Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Summary

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Abstract

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of Gαi/o-linked receptors including D₂ dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D₂ dopamine receptor cellular models (i.e. CHO-D_2L and HEK-AC6/D_2L), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Introduction

An adaptive adenylyl cyclase (AC) signaling response known as heterologous- or super-sensitization was first discovered in the laboratory of Nobel Laureate, Dr. Marshall Nirenberg. Dr. Nirenberg proposed that the observed increased AC responsiveness following chronic δ opioid receptor activation was a mechanism involved in opiate tolerance and dependence¹. In addition to chronic δ opioid receptor activation, this neuroadaptive response of AC signaling also occurs following persistent activation of several other Gαi/o-coupled receptors². Notably, many of these receptors are associated with pain, neuropsychiatric and neurological dis....

Protocol

1. Expansion and Cryopreservation of Assay Ready Cells

1. Culture CHO-K1-DRD_2L (CHO-D_2L) cells on a 15 cm² cell culture dish in Ham's F12 media supplemented with 1.0 μM L-glutamine, 800 μg/μl G418, 300 μg/μl hygromycin, 100 u/μl penicillin, 100 μg/μl streptomycin, and 10% fetal bovine serum (FBS).
2. Incubate the cells at 37 °C in a humidified incubator with 5% CO₂ until the cells are 90-95% confluent. Wash the cells with 10 μl Phosphate Buffered Saline (PBS), and harvest the cells by adding 3 μl of cell dissociation buffer for 5 min at 37 °C. Re....

Results

Part I. Developing a 384 well heterologous sensitization assay for identifying small molecule inhibitors using a commercially available cell model.

To study heterologous sensitization in a cell model, we made a number of improvements that enabled us to streamline the assay into a "mix and read" format. Several of the key modifications are highlighted below, and are described in more detail in the discussion. The first key step was modifying our cell culture from continuously c.......

Discussion

In an effort to facilitate studies of heterologous sensitization, we have extensively modified our previous method achieving a streamlined "mix and read" format that is amendable to high-throughput screening and mechanism of action examination. The major modifications to our protocol can be summarized as follows: 1) the use of cryopreserved cells as "assay-ready" reagents; 2) the miniaturization of the assay into 384-well format; and 3) the utilization of the HTRF measurement of cAMP.

The adop.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CHO-K1-DRD2L cells	DiscoveRx	930579C2
Ham’s F12 media	VWR	SH30026fs
Fetal Bovine Serum	VWR	SH3007003
G418	Sigma	A1720
Hygromycin	Fisher	50-230-5556
Penicillin/streptomycin	Life Technologies	15070063
15 cm dishes	BD Falcon	353025
DMSO	Sigma	D2650
Cell dissociation buffer	Life Technologies	13150016
Phosphate Buffered Saline	Life Technologies	10010-049
Cryovials	Fisher	976171
Opti-MEM	Life Technologies	31985070
TC treated 384-well plates	Perkin-Elmer	6007688
HTRF cAMP Dynamic 2 kit	Cisbio Bioassays	62AM4PEB	http://www.htrf.com/camp-cell-based-assay
Synergy 4	Biotek	H4MLFPTAD
Prism 6	GraphPad Software	NA
3-Isobutyl-1-methylxanthine	Sigma	I5879
(±)Quinpirole	Sigma	Q111
Spiperone	Sigma	S7395
Clozapine	Sigma	C6305
Haloperidol	Sigma	H1512
S-(-)Sulpiride	Sigma	S112
Lipofectamine 2000 transfection reagent	Invitrogen	11668019
Trypan blue	Life Technologies	T10282
Water, DNase- and RNase-free	MP Biomedicals	821739
Gas siRNA	Dharmacon	custom siRNA
Nontargeting siRNA control	Dharmacon	D-001206-14-20
siGlo Red	Dharmacon	D-001630-02