Intracellular Cannabinoid Type 1 (CB1) Receptors Are Activated by Anandamide*

Recent studies have demonstrated that the majority of endogenous cannabinoid type 1 (CB1) receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. Using calcium imaging and intracellular microinjection in CB1 receptor-transfected HEK293 cells and NG108-15 neuroblastoma × glioma cells, we provide evidence that anandamide acting on CB1 receptors increases intracellular calcium concentration when administered intracellularly but not extracellularly. The calcium-mobilizing effect of intracellular anandamide was dose-dependent and abolished by pretreatment with SR141716A, a CB1 receptor antagonist. The anandamide-induced calcium increase was reduced by blocking nicotinic acid-adenine dinucleotide phosphate- or inositol 1,4,5-trisphosphate-dependent calcium release and abolished when both lysosomal and endoplasmic reticulum calcium release pathways were blocked. Taken together, our results indicate that, in CB1 receptor-transfected HEK293 cells, intracellular CB1 receptors are functional; they are located in acid-filled calcium stores (endolysosomes). Activation of intracellular CB1 receptors releases calcium from endoplasmic reticulum and lysosomal calcium stores. In addition, our results support a novel role for nicotinic acid-adenine dinucleotide phosphate in cannabinoid-induced calcium signaling.

Although most studies on the cannabinoid type 1 (CB 1 ) 3 receptor have focused on its localization to the plasma membrane, increasing evidence suggests that not only are CB 1 receptors highly expressed in intracellular compartments but also that these intracellular receptors are functional. An early study in N18TG2 neuroblastoma cells demonstrated localization of CB 1 receptors to nuclear membranes in cells not treated with agonists (1). More recent studies have suggested that the CB 1 receptor undergoes constitutive endocytosis in both neurons and transfected cells (2)(3)(4). Rozenfeld and Devi (5) found that the majority of the endogenous CB 1 receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. They further demonstrated that, during trafficking, the lysosomal CB 1 receptors may interact with G␣ i proteins. Another recent report indicated that the intracellular pool of CB 1 receptors does not contribute to cellsurface repopulation (6), and thus, it may have a distinct yet unknown function from the membrane CB 1 receptors.
Most previous studies examining cannabinoids, including anandamide, have demonstrated a nonspecific effect in that non-transfected cells also showed a calcium response (7,8).
One study showed that only the indole derivative WIN55,212-2 was able to increase intracellular calcium via CB 1 receptor coupling to G q/11 proteins (9). Many studies have shown that cannabinoids can inhibit a voltage-sensitive calcium channel present in neurons and neuroblastoma cells (e.g. Refs. 7, 10, and 11). In this study, we show that anandamide increases intracellular calcium concentration but only when injected in CB 1 receptortransfected HEK293 cells and NG108-15 neuroblastoma ϫ glioma cells and provide evidence that intracellular CB 1 receptors are functional.

EXPERIMENTAL PROCEDURES
Cell Culture-CB 1 receptor-transfected HEK293 cells have been described previously (12). Briefly, stably transfected HEK293 cell lines were created by transfection with human CB 1 -pcDNA3 using Lipofectamine reagent (Invitrogen) and selected in DMEM containing G418 (Geneticin; 1 mg/ml) and 10% fetal calf serum at 37°C in a 5% CO 2 incubator. Colonies of ϳ500 cells were picked (ϳ2 weeks post-transfection) and allowed to expand and then were tested for expression of receptor by immunostaining. Cell lines containing moderate-to-high levels of receptor expression were tested for receptor-binding properties. The cell line was maintained in DMEM and 10% fetal calf serum with 0.7 mg/ml G418 at 37°C in a 5% CO 2 incubator. NG108-15 cells (mouse neuroblastoma ϫ rat glioma) were cultured in DMEM without sodium pyruvate, 10% fetal bovine serum, 0.1 mM hypoxanthine, 400 nM aminopterin, and 0.016% thymidine as suggested by the manufacturer (American Type Culture Collection, Manassas, VA).
Calcium Imaging-Intracellular Ca 2ϩ measurements were performed as described previously (13,14). Briefly, cells were incubated with 5 M fura-2/AM (Invitrogen) in Hanks' balanced salt solution at room temperature for 45 min in the dark, washed three times with dye-free buffer, and incubated for another 45 min to allow for complete de-esterification of the dye. Coverslips (25-mm diameter) were subsequently mounted in an open bath chamber (RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope (Nikon Eclipse TiE, Optical Apparatus Co., Ardmore, PA). The microscope was equipped with a Perfect Focus System and a Photometrics CoolSNAP HQ 2 CCD camera (Roper Scientific and Optical Apparatus Co.). During the experiments, the Perfect Focus System was activated. Fura-2/AM fluorescence (emission ϭ 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 Nikon NIS-Elements AR 3.1 software (Optical Apparatus Co.). The ratio of the fluorescence signals (340/380 nm) was converted to Ca 2ϩ concentrations (15). In Ca 2ϩ -free experiments, CaCl 2 was omitted, and 2.5 mM EGTA was added.
Intracellular Microinjection-Injections were performed using Femtotips II, InjectMan NI 2, and FemtoJet systems (Eppendorf) as described previously (13,14). Pipettes were back-filled with an intracellular solution composed of 110 mM KCl, 10 mM NaCl, and 20 mM HEPES (pH 7.2) or the drugs to be studied. The injection time was 0.4 s at 60 hectopascals with a compensation pressure of 20 hectopascals to ensure that Ͻ0.1% of the cell volume was microinjected.
Immunocytochemistry-HEK293 cells were transfected with CB 1 -GFP cDNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature and processed for LAMP-1 (lysosome-associated membrane protein 1) (16) immunoreactivity. Cells were washed three times with PBS and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. Cells were blocked with 3% BSA in PBS for 30 min and incubated with LAMP-1 (mouse IgG, 1:500 dilution; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) for 2 h at room temperature. Cells were washed three times with PBS and then incubated with Alexa 568-labeled goat anti-mouse antibodies (1:2000 dilution; Molecular Probes, Eugene, OR) for 1 h. After washing three times with PBS, cells were mounted with DAPI Fluoromount G (Southern-Biotech, Birmingham, AL) and examined under a confocal laser scanning microscope (Leica TCS SP5) with excitation wavelengths set to 405 nm for DAPI, 488 nm for GFP, and 561 nm for Alexa 568 in the sequential mode. 1 Receptors-HEK293 cells stably expressing the human CB 1 receptor (12) were examined for their responses to anandamide; non-transfected HEK293 cells served as a control. We also tested the responses to anandamide in NG108-15 cells, which endogenously express CB 1 but not CB 2 receptors (17). The basal cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) was 65 Ϯ 0.6 nM (n ϭ 124) in CB 1 receptor-expressing HEK293 cells, 66 Ϯ 0.8 nM (n ϭ 75) in non-transfected HEK293 cells, and 64 Ϯ 0.7 nM (n ϭ 84) in NG108-15 cells. Extracellular administration of anandamide did not produce a significant increase in [Ca 2ϩ ] i , whereas the cells responded to ATP (10 M) administration by an increase in [Ca 2ϩ ] i of 599 Ϯ 34 nM (n ϭ 46) (Fig. 1). The response to ATP was used as proof of integrity of G q -dependent pathways in these cells, as HEK293 cells endogenously express P2Y receptors (18). Similar results were obtained when anand- amide was extracellularly administered to non-transfected HEK293 cells (supplemental Fig. 1).

Intracellular but Not Extracellular Administration of Anandamide Increases Cytosolic Ca 2ϩ Concentration in Cells Expressing CB
Anandamide (10, 100, and 1000 nM) administered by intracellular injection produced concentration-dependent increases in [Ca 2ϩ ] i of 126 Ϯ 22, 567 Ϯ 76, and 854 Ϯ 212 nM, respectively (n ϭ 6 for each concentration tested). The Ca 2ϩ response to intracellular administration of anandamide was fast, robust, and transitory, similar to that induced by the injection of second messengers (see Fig. 5). Fig. 2 shows examples of representative experiments (A and B) and the dose-response relationship (C and D). The effect was CB 1 receptor-specific, as pretreat-ment with SR141716A (1 M, 15 min), a CB 1 receptor antagonist (19), prevented the anandamide-induced increase in [Ca 2ϩ ] i (Fig. 2, C and D). Moreover, intracellular injection of anandamide in non-transfected HEK293 cells did not produce an increase in [Ca 2ϩ ] i higher than the control injection (supplemental Fig. 2).
Functional CB 1 Receptors Are Localized on Lysosomes-The increase in [Ca 2ϩ ] i produced by intracellular administration of anandamide was markedly reduced by pretreatment with bafilomycin A1 (1 M, 1 h), a blocker of lysosomal Ca 2ϩ release, but was not affected by pretreatment with brefeldin A (10 M, 1 h), which disrupts the Golgi apparatus (Fig. 3, A and B). In cells pretreated with brefeldin A, anandamide (100 nM) increased [Ca 2ϩ ] i by 579 Ϯ 71 nM (n ϭ 6), whereas pretreatment with bafilomycin A1 markedly reduced the response to anandamide to 37 Ϯ 14 nM (Fig. 3B). Bafilomycin A1, a V-type ATPase inhibitor (20), blocks the release of Ca 2ϩ from lysosome-like acidic stores in a variety of other cell types (21)(22)(23)(24)(25). In CB 1 -GFPtransfected HEK293 cells, in which lysosomes were labeled with LAMP-1 (16), intracellular CB 1 receptors were colocalized with lysosomes ( Fig. 3, C-F (Fig. 4). Adding monensin (50 M) on the plateau of the thapsigargin-induced response produced a supplemental increase in [Ca 2ϩ ] i (Fig. 4). These results indicate that HEK293 cells stably transfected with the CB 1 receptor possess both endoplasmic reticulum and lysosomal Ca 2ϩ stores.
Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca 2ϩ from lysosomes (26), whereas inositol 1,4,5-trisphosphate (IP 3 ) releases Ca 2ϩ from the endoplasmic reticulum via IP 3 receptor activation (27). Intracellular administration of NAADP (50 nM) produced a fast and transitory increase in [Ca 2ϩ ] i of 296 Ϯ 54 nM (n ϭ 6) that was prevented by pretreatment with Ned-19 (5 M, 15 min), an inhibitor of NAADP signaling (28). Intracellular injection of IP 3 (50 nM) increased [Ca 2ϩ ] i by 511 Ϯ 41 nM; co-administration of IP 3 and heparin (100 g/ml) in cells pretreated with xestospongin C (10 M, 15 min) did not increase [Ca 2ϩ ] i (Fig. 5).   6), which was lower than that produced by anandamide in untreated cells (567 Ϯ 76 nM) (Fig. 6, B and D). The response to anandamide was reduced by blocking IP 3 receptors with xestospongin C and heparin (133 Ϯ 29 nM, n ϭ 6) (Fig. 6C) and abolished when the Ca 2ϩ release from both NAADP-and IP 3sensitive Ca 2ϩ stores was blocked (Fig. 6, C and D). Because the amplitude of the Ca 2ϩ response to anandamide was higher than that accounted for by the NAADP-dependent and IP 3 -dependent Ca 2ϩ release together, a Ca 2ϩ -induced Ca 2ϩ release  mechanism is likely responsible for the difference. In addition, pretreatment with U73122, a phospholipase C (PLC) inhibitor (29), reduced but did not abolish the Ca 2ϩ response to anandamide (Fig. 6C).
Next, to validate our results obtained in CB 1 receptor-transfected cells in endogenously expressing cells, we carried out calcium imaging experiments in NG108-15 cells, which endogenously express CB 1 but not CB 2 receptors (17). In NG108-15 cells, extracellular administration of anandamide did not produce a significant increase in [Ca 2ϩ ] i , whereas the administration of ATP (10 M) increased [Ca 2ϩ ] i by 432 Ϯ 57 nM (n ϭ 52) (Fig. 7).

DISCUSSION
Endocannabinoids are lipid messengers generated intracellularly; they cannot be stored in vesicles (30,31). As a result, they may target intracellular or extracellular cannabinoid receptors from the same cells or in neighboring cells. On the basis of the fact that other G protein-coupled receptors such as those for angiotensin II (32,33) are active when stimulated from inside the cells, we tested whether or not CB 1 receptors can be activated intracellularly. We addressed this question in cells stably transfected with or endogenously expressing CB 1 receptors by monitoring the [Ca 2ϩ ] i of fura-2/AM-loaded cells in response to extracellular and intracellular administration of anandamide.
Although extracellular administration of anandamide did not enhance [Ca 2ϩ ] i , intracellular injection of anandamide elicited a dose-dependent increase in [Ca 2ϩ ] i , supporting a functional intracellular location of CB 1 receptors. The anandamideinduced [Ca 2ϩ ] i increase was absent after pretreatment with a CB 1 receptor antagonist or in cells lacking the CB 1 receptor (non-transfected HEK293 cells), further supporting the specificity of the response.
The next series of experiments were designed to characterize the intracellular localization of functional CB 1 receptors. Previous reports suggested the association of CB 1 receptors with endolysosomal compartments (5,6). The Golgi apparatus has also been involved in receptor internalization. To test which of these organelles are involved, we disrupted lysosomes with bafilomycin A 1 or the Golgi apparatus with brefeldin A. Pretreatment with bafilomycin A 1 markedly reduced the response to anandamide, whereas brefeldin A did not significantly affect it; these results indicate a critical role for acid-filled lysosomal Ca 2ϩ stores in the anandamide-induced increase in [Ca 2ϩ ] i and suggest the localization of CB 1 receptors on endolysosomes. We also sought morphological evidence for localization of CB 1 receptors on lysosomes labeled with LAMP-1 and found that intracellular CB 1 receptors are indeed localized on lysosomes in CB 1 -GFP-transfected HEK293 cells.
Next, we examined the presence of endoplasmic reticulum and lysosomal Ca 2ϩ stores in CB 1 receptor-transfected HEK293 cells. In Ca 2ϩ -free Hanks' balanced salt solution supplemented with the Ca 2ϩ chelator EGTA, thapsigargin (a sarcoplasmic/endoplasmic reticulum ATPase inhibitor) produced an increase in [Ca 2ϩ ] i. Monensin, which collapses pH gradients across acidic organelles (34), added on the plateau of the thapsigargin-induced response produced a supplemental increase in [Ca 2ϩ ] i . These results indicate that both endoplasmic reticulum and lysosomal Ca 2ϩ stores are present in CB 1 receptortransfected HEK293 cells.
The anandamide-induced [Ca 2ϩ ] i increase was not affected when experiments were carried out in Ca 2ϩ -free saline, indicating that Ca 2ϩ influx is not involved in this response. We further characterized the involvement of intracellular stores in the anandamide-induced [Ca 2ϩ ] i increase. NAADP, a potent Ca 2ϩ -mobilizing second messenger, releases Ca 2ϩ from acidic lysosome-like Ca 2ϩ stores via recently identified two-pore channels (13,(35)(36)(37). Intracellular injection of NAADP elevated [Ca 2ϩ ] i ; the response was blocked by pretreatment with Ned-19, a specific inhibitor of NAADP signaling (28). IP 3 releases Ca 2ϩ from the endoplasmic reticulum via IP 3 recep-tors, well characterized Ca 2ϩ channels located on the endoplasmic reticulum (27). The increase in [Ca 2ϩ ] i produced by intracellular injection of IP 3 was blocked by xestospongin C and heparin, antagonists of IP 3 receptors.
Having established that lysosomes and the endoplasmic reticulum are not only present but also functional in CB 1 receptor-transfected HEK293 cells, we assessed their involvement in the Ca 2ϩ -mobilizing effect of anandamide. We used two different approaches to assess the role of lysosomes. Besides disrupting the lysosomes with bafilomycin A1, we assessed also the effect of Ned-19, which blocks the NAADP-dependent lysosomal Ca 2ϩ release without disrupting the lysosomes. Pretreatment with Ned-19 reduced the anandamide-induced Ca 2ϩ response to a lesser extent than bafilomycin A 1 . The difference in the magnitude of the Ca 2ϩ responses to anandamide in the presence of bafilomycin A1 and Ned-19 reflects the different mechanisms employed by these two agents in interfering with the lysosomal Ca 2ϩ release; it also indicates that intact lysosomes are important for the anandamide-induced Ca 2ϩ responses. These results support the involvement of NAADP-dependent lysosomal Ca 2ϩ stores in the response to anandamide.
As earlier reports indicated that CB 1 receptor coupling to G q/11 proteins may increase [Ca 2ϩ ] i (9), we tested whether the PLC-IP 3 pathway is involved in the effect of anandamide in CB 1 receptor-transfected HEK cells. Pretreatment with U73122, a PLC inhibitor (29), reduced but did not abolish the anandamide-induced increase in [Ca 2ϩ ] i . The response was similar to that produced by anandamide in the presence of the IP 3 receptor antagonists xestospongin C and heparin and indicates the involvement of the PLC-IP 3 pathway, as reported previously (29). The amplitude of the increase in [Ca 2ϩ ] i produced by anandamide was higher than the sum of the NAADP-dependent and IP 3 -dependent Ca 2ϩ release, thus suggesting the participation of a Ca 2ϩ -induced Ca 2ϩ release mechanism in this response. Similarly, activation of NAADP receptors has been associated with the recruitment of inositol trisphosphate and/or ryanodine receptors via Ca 2ϩ -induced Ca 2ϩ release (38 -43). However, because HEK293 cells lack ryanodine receptors (44), we suggest the involvement of the IP 3 receptor and a Ca 2ϩ -induced Ca 2ϩ release mechanism.
Several studies have revealed the presence of intracellular CB 1 receptors, including one showing functional CB 1 receptors in endolysosomes (reviewed in Ref. 45). Moreover, a recent study indicated that the intracellular pool of CB 1 receptors does not contribute to repopulation of membrane receptors (6), and thus, it may have a distinct function from the membrane CB 1 receptors.
Immunogold labeling studies have demonstrated the presence of CB 1 receptors intracellularly in axons of the basal ganglia (46) and locus coeruleus (47). Thus, if these intracellular receptors are active, they could play important roles in neuronal signaling. This is also supported by our novel finding that anandamide activates NAADP-dependent Ca 2ϩ pathways. NAADP has been implicated in important neuronal functions, such as neurosecretion (48,49), neurite outgrowth (42), neuronal differentiation (23), and membrane depolarization (50).
In sum, our findings indicate that intracellular CB 1 receptors are functional and that their activation mobilizes Ca 2ϩ from the endoplasmic reticulum and lysosomes; the proposed model is summarized in Fig. 9. We have also reported, for the first time, a role for NAADP in cannabinoid-induced Ca 2ϩ signaling.