MTT-Based Assessment of the Effects of NPS and SHA68 on HEK293 Cell Viability

The Neuropeptide S receptor (NPSR) is a class A G protein-coupled receptor (GPCR) activated by the endogenous ligand Neuropeptide S (NPS). NPSR activation engages both Gs- and Gq-mediated signalling pathways, resulting in increased intracellular cAMP production and calcium mobilization. Through these signalling mechanisms, the NPS–NPSR system contributes to the regulation of several physiological processes, including arousal, anxiety, learning, memory consolidation, stress responses, and nociception. Its broad involvement in central nervous system functions has therefore made NPSR a potential pharmacological target for the development of treatments for neuropsychiatric disorders. The aim of this study was to evaluate whether exposure to the endogenous agonist NPS or the selective NPSR antagonist SHA68 affected the viability and metabolic activity of human embryonic kidney 293 (HEK293) cells expressing NPSR. Wild-type HEK293 cells were tested in parallel to distinguish NPSR-dependent effects from non-receptor-mediated cellular responses. NPSR-expressing and wild-type HEK293 cells were exposed to increasing concentrations of NPS, either alone or in the presence of 1 μM SHA68. Triton X-100 was used as a positive control for cytotoxicity. Cellular metabolic activity was evaluated using the classical 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, which measures the reduction of MTT to insoluble formazan by metabolically active cells. Following solubilization of the formazan crystals, absorbance was measured at 570 nm using a Victor microplate reader. Results were expressed as a percentage of the response measured in vehicle-treated control cells. Triton X-100 produced a marked concentration-dependent reduction in the MTT signal, confirming the responsiveness of the assay to cytotoxic treatment. NPS produced a modest reduction in MTT-derived metabolic activity after 24 h of treatment. This effect was prevented by co-treatment with SHA68, consistent with pharmacological antagonism of the NPS effect. However, variability in wild-type HEK293 experiments prevents a definitive conclusion regarding NPSR dependence. Further studies using complementary functional and proliferation assays will be required to clarify the biological significance of these findings.