Paracrine Transactivation of the CB1 Cannabinoid Receptor by AT1 Angiotensin and Other Gq/11 Protein-coupled Receptors*

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT1 receptor (AT1R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB1Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT1 angiotensin receptors by determining CB1 cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB1 receptors was visualized by detecting translocation of green fluorescent protein-tagged β-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of Gq/11-coupled M1, M3, M5 muscarinic, V1 vasopressin, α1a adrenergic, B2 bradykinin receptors, but not Gi/o-coupled M2 and M4 muscarinic receptors, also led to paracrine transactivation of CB1 receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of Gq/11-coupled receptors.

Hormones, neurotransmitters, and other chemical mediators acting on G protein-coupled receptors (GPCRs) 2 exert their effects on the target cells by stimulating G protein-dependent and independent intracellular signaling pathways (1)(2)(3)(4). Activation of G q/11 protein-coupled receptors causes phospholipase C activation, which produces inositol-trisphosphate and diacylglycerol from phosphatidylinositol (4,5)bisphosphate, leading to Ca 2ϩ -signal generation and protein kinase C activation. However, the concerted response of tissues to chemical mediators frequently also involves the activation of cells adjacent to the target cells, due to the release of paracrine mediators. A well known example is NO, which can be released from activated endothelial cells to cause relaxation of adjacent vascular smooth muscle cells. Lipid mediators can also act as intercellular messengers. For example, endocannabinoids released from postsynaptic neurons after depolarization act as retrograde transmitters by binding to and stimulating presynaptic cannabinoid receptors, which leads to inhibition of ␥-aminobutyric acid release (an event termed depolarizationinduced suppression of inhibition, DSI) (5)(6)(7).
Cannabinoid receptors were first identified based on their ability to selectively recognize marijuana analogs. To date, two cannabinoid receptors have been identified by molecular cloning, CB 1 and CB 2 receptors (CB 1 R and CB 2 R, respectively) (5,8,9), although additional GPCRs have also been proposed to function as cannabinoid receptors (10,11). Cannabinoid receptors also recognize certain lipids present in animal tissues termed endocannabinoids, such as arachidonylethanolamide (anandamide), 2-arachidonoylglycerol (2-AG), and 2-arachidonoylglyceryl ether (noladin ether) (7,(12)(13)(14)(15)(16). In adult and fetal neural tissues, the two major endocannabinoids, anandamide and 2-AG, are produced on demand, usually after depolarization of postsynaptic cells or following stimulation of G q -coupled metabotropic glutamate or muscarinic acetylcholine receptors (7,12,(17)(18)(19)(20). Enzymes responsible for 2-AG production and metabolism in tissues are localized to well defined structures at synapses, near the axon terminals of CB 1 R-expressing cells (5,7). In contrast, in peripheral tissues baseline levels of endocannabinoid production usually manifest as "endocannabinoid tone," with poorly understood localization of the various components of the endocannabinoid system. 2-AG levels in brain homogenates and in many peripheral tissues are near its K d for the CB 1 R (19), suggesting that function of endocannabinoids may not be limited to localized synaptic signaling.
There is mounting evidence that endocannabinoids play important roles in peripheral cardiovascular, inflammatory, intestinal, and metabolic regulation (21)(22)(23)(24). 2-AG is produced by diacylglycerol-lipase (DAGL) after cleavage of the fatty-acid in the sn-1 position of diacylglycerol (DAG) (19,25). Phospholipase C activation by G q/11 protein-coupled receptors produces DAG, which can serve as a substrate for DAGL. Plasma membrane phosphoinositides are enriched in arachidonic acid in the sn-2 position (26), and DAGL is expressed ubiquitously (27), which suggests that phospholipase C-mediated cleavage of polyphosphoinositides may routinely lead to the formation of 2-AG. In accordance with this hypothesis, we have recently shown that angiotensin II-(Ang II)-mediated activation of the G q/11 -coupled AT 1 angiotensin receptor (AT 1 R) leads to DAGLdependent activation of CB 1 Rs expressed in Chinese hamster ovary (CHO) cells (28).
Here our aim has been to examine the possibility that 2-AG serves as a common paracrine signal generated via activation of G q/11 protein-coupled, Ca 2ϩ -mobilizing receptors. Accordingly, we co-expressed CB 1 Rs and BRET-based sensors of G protein activation in CHO cells, and used these cells to detect endocannabinoid release from adjacent cells that express AT 1 R or other Ca 2ϩ -mobilizing GPCRs. We have further shown that activation of AT 1 R by Ang II increases 2-AG levels in CHO cells. These findings suggest that 2-AG is commonly released following activation of Ca 2ϩ -mobilizing GPCRs and serves as a paracrine signal to activate CB 1 R in neighboring cells.
Plasmid Constructs and Transfection-RFP-tagged CB 1 R was constructed by subcloning the CB 1 R cDNA into an mRFP containing vector (provided by Dr. R. Tsien, University of California, San Diego, CA). EYFP-␤ 1 was generated by subcloning human ␤ 1 subunit into the mammalian expression vector pEYFP-C1 (Clontech). ␣ o -Rluc was constructed by replacing the CFP coding region in ␣ o -CFP with Renilla luciferase. Rat HA-AT 1 R receptor and AT 1 -EYFP were constructed as described earlier (32). AT 1 R-Cerulean was constructed by replacing the cDNA of EYFP with Cerulean coding region (provided by Dr. R. Tsien). CHO cells were transfected with Lipofectamine 2000 according to manufacturer's suggestions using 2 or 16-24 g of DNA and 2 or 16 l of Lipofectamine 2000 in 6-well plates or 100-mm tissue culture plates, respectively. CHO cells were maintained in Ham's F-12 supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, and 100 IU/ml penicillin. COS-7 kidney fibroblast cells and HEK293 human embryonic fibroblast cells were grown in complete Dulbecco's modified Eagle's medium containing glucose, glutamine, sodium bicarbonate, and supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, 100 IU/ml penicillin.
Site-directed Mutagenesis-Mutations in the rat CB 1 R (D214A and R215A) were performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's suggestions and verified using automated sequencing.
Confocal Laser-scanning Microscopy-CHO cells were grown on glass coverslips and transfected with labeled CB 1 R and AT 1 R and GFP-labeled ␤-arrestin2, as described above, 48 h prior to measurement. In transactivation experiments, the cells were detached with Versene 1 day after transfection; the indicated cells were mixed and placed back to coverslips. Cerulean, EGFP, and RFP were excited with the 458 and 488 nm lines of the argon laser and the 543 nm line of the helium/neon laser, respectively; their emitted fluorescence was detected in multitrack mode with 480 -520 nm, 500 -530 nm band pass and 560 nm long pass filters, respectively. Because GFP caused a weak cross-talk in this setup in the 480 -520 nm band pass channel used for Cerulean, the full images showing Cerulean were corrected for the cross-talk of the GFP signals.
BRET Assay of G Protein Activation-Energy transfer between G protein subunits was measured using ␣ o G protein subunit fused with Renilla luciferase (␣ o -Rluc) and ␤ 1 subunit labeled with enhanced yellow fluorescent protein (EYFP-␤ 1 ). Medium was changed to fetal bovine serum-supplemented Ham's F-12 6 h following transfection and incubated overnight. Before the experiments the cells were detached with Versene and centrifuged. Cells were suspended in a modified Krebs-Ringer buffer containing (in mM): 120 NaCl, 4.7 KCl, 1.2 CaCl 2 , 0.7 MgSO 4 , 10 glucose, 10 sodium Hepes, pH 7.4 (containing 1 g/liter albumin in paracrine transactivation studies) and transferred to white 96-well plates. The cell density was between 100,000 and 200,000 cells/well. Coelenterazine h was added to a final concentration of 5 M, and readings were collected using a Mithras LB 940 Multilabel Reader (Berthold Technologies, Bad Wildbad, Germany). BRET ratio was defined as (emission at 530 nm)/(emission at 485), and the normalized BRET ratio was calculated as the BRET ratio for the co-expressed EYFP-tagged and Rluc-tagged molecules minus the BRET ratio for the coexpressed non-tagged and Rluc-tagged molecules. Data for G protein activity are shown as the percent changes in normalized BRET ratios compared with the mean of the four control BRET ratio points before the first stimulation (BRET ratio, percent of control).
Measurement of 2-AG and Anandamide Levels-CHO cells transfected with AT 1 R were maintained in Ham's F-12 medium in 10-cm tissue culture plates as described above. Aliquots of 4 ϫ 10 6 cells in 10-cm tissue culture plates containing 3.5 ml of modified Krebs-Ringer buffer were incubated with vehicle or 100 nM angiotensin II for the indicated times, following which the cells plus medium were extracted in 2 volumes of ice-cold chloroform:methanol (2:1, v/v) containing 7 ng of 4 H 2 -anandamide as internal standard. The chloroform phase was separated and re-extracted twice and finally dried under a stream of nitrogen. The dried residue was reconstructed in 100 l of chloroform, deproteinated with 2 ml of ice-cold acetone, centrifuged, and the clear supernatant was evaporated to dryness. Samples were resuspended in 50 l of methanol for analysis of endocannabinoid content by liquid chromatography/in line mass spectrometry, as described (33).
Statistical Analysis-All data are presented as means Ϯ S.E. Differences between groups were analyzed by one-or two-way repeated measures analysis of variance combined with Holm-Sidak test using the software SigmaStat for Windows 3.5 (Systat Software Inc., Richmond, CA). The value of p Ͻ 0.05 was considered significant.

Stimulation of AT 1 Rs Expressed in CHO, HEK, and COS Cells
Leads to Paracrine Transactivation of CB 1 Rs-We have previously shown that CB 1 R can be transactivated with Ang II in cells co-expressing CB 1 R and AT 1 R (28). We therefore postulated that, if the transactivation of CB 1 R was caused by a released endocannabinoid, it would also occur if AT 1 R and CB 1 R were expressed in separate, adjacent cells. To examine this possibility, we monitored the activation of CB 1 Rs expressed in CHO cells to detect Ang II-induced endocannabinoid release by other cells mixed to the cells expressing CB 1 R. CB 1 R activity was quantified by detecting bioluminescence resonance energy transfer (BRET) between G o protein subunits (Renilla luciferase-tagged ␣ o and EYFP-tagged ␤ 1 , ␣ o -Rluc, and EYFP-␤ 1 , respectively) as described previously (28). In this experimental setting, activation of CB 1 R leads to a decrease of the BRET signal resulting from the dissociation of activated G o protein subunits, whereas inactivation by an antagonist increases the BRET signal, because it leads to the association of G protein subunits (28,29). CHO cells were transfected either with expression plasmids of AT 1 R, or were co-transfected with plasmids of CB 1 R and tagged G protein subunits. Twenty-four hours later, the cells were mixed and CB 1 R activity was measured by detecting BRET interaction between G protein subunits, as a way to monitor endocannabinoid release. Stimulation of the cells with Ang II led to a decrease in BRET signal indicating activation of CB 1 R (Fig. 1A, filled circles). Administration of the CB 1 R inverse agonist AM251 caused an increase in the BRET signal, reflecting the inhibition of the basal activity of the receptor. AM251 also blocked the Ang II-induced transactivation (Fig. 1A, open circles). A slight baseline shift of the BRET signal occurred during treatment, which might have been caused by the small increase in volume; and the kinetics of the response was too fast to analyze its initial phase with manual stimulation. Therefore, an automated injector was used in separate experiments to analyze the initial phase of the Ang II-induced response in higher time-resolution (Fig. 1, B and C). The initial baseline shift occurred promptly both in control and stimulated cells. In Ang II-stimulated cells (filled circles) after a lag time of ϳ15-20 s the BRET signal decreased rapidly (Fig. 1B), which was not observed in cells treated with medium (control cells, open squares). AM251 fully prevented the effect of Ang II stimulation on the BRET signal (Fig. 1C).
When AT 1 R-transfected cells were mixed with cells expressing the G o sensor, but no CB 1 R, G o protein activation did not occur, which provides additional evidence that it was mediated through CB 1 R (Fig. 1D). Similarly, when cells expressing CB 1 R, but not AT 1 R, were stimulated with Ang II, the BRET signal remained unaffected (Fig. 1E). In other experiments, the original 1:1 ratio of AT 1 R or CB 1 R-expressing cells was increased to 5:1 or decreased to 0.1:1, keeping the number of CB 1 R-expressing cells constant at 100,000 cells/well, which resulted in a corresponding increase or decrease, respectively, in G o protein activation, approaching a plateau between 1:1 and 5:1 cell ratios (Fig. 1F). These findings suggest that G o protein activation in CB 1 R-expressing cells was mediated by Ang II-induced endocannabinoid release from CHO cells expressing AT 1 R. Transactivation of CB 1 R was also inhibited by pretreatment of the cells expressing AT 1 R with the DAGL inhibitor tetrahydrolipstatin (1 M) ( Fig. 2A, open triangles), suggesting that DAGL plays a role in the process. Statistical analysis of the data is shown in Fig. 2B.
Transactivation of CHO cells expressing CB 1 Rs was also detected when these cells were mixed with equal number of HEK293 or COS7 cells expressing AT 1 Rs, and stimulated with Ang II. These data demonstrate that the AT 1 R-mediated paracrine transactivation of CB 1 Rs is not restricted to CHO cells and may be a common mechanism in different cell types (Fig.  3). Slightly increased transactivation of CB 1 Rs was observed, when these AT 1 R-expressing cells were added in a 5-fold excess, suggesting that the response was almost maximal under these conditions (supplemental Fig. S1).
AT 1 R Activation Leads to ␤-Arrestin Translocation to CB 1 R in Adjacent Cells-To provide additional evidence that Ang II-induced activation of AT 1 R leads to the formation and release of endocannabinoids, ␤-arrestin translocation to transactivated CB 1 Rs was measured in CHO cells. ␤-Arrestin coupling to GPCRs has often been used as an indicator of receptor activation (31,32,34). When RFP-tagged CB 1 R (CB 1 R-RFP) was co-expressed with GFP-tagged ␤-arrestin2 (␤-arr2GFP), CB 1 R-RFP localized both to the cell membrane and to intracellular vesicles (Fig. 4A), which is consistent with the previously reported constitutive internalization of this receptor, which may be caused by constitutive receptor activity, endocannabinoid formation, or other mechanisms (28,30,35). Following stimulation with the synthetic cannabinoid agonist WIN 55,212-2, ␤-arr2-GFP translocated to the activated CB 1 Rs at the cell surface, and was detected in punctate structures at the plasma membrane (Fig. 4A). The conserved DRY motif in the second intracellular loop of GPCRs has been previously implicated in ␤-arrestin binding of some GPCRs (36 -38). In other GPCRs, mutations in the conserved DRY region caused enhanced ␤-arrestin binding (39,40). The enhanced ␤-arrestin binding of these receptors may be caused by their reduced G protein coupling (41)(42)(43). We have generated mutations in the conserved DRY motif within the second intracellular loop of CB 1 R (D214A and R215A) (CB 1 R[DRY/AAY]). To study the interaction of the mutant CB 1 R with ␤-arrestin, RFP-tagged CB 1 R(DRY/AAY) (CB 1 R(DRY/AAY)-RFP) was co-expressed with ␤-arr2-GFP in CHO cells. In non-stimulated cells, ␤-arr2-GFP was distributed diffusely in the cytoplasm, but basal activity of this mutant CB 1 R caused its appearance in punctate structures at the plasma membrane (Fig. 4B, left). Stimulation with WIN55,212-2 caused robust translocation of ␤-arr2-GFP to the plasma membrane, with only very faint fluorescence remaining in the cytoplasm (Fig. 4B, middle). These data demonstrate that co-expression of CB 1 R(DRY/AAY)-RFP with ␤-arr2-GFP is a sensitive tool for visualization of both basal and agonist-induced CB 1 R activity.
To test if endocannabinoid release could be detected by ␤-arr2-GFP translocation to CB 1 R, CB 1 R(DRY/AAY)-RFP and ␤-arr2-GFP were co-expressed in CHO cells, and a separate pool of cells were transfected independently with AT 1 R-Cerulean. Twenty-four hours after transfection, the cells were mixed and placed on coverslips, and another day later they were visualized using confocal microscopy. As shown in Fig. 5, after Ang II-induced stimulation of the mixed CHO cell population expressing CB 1 R(DRY/AAY)-RFP and ␤-arr2-GFP or AT 1 R-Cerulean, translocation of ␤-arr2-GFP to the plasma membrane was observed in cells expressing CB 1 R(DRY/AAY)-RFP. This response was observed in 67 Ϯ 7% of cells (n ϭ 3, 86 cells total) with neighboring AT 1 R-Cerulean-expressing cells; whereas in parallel experiments, when cells expressing CB 1 R(DRY/AAY)-RFP and ␤-arr2-GFP were stimulated with Ang II in the absence of AT 1 R-expressing cells, no translocation of ␤-arr2-GFP was detected (Fig. 4C). These data provide additional evidence that a paracrine endocannabinoid mediator is released as a result of AT 1 R activation. 1 Rs-To verify whether the Ang II-induced decrease in BRET signal detected by confocal microscopy was, in fact, due to endocannabinoid release, we measured 2-AG and anandamide levels in control and Ang II-stimulated AT 1 R-expressing cells. As shown in Fig. 6, stimulation of cells with Ang II caused a statistically significant sustained elevation of 2-AG levels with similar kinetics to that of CB 1 R transactivation measured with BRET (Fig.  1A). Anandamide levels were very low and did not show statistically significant changes in response to Ang II stimulation (supplemental Fig. S2.). These data are consistent with our hypothesis that G q/11 -mediated DAG formation serves as a source of endocannabinoid release and suggest that the endocannabinoid involved in Ang II-induced paracrine CB 1 R transactivation is 2-AG.   pendent on Intracellular Ca 2ϩ Signal Generation-2-AG production in neural cells can be stimulated either by the Ca 2ϩ signal caused by activation of ionotropic receptors, by stimulation of G q activating (metabotropic) cholinergic or glutamatergic receptors, or by the coincidence of two signals (44). 2-AG production can be stimulated in cultured cell lines overexpressing DAGLs by inducing a Ca 2ϩ signal with ionomycin (27,44). Therefore we asked if transactivation of CB 1 R by AT 1 R is dependent on Ca 2ϩ . In control cells, G protein activation occurred after Ang II stimulation and a Ca 2ϩ signal was detected (Fig. 7, A and D). In cells preincubated with 1,2bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM; 60 M) and preloaded with FURA-2, Ang II-induced Ca 2ϩ elevation was not detected even though transactivation of CB 1 R still occurred (Fig. 7, B and D). Moreover, administration of ionomycin (1 M) was not able to fully mimic the effect of AT 1 R stimulation, although a small, reproducible, but in this set of experiments statistically not significant G o protein activation was detectable (Fig. 7, C and D). The averaged BRET data are presented in Fig.  7D. Because, under these conditions, ionomycin causes larger elevations of cytoplasmic [Ca 2ϩ ] than those caused by Ang II (Fig. 7, A and C, inset), these results suggest that transactivation was not primarily mediated by intracellular Ca 2ϩ elevation.

2-AG Formation Is Enhanced by Stimulation of AT
G q/11 -coupled GPCRs Cause Paracrine Transactivation of CB 1 R-It has been demonstrated previously that activation of G q -coupled muscarinic or metabotropic glutamate receptors induces endocannabinoid release in neurons (17)(18)(19). To test if endocannabinoid release common occurs after stimulation of G q -coupled receptors, we expressed M 1 , M 3 , and M 5 cholinergic (M 1 R, M 3 R, and M 5 R, respectively), V 1 vasopressin (V 1 R), ␣ 1A adrenergic (␣ 1A AR), and B 2 bradykinin (B 2 R) receptors in CHO cells, and tested if paracrine transactivation of CB 1 R occurs. We also tested the effect of stimulation of G i/o -coupled M 2 and M 4 muscarinic cholinergic receptors (M 2 R and M 4 R, respectively). G q/11 -or G i/o -activating receptors were expressed in one set of cells, and CB 1 R and G o sensor were expressed in separate population of cells. The two populations of cells were mixed and stimulated with the appropriate ligands. As shown in Fig. 8, CB 1 R was transactivated when G q/11 protein-activating M 1 R, M 3 R, M 5 R, ␣ 1A AR, V 1 R, or B 2 R-expressing cells were stimulated with the appropriate ligands, but no transactivation was detected when G i/o protein-coupled M 2 R or M 4 R were stimulated. The degree of transactivation in the former groups was similar to that measured with cells expressing AT 1 R (Fig. 2B). Transactivation did not occur in the absence of either CB 1 R or the G q/11 -activating receptor (supplemental Fig. S3). These findings demonstrate that paracrine CB 1 R transactivation by endocannabinoids is not specific to AT 1 R and can be also initiated in cells that express other G q/11activating GPCRs.

DISCUSSION
Seven transmembrane GPCRs constitute the largest group of membrane receptors (45). They respond to a large variety of stimuli and transduce various signals across the plasma membrane by coupling to heterotrimeric G proteins (46,47). There are about 1000 GPCRs in the human genome, and about half of the medications used in current clinical practice modify the biological activity of GPCRs (48,49). Stimulation of GPCRs leads to activation of heterotrimeric G proteins (50) composed of ␣, ␤, and ␥ subunits, and GPCR activation causes dissociation or conformational rearrangement of the ␣ subunit from the ␤␥ complex (51). This is followed by intracellular responses depending on the composition of the heterotrimer. In the case of G q/11 -coupled receptors, such as the AT 1 R, early events include cleavage of phosphatidylinositol 1,4,5-bisphosphate by phospholipase C␤ and formation of second messengers such as inositol 1,4,5-trisphosphate and DAG (2, 3). Inositol 1,4,5trisphosphate initiates Ca 2ϩ release from intracellular stores, whereas DAG activates protein kinase C (52). Because DAG is a common signaling molecule in different cell types and DAGL is found in almost every tissue (27), we hypothesized that 2-AG formation can be a common early signaling event in most cells. To detect the formation and release of endocannabinoids as a general consequence of G q/11 protein activation, we expressed AT 1 R in CHO, HEK293, and COS7 cell lines, which do not express cannabinoid receptors constitutively.
CHO cells expressing CB 1 R and a G i/o BRET sensor were used to monitor endocannabinoid release (28,29,51). Mixing these cells with cells expressing other GPCRs allowed us to detect the endocannabinoid release caused by these GPCRs. An advantage of using these sensors is that the signal is less affected by the rapid degradation of endocannabinoids. Our findings clearly indicate that activation of AT 1 Rs leads to endocannabinoid formation and release, with the subsequent activation of CB 1 Rs. Our data also indicate that activation of G q/11 -coupled receptors can cause paracrine activation of adjacent cells, because in our experiments the stimulated G q/11coupled receptors and the CB 1 R were expressed in different cells. We have also demonstrated that Ang II increased the level of 2-AG in CHO cells expressing AT 1 R. In control cells, 2-AG was also detected, which is consistent with the possible role of this molecule in the basal activity of CB 1 R (28). These data support our hypothesis that the Ang II-induced DAGL-dependent transactivation of CB 1 Rs is mediated by 2-AG. Therefore, 2-AG is the most likely candidate to mediate the observed paracrine effects in our cells. The release of endocannabinoids from cells may occur via unidentified transporter molecules, which is consistent with their proposed paracrine mediator role (53). However, we could not detect transactivation when the supernatant from the stimulated cell population was transferred to cells expressing CB 1 Rs. 3 It is possible that endocannabinoids, being lipophilic molecules, remain membrane-associated and require direct contact with adjacent CB 1 R-expressing cells for receptor activation.
We have also tested other G q/11 -activating GPCRs, including muscarinic acetylcholine receptors, which are known to cause 2-AG release in neural tissues, as well as ␣ 1 ARs, V 1 Rs, and B 2 Rs. Our data suggest that 2-AG release is a common consequence of the activation of various G q/11 -coupled receptors. Because 2-AG can be released by cells (54), its release can cause paracrine regulation of adjacent cells. Based on our data we propose that activation of G q/11 -coupled receptors results in a coordinated intracellular (inositol 1,4,5-trisphosphate and DAG) and intercellular (2-AG) signaling, and the phospholipase C-catalyzed hydrolysis of polyphosphoinositides serves as a source of both types of messengers.
Blockade or stimulation of cannabinoid receptors has wideranging effects in a number of organ systems, including brain, cardiovascular system, adipose tissue, liver, immune system, and the eye (12,21,22). The present results indicate that endocannabinoid activation of CB 1 R can also be triggered indirectly via G q/11 -coupled receptors, which means that the primary 3 G. Turu and L. Hunyady, unpublished observation.  CHO cells (4 million cells/10-cm tissue culture plates with 3.5 ml of medium) expressing AT 1 R were treated with 100 nM AngII for indicated times, and lipids were extracted from cells and the medium. Tissue levels of 2-AG was quantified by liquid chromatography/in-line mass spectrometry and statistical analysis of the data with analysis of variance was performed as described under "Experimental Procedures" (n ϭ 3; *, p Ͻ 0.05; **, p Ͻ 0.01).
response to agonists of these receptors can be modulated by endocannabinoids. Furthermore, some of the physiological effects observed following CB1 receptor blockade may be due to removal of such a modulation of tonically active Gq/11-coupled receptor signaling. Furthermore, these data also suggest that endocannabinoid production is not restricted to small, well defined regions/tissues in the organism, but is a general property of many, if not all, tissues. Although CB 1 R is expressed mainly in the central nervous system, it is also found in many peripheral tissues, and stimulation or blocking of these receptors has marked effects on their function (12,21,22).
In addition, 2-AG released as a paracrine mediator may activate CB 2 R, because it is a full agonist at both CB 1 R and CB 2 R (19) and may also activate additional receptors (12). We have also tested whether G q protein activation leads to endocannabinoid release in different types of cell lines. CHO cells are ovarian, whereas HEK293 cells are from the kidney and COS cells are fibroblasts. We detected CB 1 R activation after stimulation of G q protein-activating AT 1 R expressed in all three cell lines tested and also following stimulation of other G q protein-activating receptors transfected into these cells. This strongly suggests that endocannabinoid release is not dependent of cell type or specific G q/11 -coupled receptors and may be a general property of all tissues upon G q protein activation. Endocannabinoid release triggered by activation of G q/11 -coupled receptors could be inhibited by a DAGL inhibitor, suggesting that the underlying mechanism was an increase in the DAGL-catalyzed hydrolysis of DAG to yield 2-AG. However, we cannot exclude the alternative possibility that the increased cellular levels of 2-AG result, at least in part, from inhibition of its degradation via monoacylglycerol lipase. Thus, the magnitude of the basal FIGURE 7. Transactivation of CB 1 R by coexpressed AT 1 R is independent of cytoplasmic Ca 2؉ signal generation. CB 1 R activity was measured by BRET between Go protein subunits in cells coexpressing CB 1 R and AT 1 R. Insets: to study the cytoplasmic Ca 2ϩ signal generation, the cells were loaded with FURA 2 for 45 min, and Ca 2ϩ was measured in suspended cells by detecting the ratio of fluorescent emissions ratio at 510 nm during excitation at two wavelengths (380/340 nm). A, cells were treated with vehicle (veh, empty squares) or Ang II (100 nM, filled circles) at the time indicated by the first arrow, and BRET signal was measured in cells expressing CB 1 R. At the second arrow the Ang II-stimulated cells were also treated with WIN55 (1 M). B, cells expressing the same constructs were preloaded with 60 M BAPTA-AM for 45 min on room temperature, and G o activation was measured. C, the effect of ionomycin (1 M) on G o protein activation in cells expressing the G o protein sensors. D, quantification of data in A-C: mean values of five time points after stimulation with vehicle (white bars), Ang II or ionomycin (black bars) (n ϭ 3; *, p Ͻ 0.01). "endocannabinoid tone" and endocannabinoid release may depend on relative activity of these enzymes.
In conclusion, our data show that 2-AG formation and release is a general paracrine signaling mechanism of G q/11coupled GPCRs. Based on these results we propose that, in addition to inositol 1,4,5-trisphosphate-and DAG-mediated intracellular signaling, 2-AG formed from the DAG generated during activation of G q/11 proteins is released and acts as a paracrine signal for cannabinoid receptors on adjacent cells, which would modulate the primary response to Ca 2ϩ -mobilizing hormones in a region-specific manner. Because 2-AG has been implicated in a variety of physiological functions and the enzymes responsible for its biosynthesis are present in most tissues, we are proposing that the previously recognized retrograde transmitter role of endocannabinoids is part of a much broader paracrine signaling role of these mediators.