Evaluating Opioid-Mediated Adenylyl Cyclase Inhibition in Live Cells Using a BRET-Based Assay

Manandhar, Preeti; Sachdev, Shivani; Santiago, Marina

doi:10.1007/978-1-0716-0884-5_11

Cited by 1 publication

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“…Bioluminescence based cAMP assay was performed in HEK-µ cells to investigate the effect of pregabalin or gabapentin on morphine mediated inhibition of cAMP production (Sachdev et al, 2019;Manandhar, Sachdev & Santiago, 2020). The cells for the assay were detached from the flask with trypsin/EDTA and seeded in a 10 cm plate at a density of 6 million cells to reach 60-70% confluency in 24 h. Next day, pcDNA3L-His-CAMYEL plasmid was transiently transfected into the cells using linear polyethyleneimine (PEI) at a ratio of 1:6 (DNA: PEI).…”

Section: Assay For Camp Measurementmentioning

confidence: 99%

Do gabapentin or pregabalin directly modulate the µ receptor?

Manandhar

Murnion

Grimsey

et al. 2021

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Background Pregabalin and gabapentin improve neuropathic pain symptoms but there are emerging concerns regarding their misuse. This is more pronounced among patients with substance use disorder, particularly involving opioids. Co-ingestion of gabapentinoids with opioids is increasingly identified in opioid related deaths, however, the molecular mechanism behind this is still unclear. We have sought to determine whether pregabalin or gabapentin directly modulates acute μ receptor signaling, or μ receptor activation by morphine. Methods The effects of pregabalin and gabapentin were assessed in HEK 293 cells stably transfected with the human μ receptor. Their effect on morphine induced hyperpolarization, cAMP production and ERK phosphorylation were studied using fluorescent-based membrane potential assay, bioluminescence based CAMYEL assay and ELISA assay, respectively. Pregabalin/gabapentin effects on morphine-induced hyperpolarization were also investigated in AtT20 cells. Results Pregabalin or gabapentin (1 µM, 100 µM each) did not activate the µ receptor or affect K channel activation or ERK phosphorylation produced by morphine. Neither drug affected the desensitization of K channel activation produced by prolonged (30 min) application of morphine. Gabapentin (1 µM, 100 µM) and pregabalin (1 µM) did not affect inhibition of forskolin-stimulated cAMP production by morphine. However, pregabalin (100 µM) potentiated forskolin mediated cAMP production, although morphine still inhibited cAMP levels with a similar potency to control. Discussion Pregabalin or gabapentin did not activate or modulate µ receptor signaling in three different assays. Our data do not support the hypothesis that gabapentin or pregabalin augment opioid effects through direct or allosteric modulation of the µ receptor. Pregabalin at a high concentration increases cAMP production independent of morphine. The mechanism of enhanced opioid-related harms from co-ingestion of pregabalin or gabapentin with opioids needs further investigation.

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Section: Assay For Camp Measurementmentioning

confidence: 99%