ABSTRACT
BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.
Subject(s)
Ion Channels , Nitric Oxide , Portal Vein , TRPV Cation Channels , Animals , Male , Mice , Endothelium/metabolism , Nitric Oxide Synthase/metabolism , Osmotic Pressure , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , Vasodilation , Ion Channels/genetics , Ion Channels/metabolismABSTRACT
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.
Subject(s)
Calcium , Mechanotransduction, Cellular , Mice , Humans , Animals , Calcium/metabolism , Reproducibility of Results , Mechanotransduction, Cellular/physiology , Binding Sites , Calcium Channels/metabolism , Ion Channels/metabolismABSTRACT
[Figure: see text].
Subject(s)
Adipose Tissue/metabolism , Diabetes Mellitus, Type 2/metabolism , Endothelium, Vascular/metabolism , Glucose/metabolism , Muscle, Skeletal/metabolism , Paracrine Communication , Animals , Cells, Cultured , Humans , Hydrogen Peroxide/metabolism , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , NADPH Oxidase 4/genetics , NADPH Oxidase 4/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolismABSTRACT
Mechanical force is a determinant of Notch signalling but the mechanism of force detection and its coupling to Notch are unclear. We propose a role for Piezo1 channels, which are mechanically-activated non-selective cation channels. In cultured microvascular endothelial cells, Piezo1 channel activation by either shear stress or a chemical agonist Yoda1 activated a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a Ca2+-regulated transmembrane sheddase that mediates S2 Notch1 cleavage. Consistent with this observation, we found Piezo1-dependent increase in the abundance of Notch1 intracellular domain (NICD) that depended on ADAM10 and the downstream S3 cleavage enzyme, γ-secretase. Conditional endothelial-specific disruption of Piezo1 in adult mice suppressed the expression of multiple Notch1 target genes in hepatic vasculature, suggesting constitutive functional importance in vivo. The data suggest that Piezo1 is a mechanism conferring force sensitivity on ADAM10 and Notch1 with downstream consequences for sustained activation of Notch1 target genes and potentially other processes.
Subject(s)
ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Endothelial Cells/metabolism , Ion Channels/metabolism , Membrane Proteins/metabolism , Receptor, Notch1/metabolism , Animals , Cells, Cultured , Enzyme Activation , Gene Expression Regulation , Humans , Ion Channels/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Protein Domains , Stress, Mechanical , Transcription Factor HES-1/geneticsABSTRACT
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.
Subject(s)
Aorta, Thoracic/drug effects , Ion Channels/antagonists & inhibitors , Pyrazines/pharmacology , Animals , Aorta, Thoracic/metabolism , CHO Cells , Cells, Cultured , Cricetulus , HEK293 Cells , Humans , Ion Channels/metabolism , Mice , Pyrazines/chemical synthesis , Pyrazines/chemistry , Structure-Activity RelationshipABSTRACT
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.