Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion

Lieb, Sebastian; Michaelis, Stefanie; Plank, Nicole; Bernhardt, Günther; Buschauer, Armin; Wegener, Joachim

doi:10.1016/j.phrs.2016.04.026

Cited by 24 publications

(18 citation statements)

References 33 publications

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“…This convoluted time-course signal can then be deciphered to determine the mechanism of action of drugs by acquiring the profile of an individual compound from a library. The most prominent label-free approach to assess drug polypharmacology is currently the optical measurement of dynamic mass redistribution with nanogratings and now impedance sensing is rapidly emerging as an alternative method [43]. They are both sensitive to cell density, cell-substrate adhesion and morphological changes.…”

Section: (Ii) Pluripotent Stem Cellsmentioning

confidence: 99%

Impedance-based cellular assays for regenerative medicine

Gamal

Underwood

et al. 2018

Phil. Trans. R. Soc. B

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Therapies based on regenerative techniques have the potential to radically improve healthcare in the coming years. As a result, there is an emerging need for non-destructive and label-free technologies to assess the quality of engineered tissues and cell-based products prior to their use in the clinic. In parallel, the emerging regenerative medicine industry that aims to produce stem cells and their progeny on a large scale will benefit from moving away from existing destructive biochemical assays towards data-driven automation and control at the industrial scale. Impedance-based cellular assays (IBCA) have emerged as an alternative approach to study stem-cell properties and cumulative studies, reviewed here, have shown their potential to monitor stem-cell renewal, differentiation and maturation. They offer a novel method to non-destructively assess and quality-control stem-cell cultures. In addition, when combined with disease models they provide complementary insights as label-free phenotypic assays. IBCA provide quantitative and very sensitive results that can easily be automated and up-scaled in multi-well format. When facing the emerging challenge of real-time monitoring of three-dimensional cell culture dielectric spectroscopy and electrical impedance tomography represent viable alternatives to two-dimensional impedance sensing.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

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Section: (Ii) Pluripotent Stem Cellsmentioning

confidence: 99%

Impedance-based cellular assays for regenerative medicine

Gamal

Underwood

et al. 2018

Phil. Trans. R. Soc. B

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“…1A). The cells adhered well to the bottom of the wells on the gold-coated electrodes, which is essential for sufficient measurements of impedance [41]. Therefore no additional well coatings had to be used.…”

Section: Endogenous Glutamate Reduced Agonist Yet Enhanced Antagonistmentioning

confidence: 99%

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

Doornbos

Linden

Vereyken

et al. 2018

Biochemical Pharmacology

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Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a label-free whole cell impedance-based biosensor system (i.e. xCELLigence) to study the pharmacology of one GPCR in particular, the mGlu 2 receptor. This receptor is a target for the treatment of several psychiatric diseases such as schizophrenia and depression.After optimization of assay conditions to prevent interference of endogenous glutamate in the culture medium, detailed pharmacological assessments were performed. Concentration-response curves showed a concentration-dependent increase in impedance for agonists and positive allosteric modulators, whereas receptor inhibition by an antagonist or negative allosteric modulator resulted in a concentration-dependent decrease in cellular impedance. Interestingly, constitutive receptor activity was observed that was decreased by LY341495, which therefore behaved as an inverse agonist here, a property that was heretofore unappreciated. This was confirmed by concentration-dependent modulation of LY341495 potency and efficacy by a allosteric modulators.In summary, the use of the xCELLigence system to study mGlu 2 receptor pharmacology was validated. This is the first class C GPCR to be characterized extensively by such method, opening new avenues to study receptor pharmacology including inverse agonism and demonstrating its value for future drug discovery efforts of mGlu receptors as well as other GPCRs.

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“…NPS fragments (NPS (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), NPS (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), NPS(1-6), NPS(1-10) 23…”

Section: Drugs and Reagentsunclassified

“…These results are summarized in Table 1. Specifically, NPS (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and [Bip 2 ]NPS at 10 μmol L −1 , but not at 1 μmol L −1 , elicited a significant DMR response in wild-type cells, thus for these compounds, 1 μmol L −1 was selected as highest concentration for further studies. PWT1-NPS caused a significant DMR effect at 1 and 0.1 μmol L −1 in wild-type cells, thus further experiments in HEK293 mNPSR were performed using 0.01 μmol L −1 as highest concentration for this ligand.…”

Section: Dmr Effects Of Npsr Ligandsmentioning

confidence: 99%

“…Several intracellular events can lead to changes in the cells refractive index, that is, protein recruitment, receptor internalization and recycling, second messenger alternation, cytoskeletal remodeling, and cell adhesion changes . DMR has been already applied to study the pharmacological properties of new ligands acting at various GPCRs, such as histamine H 1 , β 2 adrenergic, muscarinic M 3 , purinergic P2Y, formyl peptide, and protease‐activated receptors. Classical opioid and the nociceptin/orphanin FQ peptide (NOP) receptors were also investigated in DMR studies.…”

Section: Introductionmentioning

confidence: 99%

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Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

Ruzza

Ferrari

Guerrini

et al. 2018

Pharmacology Res & Perspec

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Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label‐free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS‐evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq‐dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.

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Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion

Cited by 24 publications

References 33 publications

Impedance-based cellular assays for regenerative medicine

Impedance-based cellular assays for regenerative medicine

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

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