Assessment of Morphine Tolerance Using cAMP‐GloSensor&[trade] Real‐time Detection Assay in SH‐SY5Y Human Neuroblastoma Cells
Authors: Bhalla Shaifali , Karlinski Zachary , Bosco Kylie , Zhang Zhong , Gilchrist Annette 
Published: April 2020

Purpose

Activation of GPCRs by morphine triggers downstream effects including inhibition of adenylyl cyclase and decrease in 3′,5′‐cyclic adenosine monophosphate (cAMP). cAMP upregulation is observed in morphine tolerance and withdrawal. Endothelin‐A receptor (ETAR) antagonist, BQ123 potentiates morphine analgesia, reverses tolerance, and reduces withdrawal symptoms. However, signaling mechanisms involved in these phenomena are unknown. The objective of this study was to determine morphine‐ and BQ123‐induced changes in cAMP signaling in SH‐SY5Y neuroblastoma cells which endogenously express mu‐opioid receptors (MORs) and ETARs.

Methods

SH‐SY5Y cells (ATCC®) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37°C/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104 cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and 30% transfection efficiency was achieved. Stably transfected SH‐SY5Y cell line was established using GloSensor™ −23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Promega’s real‐time detection system in live cells, the cAMP‐GloSensor™ assay was used to measure cAMP responses. Stably 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 an EnSpire™ multimode microplate reader (PerkinElmer). In acute studies, cells were treated for 2 h with morphine (10–10−5 M) and BQ123 (1 μM). In chronic studies, cells were treated for 24 h with morphine (1 μM) in the presence and absence of BQ123 (1 μM). After induction of tolerance for 24 h, two challenge doses of morphine (10 μM and 20 μM) were tested. Isoproterenol was used as a positive control (1–10−8 M). Each experiment was repeated in triplicate and data was analyzed in GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA).

Results

A concentration‐dependent increase in cAMP was observed with isoproterenol in SH‐SY5Y cells stably transfected with −23F cAMP plasmid. Acute morphine treatment (10–10−5 M for 2 h) produced concentration‐dependent decrease in cAMP, while acute treatment with BQ123 (1–10−5 M) increased cAMP levels. Of interest, BQ123 (1 μM) appeared to enhance morphine‐induced decrease in cAMP response. Chronic morphine treatment (1 μM for 24 h) increased cAMP response, indicative of tolerance. Cells treated with combination of morphine (1 μM) and BQ123 (1 μM) for 24 h showed dose‐dependent inhibition of cAMP levels with challenge doses of morphine.

Conclusion

To better understand the mechanism by which ETAR antagonists reverse morphine tolerance and reduce withdrawal symptoms, signaling mechanisms were explored in SH‐SY5Y cells endogenously expressing ETA receptors and MORs. We found that BQ123 appears to enhance morphine‐induced cAMP signaling (measured by Glo‐Sensor™ cAMP assay) through Gi‐coupled MORs in both acute and chronic studies, which support our previous work. We will further explore the involvement of beta‐arrestin and GPCR kinases (GRKs) in these phenomena.

Support or Funding Information

Chicago College of Pharmacy Faculty Research Grant; College of Graduate Studies, Biomedical Sciences, Midwestern University, Downers Grove IL.