Multiplexed G-Protein–Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening

Miret, Juan J.; Zhang, Jiansu; Min, Hyunsuk; Lewis, Karina; Roth, Mark B.; Charlton, Maura E.; Bauer, Paul H.

doi:10.1177/1087057105279493

Cited by 15 publications

(8 citation statements)

References 23 publications

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“…Mouse ES Cell-derived Neurons-The Molecular Devices automated FLiPR assay for measuring intracellular Caϩ fluxes is a commonly used platform technology for screening agonists and antagonists of G-protein coupled receptors (15,16). For the HTS, the adherent mES cell-derived neural cultures were plated into 384-well poly-D-lysine-coated tissue culture dishes (BD Biosciences) by the automated SelecT (The Automation Partnerships, Wilmington, DE) at a cell density of 6 ϫ 10 3 cells/ well in differentiation medium (see above).…”

Section: Fluorimetric Imaging Plate Reader (Flipr) Assay Usingmentioning

confidence: 99%

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

McNeish

Roach

Hambor

et al. 2010

Journal of Biological Chemistry

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Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, ␣-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 ؋ 10 6 compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10 6 compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.Cognitive impairment is a fundamental trademark of many CNS 2 diseases such as Alzheimer disease, schizophrenia, and attention deficit hyperactivity disorder. Unfortunately few pharmaceutical agents have proven to be efficacious in treating cognitive impairment. As such, the medical need for new therapeutic approaches for improving cognitive deficits has emerged as an important frontier in treating central nervous system diseases. Glutamate mediates the vast majority of excitatory neurotransmission in the CNS with post-synaptic signaling regulated by either ligand-gated cation channels (ionotropic) or G-protein-coupled receptors (metabotropic). Ionotropic ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are tetrameric aggregates composed of combinations of one or more of four subunits (GluR1-4) that can each bind a glutamate neurotransmitter molecule. Upon binding of glutamate, the associated ion channel pore opens, allowing cations to cross the membrane and depolarize the postsynaptic cell. Termination of the postsynaptic signal is driven primarily by the removal of glutamate from the synaptic cleft, reducing the occupancy of the glutamate binding site and subsequent channel closure (deactivation). Additionally, the conduction pathway will close in the continued presence of bound agonist as a result of conformational changes secondary to glutamate binding (desensitization).AMPA receptor pharmacology has identified agonists, competitive and non-competitive antagonists, and positive allosteric modulators (3). This latter class of pharmacologic agen...

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Section: Fluorimetric Imaging Plate Reader (Flipr) Assay Usingmentioning

confidence: 99%

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

McNeish

Roach

Hambor

et al. 2010

Journal of Biological Chemistry

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“…The cells were loaded with 4 M Fluo-4-AM fluorescent indicator dye (Invitrogen) for 1 h in assay buffer (Hanks' balanced salt solution containing 20 mM HEPES, 2.5 mM probenecid, and 1% fetal calf serum) and then washed four times in assay buffer without fetal calf serum. Changes in the [Ca 2ϩ ] i were assayed using a fluorometric imaging plate reader system (FLIPR96; Molecular Devices, Sunnyvale, CA) (Miret et al, 2005;Mori et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop

Ushiyama

Ikeda²,

Sugawara³

et al. 2007

Mol Pharmacol

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Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide first isolated from ovine hypothalamus, occurs in two forms: one consists of 38 amino acid residues (PACAP-38) and the other contains 27 amino acid residues (PACAP-27) (Miyata et al., 1989. The actions of PACAP are mediated through G-protein-coupled receptors (GPCRs) that belong to group II of the secretin receptor family. Three PACAP/vasoactive intestinal polypeptide (VIP) receptor genes have been cloned: one encodes the PACAP-preferring receptor PAC1 (Hashimoto et al., 1993;Hosoya et al., 1993;Morrow et al., 1993;Pisegna and Wank, 1993;Spengler et al., 1993;Aino et al., 1995;Vaudry et al., 2000;Harmar, 2001), whereas the other two encode receptors that respond equally to PACAP and VIP (VPAC1 and VPAC2) (Ishihara et al., 1992;Lutz et al., 1993Lutz et al., , 1999. Typically, group II receptors signal through the protein kinase A (PKA) pathway, although PAC1 not only activates the PKA pathway but is also coupled to the phospholipase C (PLC) pathway, resulting in changes in the intracellular calcium concentration ([Ca 2ϩ ] i ). In mice, the PAC1 gene contains more than 18 exons, and more splice variants of PAC1 have been identified than for most of the other GPCRs (Aino et al., 1995). Most of the PAC1 isoforms are formed as a result of altern...

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“…While in vivo Ca 2+ imaging has become a centrepiece of research in physiological and cell biological processes, it is only rarely used as a tool in pharmacological research. High‐throughput Ca 2+ imaging is increasingly utilized in multi‐well sample configurations in drug discovery research, particularly for screening of unknown compounds (Miret et al ., 2005). In vivo Ca 2+ imaging could potentially play an important role in pharmacological characterization of potential drugs.…”

Section: Indicator Loading Techniquesmentioning

confidence: 99%

Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology

Russell

2011

British J Pharmacology

122

100

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The design and engineering of organic fluorescent Ca 2+ indicators approximately 30 years ago opened the door for imaging cellular Ca 2+ signals with a high degree of temporal and spatial resolution. Over this time, Ca 2+ imaging has revolutionized our approaches for tissue-level spatiotemporal analysis of functional organization and has matured into a powerful tool for in situ imaging of cellular activity in the living animal. In vivo Ca 2+ imaging with temporal resolution at the millisecond range and spatial resolution at micrometer range has been achieved through novel designs of Ca 2+ sensors, development of modern microscopes and powerful imaging techniques such as two-photon microscopy. Imaging Ca 2+ signals in ensembles of cells within tissue in 3D allows for analysis of integrated cellular function, which, in the case of the brain, enables recording activity patterns in local circuits. The recent development of miniaturized compact, fibre-optic-based, mechanically flexible microendoscopes capable of two-photon microscopy opens the door for imaging activity in awake, behaving animals. This development is poised to open a new chapter in physiological experiments and for pharmacological approaches in the development of novel therapies. LINKED ARTICLESThis article is part of a themed section on Imaging. To view the other articles in this section visit http://dx.doi.org/10.1111/ bph.2011.163.issue-8 BJP has previously published an Imaging in Pharmacology themed section, edited by A Davenport and C Daly. To view this section visit http://dx

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Multiplexed G-Protein–Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening

Cited by 15 publications

References 23 publications

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop

Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology

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