the pyrazine of Yoda1 and its potassium salt (KC289) have equivalent or improved reliability, efficacy and potency, compared with Yoda1 in functional assays. Tested against overexpressed mouse PIEZO1 in calcium assays, the order of potency (as EC50 values, nM) was KC289, 150 > KC159 280 > Yoda1, 600). These compounds were selective for PIEZO1 over other membrane proteins, and the physicochemical properties were more suited to physiological conditions than those of Yoda1. The vasorelaxant effects were consistent with PIEZO1 agonism. In contrast, substitution with 2-benzoic acid failed to generate a modulator. CONCLUSION AND IMPLICATIONS: 4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.


Asunto(s)
Calcio , Mecanotransducción Celular , Ratones , Humanos , Animales , Calcio/metabolismo , Reproducibilidad de los Resultados , Mecanotransducción Celular/fisiología , Sitios de Unión , Canales de Calcio/metabolismo , Canales Iónicos/metabolismo
3.
Sci Rep ; 9(1): 16876, 2019 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-31727906

RESUMEN

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic ß-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat ß-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both ß-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from ß-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from ß-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.


Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Canales Iónicos/genética , Proteínas de la Membrana/genética , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Gadolinio/farmacología , Regulación de la Expresión Génica , Glucosa/metabolismo , Heterocigoto , Secreción de Insulina/genética , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Mecanotransducción Celular , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Rojo de Rutenio/farmacología , Tiadiazoles/farmacología , Técnicas de Cultivo de Tejidos
4.
Br J Pharmacol ; 175(10): 1744-1759, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29498036

RESUMEN

BACKGROUND AND PURPOSE: The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist, but the pharmacology of these channels is otherwise limited. EXPERIMENTAL APPROACH: Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta. KEY RESULTS: Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 µM Yoda1-induced Ca2+ -entry with IC50 s of 1.3 µM (HEK 293 cells) and 1.5 µM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca2+ elevation or store-operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings, which was mediated by an endothelium- and NO-dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1. CONCLUSION AND IMPLICATIONS: Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.


Asunto(s)
Aorta Torácica/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Pirazinas/farmacología , Animales , Aorta Torácica/metabolismo , Células CHO , Células Cultivadas , Cricetulus , Células HEK293 , Humanos , Canales Iónicos/metabolismo , Ratones , Pirazinas/síntesis química , Pirazinas/química , Relación Estructura-Actividad
5.
Nat Commun ; 8(1): 350, 2017 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-28839146

RESUMEN

Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.


Asunto(s)
Canales Iónicos/fisiología , Condicionamiento Físico Animal/fisiología , Animales , Presión Sanguínea , Señalización del Calcio , Células Cultivadas , Células Endoteliales/metabolismo , Células HEK293 , Homeostasis/genética , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Masculino , Ratones , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Vasoconstricción/fisiología
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