RESUMEN
Lay summary: Friction caused by blood flowing across cells that line blood vessels (endothelial cells) activates sensors of mechanical force. This produces nitric oxide (NO) which widens placental blood vessels, enabling more blood flow to the baby. This study sought to determine whether the mechanical sensor, Piezo1, is important for NO production in fetoplacental endothelial cells (FpECs) and whether the steps in this pathway are different in small for gestational age (SGA) babies, where placental blood flow is often altered. We showed that in healthy FpECs, blood flow increased NO signalling. We suggest that in SGA babies, FpECs have an increase in baseline levels of NO signalling, suggestive of a compensatory drive. Treating healthy and SGA cells with a Piezo1 chemical activator, Yoda1, upregulated NO signalling. This shows that Piezo1 is linked to NO and that in SGA, FpECs have the capacity to further increase NO. Further research will establish whether Piezo1 enhancement leads to increased blood flow in the placenta. If so, Piezo1 could be a new target for developing treatments to prevent poor growth of babies in the womb.
Asunto(s)
Células Endoteliales , Placenta , Embarazo , Femenino , Animales , Células Endoteliales/metabolismo , Placenta/metabolismo , Fosforilación , Edad Gestacional , Óxido Nítrico Sintasa/metabolismo , Endotelio/metabolismoRESUMEN
The mechanical force of blood flow is a fundamental determinant of vascular homeostasis. This frictional stimulation of cells, fluid shear stress (FSS), is increasingly recognised as being essential to placental development and function. Here, we focus on the role of FSS in regulating fetoplacental circulatory flow, both in normal pregnancy and that affected by fetal growth restriction (FGR). The fetus is reliant on placental perfusion to meet its circulatory and metabolic demands. Failure of normal vascular adaptation and the mechanisms enabling responsive interaction between fetoplacental and maternal circulations can result in FGR. FSS generates vasodilatation at least partly through the release of endothelial nitric oxide, a process thought to be vital for adequate blood flow. Where FGR is caused by placental dysfunction, placental vascular anatomy is altered, alongside endothelial dysfunction and hypoxia, each impacting upon the complex balance of FSS forces. Identifying specific mechanical sensors and the mechanisms governing how FSS force is converted into biochemical signals is a fast-paced area of research. Here, we raise awareness of Piezo1 proteins, recently discovered to be FSS-sensitive in fetoplacental endothelium, and with emerging roles in NO generation, vascular tone and angiogenesis. We discuss the emerging concept that activating mechanosensors such as Piezo1 ultimately results in the orchestrated processes of placental vascular adaptation. Piecing together the mechanisms governing endothelial responses to FSS in placental insufficiency is an important step towards developing new treatments for FGR.
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Retardo del Crecimiento Fetal/fisiopatología , Circulación Placentaria , Animales , Femenino , Feto/irrigación sanguínea , Hemodinámica , Humanos , Embarazo , Arterias Umbilicales/fisiopatologíaRESUMEN
Blood flow, and the force it generates, is critical to placental development and function throughout pregnancy. This mechanical stimulation of cells by the friction generated from flow is called shear stress (SS) and is a fundamental determinant of vascular homeostasis, regulating remodelling and vasomotor tone. This review describes how SS is fundamental to the establishment and regulation of the blood flow through the uteroplacental and fetoplacental circulations. Amongst the most recent findings is that alongside the endothelium, embryonic stem cells and the villous trophoblast are mechanically sensitive. A complex balance of forces is required to enable effective establishment of the uteroplacental circulation, while protecting the embryo and placental villi. SS also generates flow-mediated vasodilatation through the release of endothelial nitric oxide, a process vital for adequate placental blood flow. The identification of SS sensors and the mechanisms governing how the force is converted into biochemical signals is a fast-paced area of research, with multiple cellular components under investigation. For example, the Piezo1 ion channel is mechanosensitive in a variety of tissues including the fetoplacental endothelium. Enhanced Piezo1 activity has been demonstrated in response to the Yoda1 agonist molecule, suggesting the possibility for developing tools to manipulate these channels. Whether such agents might progress to novel therapeutics to improve blood flow through the placenta requires further consideration and research.
Asunto(s)
Mecanotransducción Celular/fisiología , Placenta/metabolismo , Placentación/fisiología , Células Endoteliales/metabolismo , Femenino , Humanos , Mecanotransducción Celular/genética , Placenta/citología , Placentación/genética , Embarazo , Estrés MecánicoRESUMEN
STUDY QUESTION: Does the shear stress sensing ion channel subunit Piezo1 have an important mechanotransduction role in human fetoplacental endothelium? SUMMARY ANSWER: Piezo1 is present and functionally active in human fetoplacental endothelial cells, and disruption of Piezo1 prevents the normal response to shear stress. WHAT IS KNOWN ALREADY: Shear stress is an important stimulus for maturation and function of placental vasculature but the molecular mechanisms by which the force is detected and transduced are unclear. Piezo1 channels are Ca2+-permeable non-selective cationic channels which are critical for shear stress sensing and maturation of murine embryonic vasculature. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated the relevance of Piezo1 to placental vasculature by studying human fetoplacental endothelial cells (FpECs) from healthy pregnancies. Endothelial cells were isolated from placental cotyledons and cultured, for the study of tube formation and cell alignment to shear stress. In addition, human placental arterial endothelial cells were isolated and studied immediately by patch-clamp electrophysiology. MAIN RESULTS AND THE ROLE OF CHANCE: The synthetic Piezo1 channel agonist Yoda1 caused strong elevation of the intracellular Ca2+ concentration with a 50% effect occurring at about 5.4 µM. Knockdown of Piezo1 by RNA interference suppressed the Yoda1 response, consistent with it being mediated by Piezo1 channels. Alignment of cells to the direction of shear stress was also suppressed by Piezo1 knockdown without loss of cell viability. Patch-clamp recordings from freshly isolated endothelium showed shear stress-activated single channels which were characteristic of Piezo1. LIMITATIONS, REASONS FOR CAUTION: The in vitro nature of fetoplacental endothelial cell isolation and subsequent culture may affect FpEC characteristics and PIEZO1 expression. In addition to Piezo1, alternative shear stress sensing mechanisms have been suggested in other systems and might also contribute in the placenta. WIDER IMPLICATIONS OF THE FINDINGS: These data suggest that Piezo1 is an important molecular determinant of blood flow sensitivity in the placenta. Establishing and manipulating the molecular mechanisms regulating shear stress sensing could lead to novel therapeutic strategies to improve blood flow in the placenta. LARGE-SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTEREST(S): LCM was funded by a Clinical Research Training Fellowship from the Medical Research Council and by the Royal College of Obstetricians and Gynaecologists, and has received support from a Wellcome Trust Institutional Strategic Support Fund. JS was supported by the Wellcome Trust and a BHF Intermediate Research Fellowship. HJG, CW, AJH and PJW were supported by PhD Studentships from BHF, BBSRC and the Leeds Teaching Hospitals Charitable Foundation respectively. All authors declare no conflict of interest.
Asunto(s)
Células Endoteliales/metabolismo , Canales Iónicos/metabolismo , Placenta/citología , Placenta/metabolismo , Células Cultivadas , Femenino , Humanos , Canales Iónicos/genética , Mecanotransducción Celular/fisiología , Embarazo , Estrés MecánicoRESUMEN
A critical point in mammalian development occurs before mid-embryogenesis when the heart starts to beat, pushing blood into the nascent endothelial lattice. This pushing force is a signal, detected by endothelial cells as a frictional force (shear stress) to trigger cellular changes that underlie the essential processes of vascular remodeling and expansion required for embryonic growth. The processes are complex and multifactorial and Piezo1 became a recognized player only 2years ago, 4years after Piezo1's initial discovery as a functional membrane protein. Piezo1 is now known to be critical in murine embryonic development just at the time when the pushing force is first detected by endothelial cells. Murine Piezo1 gene disruption in endothelial cells is embryonic lethal and mutations in human PIEZO1 associate with severe disease phenotype due to abnormal lymphatic vascular development. Piezo1 proteins coassemble to form calcium-permeable nonselective cationic channels, most likely as trimers. They are large proteins with little if any resemblance to other proteins or ion channel subunits. The channels appear to sense mechanical force directly, including the force imposed on endothelial cells by physiological shear stress. Here, we review current knowledge of Piezo1 in the vascular setting and discuss hypotheses about how it might serve its vascular functions and integrate with other mechanisms. Piezo1 is a new important player for investigators in this field and promises much as a basis for better understanding of vascular physiology and pathophysiology and perhaps also discovery of new therapies.
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Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular , Estrés Mecánico , Animales , HumanosRESUMEN
Ovarian cancer constitutes the second most common gynecological cancer with a five-year survival rate of 40%. Among the various histotypes associated with hereditary ovarian cancer, high-grade serous epithelial ovarian carcinoma (HGSEOC) is the most predominant and women with inherited mutations in BRCA1 have a lifetime risk of 40-60%. HGSEOC is a challenge for clinical oncologists, due to late presentation of patient, diagnosis and high rate of relapse. Ovarian tumors have a wide range of clinical presentations including development of ascites as a result of deregulated endothelial function thereby causing increased vascular permeability of peritoneal vessels. The molecular mechanisms remain elusive. Studies have shown that fallopian tube cancers develop in women with BRCA1 gene mutations more often than previously suspected. Recent studies suggest that many primary peritoneal cancers and some high-grade serous epithelial ovarian carcinomas actually start in the fallopian tubes. In this article we have addressed the molecular pathway of a recently identified potential biomarker Ubc9 whose deregulated expression due to BRCA1 dysfunction can result in HGSEOC with peritoneal permeability and formation of ascites. We also discuss the role of downstream targets Caveolin-1 and Vascular Endothelial Growth Factor (VEGF) in the pathogenesis of ascites in ovarian carcinomas. Finally we hypothesize a signaling axis between Ubc9 over expression, loss of Caveolin-1 and induction of VEGF in BRCA1 mutant HGSEOC cells. We suggest that Ubc9-mediated stimulation of VEGF as a novel mechanism underlying ovarian cancer aggressiveness and ascites formation. Agents that target Ubc9 and VEGF signaling may represent a novel therapeutic strategy to impede peritoneal growth and spread of HGSEOC.
RESUMEN
Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn(2+) level ([Zn(2+)]c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia-reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn(2+)]c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn(2+)]c but abolished the cytosolic Zn(2+) accumulation during reperfusion as well as ROS-elicited increases in the [Zn(2+)]c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn(2+)]c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Citosol/metabolismo , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Canales Catiónicos TRPM/deficiencia , Zinc/metabolismo , Animales , Muerte Celular , Peróxido de Hidrógeno/toxicidad , Ataque Isquémico Transitorio/complicaciones , Masculino , Trastornos de la Memoria/etiología , Trastornos de la Memoria/prevención & control , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Canales Catiónicos TRPM/metabolismoRESUMEN
Transient receptor potential canonical (TRPC) channels are the canonical (C) subset of the TRP proteins, which are widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium and sodium entry and have wide-ranging functions that include regulation of cell proliferation, motility and contraction. The channels are modulated by a multiplicity of factors, putatively existing as integrators in the plasma membrane. This review considers the sensitivities of TRPC channels to lipids that include diacylglycerols, phosphatidylinositol bisphosphate, lysophospholipids, oxidized phospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate, cholesterol and some steroidal derivatives and other lipid factors such as gangliosides. Promiscuous and selective lipid sensing have been detected. There appear to be close working relationships with lipids of the phospholipase C and A(2) enzyme systems, which may enable integration with receptor signalling and membrane stretch. There are differences in the properties of each TRPC channel that are further complicated by TRPC heteromultimerization. The lipids modulate activity of the channels or insertion in the plasma membrane. Lipid microenvironments and intermediate sensing proteins have been described that include caveolae, G protein signalling, SEC14-like and spectrin-type domains 1 (SESTD1) and podocin. The data suggest that lipid sensing is an important aspect of TRPC channel biology enabling integration with other signalling systems.
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Lípidos de la Membrana/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPC/metabolismo , Animales , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Colesterol/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Ratones , Fosfolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Mammals contain 28 genes encoding Transient Receptor Potential (TRP) proteins. The proteins assemble into cationic channels, often with calcium permeability. Important roles in physiology and disease have emerged and so there is interest in whether the channels might be suitable therapeutic drug targets. Here we review selected members of three subfamilies of mammalian TRP channel (TRPC5, TRPM2 and TRPA1) that show relevance to sensing of adversity by cells and biological systems. Summarized are the cellular and tissue distributions, general properties, endogenous modulators, protein partners, cellular and tissue functions, therapeutic potential, and pharmacology. TRPC5 is stimulated by receptor agonists and other factors that include lipids and metal ions; it heteromultimerises with other TRPC proteins and is involved in cell movement and anxiety control. TRPM2 is activated by hydrogen peroxide; it is implicated in stress-related inflammatory, vascular and neurodegenerative conditions. TRPA1 is stimulated by a wide range of irritants including mustard oil and nicotine but also, controversially, noxious cold and mechanical pressure; it is implicated in pain and inflammatory responses, including in the airways. The channels have in common that they show polymodal stimulation, have activities that are enhanced by redox factors, are permeable to calcium, and are facilitated by elevations of intracellular calcium. Developing inhibitors of the channels could lead to new agents for a variety of conditions: for example, suppressing unwanted tissue remodeling, inflammation, pain and anxiety, and addressing problems relating to asthma and stroke.
Asunto(s)
Calcio/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Modelos Animales de Enfermedad , Humanos , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
BACKGROUND AND PURPOSE: Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids. EXPERIMENTAL APPROACH: Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca2+ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells. KEY RESULTS: Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1-2min. Dehydroepiandrosterone sulphate or 17ß-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid. CONCLUSIONS AND IMPLICATIONS: Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.
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Hormonas Esteroides Gonadales/farmacología , Canales Catiónicos TRPC/antagonistas & inhibidores , Calcio/metabolismo , Células Cultivadas , Dihidrotestosterona/farmacología , Estradiol/farmacología , Células HEK293 , Humanos , Lisofosfolípidos/farmacología , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Fosfolípidos/metabolismo , Pregnenolona/farmacología , Progesterona/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The transient receptor potential melastatin-3 (TRPM3) channel forms calcium-permeable, non-selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance. EXPERIMENTAL APPROACH: We used TRPM3 channels over-expressed in HEK 293 cells, with intracellular calcium measurement and whole-cell patch-clamp recording techniques. KEY RESULTS: The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3-dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (beta) configuration of the side group, revealing previously unrecognized stereo-selectivity and supporting existence of a specific binding site. A cis-oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non-steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects. CONCLUSIONS AND IMPLICATIONS: Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.
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Pregnenolona/química , Pregnenolona/farmacología , Canales Catiónicos TRPM/agonistas , Animales , Calcio/metabolismo , Línea Celular , Humanos , Ratones , Técnicas de Placa-Clamp , Estereoisomerismo , Relación Estructura-Actividad , Canales Catiónicos TRPM/genética , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. EXPERIMENTAL APPROACH: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca(2+) measurement. KEY RESULTS: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca(2+) entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. CONCLUSIONS AND IMPLICATIONS: The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.
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Anticuerpos/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular , Colorantes Fluorescentes , Fura-2/metabolismo , Humanos , Riñón/metabolismo , Técnicas de Placa-Clamp/métodos , Unión ProteicaRESUMEN
BACKGROUND AND PURPOSE: TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium-permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5. EXPERIMENTAL APPROACH: Human TRPC5 channels were expressed in HEK 293 cells and studied using fura-2 and patch-clamp recording to measure intracellular calcium and membrane currents respectively at room temperature. Human TRPM2 channels were studied for comparison. KEY RESULTS: TRPC5 activation by carbachol, Gd3+ or LPC was inhibited by halothane and chloroform at > or =0.1 and 0.2 mM respectively. Neither agent inhibited TRPM2. Propofol had an initial stimulatory effect on TRPC5 (evident in patch-clamp recordings only) and an inhibitory effect at > or =10 microM. TRPM2 was not affected by propofol. Propofol inhibited activation of TRPC5 by Gd3+ but not LPC, suggesting the effect was not directly on the channel. Propofol's anti-oxidant property was not necessary for its inhibitory effect because di-isopropyl benzene, a propofol analogue that lacks the hydroxyl group, also inhibited TRPC5. CONCLUSIONS AND IMPLICATIONS: The data show the sensitivity of TRPC5 channel to general anaesthetics and suggest that some of the effects could have clinical relevance. The effects may be explained in part by the sensitivity of the channel to biophysical properties of the lipid bilayer.
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Anestésicos por Inhalación/farmacología , Anestésicos Intravenosos/farmacología , Canales Catiónicos TRPC/efectos de los fármacos , Anestésicos por Inhalación/administración & dosificación , Anestésicos Intravenosos/administración & dosificación , Calcio/metabolismo , Línea Celular , Cloroformo/administración & dosificación , Cloroformo/farmacología , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Fura-2 , Halotano/administración & dosificación , Halotano/farmacología , Humanos , Técnicas In Vitro , Elementos de la Serie de los Lantanoides/farmacología , Lisofosfatidilcolinas/farmacología , Técnicas de Placa-Clamp , Propofol/administración & dosificación , Propofol/farmacología , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPM/efectos de los fármacos , Canales Catiónicos TRPM/metabolismoRESUMEN
Blood vessels are essential for animal life, allowing flow of oxygen and nutrients to tissues and removal of waste products. Consequently, inappropriate remodelling of blood vessels, resulting in occlusion, can lead to disabling or catastrophic events: heart attacks, strokes and claudication. An important cell type of remodelling is the VSMC (vascular smooth-muscle cell), a fascinating cell that contributes significantly to occlusive vascular diseases by virtue of its ability to 'modulate' to a cell that no longer contracts and arranges radially in the medial layer of the vessel wall but migrates, invades, proliferates and adopts phenotypes of other cells. An intriguing aspect of modulation is switching to different ion transport systems. Initial events include loss of the Ca(V)1.2 (L-type voltage-gated calcium) channel and gain of the K(Ca)3.1 (IKCa) potassium channel, which putatively occur to enable membrane hyperpolarization that increases rather than decreases a type of calcium entry coupled with cell cycle activity, cell proliferation and cell migration. This type of calcium entry is related to store- and receptor-operated calcium entry phenomena, which, in VSMCs, are contributed to by TRPC [TRP (transient receptor potential) canonical] channel subunits. Instead of being voltage-gated, these channels are chemically gated - importantly, by key phospholipid factors of vascular development and disease. This brief review focuses on the hypothesis that the transition to a modulated cell may require a switch from predominantly voltage- to predominantly lipid-sensing ion channels.
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Músculo Liso Vascular/metabolismo , Canales de Potasio/metabolismo , Enfermedades Vasculares/metabolismo , Silenciador del Gen , Humanos , Activación del Canal Iónico , Músculo Liso Vascular/patología , Transcripción Genética , Enfermedades Vasculares/patologíaRESUMEN
Canonical transient receptor potential 5 TRPC5 (also TrpC5, trp-5 or trp5) is one of the seven mammalian TRPC proteins. Its known functional property is that of a mixed cationic plasma membrane channel with calcium permeability. It is active alone or as a heteromultimeric assembly with TRPC1; TRPC4 and TRPC3 may also be involved. Multiple activators of TRPC5 are emerging, including various G protein-coupled receptor agonists, lysophospholipids, lanthanide ions and, in some contexts, calcium store depletion. Intracellular calcium has complex impact on TRPC5, including a permissive role for other activators, as well as inhibition at high concentrations. Protein kinase C is inhibitory and mediates desensitisation following receptor activation. Tonic TRPC5 activity is detected and may reflect the presence of constitutive activation signals. The channel has voltage dependence but the biological significance of this is unknown; it is partially due to intracellular magnesium blockade at aspartic acid residue 633. Protein partners include calmodulin, CaBP1, enkurin, Na(+)-H+ exchange regulatory factor (NHERF) and stathmin. TRPC5 is included in local vesicular trafficking regulated by growth factors through phosphatidylinositol (PI)-3-kinase, Rac1 and PIP-5-kinase. Inhibition of myosin light chain kinase suppresses TRPC5, possibly via an effect on trafficking. Biological roles of TRPC5 are emerging but more reports on this aspect are needed. One proposed role is as a mediator of calcium entry and excitation in smooth muscle, another as an inhibitor of neuronal growth cone extension. The latter is intriguing in view of the original cloning of the human TRPC5 gene from a region of the X chromosome linked to mental retardation. TRPC5 is a broadly expressed calcium channel with capability to act as an integrator of extracellular and intracellular signals at the level of calcium entry.
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Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/fisiología , Animales , Humanos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Proteínas/metabolismo , Canales Catiónicos TRPC/efectos de los fármacosRESUMEN
TRPC5 [TRP (transient receptor potential) canonical (or classical) 5] is a widely expressed mammalian homologue of Drosophila TRP, forming a calcium- and sodium-permeable channel in the plasma membrane either as a homomultimer or heteromultimer with other proteins (e.g. TRPC1). Although several factors are known to stimulate the channel, understanding of its endogenous activators and functions is limited. This paper provides a brief and focused review of our latest findings that show that TRPC5 is a sensor of important signalling phospholipids, including lysophosphatidylcholine and sphingosine 1-phosphate, acting extracellularly or intracellularly. Underlying mechanisms of action and biological relevance are discussed.
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Fosfolípidos/química , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/fisiología , Animales , Calcio/química , Calcio/metabolismo , Membrana Celular/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Lípidos/química , Lisofosfatidilcolinas/química , Lisofosfolípidos/química , Modelos Biológicos , Transducción de Señal , Sodio/química , Esfingosina/análogos & derivados , Esfingosina/químicaRESUMEN
Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.
Asunto(s)
Canales Catiónicos TRPC/fisiología , Túnica Íntima/patología , Enfermedades Vasculares/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Hiperplasia , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Ratas , Ratas Endogámicas WKY , Vena Safena/patología , Porcinos , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Regulación hacia Arriba , Enfermedades Vasculares/tratamiento farmacológicoRESUMEN
Throughout the body there are smooth muscle cells controlling a myriad of tubes and reservoirs. The cells show enormous diversity and complexity compounded by a plasticity that is critical in physiology and disease. Over the past quarter of a century we have seen that smooth muscle cells contain--as part of a gamut of ion-handling mechanisms--a family of cationic channels with significant permeability to calcium, potassium and sodium. Several of these channels are sensors of calcium store depletion, G-protein-coupled receptor activation, membrane stretch, intracellular Ca2+, pH, phospholipid signals and other factors. Progress in understanding the channels has, however, been hampered by a paucity of specific pharmacological agents and difficulty in identifying the underlying genes. In this review we summarize current knowledge of these smooth muscle cationic channels and evaluate the hypothesis that the underlying genes are homologues of Drosophila TRP (transient receptor potential). Direct evidence exists for roles of TRPC1, TRPC4/5, TRPC6, TRPV2, TRPP1 and TRPP2, and more are likely to be added soon. Some of these TRP proteins respond to a multiplicity of activation signals--promiscuity of gating that could enable a variety of context-dependent functions. We would seem to be witnessing the first phase of the molecular delineation of these cationic channels, something that should prove a leap forward for strategies aimed at developing new selective pharmacological agents and understanding the activation mechanisms and functions of these channels in physiological systems.
Asunto(s)
Proteínas de Drosophila/fisiología , Canales Iónicos/fisiología , Músculo Liso/fisiología , Secuencia de Aminoácidos , Animales , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Canales Iónicos/química , Canales Iónicos/genética , Datos de Secuencia MolecularRESUMEN
This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding K(V)alpha1 subunits as well as K(V)beta1, K(V)alpha2.1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Ca)beta1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding K(V)alpha2.1 but less or similar amounts encoding K(V)beta1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Cabeta)1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant K(V)alpha1 K(+) current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K(+) current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.
Asunto(s)
Arterias Mesentéricas/fisiología , Familia de Multigenes , Músculo Liso Vascular/fisiología , Canales de Potasio/genética , Canales de Potasio/metabolismo , Resistencia Vascular , Animales , Aorta Torácica/metabolismo , Conductividad Eléctrica , Expresión Génica , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/metabolismo , Regulación hacia ArribaRESUMEN
1. In this study, we determined a pharmacological profile of store-operated channels (SOCs) in smooth muscle cells of rabbit pial arterioles. Ca(2+)-indicator dyes, fura-PE3 or fluo-4, were used to track [Ca(2+)](i) and 10 micro M methoxyverapamil (D600) was present in all experiments on SOCs to prevent voltage-dependent Ca(2+) entry. Store depletion was induced using thapsigargin or cyclopiazonic acid. 2. SOC-mediated Ca(2+) entry was inhibited concentration dependently by Gd(3+) (IC(50) 101 nM). It was also inhibited by 10 micro M La(3+) (70% inhibition, N=5), 100 micro M Ni(2+) (57% inhibition, N=5), 75 micro M 2-aminoethoxydiphenylborate (66% inhibition, N=4), 100 micro M capsaicin (12% inhibition, N=3) or preincubation with 10 micro M wortmannin (76% inhibition, N=4). It was completely resistant to 1 micro M nifedipine (N=5), 10 micro M SKF96365 (N=6), 10 micro M LOE908 (N=14), 10-100 micro M ruthenium red (N=1+2), 100 micro M sulindac (N=4), 0.5 mM streptomycin (N=3) or 1 : 10,000 dilution Grammostolla spatulata venom (N=4). 3. RT-PCR experiments on isolated arteriolar fragments showed expression of mRNA species for TRPC1, 3, 4, 5 and 6. 4. The pharmacological profile of SOC-mediated Ca(2+) entry in arterioles supports the hypothesis that these SOCs are distinct from tonically active background channels and several store-operated and other nonselective cation channels described in other cells. Similarities with the pharmacology of TRPC1 support the hypothesis that TRPC1 is a subunit of the arteriolar smooth muscle SOC.