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

Abstract

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.

A Dynamic Mass Redistribution Assay for the Human Sweet Taste Receptor Uncovers G-Protein Dependent Biased Ligands

“…Measuring the peak DMR response turned out to provide a much more robust assay window than measuring the final and sustained DMR response ( Figure 2D ). For this reason, subsequent data depicted in this report was generated with the peak DMR responses, as reported in other DMR studies with different GPCRs ( Schrage et al, 2013 ; Malfacini et al, 2018 ; Ruzza et al, 2018 ). Under these conditions, sucralose generated an EC 50 of 163 μM in this assay ( Figure 2E ; Table 1 ) (pEC 50 3.825 ± 0.174) and only the highest concentration of sucralose (≥2 mM) produced a smaller non-specific effect on the parental U2OS cells [typically around 20–40 pm (pm), Figures 2B,E ].…”

The Neural Network of Neuropeptide S (NPS): Implications in Food Intake and Gastrointestinal Functions

“…In vitro pharmacological studies, performed on cell lines stably expressing human and murine NPSRs, revealed that NPS acts as an excitatory neurotransmitter, in light of the ability to elevate intracellular-free Ca 2+ and to stimulate cAMP synthesis, at nanomolar concentrations and in a dose-dependent manner, indicating that the NPSR couples to both G q and G s protein, and thus increases cellular excitability [ 15 , 25 , 29 , 30 ]. Additionally, radioligand binding assays demonstrated that the radiolabeled NPS analog ( 125 I-labeled Tyr 10 -NPS) shows a displaceable binding with increasing concentrations of NPS (Kd = 0.3 nm), suggesting that NPS behaves as a typical neurotransmitter, binding and activating its cognate receptor with high potency and specificity [ 15 , 28 ].…”

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

“…( A ) Interference of the G protein modulators FR, CTX, PTX and gallein with histamine receptor mediated signaling. FR (alias UBO − QIC) selectively silences Gα q/11 signaling by blocking the GDP − release at concentrations 0.1 to 1.0 µM [ 18 , 41 , 44 , 45 , 46 , 47 ]. PTX selectively and irreversibly silences Gα i/o at a concentration of 100 ng/mL by ADP − ribosylation at the Gα − subunit [ 20 , 33 , 37 , 40 , 41 , 42 ].…”

“…As ribosylation leads to a maximum activation of the Gα s protein rather than inhibition of the Gα s protein, the results should be interpreted with caution. FR (alias UBO-QIC) selectively silences Gα q/11 signaling by blocking the GDP-release in the Gα subunit [ 18 , 41 , 44 , 45 , 46 , 47 ], which is a mandatory step in G protein activation. The advantage of such studies is that they can be performed with the same cell model under identical experimental conditions, thereby precluding cell bias.…”

“…The advantage of such studies is that they can be performed with the same cell model under identical experimental conditions, thereby precluding cell bias. Such investigations have already been performed for a variety of GPCRs [ 37 , 38 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Another approach to investigate the involvement of G proteins in signal transduction is to prevent their expression by knocking out the corresponding genes [ 41 , 54 , 55 ].…”