Photorelease of 2-Arachidonoylglycerol in Live Cells

Article abstract of DOI:10.1021/jacs.9b05978

2-Arachidonoylglycerol (2-AG) is acting as a full agonist of cannabinoid receptor 1 and 2. Direct manipulation of 2-AG levels is a challenging task. The amphiphilic properties and the instability of 2-AG in aqueous media complicate its use as a drug-like molecule. Additionally, inhibition of the protein machinery that regulates 2-AG levels may also affect other monoacylglycerols. Therefore, we developed a novel method to elevate 2-AG levels with a flash of light. The resulting tool is a photoactivatable "caged" 2-arachidonoylglycerol (cg2-AG) allowing for the rapid photorelease of the signaling lipid in live cells. We characterized the mechanism of uncaging and the effect of 2-AG on the regulation of the β-cell signaling network. After uncaging of 2-AG, we monitored calcium levels, CB1-GIRK channel coupling, and CB1-mediated inhibition of adenylate cyclase and protein kinase A activity.

Full text of DOI:10.1021/jacs.9b05978

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Communication
Photo-Release of 2-Arachidonoylglycerol in Live Cells
Aurelien Laguerre, Sebastian Hauke, Jian Qiu, Martin J. Kelly, and Carsten Schultz
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05978 • Publication Date (Web): 27 Sep 2019
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Photo-Release of 2-Arachidonoylglycerol in Live Cells.
Aurélien Laguerre, Sebastian Hauke§, Jian Qiu, Martin J. Kelly and Carsten Schultz*§.
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Aurélien Laguerre, Jian Qiu, Martin Kelly, Carsten Schultz, Dept. of Chemical Physiology & Biochemistry, OHSU,
Portland, OR, USA. § Sebastian Hauke, Carsten Schultz, European Molecular Biology Laboratory, Cell Biology and
Biophysics Unit, 69117 Heidelberg, Germany.
Supporting Information Placeholder
[11]. Caged lipids possess a photo-removable protecting group
ABSTRACT: 2-Arachidonoylglycerol (2-AG) is the only
(PRPG) covalently attached to one of the crucial functional groups
of the lipid, thereby preventing its recognition by the cellular
machinery. A flash of UV light is sufficient for the PRPG to release
the signaling molecule. Here, this concept is applied to 2-AG,
allowing the exclusive manipulation of 2-AG levels and its
downstream effectors with high spatio-temporal resolution without
interfering with the genetic or the protein machinery. We
monitored calcium levels and CB1-mediated inhibition of
adenylate cyclase and protein kinase A activity via confocal
monoacylglycerol acting as a full agonist of cannabinoid receptor
1 and 2. Direct manipulation of 2-AG levels is a challenging task.
The amphiphilic properties and the instability of 2-AG in aqueous
media complicate its use as a drug-like molecule. Additionally,
inhibition of the protein machinery that regulates 2-AG levels may
also affect other monoacylglycerols. Therefore, we developed a
novel method to elevate 2-AG levels with a flash of light. The
resulting
tool
is
a
photo-activatable
“caged”
2-
arachidonoylglycerol (cg2-AG) allowing for the rapid photo-
release of the signaling lipid in live cells. We characterized the
mechanism of uncaging and the effect of 2-AG on the regulation of
the β-cell signaling network. After uncaging of 2-AG, we
monitored calcium levels, CB1-GIRK channel coupling and CB1-
mediated inhibition of adenylate cyclase and protein kinase A
activity.
The
endocannabinoid
lipids
anandamide
and
2-
arachidonoylglycerol (2-AG) stimulates the two G-protein coupled
cannabinoid receptor subtypes 1 and 2 (CB1 and CB2) [1, 2]. The
downstream signaling network regulates a multitude of processes
such as mood, appetite, pain-sensation, memory, and insulin
secretion [3, 4]. 2-AG is produced on demand via the activation of
diacylglycerol lipases α and β but also depends on the
dephosphorylation of lysophosphatidic acid to 2-AG. 2-AG levels
are further regulated by several monoacylglycerol lipases (MAGL,
ABHD6 and ABHD12) that degrade 2-AG into glycerol and
arachidonic acid [5]. All three enzyme are located at the plasma
membrane but MAGL also resides in the cytosol [6]. 2-AG likely
targets its receptors in the producing cell as well as triggering CB1
and CB2 in neighboring cells. [7]. CB1 and CB2 act via the trimeric
Gi/o protein but also open the G protein-coupled inwardly-
rectifying potassium channel (GIRK) via the G-protein β/γ subunit
[8]. The physicochemical properties of 2-AG complicate its use as
a drug-like molecule for external administration, due to its
amphiphilic properties, its low solubility in aqueous solutions and
its fast molecular rearrangement thermodynamically favoring the
formation of the primary ester in 1(3)-AG [9]. As a result,
commercially available 2-AG is composed of a 9:1 ratio of the two
microscopy as well as CB1-GIRK channel coupling by
Figure 1. A. Chemical synthesis of caged 2-arachidonoylglycerol
(cg2-AG) and proposed solvent-assisted mechanism of photo-
release. B. Confocal micrographs of live MIN6 treated with 10 µM
cg2-AG or alcohol 4, before and after flash photolysis in a circular
region of interest (λex=405 nm, λuncaging=375 nm). Normalized
fluorescence intensity of live MIN6 cells treated with cg2-AG (10
µM) or alcohol 4 in ±UV. The reduction in fluorescence after
photolysis is likely due to C. UV/VIS spectra of cg2-AG (10 µM
in H2O) ±UV light photolysis (from 1 to 3 min irradiation, 1 kW
Xenon lamp equipped with a 350 nm long-pass filter). Top right
inlet. UV/VIS spectra of alcohol 4 under the same conditions is not
affected by the presence of light. D. Fluorescence emission spectra
of cg2-AG (10 µM in H2O, λex=405 nm), ±UV light photolysis.
isomers. Therefore,
a
strategy that prevents premature
isomerization will be beneficial. Other strategies to manipulate
endogenous 2-AG levels make use of lipase inhibitors, albeit in an
unspecific fashion [10]. Effects on other monoacylglycerols may
activate undesired protein targets and complicate the interpretation
of experimental observations. A more direct approach is the use of
caged lipids, which offer a unique spatio-temporal precision to
release natural lipids inside the cell, with a simple flash of light
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electrophysiology. It should be mentioned that for activating CB1,
350 nm did not affect UV/VIS absorbance and fluorescence
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Westphal et al. recently prepared and tested photoswitchable
tetrahydrocannabinol derivatives [12]. Herein, we aimed at
stimulating the cannabinoid receptors by its endogenous ligand,
which required the ability to selectively manipulate 2-AG
concentration in live cells.
emission intensity of 4 (Figures 1C and S11B).
The mouse β-cell line MIN6 is known to endogenously express
CB1 and CB2 and served as a model for studying photo-activation
of cg2-AG and its effect on cannabinoid receptors in live cells [18].
The insertion of a lipophilic PRPG onto both of hydroxyl groups of
the glycerol decreased the amphiphilic nature of 2-AG, prohibited
migration of the fatty acid and facilitated the cellular internalization
PRPGs such as nitrobenzyl groups and coumarins have been widely
used to prepare “caged” lipid derivatives [13, 14, 15]. However, the
protection of 1,3-diols has been a difficult task. Previous attempts
described by the Lawrence group showed that 6-membered ring
acetals formed with 6-bromo-7-hydroxy-4-formylcoumarin were
resistant to photo-cleavage [16]. We hypothesized that the presence
of an ester on the six-membered ring of the acetal of caged 2-AG
(cg2-AG) would reduce the electron-density on the oxygen-bearing
glycerol leaving group. We investigated 7-diethylamino coumarin
as a scaffold for caging 2-AG. Its fluorescent properties allow for
the visualization in single cells prior to photolysis. Unlike with
classic caging strategies, the uncaging mechanism of
photosensitive cyclic acetals would impose the departure of a
chemically altered chromophore after illumination providing the
opportunity to follow the uncaging process by simply monitoring
changes in fluorescence emission. A short and straightforward
synthetic pathway allowed for the synthesis of cg2-AG in only four
steps (Figure 1A). The first synthetic steps are directly inspired by
the described synthesis of the 7-diethylamino-4-hydroxymethyl-
coumarin [17]. 7-Diethylamino-4-formyl-coumarin 3 was used for
acetal formation with glycerol. Alcohol 4 provided a versatile
platform for the attachment of fatty acids and was used to couple
arachidonic acid, thereby providing cg2-AG.
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Figure 2. A. Representative traces of fluorescence increase F/F0
of live MIN6 pre-treated with the cell permeant calcium dye Fluo4-
AM (5 µM) in presence of cg2-AG ([cg2-AG]=10 µM, n=6, cyan
trace) and subjected to flash photolysis (λex=488 nm, λuncaging=375
nm) with and without the CB1 antagonist rimonabant (1 µM; [cg2-
AG]=10 µM, n=6, pink trace). B. Bar graphs summarizing
fluorescence intensity increase F/F0 of the calcium dye Fluo4/AM
(5 µM) in MIN6, treated with cg2-AG and subjected to flash
photolysis in the absence ([cg2-AG]=10 µM, n=136) or presence
of rimonabant (1 µM; [cg2-AG]=10 µM, n=79). The CB1 agonist
WIN55 was used as positive control ([WIN55]=10 µM, n=84) C.
Calcium imaging of MIN6 cells upon the uncaging of cg2-AG (10
µM) -/+ pre-incubation with rimonabant (1 µM). D.
Photomicrograph showing a recording pipette patched onto a MIN6
cell dispersed on a cover slip. E. Currents generated in a whole-cell
recording of a MIN6 cell elicited by holding the cell at –60 mV and
giving a series of 1 sec prepulses ranging from -50 mV to -140 mV
(in 10 mV increments) and stepping back to -60 mV before, after
photo-stimulation (0.6 mW for 1 min, and during perfusion with
300 μM diazoxide). F. Representative traces of photo-induced
currents by released 2-AG, KATP opener diazoxide or GIRK
channel blocker tertiapin. (Upper trace) After a seal was formed
and the whole-cell configuration was obtained, cells were perfused
with cg2-AG (10 μM) for 10 min before activating with a 375 nm
light pulse. After the current returned to resting level, the cells were
perfused with diazoxide, which generated a robust outward current.
Vhold= −60 mV. (Lower trace) The outward current induced by the
uncaging of cg2-AG was blocked by pretreatment with GIRK
channel blocker tertiapin (100 nM) for 15 min. G. I/V curves
generated in MIN6 cells in the presence of cg2-AG (10 μM, ±UV
light) or diazoxide (300 μM). H. Bar graphs summarizing the
effects of cg2-AG in absence or presence of tertiapin. Data points
represent the mean ±SEM. Cell numbers are indicated. Un-paired
t-test, t=2.551, P=0.0447.
Our first attempts to characterize the uncaging of cg2-AG in
organic solvents such as chloroform, dimethyl sulfoxide, ethanol or
methanol were unsuccessful. Using a 1kW Xenon lamp equipped
with a 350 nm long-pass filter, nuclear magnetic resonance
experiments showed that cg2-AG was photo-resistant in organic
solvents even after 15 minutes of irradiation (Figure S6). Following
the release mechanism proposed by Lawrence and co-workers [16],
we hypothesized that the presence of water could be a requirement
for proper uncaging. Satisfactorily, liquid chromatography / mass
spectrometry (LC/MS) analysis of a 10 µM aqueous solution
confirmed the photo-release of 2-AG in water (Figure S7 and S8).
Further UV/VIS HPLC analysis demonstrated the photo-release of
intermediate 3 and its hydrated form as well as 2-AG (Figure S9).
We confirmed that the caged lipid did not precipitate over time. UV
irradiation had a strong effect on the sample, triggering a rapid and
drastic reduction of the absorbance (Figure 1C). Fluorescence
emission intensity measurements of cg2-AG under the same
conditions showed the same tendency (Figure 1D, Figure S9).
Prolonged light exposure resulted in a stable fluorescence intensity,
indicating that the photo-reaction came to completion and produced
a new photo-stable compound (Figure S10C and S10D). While the
expected aldehyde 3 is highly fluorescent (Figure S9E), we
identified the hydrate of 3 as the low-fluorescent species (Figure
S9F). It should be mentioned that in cells, the formation of Schiff’s
bases and glutathione adducts might contribute to the observed
reduction in fluorescence.[13] Similar experiments in ethanol
showed no fluorescence decrease of the caged compound after
illumination (Figure S11C), further indicating the essential
participation of water molecules in the uncaging mechanism. To
confirm our hypothesis that the ester would reduce the electron-
density and encourage the departure of the leaving group, the
experiments were performed with alcohol 4 (Figure 1A-C). The
absorbance of 4 was red-shifted towards lower energy wavelengths
(401 nm vs. 389 nm for cg2-AG) and a stoke shift of 103 nm (vs.
87 nm for cg2-AG), demonstrating the electron-withdrawing effect
of the ester. As expected from Lawrence’s work, illumination at
of the caged compound leading to rapid and complete cellular
accumulation of cg-2AG in internal membranes at 1 to 10 µM
extracellular concentration (Figure S12. As previously observed in
vitro, flash photolysis at 375 nm in live MIN6 β-cells triggered a
strong decrease (62.0 %, n=25) of the fluorescence emission
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intensity (Figure 1B and S12A). Illumination of control alcohol 4
cells, confirming that cg2-AG was not metabolized by the cells
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(Figure 1B) was ineffective. Unlike many other diethyl amino-
coumarin cages, cg2-AG was almost completely resistant to
photolysis at 405 nm in cells (Figure S12B). This feature allowed
monitoring the amount of caged lipid by confocal microscopy with
405 nm illumination during the experiment without triggering
uncaging. At 375 nm, we followed the release of the
monoacylglycerol by directly measuring the drop in fluorescence
intensity at a single cell level.
over time.
Upon activation, CB1 is known to interact with the G-protein
inwardly rectifying potassium channel (GIRK) through the β/γ
subunit [20], triggering opening of the channel and membrane
hyperpolarization. Therefore, we performed whole-cell patches in
voltage clamp and current clamp mode on MIN6 cells under the
same buffer conditions as for the calcium imaging experiments
described above (Figure 2C). Before the recording, MIN6 cells
were incubated with 10 µM of cg2-AG, added directly to the
culture media. After 10 min, the media was replaced with imaging
buffer containing 11 mM glucose. In the absence of light, the caged
endocannabinoid was unable to activate the potassium channel
opening (Figure 2D). UV light irradiation at 375 nm transiently
triggered the opening of GIRK channels (Figure 2D, middle panel
and 2E), leading to an outward current (13.9 pA ±3.9, n=5) that was
inhibited with the selective GIRK channel blocker tertiapin (100
nM, 3.2 pA ±0.4, n=4, Figure 2D, lower trace and Figure 2G).
GIRK opening by the agonist diazoxide served as a positive control
(Figures 2D, 2E, upper trace and 2F).
Endocannabinoid lipids are known to induce a transient elevation
of intracellular Ca2+ levels ([Ca2+]i) through activation of CB1 [19].
Accordingly, MIN6 cells in the presence of 11 mM glucose at 37
°C were treated with permeant cg2-AG (10 µM) as well as the
calcium indicator Fluo-4/AM (5 µM) for 20 min. Fluctuations of
[Ca2+]i were monitored through excitation at 488 nm and emission
above 515 nm (F/F0) on the confocal microscope. A clear and
transient elevation of the intracellular calcium concentration [Ca2+]i
was observed among the illuminated cells ([cg2-AG]=10µM,
n=136 cells), characteristic of the activation of the CB1 (Figure 2A
and S13) and in agreement with reports made by other groups [7,
9, 17, 18]. This transient was almost completely abolished when
cells were pre-incubated with the CB1 antagonist rimonabant (1
µM; [cg2-AG]=10 µM, n=79 cells), confirming the specific
activation of the CB1 receptor upon uncaging of cg2-AG (Figure
2A, 2B and S13). The signal magnitude with 10 µM of cg2-AG was
directly comparable to the addition of the CB1 full agonist WIN55
([WIN55]=10 µM, n=84 cells, Figure S13). Addition of cg2-AG
only was unable to trigger calcium transients, demonstrating that
the coumarin efficiently prevented 2-AG activity (Figure S13G).
However, the addition of a pre-illuminated solution containing cg2-
AG triggered a calcium transient similar to the same concentration
of 2-AG (Figure S13H and S13I). Release from cg2-AG at internal
membranes is likely avoiding premature degradation by the
dominantly plasm membrane-bound lipases and could explain the
higher potency of 2-AG uncaging over 2-AG addition. Finally, we
performed HPLC-MS analysis of lipids extracts prepared from
MIN6 treated with cg2-AG in the presence or absence of UV light
and at two different points in time (30 min and 60 min after addition
of cg2-AG, see Figure S14). As expected, UV light exposure
clearly reduced the amount of cg2-AG in cells. Prolonged
incubation times did not reduce the quantity of caged lipid inside
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CB1 receptor signals through Gi/o protein activation and subsequent
inhibition of adenylate cyclase (AC) [21]. Alterations of the AC
activity has been reported to disrupt the Ca2+-cAMP-PKA
oscillatory circuit in MIN6 cells [22]. Therefore, we monitored the
inhibition of forskolin-stimulated adenylate cyclase (AC) and
protein kinase A (PKA) activity after CB1 activation via cg2-AG
uncaging. We used a genetically encoded EPAC-based cAMP
sensor [23] and the A-kinase FRET sensor AKAR4 [24]. After 48
h transfection, cells were incubated in the presence or absence of
cg2-AG (10 µM), and the culture media was exchange with 11mM
glucose imaging buffer. After a 10 min resting period, cells were
mounted on the microscope (λex=440 nm) and the ratio CFP/YFP
was monitored during stimulation with forskolin (50 µM). After
flash photolysis, we found a significant reduction in forskolin-
induced cAMP levels as well as PKA activation compared to
illumination in the absence of cg2-AG (Figure 3A,B,D) (AC
inhibition: 27.9 % ±1.4, n=46 cells, non-treated cells n=17; PKA
inhibition: 19.0 % ±0.9, n=34 and n=23, respectively). It is
important to note that cg2-AG showed no fluorescence at 440 nm.
Therefore, the caged lipid does not interfere with CFP/YFP-based
FRET sensors (Figure 3C).
In summary, we introduced a new strategy allowing for a fully
controlled spatio-temporal release of 2-AG in live cells without
altering the enzymatic machinery nor using genetic modifications.
The mechanism of photo-release allowed for the direct
visualization of the uncaging by monitoring fluorescence emission
of the cage. We established the proof-of-concept that cg2-AG
uncaging is useful to activate CB1 and its downstream effectors in
live β-cells.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications
website.
Figure 3. Normalized FRET change ratio of the genetically
encoded EPAC-based cAMP sensor (A.) and the A-kinase FRET
sensor AKAR4 (B.) in MIN6 cells upon stimulation with forskolin
(FSK, 50 μM), in presence or absence of cg2-AG (10 μM). C.
Fluorescence emission of cg2-AG before and after photo-uncaging
(left panel) and FRET ratio change of the EPAC-based sensor,
before and after stimulation with FSK (50 μM, right panel). D. Bar
graphs summarizing CB1-mediated inhibition of adenylate cyclase
and protein kinase A activity upon cg2-AG uncaging.
Figures S1 – S14, experimental details, characterization data, NMR
spectra (PDF).
AUTHOR INFORMATION
Corresponding Author
*schulcar@ohsu.edu
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Derivatives Enables Optical Control of Cannabinoid Receptor 1
Signaling. J Am Chem Soc, 139, 18206.
Notes
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The authors declare no competing financial interests.
13.
14.
Hoeglinger, D.; Nadler, A.; Schultz, C. (2014) Caged lipids as
tools for investigating cellular signaling. Biochim Biophys Acta,
1841, 1085.
Nadler, A.; Yushchenko, D.A.; Müller, R.; Stein, F.; Feng, S.;
Mulle, C.; Carta, M.; Schultz, C. (2015) Exclusive photorelease
of signalling lipids at the plasma membrane. Nat Commun, 6,
10056.
Höglinger, D.; Haberkant, P.; Aguilera-Romero, A.; Riezman,
H.; Porter, F.D.; Platt, F.M.; Galione, A.; Schultz, C. (2015)
Intracellular sphingosine releases calcium from lysosomes.
eLife;4:e10616 DOI: 10.7554/eLife.10616
Lin, W.; Lawrence, D.S. (2002) A Strategy for the Construction
of Caged Diols Using a Photolabile Protecting Group. The
Journal of Organic Chemistry, 67, 2723.
Geißler, D.; Antonenko, Y.N.; Schmidt, R.; Keller, S.; Krylova,
O.O.; Wiesner, B.; Bendig, J.; Pohl, P.; Hagen, W. (2005)
(Coumarin-4-yl)methyl esters as highly efficient, ultrafast
phototriggers for protons and their application to acidifying
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Li, C.; Jones, P.M.; Persaud, S.J. (2010) Cannabinoid Receptors
are Coupled to Stimulation of Insulin Secretion from Mouse
MIN6 β-cells. Cell. Physiol. Biochem., 26, 187.
Hegyi, Z.; Oláh, T.; Kőszeghy, A.; Piscitelli, F.; Holló, K.; Pál,
B.; Csernoch, L.; Di Marzo, V.; Antal, M. (2018) CB1 receptor
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Guo, J.; Ikeda, S.R. (2003) Endocannabinoids Modulate N-Type
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ACKNOWLEDGMENT
C.S. is grateful for financial support by OHSU, the EMBL and the
Deutsche Forschungsgemeinschaft (TRR186, projects A3 and Z1).
M.J.K. recognizes the National Institutes of Health for continued
support (NIH grants: R01 NS 038809 and DK 068098).
15.
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