Differential Activation of Intracellular versus Plasmalemmal CB₂ Cannabinoid Receptors

Abstract

The therapeutic and psychoactive properties of cannabinoids have long been recognized. The type 2 receptor for cannabinoids (CB₂) has emerged as an important therapeutic target in several pathologies, as it mediates beneficial effects of cannabinoids while having little if any psychotropic activity. Difficulties associated with the development of CB₂-based therapeutic agents have been related to its intricate pharmacology, including the species specificity and functional selectivity of the CB₂-initiated responses. We postulated that a plasmalemmal or subcellular location of the receptor may contribute to the differential signaling pathways initiated by its activation. To differentiate between these two, we used extracellular and intracellular administration of CB₂ ligands and concurrent calcium imaging in CB₂-expressing U2OS cells. We found that extracellular administration of anandamide was ineffective, whereas 2-arachidonoyl glycerol (2-AG) and WIN55,212-2 triggered delayed, CB₂-dependent Ca²⁺ responses that were Gq protein-mediated. When microinjected, all agonists elicited fast, transient, and dose-dependent elevations in intracellular Ca²⁺ concentration upon activation of Gq-coupled CB₂ receptors. The CB₂ dependency was confirmed by the sensitivity to AM630, a selective CB₂ antagonist, and by the unresponsiveness of untransfected U2OS cells to 2-AG, anandamide, or WIN55,212-2. Moreover, we provide functional and morphological evidence that CB₂ receptors are localized at the endolysosomes, while their activation releases Ca²⁺ from inositol 1,4,5-trisphosphate-sensitive- and acidic-like Ca²⁺ stores. Our results support the functionality of intracellular CB₂ receptors and their ability to couple to Gq and elicit Ca²⁺ signaling. These findings add further complexity to CB₂ receptor pharmacology and argue for careful consideration of receptor localization in the development of CB₂-based therapeutic agents.

Although cannabinoids are active at several G protein-coupled receptors and ion channels, only two “true” cannabinoid receptors are recognized, namely CB₁ and CB₂.¹ Interest in the latter has sparked as it appeared as an important therapeutic target in inflammatory and painful conditions,^2,3 while not being involved in the psychoactive cannabinoid effects, which are mainly CB₁-mediated. As such, increasing effort is being spent in the development of CB₂-based therapeutic agents.^4,5 Nonetheless, controversies exist, for instance, in CB₂ pharmacology and distribution.⁶⁻⁸ At least two CB₂ receptor isoforms have been identified, with tissue- and species-specific expression patterns.^8,9 It has been found that CB₂ agonists may elicit distinct responses at CB₂ receptors from different species.¹⁰ Moreover, functional selectivity, defined as the ability of a receptor to couple to different signaling pathways depending on the ligand that stimulates it,¹¹ has been reported for CB₂.⁷ Further complexity is added to the CB₂ receptor pharmacology with the recent finding that their intracellular activation modulates neuronal function.¹² Because CB₂ receptors have been found to signal through Ca²⁺,¹²⁻¹⁵ we used calcium imaging and extracellular and intracellular administration of cannabinoid ligands to investigate the functionality of plasmalemmal versus intracellular CB₂ receptors in U2OS cells stably expressing CB₂.

Experimental Procedures

Chemicals

Anandamide, AM630, WIN55,212-2, 2-arachidonoyl glycerol (2-AG), and d-[Trp7,9,10]-substance P were obtained from Tocris Bioscience (R&D Systems, Minneapolis, MN). All other chemicals were from Sigma (St. Louis, MO).

Cell Culture

The CB₂-β-arrestin2-GFP-U2OS (CB₂-U2OS) cell line was kindly provided by M. Caron and L. S. Barak (Duke University, Durham, NC); the CB₂ receptor sequence is the CNR2_Human sequence (GenBank accession code P34972). CB₂-U2OS cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 mg/mL Zeocin, and 200 μg/mL G418 at 37 °C in a humidified incubator with 5% CO₂. The serum was removed 24 h prior to experimentation. In experiments that aimed to evaluate Gq-dependent signaling, cell starvation was concomitant with incubation of d-[Trp7,9,10]-substance P (24 h).

Immunocytochemistry and Confocal Imaging Studies

U2OS cells transiently transfected with the GFP-tagged CB₂ receptor (kindly provided by M. Caron and L. S. Barak) and with Rab7-RFP (Addgene, Cambridge, MA) 48 h earlier were fixed with 4% paraformaldehyde, washed in phosphate-buffered saline, and mounted with DAPI Fluoromont G (Southern Biotech, Birmingham, AL). Cells were imaged using a Carl Zeiss 710 two-photon confocal microscope with a 63× oil immersion objective, using a 1× digital zoom, with excitations set for DAPI, GFP, and DsRed at 405, 488, and 561 nm, respectively. Images were analyzed using Zen 2010 (Zeiss), as previously reported.¹⁶

Calcium Imaging

Measurements of [Ca²⁺]_i were performed as previously described.¹⁶⁻¹⁹ Briefly, cells were incubated with 5 μM Fura-2 AM (Invitrogen) in HBSS at room temperature for 45 min, washed with dye-free HBSS, and incubated for an additional 45 min to allow dye de-esterification. Coverslips were mounted in an open bath chamber (RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope (Nikon Eclipse TiE, Nikon Inc., Melville, NY), equipped with a Perfect Focus System and a Photometrics CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). During the experiments, the Perfect Focus System was activated. Fura-2 AM fluorescence (emission at 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR version 3.1 (Nikon Inc.). After appropriate calibration with ionomycin and CaCl₂, and Ca²⁺-free and EGTA, respectively, the ratio of the fluorescence signals (340 nm to 380 nm) was converted to Ca²⁺ concentration.²⁰

Intracellular Microinjection

Injections were performed using Femtotips II, InjectMan NI2, and FemtoJet systems (Eppendorf) as previously reported.¹⁶⁻¹⁹ Pipettes were backfilled with an intracellular solution containing 110 mM KCl, 10 mM NaCl, and 20 mM HEPES (pH 7.2) or the compounds to be tested. The injection time was 0.4 s at 60 hectoPascal with a compensation pressure of 20 hectoPascal to ensure that the microinjected volume was <1% of the cell volume. The intracellular concentration of chemicals was determined on the basis of the concentration in the pipet and the volume of injection. The cells to be injected were Z-scanned before injection, and the cellular volume was automatically calculated by NIS-Elements AR version 3.1 (Nikon Inc.).

Statistics

Data are expressed as means and the standard error of the mean. One-way analysis of variance, followed by post hoc Bonferroni and Tukey tests, was used to assess significant differences between groups; P < 0.05 was considered statistically significant.

Results

Effects of Extracellular versus Intracellular Administration of CB₂ Agonist 2-Arachidonoyl Glycerol on the Cytosolic Ca²⁺ Concentration of CB₂-Expressing U2OS Cells

We evaluated the Ca²⁺ response of U2OS cells stably expressing the CB₂ receptor to bath application and intracellular microinjection of the endocannabinoid 2-arachidonoyl glycerol (2-AG). Increasing concentrations of 2-AG (0.01, 0.1, and 1 μM) were applied extracellularly to CB₂-U2OS cells, which elevated the intracellular Ca²⁺ concentration, [Ca²⁺]_i, by 7 ± 3.4 nM (n = 31 cells), 12 ± 5.1 nM (n = 43 cells), and 87 ± 3.1 nM (n = 56 cells), respectively (Figure 1A). The effect of the latter concentration of 2-AG was statistically significant (P < 0.05) and sensitive to CB₂ blockade with AM630 (1 μM, 10 min). In the presence of AM630, Δ[Ca²⁺]_i was reduced to 14 ± 3.6 nM (n = 49) (Figure 1A,B). The effect of 2-AG on [Ca²⁺]_i was delayed by 1–2 min and increased gradually (Figure 1B).

Figure 1.

Extracellular administration of 2-arachidonoyl glycerol (2-AG) to CB₂-U2OS cells elevates [Ca²⁺]_i. (A) Comparison of the increases in [Ca²⁺]_i produced by extracellular administration of 2-AG (0.01–1 μM) and 1 μM 2-AG in the presence of CB₂ receptor antagonist AM630 (1 μM); P < 0.05 compared with basal levels (∗) or with 1 μM 2-AG (#). (B) Representative recordings of increases in [Ca²⁺]_i in response to 1 μM 2-AG in absence or presence of 1 μM CB₂ antagonist AM630.

In experiments using the intracellular microinjection technique, injection of 0.01, 0.1, and 1 μM 2-AG (final concentrations inside the cell) induced robust and significant increases in [Ca²⁺]_i of CB₂-U2OS cells of 264 ± 11 nM (n = 6 cells), 653 ± 19 nM (n = 6 cells), and 938 ± 22 nM (n = 6 cells), respectively, while control buffer microinjection had an insignificant effect of 49 ± 4 nM (n = 6 cells) (Figure 2A–D). In the presence of co-injected CB₂ antagonist AM630 (1 μM), 0.1 μM 2-AG elevated [Ca²⁺]_i by 58 ± 4 nM (n = 6 cells), similar to that of control buffer microinjection; the effect of 0.1 μM 2-AG on [Ca²⁺]_i of untransfected U2OS cells was also insignificant, measuring 34 ± 5 nM (n = 6 cells) (Figure 2A,B,E,F).

Figure 2.

Intracellular microinjection of 2-AG into CB₂-U2OS cells produces CB₂-dependent Ca²⁺ elevation. (A) Averaged increases in [Ca²⁺]_i produced by intracellular administration of control buffer (top left) or 2-AG (0.01, 0.1, and 1 μM, final concentrations inside the cell, top right), co-injection of 0.1 μM 2-AG with CB₂ antagonist AM630 (1 μM, bottom left) in CB₂-U2OS cells, or microinjection of 0.1 μM 2-AG into control U2OS cells (bottom right). (B) Comparison of the Ca²⁺ responses elicited by the treatments of CB₂-U2OS or control untransfected U2OS cells mentioned above; P < 0.05 when compared with the control (∗) or with 2-AG alone (#). (C–F) Characteristic fluorescence images of Fura-2 AM-loaded CB₂-U2OS cells before (left), during (middle), and 6 min after (right) intracellular administration of control buffer (C), 0.1 μM 2-AG alone (D), or 0.1 μM 2-AG in the presence of 1 μM AM630 (E) or of U2OS cells treated with 0.1 μM 2-AG (F). Arrows denote the injected cells; the fluorescence scale (0–3) is illustrated in each panel and magnified in the left panel of part C.

In all series of experiments using intracellular injection, CB₂-U2OS cells were pretreated for 1 min with 1 μM AM630 (applied extracellularly), to exclude the possibility that the injected cannabinoid ligands leaked from the pipet and triggered plasma membrane receptor-mediated effects.

Functional and Morphological Evidence of Localization of CB₂ to Endolysosomes in CB₂-Transfected U2OS Cells

CB₂-U2OS cells were incubated for 1 h with either 1 μM bafilomycin A1, a V-type ATPase that prevents lysosomal acidification,²¹ or 30 μM rapamycin, which blocks the last step of the engulfment of molecules by endolysosomes via microautophagy.²² Under the conditions described above, the effect of 0.1 μM 2-AG on [Ca²⁺]_i, initially measuring 653 ± 19 nM, was drastically reduced to 36 ± 4.8 nM (n = 6 cells) and 67 ± 9.1 nM (n = 6 cells) (Figure 3A–E). In U2OS cells co-expressing GFP-tagged CB₂ receptors and RFP-tagged Rab7, a small GTPase associated with both endosomes and lysosomes,²³ we observed an extensive colocalization of CB₂ and Rab7 (Figure 3F). Lysosomal disruption using bafilomycin A1 greatly reduced the extent of the merged signal of CB₂ and Rab7 (Figure 3F).

Figure 3.

Intracellular CB₂ receptors are located at endolysosomes in CB₂-expressing U2OS cells. (A) Averaged Ca²⁺ responses of CB₂-U2OS cells to intracellular administration of 0.1 μM 2-AG in the absence (left) or presence of lysosomal disruptor bafilomycin A1 (1 μM, incubation for 1 h, middle) or microautophagy inhibitor rapamycin (30 μM, incubation for 1 h, right). (B) Comparison of the increases in [Ca²⁺]_i elicited by 2-AG under the conditions described above; P < 0.05 when compared with 2-AG microinjection (∗). (C–E) Representative fluorescence images of Fura-2 AM-loaded CB₂-U2OS cells before (left), during (middle), and 6 min after (right) intracellular administration of 0.1 μM 2-AG in the absence (C) and presence of bafilomycin A1 (D) or rapamycin (E). (F) Confocal images (top row) showing the colocalization of the GFP-tagged CB₂ receptor and RFP-tagged Rab7, an endolysosomal marker, in GFP-CB₂- and RFP-Rab7-transfected U2OS cells; the nuclei are labeled with DAPI (blue). Lysosomal disruption (bottom row) with bafilomycin A1 markedly reduces the extent of the merged immunostaining of CB₂ and Rab7.

Intracellular, but Not Extracellular, Administration of Anandamide Elevates the Cytosolic Ca²⁺ Concentration of CB₂-Expressing U2OS Cells

CB₂-U2OS cells extracellularly treated with progressive concentrations of anandamide (0.01, 0.1, and 1 μM) failed to respond with a significant increase in [Ca²⁺]_i (Figure 1 of the Supporting Information); incubation with CB₂ antagonist AM630 (1 μM) did not modify the response to 1 μM anandamide (Figure 1 of the Supporting Information).

However, CB₂-U2OS cells responded to intracellular administration of anandamide (0.01, 0.1, and 1 μM, final concentrations inside the cell) with significant and concentration-dependent elevations of [Ca²⁺]_i: 124 ± 7.4, 368 ± 8.4, and 574 ± 7.2 nM, respectively [n = 6 cells for each concentration tested (Figure 4A,B)]. The anandamide-induced increase in [Ca²⁺]_i was fast and transient (Figure 4A); blockade of intracellular CB₂ receptors upon co-injection of 1 μM AM630 prevented the effect of microinjected anandamide (1 μM), reducing it to 37 ± 4.1 nM [n = 6 cells (Figure 4A–D)], a response similar to that elicited by control buffer microinjection (Figure 2). Characteristic images depicting the increases in the Fura-2 fluorescence ratio at 340 and 380 nm upon microinjection of anandamide (1 μM) in the absence or presence of AM630 (1 μM) are shown in panels C and D of Figure 4.

Figure 4.

Intracellular administration of anandamide increases [Ca²⁺]_i in CB₂-U2OS cells. (A) Averaged Ca²⁺ responses induced by increasing doses of microinjected anandamide (ANA, 0.01–1 μM, left) or by co-injection of 0.1 μM anandamide and 1 μM CB₂ antagonist AM630 (right). (B) Comparison of the increases in [Ca²⁺]_i produced by microinjected anandamide (0.01, 0.1, and 1 μM) and anandamide (0.1 μM) in the presence of AM630 (1 μM); P < 0.05 compared with the control (∗) (see Figure 2) or with 0.1 μM anandamide alone (#). (C and D) Typical fluorescence images of Fura-2 AM-loaded CB₂-U2OS cells before (left), during (middle), and 6 min after (right) intracellular administration of 0.1 μM anandamide alone (C) or in the presence of 1 μM AM630 (D). Arrows denote the injected cells; the fluorescence scale (0–3) is illustrated in each panel and magnified in the left panel of part C.

Anandamide produced negligible effects in control untransfected U2OS cells regardless of whether it was applied extracellularly (Figure 2 of the Supporting Information) or intracellularly (Figure 3 of the Supporting Information).

WIN55,212-2 Produces CB₂ Receptor-Dependent Increases in Ca²⁺ Concentration in CB₂-U2OS Cells

Extracellular administration of WIN55,212-2 (0.01, 0.1, and 1 μM) elevated [Ca²⁺]_i by 11 ± 8.1 nM (n = 27 cells), 17 ± 6.3 nM (n = 38 cells), and 262 ± 6.8 nM [n = 46 cells (Figure 5A,B)]; the latter effect was statistically significant (P < 0.05) and sensitive to CB₂ blockade with AM630 [1 μM, incubation for 10 min, Δ[Ca²⁺]_i reduced to 12 ± 6.8 nM (n = 36 cells)] (Figure 5A,B). The Ca²⁺ response elicited by WIN55,212-2 occurred with a latency of 1–2 min (Figure 5B), similar to the effect of bath-applied 2-AG (Figure 1B).

Figure 5.

Extracellular administration of WIN55,212-2 to CB₂-U2OS cells elevates [Ca²⁺]_i. (A) Comparison of the increases in [Ca²⁺]_i produced by extracellular administration of WIN55,212-2 (WIN, 0.01–1 μM) and 1 μM WIN55,212-2 in the presence of CB₂ receptor antagonist AM630 (1 μM); P < 0.05 compared with basal levels (∗) or with 1 μM WIN55,212-2 (#). (B) Representative recordings of increases in [Ca²⁺]_i in response to 1 μM WIN in the absence or presence of 1 μM CB₂ antagonist AM630.

Microinjection of WIN55,212-2 (0.01, 0.1, and 1 μM, final concentrations inside the cell) into CB₂-U2OS cells triggered fast, transient, and concentration-dependent increases in [Ca²⁺]_i of 257 ± 5.3, 635 ± 7.1, and 924 ± 17 nM (n = 6 cells for each concentration tested), whereas when 1 μM AM630 was co-injected with 1 μM WIN55,212-2, a small and insignificant increase in Ca²⁺ concentration, of 31 ± 4.6 nM (n = 6 cells), was apparent (Figure 6A,B). Representative examples of the increase in the F₃₄₀/F₃₈₀ fluorescence ratio produced by intracellular administration of WIN55,212-2 or AM630 and WIN55,212-2 are shown in panels C and D of Figure 6. The insignificant Ca²⁺ responses to extracellular and intracellular administration of WIN55,212-2 in control U2OS cells are illustrated in Figures 2 and 3 of the Supporting Information, respectively.

Figure 6.

Microinjection of WIN55,212-2 increases [Ca²⁺]_i in CB₂-U2OS cells. (A) Averaged [Ca²⁺]_i elevations produced by increasing doses of microinjected WIN55,212-2 (WIN 0.01–1 μM, left) or by co-injection of 0.1 μM WIN55,212-2 and 1 μM CB₂ antagonist AM630 (right). (B) Comparison of the increases in [Ca²⁺]_i elicited by microinjected WIN55,212-2 (0.01, 0.1, and 1 μM) and WIN55,212-2 (0.1 μM) in the presence of AM630 (1 μM); P < 0.05 compared with the control (∗) (see Figure 2) or with 0.1 μM WIN55,212-2 alone (#). (C and D) Characteristic fluorescence images of Fura-2 AM-loaded CB₂-U2OS cells before (left), during (middle), and 6 min after (right) intracellular administration of 0.1 μM WIN55,212-2 alone (C) or in the presence of 1 μM AM630 (D). Arrows denote the injected cells; the fluorescence scale (0–3) is illustrated in each panel and magnified in the left panel of part C.

Effects of Extracellular 2-AG and WIN55,212-2 Are Mediated by Gq Coupling of CB₂ Receptors

2-AG (1 μM), anandamide (1 μM), or WIN55,212-2 (1 μM) was bath-applied to CB₂-U2OS cells treated overnight with chemicals interfering with G protein signaling. After cholera toxin (CTX, 100 ng/mL) pretreatment, 2-AG produced an increase in the [Ca²⁺]_i of 83 ± 5.1 nM [n = 61 cells (Figure 7A,B)] similar to the effect of 2-AG on untreated cells. CTX irreversibly abolishes the GTPase activity of the Gs α subunit, inducing continual activation of adenylyl cyclase and increased intracellular levels of cAMP. In this manner, it hinders ligands of Gs-coupled receptors from eliciting their Gs-dependent effects. After pretreatment with the Gi/o inhibitor pertussis toxin (PTX, 100 ng/mL), the response of 2-AG was negligibly modified, measuring 85 ± 4.2 nM [n = 53 cells (Figure 7A,B)]. Conversely, pretreatment of CB₂-U2OS cells with Gq blocker d-[Trp7,9,10]-substance P (D-SP, 100 ng/mL)²⁴ greatly reduced the effect of 2-AG to 7 ± 2.7 nM [n = 67 cells (Figure 7A,B)].

Figure 7.

The Ca²⁺ responses produced by bath-applied 2-AG and WIN55,212-2 are prevented by a Gq protein inhibitor. (A) Representative examples of the Ca²⁺ responses induced by 1 μM 2-AG (top), 1 μM anandamide (ANA, middle), or 1 μM WIN55,212-2 (WIN, bottom), applied by bath to CB₂-expressing U2OS cells in the presence of cholera toxin (CTX, 100 nM, top), which occludes Gs-dependent signaling, Gi/o blocker pertussis toxin (PTX, 100 nM, middle), or Gq protein inhibitor d-[Trp7,9,10]-substance P (D-SP, 100 μM, bottom); 2-AG and WIN55,212-2 increased [Ca²⁺]_i, and the response was Gq protein-mediated. (B) Comparison of the mean amplitude of the Ca²⁺ responses produced by extracellular administration of 1 μM 2-AG (left), 1 μM anandamide (middle), or 1 μM WIN55,212-2 (right) in the absence and presence of the indicated G protein inhibitors; P < 0.05 compared with WIN55,212-2 alone (∗).

The Ca²⁺ response elicited by anandamide was largely similar and not significant regardless of whether anandamide was applied alone [Δ[Ca²⁺]_i of 9 ± 4.7 nM (n = 33 cells)] or after stimulating Gs-dependent signaling with CTX (100 ng/mL), inhibiting Gi/o with PTX (100 ng/mL), and inhibiting Gq with D-SP (100 ng/mL). The Δ[Ca²⁺]_i values were 8 ± 5.2 nM (n = 29 cells), 6 ± 4.8 nM (n = 41 cells), and 7 ± 2.3 nM (n = 36 cells), respectively (Figure 7A,B).

D-SP pretreatment drastically diminished the effect induced by WIN55,212-2, reducing it to 58 ± 3.9 nM, while in the presence of CTX or PTX, WIN55,212-2 increased [Ca²⁺]_i to similar extents as in their absence: Δ[Ca²⁺]_i values were 257 ± 6.8 nM (n = 53 cells) and 243 ± 7.9 nM (n = 47 cells) in cells pretreated with CTX and PTX, respectively, and 262 ± 6.8 nM (n = 49 cells) for WIN55,212-2 alone (Figure 7A,B).

Intracellular CB₂ Receptors Couple to Gq Proteins

Overnight treatment of CB₂-U2OS cells with CTX (100 ng/mL) or PTX (100 ng/mL) failed to significantly modify the Ca²⁺ response elicited by intracellular administration of 0.1 μM 2-AG (Figure 8A,B). Under the conditions described above, 2-AG increased [Ca²⁺]_i by 654 ± 21 nM (n = 6 cells) and 629 ± 18 nM (n = 6 cells), respectively, while the response to 2-AG alone measured 652 ± 19 nM [n = 6 cells (Figure 8A,B)]. After incubation of cells with D-SP (100 ng/mL), the effect of 2-AG was greatly decreased to 62 ± 23 nM (n = 6 cells), indicating participation of a Gq-dependent pathway in the response (Figure 8A,B).

Figure 8.

Intracellular microinjection of 2-AG, anandamide, or WIN55,212-2 produces Gq-dependent Ca²⁺ responses in CB₂-expressing U2OS cells. (A) Averaged Ca²⁺ responses induced by intracellular administration of 0.1 μM 2-AG (top), 0.1 μM anandamide (ANA, middle), or 0.1 μM WIN55,212-2 (WIN, bottom) in CB₂-U2OS cells pretreated with cholera toxin (CTX, 100 nM, top), pertussis toxin (PTX, 100 nM, middle), or Gq protein inhibitor d-[Trp7,9,10]-substance P (D-SP, 100 μM, bottom); each ligand increased [Ca²⁺]_i, and the response was sensitive to Gq protein blockade. (B) Comparison of the mean amplitude of the Ca²⁺ responses produced by intracellular administration of 0.1 μM 2-AG (left), 0.1 μM anandamide (middle), or 0.1 μM WIN55,212-2 (right) in absence and presence of the indicated G protein inhibitors; in each group of data, P < 0.05 compared with 2-AG, anandamide, or WIN55,212-2 alone (∗).

Upon pretreatment with CTX (100 ng/mL) or PTX (100 ng/mL), intracellular injection of anandamide (0.1 μM) elevated [Ca²⁺]_i of CB₂-U2OS cells by 359 ± 7.2 nM (n = 6 cells) or 355 ± 11.2 nM (n = 6 cells), respectively; these effects are similar to those triggered by microinjected anandamide alone [368 ± 8.4 nM (n = 6 cells)], indicating that Gs and Gi/o proteins are not mediating its effect. Upon D-SP (100 ng/mL) pretreatment, intracellular anandamide produced an insignificant Ca²⁺ response of 38 ± 6.3 nM [n = 6 cells (Figure 8A,B)].

Similarly, CTX and PTX (both at 100 ng/mL) pretreatment did not significantly modify the response of CB₂-U2OS cells to microinjected WIN55,212-2 (0.1 μM). Δ[Ca²⁺]_i values were 668 ± 13 nM (n = 6 cells) and 641 ± 9.8 nM (n = 6 cells) after CTX and after PTX, respectively, and 635 ± 7.1 nM for WIN55,212-2 alone. However, blocking Gq proteins with 100 ng/mL D-SP basically abolished the effect of intracellular WIN55,212-2. In this case, Δ[Ca²⁺]_i decreased to 27 ± 5.4 nM [n = 6 cells (Figure 8A,B)].

Activation of Intracellular CB₂ Mobilizes Ca²⁺ from Distinct Pools

CB₂-U2OS cells incubated with Ca²⁺-free saline responded to microinjection of 0.1 μM 2-AG with a 457 ± 5.2 nM increase in [Ca²⁺]_i (n = 6 cells), while the effect of control buffer microinjection was negligible {Δ[Ca²⁺]_i = 29 ± 1.7 nM [n = 6 cells (Figure 9A,B)]}. The response to 2-AG was reduced in Ca²⁺-free versus Ca²⁺-containing HBSS [Δ[Ca²⁺]_i = 653 ± 19 nM (Figure 2)], indicating mobilization of both intracellular and extracellular Ca²⁺ pools. Using a pharmacological approach, we further sought to identify the intracellular sources of Ca²⁺ involved in the response to microinjected 2-AG. Pretreatment of cells with ryanodine receptor blocker ryanodine (10 μM, 1 h) did not significantly modify the Ca²⁺ response to 2-AG, which measured 452 ± 5.9 nM [n = 6 cells (Figure 9A,B)]. Conversely, pretreatment with Ned-19 (5 μM, 15 min), which blocks endolysosomal Ca²⁺ release via the NAADP-sensitive two-pore channels,²⁵ decreased the extent of the Ca²⁺ elevation produced by 2-AG to 349 ± 4.3 nM (n = 6 cells); inositol 1,4,5-trisphosphate receptor (IP₃R) inhibition induced by treating CB₂-U2OS cells with heparin and xestospongin C (10 μM, 15 min) also induced a significant decrease in the response to 2-AG {Δ[Ca²⁺]_i = 168 ± 3.6 nM [n = 6 cells (Figure 9A,B)]}. When both IP₃Rs and the two-pore channels were blocked, the Ca²⁺ response to intracellular microinjection of 2-AG was completely abolished {Δ[Ca²⁺]_i = 34 ± 2.4 nM [n = 6 cells (Figure 9A,B)]}.

Figure 9.

2-AG mobilizes endoplasmic reticulum and acidic-like Ca²⁺ stores. (A) Averaged Ca²⁺ responses of CB₂-U2OS cells in Ca²⁺-free saline, microinjected with either control buffer or 0.1 μM 2-AG in the absence or presence of ryanodine receptor blocker ryanodine (Ry), two-pore channel antagonist Ned-19, IP₃R inhibitors xestospongin C (XeC) and heparin (Hep), or a combination of Ned-19, XeC, and Hep. (B) Comparison of the Ca²⁺ increases produced by the treatments described in (A); P < 0.05 compared with 2-AG microinjection (∗), 2-AG in the presence of Ned-19 (#), or 2-AG in the presence of IP₃R blockers (+).

Discussion

In addition to the effects initiated at the plasma membrane, G protein-coupled receptors (GPCRs) may also trigger signaling cascades upon their activation within the cell.²⁶ The emerging paradigm of functional intracellular GPCRs is particularly significant in the case of lipid messengers that are generated intracellularly and may target their receptors at both sites, (reviewed in ref (27)). We and others reported the functionality of intracellularly expressed CB₁ receptors,^17,28 as well as their ability to use Ca²⁺ as a second messenger.¹⁷ To evaluate whether CB₂ receptors elicit Ca²⁺ signaling upon plasmalemmal or intracellular activation, cannabinoid ligands were administered extracellularly or microinjected into U2OS cells stably expressing CB₂.

We noticed that extracellular administration of 2-AG induced a small increase in [Ca²⁺]_i of CB₂-U2OS cells only at the highest concentration tested here (1 μM). Bath application of anandamide did not elevate [Ca²⁺]_i of CB₂-U2OS cells. While 2-AG produced a gradual increase in [Ca²⁺]_i of CB₂-U2OS cells, a robust Ca²⁺ response was induced by extracellular administration of WIN55,212-2, which is a cannabinoid agonist displaying high affinity and/or intrinsic activity at this receptor.^1,29 The CB₂ specificity was indicated by the ability of CB₂ antagonist AM630²⁹ to inhibit the effects of extracellular administration of 2-AG and WIN55,212-2 in CB₂-U2OS cells and by the unresponsiveness of untransfected U2OS cells to bath application of WIN55,212-2.

Similar to CB₁, CB₂ receptors couple to Gi proteins and inhibit cAMP formation or activate MAPK.^6,29 However, coupling to Gs or Gq of CB₁ receptors has also been reported.^30,31 While a cannabinoid-dependent [Ca²⁺]_i increase may occur downstream of Gi,^32,33 we noticed that the CB₂-dependent effects of 2-AG or WIN55,212-2 were completely contingent on Gq in our paradigm. WIN55,212-2 also activates Gq downstream of CB₁;³¹ moreover, it can trigger a CB₂-dependent signaling pathway different from that elicited by other ligands.⁷ Thus, the discrepancy between the effects of extracellular administration of anandamide, 2-AG, and WIN55,212-2 in our study may be a result of the reported functional selectivity at CB₂ receptors.^7,34

In a recent study, we demonstrated that anandamide can trigger fast CB₁-dependent Ca²⁺ signaling upon microinjection but is ineffective upon extracellular administration in CB₁-transfected cells.¹⁷ To test the hypothesis of functional intracellular CB₂ receptors in CB₂-U2OS cells, intracellular injections of 2-AG, anandamide, or WIN55,212-2 were conducted. When 2-AG or anandamide was microinjected, concentration-dependent elevations of [Ca²⁺]_i were observed, and the Ca²⁺ response pattern was very fast, which is in contrast with the delayed, modest response and lack of effect, respectively, observed with the extracellular administration of these two agonists. Likewise, intracellular administration of WIN55,212-2 resulted in a concentration-dependent and robust effect. WIN55,212-2 and 2-AG produced Ca²⁺ responses with an amplitude higher than that of anandamide, which is consistent with the full agonistic activity at CB₂ receptors reported for these ligands.^1,29 The sensitivity of these responses to the intracellular blockade of CB₂ and the unresponsiveness of control, untransfected U2OS cells to microinjected cannabinoids were considered to be indicative of CB₂ specificity. We further noticed that interfering with Gq-dependent, but not with Gs- or Gi/o-dependent, signaling prevented the effect of microinjected 2-AG, anandamide, or WIN55,212-2. Moreover, the endocannabinoids and WIN55,212-2 trigger similar signaling pathways upon intracellular administration, despite their unrelated chemical structure.

The accumulating evidence pointing to endolysosomes as intracellular locations where GPCRs initiate signaling^{16,18,19,35,36} prompted us to evaluate whether this may be the case for CB₂. In an initial functional study, we found that the effect of intracellular 2-AG is abolished by lysosomal disruption. Because rapamycin reduced the effects of microinjected 2-AG, ligand microautophagy²² may be a necessary step in the activation of endolysosomally located CB₂. The localization of CB₂ to lysosomes was confirmed by an additional study, providing morphological evidence that CB₂ colocalizes with the endolysosomal-associated small GTPase Rab7. Lysosomal disruption with bafilomycin A1 markedly reduced the fluorescence intensity of both CB₂ and Rab7, supporting the findings of the functional study.

Next, we sought to examine the pools of Ca²⁺ mobilized upon activation of intracellularly located CB₂. The reduction in the amplitude of the Ca²⁺ response of CB₂-U2OS cells incubated with Ca²⁺-free saline to microinjected 2-AG indicated that both influx of extracellular Ca²⁺ and release of Ca²⁺ from intracellular stores occur downstream of intracellular CB₂ activation. Using a pharmacological approach, we further determined that 2-AG-induced stimulation of intracellular CB₂ results in Ca²⁺ mobilization from endolysosomes via NAADP-sensitive two-pore channels and from the endoplasmic reticulum via the IP₃R. This effect correlates well with the localization of CB₂ at the endolysosmes, as well as with the localization of NAADP-generating enzymes^37,38 at the membrane of acidic organelles.³⁹

Thus, we conclude that intracellular CB₂ receptors are functional, endolysosomally located, and Gq-coupled in CB₂-U2OS cells, and their activation mobilizes NAADP-sensitive acidic-like Ca²⁺ stores and inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools. In addition, we note that CB₂ agonists elicit discrepant and delayed effects and exhibit a considerably lower potency when administered extracellularly as opposed to intracellularly. Although these effects appear to be Gq-mediated, it may be that a considerable pool of plasmalemmal CB₂ receptors do not couple with Gq/Ca²⁺ signaling. Conversely, Gq coupling and associated Ca²⁺ responses may be a characteristic of intracellularly located CB₂, in which case the delay in the effects elicited by agonists upon extracellular administration may be a result of the latency necessary for membrane permeation. Nonetheless, differential activation of plasmalemmal CB₂ by the three agonists (applied extracellularly) is more likely to be a result of the functional selectivity at CB₂,^7,34 which is supported by the previous finding that 2-AG (which promotes agonist-directed trafficking at CB₂³⁴) has a low potency at eliciting CB₂-mediated Ca²⁺ responses.³⁴ Binding of a particular agonist to a GPCR results in the enrichment of a unique set of receptor conformations based on the microaffinity of the agonist for each conformation; because distinct conformations presumably couple receptors differently to specific G proteins and intracellular effectors, individual agonists ultimately produce distinct effects.¹¹ As such, anandamide, 2-AG, and WIN55,212-2 may induce conformational changes in plasmalemmal CB₂, resulting in distinct responses, as observed in the study presented here. However, we note that this is not the case with intracellular, endolysosomally located CB₂, which appears to couple to the same Gq-mediated Ca²⁺ pathway in response to intracellular administration of the three agonists; moreover, the three agonists have higher potency and efficacy at eliciting intracellularly initiated CB₂-dependent Ca²⁺ responses. Thus, functional selectivity may apply more to plasmalemmal than to intracellular CB₂.

On a different note, plasma membrane-located CB₂ is internalized upon agonist stimulation and slowly recycled back to the cell surface via a Rab11-dependent pathway, while rapid recycling, via Rab4, does not occur.⁴⁰ Moreover, in the case of CB₂, not all receptors are recycled, and chronic agonist stimulation does not promote switching from recycling to degradative pathways, supporting the hypothesis of a putative role of sequestration of the CB₂ receptor to the cytoplasm following internalization.⁴⁰

Our results, together with those of others,¹² add further complexity to the CB₂ receptor pharmacology and argue for careful consideration of receptor localization in the development of CB₂-based therapeutic agents. Moreover, because 2-AG is intracellularly produced “on demand”,⁴¹ its first target may be intracellularly located CB₂, which may be relevant both in physiological states and in pathologies with an increased level of production of endocannabinoids.

Glossary

Abbreviations

2-AG

2-arachidonoyl glycerol

[Ca²⁺]_i

intracellular Ca²⁺ concentration

CB₂-U2OS

CB₂-β-arrestin 2-green fluorescent protein-U2OS

CTX

cholera toxin

DMEM

Dulbecco’s modified Eagle’s medium

D-SP

d-[Trp7,9,10]-substance P

EGTA

ethylene glycol tetraacetic acid

GFP

green fluorescent protein

GPCR

G protein-coupled receptor

HBSS

Hank’s balanced salt solution

IP₃R

inositol 1,4,5-trisphosphate receptor

MAPK

mitogen-activated protein kinase

NAADP

nicotinic acid adenine dinucleotide phosphate

PTX

pertussis toxin

RFP

red fluorescent protein.

Supporting Information Available

Evidence of negligible effects of extracellular anandamide on CB₂-expressing U2OS cells or untransfected U2OS cells, insignificant responses produced by microinjected anandamide in untransfected U2OS cells is provided as supplemental data, and insignificant Ca²⁺ responses to extracellular or intracellular administration of WIN55,212-2 in untransfected U2OS cells. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

G.C.B. and E.D. contributed equally to this work.

This work was supported by National Institutes of Health Grants HL090804 (to E.B.) and DA035926 and DA023204 (to M.E.A.).

The authors declare no competing financial interests.

Funding Statement

National Institutes of Health, United States