Real-time analysis of the activities of GnRH and GnRH analogs in ?T3-1 cells by the Cytosensor microphysiometer

Ng, Sophia; Yu, KL; Yung, Wing‐Ho; Chow, Billy K. C.

doi:10.1002/1097-4644(20010301)80:3<304::aid-jcb20>3.0.co;2-h

Cited by 4 publications

(1 citation statement)

References 32 publications

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“…The Cytosensor microphysiometer (Hafner, 2000) was widely adopted in the late 90s to measure metabolic changes and was particularly relevant for APJ deorphanization. This technology provides a quantitative measurement of extracellular acidification rate elicited by small molecules, neuropeptides, and hormones in response to GPCR activation but also to ligand-gated ion channels and receptor tyrosine kinases (Barrett et al, 1997;Lang et al, 2001;Merkouris et al, 1997;Ng et al, 2001;Pitchford et al, 1995;Pitchford et al, 1997).…”

Section: Apelin/elabela Receptor (Apj Receptor)mentioning

confidence: 99%

Deorphanization of G Protein Coupled Receptors (GPCRs): a historical perspective.

Franchini,

Orlandi

2024

Mol Pharmacol

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Counting over 800 members, G Protein Coupled Receptors (GPCRs) form the largest family of membrane receptors encoded in the human genome. Since the discovery of G proteins and GPCRs in the late 1970s and early 1980s, a significant portion of the GPCR research has been focused on identifying ligand/receptor pairs in parallel to studies related to their signaling properties. Despite significant advancements, about a fourth of the ~400 non-odorant GPCRs are still considered orphan because their natural or endogenous ligands have yet to be identified. We should consider that every GPCR was once an orphan and that endogenous ligands have often been associated with biological effects without a complete understanding of the molecular identity of their target receptors. Within this framework, this review offers a historical perspective on deorphanization processes for representative GPCRs, including Ghrelin receptor, GABAB receptor, Apelin receptor, Cannabinoid receptors, and GPR15. It explores three main scenarios encountered in deorphanization efforts and discusses key questions and methodologies employed in elucidating ligandreceptor interactions, providing insights for future research endeavors. SIGNIFICANCE STATEMENTUnderstanding how scientists have historically approached the issue of GPCR deorphanization and pairing of biologically active ligands with their cognate receptors are relevant topics in pharmacology. In fact, the biology of each GPCR, including their pathophysiological involvement, has often been uncovered only after their deorphanization, illuminating druggable targets for various diseases. Furthermore, uncovered endogenous ligands have therapeutic value as many ligands -or derivates thereof -are developed into drugs.

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Section: Apelin/elabela Receptor (Apj Receptor)mentioning

confidence: 99%

Deorphanization of G Protein Coupled Receptors (GPCRs): a historical perspective.

Franchini,

Orlandi

2024

Mol Pharmacol

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Development of a 68Ga-peptide tracer for PET GnRH1-imaging

et al. 2016

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The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 μg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.

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A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve systemElectronic supplementary information (ESI) available: Derivation of eqn. (4). See http://www.rsc.org/suppdata/an/b3/b315007k/

Erxleben

Manion

Hockenbery

et al. 2004

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Monitoring extracellular acidification rates (ECARs) is important for the study of cellular activities, since it allows for the evaluation of factors that alter metabolic function, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. While the light addressable potentiometric sensor (Cytosensor Microphysiometer) has been the principal tool for ECARs measurement in the past, this work introduces a novel method that exploits an immobilized pH indicator on the surface of microcarrier beads (Sephadex) and is probed with a fiber optic coupled spectrophotometer. Likewise, live cells under investigation were also immobilized on microcarrier beads (Cytopore). These beads are metered, transported and monitored within a microfluidic system, termed as the Lab-on-Valve (LOV). Use of carrier beads in conjunction with Bead Injection Spectrophotometry and a Lab-on-Valve module (BIS-LOV), makes ECAR measurements reliable and automated. The feasibility of the BIS-LOV approach is demonstrated measuring ECARs of the mouse hepatocyte cell line, TABX.2S, grown on Cytopore beads packed within the central channel of the LOV system. These immobilized cells were perfused in a phosphate buffer carrier solution (capacity: 1 mmol L(-1), pH 7.4). Protons extruded from 10(5) to 10(6) cells were accumulated during a stopped flow period of 220 s followed by a pH measurement, detected by changes in absorbance of the pH indicator bonded to the microcarrier beads. Addition of metabolic inhibitors (sodium azide, oxamic acid) to the carrier buffer solution can induced an increase or decrease of the basal proton extrusion rate in a very reproducible manner. Comparison of the BIS-LOV technique to the Cytosensor microphysiometer and literature confirms the validity of this novel approach, highlighting its advantages and suggesting future improvements that will make the BIS-LOV a practical tool for routine ECARs measurement.

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Real-time analysis of the activities of GnRH and GnRH analogs in ?T3-1 cells by the Cytosensor microphysiometer

Cited by 4 publications

References 32 publications

Deorphanization of G Protein Coupled Receptors (GPCRs): a historical perspective.

Deorphanization of G Protein Coupled Receptors (GPCRs): a historical perspective.

Development of a 68Ga-peptide tracer for PET GnRH1-imaging

A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve systemElectronic supplementary information (ESI) available: Derivation of eqn. (4). See http://www.rsc.org/suppdata/an/b3/b315007k/

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