Abstract

Abstract ID 15499

Poster Board 475

Purpose: Endothelin-A receptor (ETAR) antagonist, BQ123 augments opioid analgesia, mitigates tolerance, and reduces withdrawal symptoms. Opioid-induced activation of GPCRs inhibits adenylyl cyclase and decreases 3’,5’-cyclic adenosine monophosphate (cAMP). On the other hand, cAMP upregulation is observed in opioid tolerance and withdrawal. However, the signaling mechanisms involved in the effects of BQ123 on opioids are unknown. The objective of this study was to determine the effect of BQ123 on morphine-induced changes in cAMP signaling in SH-SY5Y neuroblastoma cells.

Methods: A stably transfected SH-SY5Y cell line (ATCC®) was established with GloSensor™ -23F cAMP plasmid (Promega™) containing the hygromycin-resistance gene, using ViaFect™ (Promega™) as transfection reagent. Cells were maintained and seeded in 96-well white, opaque-bottom plates (5×10⁴ cells/well) in DMEM/10% FBS at 37ºC/5% CO₂. Promega’s real-time cAMP-GloSensor^TM assay was optimized and used to measure cAMP response in live cells. Transfected cells were incubated with equilibrium buffer [cAMP GloSensor™ reagent (2% (v/v)), 88% CO₂ independent medium, 10% FBS]. cAMP levels were determined by changes in luminescence using EnSpire^TM multimode microplate reader (PerkinElmer). Cells were treated with morphine (10^-5 – 10 uM) and BQ123 (1 uM) for 2 h in acute experiments, and with morphine (1 uM) in presence and absence of BQ123 (1 uM) for 24 h in chronic studies. Two challenge doses of morphine (10 uM and 20 uM) were tested in cells treated chronically with morphine. cAMP signaling was stimulated with isoproterenol (10^-8 – 1 uM) as positive control. Experiments were repeated in triplicate and data was analyzed in GraphPad Prism version 9.00 (GraphPad Software, San Diego, CA).

Results: The positive control, isoproterenol, produced a concentration-dependent increase in cAMP in -23F cAMP plasmid transfected SH-SY5Y cells. Acute morphine treatment (10^-5 – 10 uM for 2 h) decreased cAMP, while acute treatment with BQ123 (1 uM) further decreased cAMP response. It was intriguing to note that BQ123 (1 uM) enhanced morphine-induced reduction of cAMP responses. Chronic morphine (1 uM for 24 h) increased cAMP response by 53% (p<0.05) compared to acute treatment, confirming the development of tolerance. Chronic treatment with combination of morphine (1 uM) and BQ123 (1 uM) resulted in dose-dependent inhibition of cAMP levels in response to challenge doses of morphine.

Conclusion: The present study provided insight into the mechanism by which ETAR antagonists decrease morphine tolerance in SH-SY5Y cells. BQ123 enhanced morphine-induced cAMP signaling through G_i-coupled mu opioid receptors (MORs) in both acute and chronic studies in SH-SY5Y cells, which support our previous work. These findings suggest that cAMP is involved in the effect of BQ123 on morphine tolerance. Detection of the MOR is essential to ensure that cAMP signaling is regulated by MOR agonists. We are presently confirming these effects by detection of MORs in SH-SY5Y cells using flow cytometry. In vitro experiments are ongoing to determine effects of BQ123 on intracellular calcium response to morphine treatment using fluorescent imaging. In future studies, we plan to investigate ETAR expression and explore potential interactions between MORs and ETARs in SH-SY5Y cells.

Funding Info: Faculty Research Grant, Midwestern University College of Pharmacy; Biomedical Sciences Program, College of Graduate Studies, Downers Grove, IL.