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Method Article

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

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DOI:

10.3791/51218

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January 27th, 2014

 ,  ,  ,  , 
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 2Quantitative Biology, Eli Lilly and Company

In This Article

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-D2L and HEK-AC6/D2L), 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 disorders, and include μ/κ opioid, D2/4 dopamine, 5HT1A, and M2/4 muscarinic receptors2. In addition to Dr. Nirenberg's findings, a large body of evidence exists linking sensitization of AC signaling to chronic opioid receptor activation both in vitro and in vivo3-7. Sensitization of AC has also been associated with a variety of diseases involving D2-like dopamine receptors including schizophrenia and Parkinson's disease (for review see reference2). Despite the potential importance of sensitization, the precise mechanism(s) associated with persistent Gαi/o-coupled receptor activation that leads to increased AC responsiveness remains largely unknown.

These studies provide the rationale for examining the mechanisms for sensitization of adenylyl cyclase as an important neurobiological target. Likewise, the physiological relevance of AC signaling8 and the importance that the individual AC isoforms hold in this adaptive response should also be recognized2,9,10. In the context of our research, the general features associated with heterologous sensitization of the recombinant isoforms of AC parallel those characteristics described for studying the endogenous isoforms of AC. Specifically, previous research has found that the activation of Gαi/o proteins and subsequent release/rearrangement βγ subunits are important requirements for receptor induced sensitization of all AC isoforms. Additionally, several studies suggest that signaling from protein kinases and Gβγ subunits are involved in sensitization2,11-13. Individual ACs also display unique and distinct sensitization patterns12. For instance, persistent exposure of D2 receptors to agonists is associated with sensitization of AC1 and AC8 to Ca2+/calmodulin stimulation14,15, whereas the closely related AC3 is not sensitized2. AC2, AC4, and AC7 are closely related, however, only PKC-stimulated AC2 activity is robustly sensitized after prolonged exposure of D2 receptors to agonists7,14,16,17. Additionally, AC5 and AC6 show a marked degree of heterologous sensitization to Gas- and forskolin-stimulated cAMP accumulation following activation of D2 receptors14,18-20, but appear to differ in their requirement for Gβγ subunit-AC interactions21. Although most studies of AC sensitization have used model cell lines (e.g. HEK293 cells expressing individual AC isoforms), it appears that these findings translate to native neuronal cell models4,22. More recently, the effects of AC isoform selective small molecule inhibitors identified in HEK293 cells expressing AC isoforms can also be translated to in vivo behavioral studies23.

The lack of an identified molecular mechanism for heterologous sensitization likely reflects the complexity of the adaptive response as well as the unique regulatory properties of the individual AC isoforms12. Unraveling such complexity is further complicated by the use of cumbersome methodology that has limited academic investigators from employing unbiased approaches. For example, our previous mechanistic studies involved the use of continuously cultured cellular models using 24- and 48-well tissue culture format15. Cultured cells were typically grown for 48 hr and then subjected to agonist drug treatment (2-18 hr) followed by a series of cell washes and incubations (Figure 1). AC-isoform specific cAMP accumulation protocols were then employed followed by measurement of cAMP accumulation using a laborious and time consuming [3H]cAMP binding methodology15,24. The duration from start to finish for each assay generally required a total of four to five days from cell plating to data analysis (Figure 1). The application of new technologies and automation has led to marked enhancements for sensitization studies in the industrial and HTS center setting. For example, a group working with the National Center for Chemical Genomics reported a two day HTS assay procedure for identifying small molecule inhibitors of μ opioid receptor induced sensitization in 1,536-well format25.

The present article describes our efforts to develop an HTS assay for studies of heterologous sensitization using technologies that are available at most academic research institutions. This strategy was accomplished by incorporating the use of cryopreserved cells from cell models heterologously expressing the D2 dopamine receptor in combination with endogenous or individual recombinant adenylyl cyclase isoforms (CHO-D2L or HEK-AC6/D2L). To improve our throughput, we redesigned our 48 well sensitization assay (ca. >20 steps over 4-5 days) to a five-step, single day assay in 384-well format that was essentially "mix and read". The new format uses a commercially available homogenous time resolved fluorescence (HTRF) assay to measure cAMP accumulation in intact cells with a multi mode plate reader. The assay is robust and amenable to small molecule screening, and can be effectively applied to screen for inhibitors of heterologous sensitization. In addition, we provide data that allows the use of this assay with reverse transfection of siRNA for targeted or genome wide siRNA library screening with only a minor modification to the general approach.

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Protocol

1. Expansion and Cryopreservation of Assay Ready Cells

  1. Culture CHO-K1-DRD2L (CHO-D2L) 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. Resuspend the cells using 12 μl of the culture media and count cells using trypan blue exclusion.
  3. Centrifuge the cell suspension at 500 x g for 5 min at room temperature. Aspirate the supernatant and resuspend the cell pellet in 5 μl of freezing media (10% dimethyl sulfoxide, 90% FBS). Dilute the cell suspension to achieve the desired cell concentration (e.g. 1-20 x 106 cells/μl).
  4. Aliquot 1.0 μl of cell solution to each cryovial. Incubate the cryovials in a cell freezing container at -80 °C overnight. Transfer the cryovials to a liquid N2 tank for long term storage.

2. Plating Assay Ready Cells (and Reverse Transfection Option)

  1. Rapidly thaw a frozen cryovial of cells in a 37 °C water bath. Once cells are thawed, transfer the cells to a 15 μl conical tube containing 9 μl of Opti-MEM, and mix by inverting the tube 3-5x.
  2. Centrifuge the cells at 500 x g for 5 min at room temperature. Aspirate the supernatant and resuspend the cells in 1 μl Opti-MEM.
  3. Count the cells using trypan blue exclusion to determine cell viability and dilute the viable cells as necessary (e.g. 3 x 105 cells/μl concentration) in Opti-MEM. Plate 10 μl/well of cells in a tissue culture treated 384-well plate using a multichannel pipette.
  4. Make serial dilutions of cAMP in Opti-MEM to generate a standard curve for estimating cAMP production by the cells (according to the manufactures recommendations - see Section 7). Add 10 μl/well of the cAMP standards to the plate. Note: a single standard curve can be prepared on a separate plate or blank wells on one of the assay plates.
  5. Centrifuge the plate at 100 x g for 15 sec at room temperature, and incubate at 37 °C in a humidified incubator with 5% CO2 for 1 hr.

3. Reverse siRNA Transfection Option*

This section is optional and relevant to Figure 3.

  1. Prepare a solution of siRNA in RNase-free ddH2O (e.g. 0.4 pmol/μl). Add 5 μl to individual wells of 384-well plate, and centrifuge the plate at 100 x g for 15 sec at room temperature.
  2. Dilute Lipofectamine 2000 in Opti-MEM by a factor of 0.006 (e.g. add 6 μl of Lipofectamine 2000 to 1,000 μl of Opti-MEM), and mix by pipetting up and down.
  3. Incubate the Lipofectamine 2000/Opti-MEM solution for 5 min at room temperature. Add 5 μl of the diluted Lipofectamine 2000/Opti-MEM solution to individual wells of 384-well plate that already contains siRNA. Centrifuge the plate at 100 x g for 15 sec at room temperature.
  4. Incubate the plate for 30 min at room temperature.
  5. Plate assay ready cells as described above (Section 2). Centrifuge the plate at 100 x g for 15 sec at room temperature.
  6. Return assay plate to humidified incubator at 37 °C (5% CO2) for 48-96 hr as determined for targeted gene knock down (see discussion).

4. Small Molecule Screening

  1. Dilute the drug of interest (e.g. small molecule inhibitors) in Opti-MEM to 6x the desired final concentrations. Serial dilutions can be completed using hand-held pipettes or a liquid handling station.
  2. Add 2.5 μl/well of the test compound or buffer containing vehicle (e.g. DMSO) to the side of the wells using a multichannel pipette.
  3. Centrifuge the plate at 100 x g for 15 sec at room temperature (incubation is optional).

5. Persistent Agonist Treatment

  1. Prepare a 600 nM (i.e. 6 times the desired final concentration of 100 nM) solution of quinpirole in Opti-MEM.
  2. Add 2.5 μl/well of the 600 nM quinpirole solution to the side of the wells using a multichannel pipette.
  3. Centrifuge the plate at 100 x g for 15 sec at room temperature, and incubate at 37 °C in a humidified incubator with 5% CO2 for 2 hr.

6. Stimulation of cAMP Accumulation

  1. During the 2 hr incubation, prepare the stimulation solution in Opti-MEM. The stimulation solution is comprised of 40 μM forskolin, 2 μM 3-isobutyl-1-methyxanthine (IBMX), and 4 μM spiperone (all concentrations in step 6 are 4x the desired final concentration).
  2. Add 5 μl/well of the stimulation solution to the side of the wells using a multichannel pipette.
  3. Centrifuge the plate at 100 x g for 15 sec at room temperature, and incubate at room temperature for 1 hr.

7. Quenching and Estimation of cAMP Accumulation

  1. Production of cAMP by the cells is measured using the cAMP dynamic 2 kit according to manufacturer’s instructions. Briefly, reconstitute anti-cAMP-cryptate and cAMP-d2 in distilled water. Freeze aliquots at -20 °C for short term storage.
  2. Dilute one aliquot of anti-cAMP-cryptate and one aliquot of cAMP-d2 separately in the lysis buffer according to the manufacturer’s instructions to make working solutions.
  3. Add 10 μl/well of the anti-cAMP-cryptate working solution and 10 μl/well of the cAMP-d2 working solution to the 384-well plate using a multichannel pipette.
  4. Centrifuge the plate at 100 x g for 15 sec at room temperature, and incubate at room temperature for 1 hr.
  5. Read the plate in a fluorescence plate reader (auto scale setting for sensitivity) using an excitation of 337 nm, and measure the emissions at 620 nm and 665 nm per manufactures instructions.
  6. Apply ratiometric analysis to assess cAMP standard curve per manufactures instructions. Using the resulting values, extrapolate the estimated cAMP accumulation in the test wells using data analysis software.

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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...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge Mr. Richard Zink and Todd Wiernicki for methodological training and assay guidance. We also thank John Paul Spence for careful reading and editorial suggestions. This work was supported by the National Institute of Health MH 060397, Brain and Behavior Research Foundation, and Eli Lilly and Company through the Lilly Research Award Program (LRAP).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CHO-K1-DRD2L cellsDiscoveRx930579C2
Ham’s F12 mediaVWRSH30026fs
Fetal Bovine SerumVWRSH3007003
G418SigmaA1720
HygromycinFisher50-230-5556
Penicillin/streptomycinLife Technologies15070063
15 cm dishesBD Falcon353025
DMSOSigmaD2650
Cell dissociation bufferLife Technologies13150016
Phosphate Buffered SalineLife Technologies10010-049
CryovialsFisher976171
Opti-MEMLife Technologies31985070
TC treated 384-well platesPerkin-Elmer6007688
HTRF cAMP Dynamic 2 kitCisbio Bioassays62AM4PEBhttp://www.htrf.com/camp-cell-based-assay
Synergy 4BiotekH4MLFPTAD
Prism 6GraphPad SoftwareNA
3-Isobutyl-1-methylxanthineSigmaI5879
(±)QuinpiroleSigmaQ111
SpiperoneSigmaS7395
ClozapineSigmaC6305
HaloperidolSigmaH1512
S-(-)SulpirideSigmaS112
Lipofectamine 2000 transfection reagentInvitrogen11668019
Trypan blueLife TechnologiesT10282
Water, DNase- and RNase-freeMP Biomedicals821739
Gas siRNADharmaconcustom siRNA
Nontargeting siRNA controlDharmaconD-001206-14-20
siGlo RedDharmaconD-001630-02

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Tags

Adenylyl Cyclase SensitizationcAMP Accumulation AssayHigh Throughput Screening384 well FormatCryopreserved CellsHTRF DetectionSmall Molecule ScreeningsiRNA Reverse TransfectionD2 Dopamine ReceptorQuinpirole Treatment
Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
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