Publications by authors named "Paul J Kemp"

61 Publications

Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

Cytotherapy 2021 Feb 25;23(2):111-118. Epub 2020 Nov 25.

Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK; MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Cardiff, UK; Wales Brain Repair and Intracranial Neurotherapeutics Unit, School of Medicine, Cardiff University, Cardiff, UK. Electronic address:

Background: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells.

Results: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.
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http://dx.doi.org/10.1016/j.jcyt.2020.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822401PMC
February 2021

Characterization of Negative Allosteric Modulators of the Calcium-Sensing Receptor for Repurposing as a Treatment of Asthma.

J Pharmacol Exp Ther 2021 01 28;376(1):51-63. Epub 2020 Oct 28.

Schools of Biosciences (P.L.Y., P.H., M.W.S., R.B., P.J.K., D.R.), Pharmacy (E.J.K., W.R.F., K.J.B.), and Chemistry (B.M.K.), Cardiff University, Cardiff, United Kingdom; Institute for Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria (M.W.S.); TissueGnostics GmbH, Vienna, Austria (R.E., R.N.); School of Dental Sciences, University of Newcastle, United Kingdom (V.T.); Woolcock Institute of Medical Research, The University of Sydney, Sydney, Australia (D.T., L.G.d.R.); and School of Immunology & Microbial Sciences, King's College London, London, United Kingdom (C.J.C., J.P.T.W.)

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.
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http://dx.doi.org/10.1124/jpet.120.000281DOI Listing
January 2021

Drosophila taste neurons as an agonist-screening platform for P2X receptors.

Sci Rep 2020 05 19;10(1):8292. Epub 2020 May 19.

School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK.

The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.
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http://dx.doi.org/10.1038/s41598-020-65169-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237442PMC
May 2020

Aberrant Development Corrected in Adult-Onset Huntington's Disease iPSC-Derived Neuronal Cultures via WNT Signaling Modulation.

Stem Cell Reports 2020 03 27;14(3):406-419. Epub 2020 Feb 27.

Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA 92697, USA; Department of Neurobiology and Behavior, University of California Irvine, Irvine, CA 96267, USA; Department of Memory Impairment and Neurological Disorders, University of California Irvine, Irvine, CA 92697, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Biological Chemistry, University of California Irvine, Irvine, CA 92617, USA. Electronic address:

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1 neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.
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http://dx.doi.org/10.1016/j.stemcr.2020.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066322PMC
March 2020

Huntington's Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support.

Front Neurosci 2019 28;13:669. Epub 2019 Jun 28.

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, United States.

In Huntington's disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K currents, lengthened spontaneous Ca waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets.
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http://dx.doi.org/10.3389/fnins.2019.00669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610155PMC
June 2019

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

Pflugers Arch 2018 09 24;470(9):1359-1376. Epub 2018 May 24.

School of Biosciences, Cardiff University, The Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK.

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.
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http://dx.doi.org/10.1007/s00424-018-2155-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096767PMC
September 2018

Functional Interactions between BK-Subunit and Annexin A5: Implications in Apoptosis.

Oxid Med Cell Longev 2016 25;2016:1607092. Epub 2016 Sep 25.

Division of Pathophysiology and Repair, School of Biosciences, Sir Martin Evans Building, Museum Avenue, Cardiff University, Cardiff CF10 3AX, UK.

Proteomic studies have suggested a biochemical interaction between subunit of the large conductance, voltage- and Ca-activated potassium channel (BK), and annexin A5 (ANXA5), which we verify here by coimmunoprecipitation and double labelling immunocytochemistry. The observation that annexin is flipped to the outer membrane leaflet of the plasma membrane during apoptosis, together with the knowledge that the intracellular C-terminal of BK contains both Ca-binding and a putative annexin-binding motif, prompted us to investigate the functional consequences of this protein partnership to cell death. Membrane biotinylation demonstrated that ANXA5 was flipped to the outer membrane leaflet of HEK 293 cells early in serum deprivation-evoked apoptosis. As expected, serum deprivation caused caspase-3/7 activation and this was accentuated in BK expressing HEK 293 cells. The functional consequences of ANXA5 partnership with BK were striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BK channel Ca-sensitivity, measured in inside-out membrane patches by patch-clamp. Taken together, these data suggest a novel model of the early stages of apoptosis where membrane flippage results in removal of the inhibitory effect of ANXA5 on K channel activity with the consequent amplification of Ca influx and augmented activation of caspases.
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http://dx.doi.org/10.1155/2016/1607092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055951PMC
March 2017

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

J Physiol 2016 11;594(22):6583-6594

School of Biosciences, Cardiff University, Cardiff, UK.

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.
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http://dx.doi.org/10.1113/JP270655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108907PMC
November 2016

Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells.

Stem Cells Int 2016 26;2016:1290561. Epub 2016 May 26.

Oral and Biomedical Sciences, School of Dentistry, Cardiff University, Heath Park, Cardiff CF14 4XY, UK.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.
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http://dx.doi.org/10.1155/2016/1290561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899607PMC
June 2016

The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR.

Sci Rep 2016 Feb 25;6:21975. Epub 2016 Feb 25.

School of Biosciences, Cardiff University, CF10 3AX, United Kingdom.

Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl(-)-driven fluid secretion in organ explant cultures show that pharmacological CaSR activation by calcimimetics stimulates lung fluid secretion through CFTR, an effect which in humans, but not mice, was also mimicked by fetal hypercalcemic conditions, demonstrating that the physiological relevance of such a mechanism appears to be species-specific. Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca(2+)-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases.
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http://dx.doi.org/10.1038/srep21975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766410PMC
February 2016

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Am J Physiol Cell Physiol 2016 Apr 30;310(7):C520-41. Epub 2015 Dec 30.

School of Biosciences, Cardiff University, Cardiff, United Kingdom;

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.
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http://dx.doi.org/10.1152/ajpcell.00166.2015DOI Listing
April 2016

The Role of Kv1.2 Channel in Electrotaxis Cell Migration.

J Cell Physiol 2016 Jun 10;231(6):1375-84. Epub 2015 Dec 10.

Department of Dermatology, No. 1 Hospital of China Medical University, Shenyang, China.

Voltage-gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF-induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS-7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time-lapse imaging analysis showed that EF-induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS-7 cells to EF stimulation more than their motility. Immunocytochemistry and pull-down analyses demonstrated association of Kv1.2 channels with actin-binding protein cortactin and its re-localization to the cathode-facing membrane at EF stimulation, which confirms the mechanism of EF-induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF-induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy, and various degenerative syndromes.
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http://dx.doi.org/10.1002/jcp.25259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832312PMC
June 2016

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell-derived medium spiny neurons.

Mol Ther Methods Clin Dev 2015 16;2:15030. Epub 2015 Sep 16.

Department of Cell Biology, Immunology and Neuroscience, Faculty of Medicine, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), and Networked Biomedical Research Centre for NeuroDegenerative Disorders (CIBERNED), University of Barcelona , Barcelona, Spain.

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.
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http://dx.doi.org/10.1038/mtm.2015.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571731PMC
September 2015

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma.

Sci Transl Med 2015 Apr 22;7(284):284ra60. Epub 2015 Apr 22.

School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK.

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.
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http://dx.doi.org/10.1126/scitranslmed.aaa0282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725057PMC
April 2015

Functional plasticity of the N-methyl-d-aspartate receptor in differentiating human erythroid precursor cells.

Am J Physiol Cell Physiol 2015 Jun 18;308(12):C993-C1007. Epub 2015 Mar 18.

Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland

Calcium signaling is essential to support erythroid proliferation and differentiation. Precise control of the intracellular Ca(2+) levels in erythroid precursor cells (EPCs) is afforded by coordinated expression and function of several cation channels, including the recently identified N-methyl-d-aspartate receptor (NMDAR). Here, we characterized the changes in Ca(2+) uptake and electric currents mediated by the NMDARs occurring during EPC differentiation using flow cytometry and patch clamp. During erythropoietic maturation, subunit composition and properties of the receptor changed; in proerythroblasts and basophilic erythroblasts, fast deactivating currents with high amplitudes were mediated by the GluN2A subunit-dominated receptors, while at the polychromatic and orthochromatic erythroblast stages, the GluN2C subunit was getting more abundant, overriding the expression of GluN2A. At these stages, the currents mediated by the NMDARs carried the features characteristic of the GluN2C-containing receptors, such as prolonged decay time and lower conductance. Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor. Despite this variability, NMDARs were essential for survival of EPCs in any subject tested. Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca(2+) homeostasis from the orthochromatic erythroblast stage to circulating red blood cells.
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http://dx.doi.org/10.1152/ajpcell.00395.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469746PMC
June 2015

Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

PLoS One 2013 25;8(11):e80294. Epub 2013 Nov 25.

Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom.

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080294PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840017PMC
January 2015

Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons.

PLoS One 2013 22;8(11):e81031. Epub 2013 Nov 22.

Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom.

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca(2+) channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca(2+) imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca(2+) channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca(2+) or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca(2+) channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca(2+) channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081031PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838360PMC
May 2015

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells.

J Cell Sci 2013 Oct 25;126(Pt 19):4490-501. Epub 2013 Jul 25.

Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, BE-2020 Antwerp, Belgium.

The Ca(2+)-sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca(2+)]o. In addition to sensing [Ca(2+)]o, CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca(2+)]o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67-GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca(2+)]o with a rise in intracellular Ca(2+) ([Ca(2+)]i); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex-231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca(2+)]i, suggesting that Ca(2+) influx through TRPC channels contributes to the total [Ca(2+)]i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca(2+)]i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche.
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http://dx.doi.org/10.1242/jcs.131656DOI Listing
October 2013

Oxygen sensing by the carotid body: is it all just rotten eggs?

Antioxid Redox Signal 2014 Feb 24;20(5):794-804. Epub 2013 Jul 24.

Division of Pathophysiology and Repair, School of Biosciences, Cardiff University , Cardiff, United Kingdom .

Significance: Ventilatory responses to hypoxia are initiated by the carotid body, where inhibition of specific K(+) channels causes cell depolarization, voltage-gated Ca(2+) influx, and neurotransmitter release. The identity of the upstream oxygen (O2) sensor is still controversial.

Recent Advances: The activity of BKCa channels is regulated by O2, carbon monoxide (CO), and hydrogen sulfide (H2S), suggesting that integration of these signals may be crucial to the physiological response of this tissue. BKCa is colocalized with hemeoxygenase-2, an enzyme that generates CO in the presence of O2, and CO is a BKCa channel opener. Reduced CO during hypoxia results in channel closure, conferring a degree of O2 sensitivity to the BKCa channel. Conversely, H2S is a potent BKCa inhibitor. H2S is produced endogenously by cystathionine-β-synthase and cystathionine-γ-lyase in the rat carotid body, and its intracellular concentration is dependent upon the balance between its enzymatic generation and its mitochondrial breakdown. During hypoxia, mitochondrial oxidation of H2S in many tissues is reduced, leading to hypoxia-evoked rises in its concentration. This may be sufficient to inhibit K(+) channels and lead to carotid body excitation.

Critical Issues: Carotid body function is heavily dependent upon regulated production and breakdown of CO and H2S and integration of signals from these newly emerging gasotransmitters, in combination with several other proposed mechanisms, may refine, or even define, responses of this tissue to hypoxia.

Future Directions: Since several other sensors have been postulated, the challenge of future research is to begin to integrate each in a unifying mechanism, as has been attempted for the first time herein.
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http://dx.doi.org/10.1089/ars.2013.5377DOI Listing
February 2014

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

Eur Heart J 2014 Apr 6;35(16):1078-87. Epub 2013 Mar 6.

Wolfson Centre for Stem Cells, Tissue Engineering and Modelling (STEM), University of Nottingham, Nottingham NG7 2RD, UK.

Aims: Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS.

Methods And Results: We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations).

Conclusions: These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.
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http://dx.doi.org/10.1093/eurheartj/eht067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992427PMC
April 2014

The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease.

Annu Rev Physiol 2012 17;74:271-97. Epub 2011 Oct 17.

Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff, CF10 3AX, United Kingdom.

The extracellular calcium-sensing receptor (CaSR) is the first identified G protein-coupled receptor to be activated by an ion, extracellular calcium (Ca(2+)). Since the identification of the CaSR in 1993, genetic mutations in the CaSR gene, and murine models in which CaSR expression has been manipulated, have clearly demonstrated the importance of this receptor in the maintenance of stable, free, ionized Ca(2+) concentration in the extracellular fluids. These functions have been extensively reviewed elsewhere. However, the distribution pattern and expression of the CaSR in lower vertebrates strongly suggest that the CaSR must play a role that is independent of mineral cation metabolism. This review addresses the involvement of the CaSR in nutrient sensing; its putative and demonstrated functions during conception, embryonic development, and birth; and its contributions to adult physiology and disease, with reference to CaSR-based therapeutics. Recent ongoing developments concerning the role of the CaSR in stem cell differentiation are also reviewed.
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http://dx.doi.org/10.1146/annurev-physiol-020911-153318DOI Listing
June 2012

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Pflugers Arch 2011 Aug 11;462(2):267-79. Epub 2011 May 11.

Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Museum Avenue, Cardiff, UK.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.
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http://dx.doi.org/10.1007/s00424-011-0971-0DOI Listing
August 2011

Carbon monoxide: an emerging regulator of ion channels.

J Physiol 2011 Jul 26;589(Pt 13):3055-62. Epub 2011 Apr 26.

Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK.

Carbon monoxide is rapidly emerging as an important cellular messenger, regulating a wide range of physiological processes. Crucial to its role in both physiology and disease is its ability differentially to regulate several classes of ion channels, including examples from calcium-activated K(+) (BK(Ca)), voltage-activated K(+) (K(v)) and Ca(2+) channel (L-type) families, ligand-gated P2X receptors (P2X2 and P2X4), tandem P domain K(+) channels (TREK1) and the epithelial Na(+) channel (ENaC). The mechanisms by which CO regulates these ion channels are still unclear and remain somewhat controversial. However, available structure-function studies suggest that a limited range of amino acid residues confer CO sensitivity, either directly or indirectly, to particular ion channels and that cellular redox state appears to be important to the final integrated response. Whatever the molecular mechanism by which CO regulates ion channels, endogenous production of this gasotransmitter has physiologically important roles and is currently being explored as a potential therapeutic.
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http://dx.doi.org/10.1113/jphysiol.2011.206706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145923PMC
July 2011

An exon 5-less splice variant of the extracellular calcium-sensing receptor rescues absence of the full-length receptor in the developing mouse lung.

Exp Lung Res 2011 Jun 26;37(5):269-78. Epub 2011 Feb 26.

Center for Cardiovascular Sciences, Institute for Biomedical Research, University of Birmingham, Edgbaston, United Kingdom.

The authors have recently demonstrated that, in the developing mouse lung, fetal plasma Ca(2+) suppresses branching morphogenesis and cell proliferation while promoting fluid secretion via activation of the extracellular Ca(2+)-sensing receptor (CaSR). The aim of the current study was to further elucidate the role of Ca(2+) in lung development by studying the effects of extracellular Ca(2+) on fetal lung development in mice lacking the CaSR. These mice were produced by exon 5 deletion in the CaSR gene. Since such a maneuver has been known to induce the expression of an exon 5-less splice variant of the CaSR in some tissues, the molecular and functional expression of this splice variant in the developing mouse lung was also investigated. Whereas there was a mild in vivo phenotype observed in these mice, in vitro sensitivity of Casr(-/-) lung explants to specific activators of the CaSR was unaffected. These results imply that compensatory expression of an exon 5-less splice variant rescues CaSR function in this mouse model and therefore a lung-specific, complete CaSR knockout model must be developed to fully appreciate the role for this receptor in lung development and the contribution of its ablation to postnatal respiratory disease.
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http://dx.doi.org/10.3109/01902148.2010.545471DOI Listing
June 2011

Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

Pflugers Arch 2011 Jun 8;461(6):665-75. Epub 2011 Feb 8.

Division of Pathophysiology & Repair, School of Biosciences, Museum Avenue, Cardiff University, UK.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).
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http://dx.doi.org/10.1007/s00424-011-0924-7DOI Listing
June 2011

Lung organogenesis.

Curr Top Dev Biol 2010 ;90:73-158

The Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California, USA.

Developmental lung biology is a field that has the potential for significant human impact: lung disease at the extremes of age continues to cause major morbidity and mortality worldwide. Understanding how the lung develops holds the promise that investigators can use this knowledge to aid lung repair and regeneration. In the decade since the "molecular embryology" of the lung was first comprehensively reviewed, new challenges have emerged-and it is on these that we focus the current review. Firstly, there is a critical need to understand the progenitor cell biology of the lung in order to exploit the potential of stem cells for the treatment of lung disease. Secondly, the current familiar descriptions of lung morphogenesis governed by growth and transcription factors need to be elaborated upon with the reinclusion and reconsideration of other factors, such as mechanics, in lung growth. Thirdly, efforts to parse the finer detail of lung bud signaling may need to be combined with broader consideration of overarching mechanisms that may be therapeutically easier to target: in this arena, we advance the proposal that looking at the lung in general (and branching in particular) in terms of clocks may yield unexpected benefits.
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http://dx.doi.org/10.1016/S0070-2153(10)90003-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340128PMC
November 2010

Mechanism of inhibition by hydrogen sulfide of native and recombinant BKCa channels.

Respir Physiol Neurobiol 2010 Jul 16;172(3):169-78. Epub 2010 May 16.

Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff, UK.

Recent evidence suggests that H(2)S contributes to activation of the carotid body by hypoxia by inhibiting K(+) channels. Here, we determine both the molecular identity of the K(+) channel target within the carotid body and the biophysical characteristics of the H(2)S-evoked inhibition by analyzing native rat and human recombinant BK(Ca) channel activity in voltage-clamped, inside-out membrane patches. Rat glomus cells express the enzymes necessary for the endogenous generation of H(2)S, cystathionine-beta-synthase and cystathionine-gamma-lyase. H(2)S inhibits native carotid body and human recombinant BK(Ca) channels with IC(50) values of around 275 microM. Inhibition by H(2)S is rapid and reversible, works by a mechanism which is distinct from that suggested for CO gas regulation of this channel and does not involve an interaction with either the "Ca bowl" or residues distal to this Ca(2+)-sensing domain. These data show that BK(Ca) is a K(+) channel target of H(2)S, and suggest a mechanism to explain the H(2)S-dependent component of O(2) sensing in the carotid body.
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http://dx.doi.org/10.1016/j.resp.2010.05.016DOI Listing
July 2010

Enzyme-linked oxygen sensing by potassium channels.

Ann N Y Acad Sci 2009 Oct;1177:112-8

School of Biosciences, Cardiff University, Cardiff, United Kingdom.

The ability of ion channels to respond to an acute perturbation in oxygen tension is a widespread phenomenon, which encompasses many of the major ion channel families. Integral to the ability of several ion channels to respond to acute hypoxic challenge is modulation by upstream enzymatic reactions, suggesting that many ion channels sense oxygen via enzyme-linked processes. Several enzyme-linked oxygen sensing systems have been proposed, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent production of hydrogen peroxide, hemoxygenase-dependent generation of carbon monoxide, adenosine monophosphate (AMP) kinase-dependent channel phosphorylation, and src-Lck protein tyrosine kinase, via a currently undetermined mechanism. Each of these enzymes has been shown to endow specific ion channels with the ability to respond to changes in oxygen, with hypoxia exclusively evoking channel inhibition. This article reviews these proposed mechanisms and presents new insights into how one system, hemeoxygenase-2, confers oxygen sensitivity to large conductance, voltage- and calcium-activated potassium channels.
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http://dx.doi.org/10.1111/j.1749-6632.2009.05025.xDOI Listing
October 2009

Novel regulatory aspects of the extracellular Ca2+-sensing receptor, CaR.

Pflugers Arch 2009 Oct 30;458(6):1007-22. Epub 2009 May 30.

Cardiff School of Biosciences, Cardiff University, Cardiff, UK.

The capacity to sense and adapt to changes in environmental cues is of paramount importance for every living organism. From yeast to man, cells must be able to match cellular activities to growth environment and nutrient availability. Key to this process is the development of membrane-bound systems that can detect modifications in the extracellular environment and to translate these into biological responses. Evidence gathered over the last 15 years has demonstrated that many of these cell surface "sensors" belong to the G protein-coupled receptor superfamily. Crucial to our understanding of nutrient sensing in mammalian species has been the identification of the extracellular Ca(2+)/cation-sensing receptor, CaR. CaR was the first ion-sensing molecule identified in man and genetic studies in humans have revealed the importance of the CaR in mineral ion metabolism. Latter, it has become apparent that the CaR also plays an important role outside the Ca(2+) homeostatic system, as an integrator of multiple environmental signals for the regulation of many vital cellular processes, from cell-to-cell communication to secretion and cell survival/cell death. Recently, novel aspects of receptor function reveal an unexpected role for the CaR in the regulation of growth and development in utero.
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http://dx.doi.org/10.1007/s00424-009-0681-zDOI Listing
October 2009

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

FASEB J 2009 Apr 2;23(4):1153-60. Epub 2008 Dec 2.

Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.
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http://dx.doi.org/10.1096/fj.08-109579DOI Listing
April 2009