WIN55212-2 Modulates Intracellular Calcium via CB₁ Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells

Abstract

The CB₁ cannabinoid receptor (CB₁R) and extracellular calcium (eCa²⁺)-stimulated Calcium Sensing receptor (CaSR) can exert cellular signaling by modulating levels of intracellular calcium ([Ca²⁺]_i). We investigated the mechanisms involved in the ([Ca²⁺]_i) increase in N18TG2 neuroblastoma cells, which endogenously express both receptors. Changes in [Ca²⁺]_i were measured in cells exposed to 0.25 or 2.5 mM eCa²⁺ by a ratiometric method (Fura-2 fluorescence) and expressed as the difference between baseline and peak responses (ΔF_340/380). The increased ([Ca²⁺]_i) in cells exposed to 2.5 mM eCa²⁺ was blocked by the CaSR antagonist, NPS2143, this inhibition was abrogated upon stimulation with WIN55212-2. WIN55212-2 increased [Ca²⁺]_i at 0.25 and 2.5 mM eCa²⁺ by 700% and 350%, respectively, but this increase was not replicated by CP55940 or methyl-anandamide. The store-operated calcium entry (SOCE) blocker, MRS1845, attenuated the WIN55212-2-stimulated increase in [Ca²⁺]_i at both levels of eCa²⁺. Simultaneous perfusion with the CB₁ antagonist, SR141716 or NPS2143 decreased the response to WIN55212-2 at 0.25 mM but not 2.5 mM eCa²⁺. Co-perfusion with the non-CB₁/CB₂ antagonist O-1918 attenuated the WIN55212-2-stimulated [Ca²⁺]_i increase at both eCa²⁺ levels. These results are consistent with WIN55212-2-mediated intracellular Ca²⁺ mobilization from store-operated calcium channel-filled sources that could occur via either the CB₁R or an O-1918-sensitive non-CB₁R in coordination with the CaSR. Intracellular pathway crosstalk or signaling protein complexes may explain the observed effects.

1. Introduction

Phytocannabinoids, endocannabinoids, synthetic cannabinoids, and aminoalkylindole agonists regulate neuronal activity by activating the CB₁ receptor (CB₁R) to signal via Gi/o and other G proteins, but little is known about modulating intracellular calcium concentration ([Ca²⁺]_i). Although initial studies could not detect Ca²⁺ mobilization in cultured cell models, evidence indicates that the effects of cannabinoid receptors on [Ca²⁺]_i depend on the agonist and the cell type tested. In CHO cells, the synthetic cannabinoid HU210 and its non-CB₁-binding isomer HU211 (10 μM) both induced only a non-receptor-mediated increase in [Ca²⁺]_i in untransfected, CB₁R-expressing, or CB₂R-expressing cells, using a Fura-2 method that readily detected muscarinic receptor-mediated Ca²⁺ mobilization [1,2]. In the murine neuroblastoma cell line N18TG2 endogenously expressing CB₁R (but not CB₂R), treatment with the non-selective CB_1/2R agonists 2-arachidonoylglycerol (2-AG), CP55940, or WIN55212-2 (up to 1 μM) failed to evoke Ca²⁺ mobilization using Fluo-4 fluorescence able to detect a bradykinin-mediated response [3]. The Sugiura laboratory investigated Ca²⁺-deprived suspensions of HL60 monocytic cells endogenously expressing CB₂R or NG108-15 neuro-glioma hybrid cells endogenously expressing CB₁R. In this setting, Ca²⁺ mobilization was stimulated by 1 mM eCa²⁺ followed by 2-AG, CP55940, or WIN55212-2 (up to 10 μM) and detected with Fura-2 [4,5]. Subsequent studies showed that the aminoalkylindole WIN55212-2, but not Δ⁹-tetrahydrocannabinol (Δ⁹-THC), HU210, CP55940, 2-AG, or methanandamide, increased [Ca²⁺]_i in HEK293 cells exogenously expressing CB₁R [6]. The WIN55212-2-stimulated Ca²⁺ mobilization occurred in a CB₁-dependent manner requiring Gα_q activation and release of Ca²⁺ from thapsigargin-sensitive endoplasmic reticulum (ER) stores [6]. In addition, GPR55 and GPR18 receptors have been shown to modulate [Ca²⁺]_i in neurons and other cell types in response to lipid mediators, including atypical cannabinoids [7].

Mounting evidence supports intracellular Ca²⁺ as an important second messenger for excitable and non-excitable cells, with the inositol trisphosphate (IP₃)/Calcium signaling pathway playing a vital role in linking extracellular signals to [Ca²⁺]_i [8]. Thus, one of the receptor systems involved, the G protein-coupled calcium sensing receptor (CaSR) detects extracellular Ca²⁺ (eCa²⁺) concentration, linking it to intracellular signaling affecting cell function [9]. The CaSR can couple to more than one type of Gα subunit and influence the properties of Gβγ signaling [10]. CaSR actions have been reported to act through Gαi, Gα_q, and Gβγ, with activation of phospholipase C, production of IP₃ through Gα_q, and Ca²⁺ release from the ER, being one of the major effects of CaSR activation [11]. In N18TG2 neuronal cells, stimulation of CaSR with the positive allosteric modulator calindol increased [Ca²⁺]_i in a response dependent on Gα_i/o and modulated by Gα_q [12]. Modulating [Ca²⁺]_i also seems dependent on PKC activity and localization [13]. It appears that the CaSR intracellular pathways activated by eCa²⁺ proceed via Gα_s and Gα_q, whereas activation by calcimimetics occurs via Gα_i [9].

In the present study, we aimed to explore neuronal mechanisms involved in WIN55212-2-mediated Ca²⁺ mobilization as observed by Lauckner and colleagues [6], with a focus on the extracellular [Ca²⁺] influence associated with the Sugiura procedure [4,5]. We were particularly interested in the cannabinoid receptors mediating the WIN55212-2-dependent responses in [Ca²⁺]_i and the role of CaSR activation on those responses. [Ca²⁺]_i regulation has relevant physiological significance, for example, in muscle [14] and brain tissue [15], where a role for CB₁Rs has been demonstrated. Since the CaSR monitors the extracellular Ca²⁺ environment, our studies were performed in the N18TG2 neuroblastoma cell model that endogenously expresses both CB₁R and CaSR.

2. Materials and Methods

2.1. Cells

Mouse N18TG2 neuroblastoma cells were cultured as described [16], maintained in complete media containing Dulbecco’s Modified Eagle’s Medium (DMEM): Ham’s F-12 (1:1) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) and 10% heat-inactivated bovine serum. Cells were grown in 75-cm² flasks at 37 °C in a humidified atmosphere (5% CO₂), harvested at sub-confluency, and transferred to 12 mm glass coverslips (Fisher Scientific Co., Waltham, MA, USA). At 50–75% confluence, cells were loaded for 15 min with Fura-2 (5 µM) in Krebs–Henseleit Buffer (KHB) containing (in mM) NaCl 118, KCl 4.47, NaHCO₃ 25, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂·2H₂O 0.25, glucose 5.5. Cells were incubated in two different extracellular [Ca²⁺] (eCa²⁺) and the responses in intracellular [Ca²⁺] ([Ca²⁺]_i) were measured.

2.2. Imaging

Coverslips were transferred to an imagining chamber on an inverted Olympus BBX51WI microscope equipped with a 40× objective, a xenon arc lamp (Sutter Instruments, Novato, CA, USA), and a manual stage, and a cooled charge-couple device (CCD) camera (Hamamatsu Orka II). For ratiometric imaging, the microscope was computer-controlled by HCImage software (Hamamatsu Corporation, Middlesex, NJ, USA). Cells on the field were manually marked for analysis, and F₃₄₀ and F₃₈₀ were measured for one min. KHB containing 0.25 mM Ca²⁺ (Low eCa²⁺) was passed through the imaging chamber for 5 min after which eCa²⁺ was changed to 2.5 mM (High eCa²⁺) using a perfusion valve control system (VC-6, Six Channel Perfusion Valve Control Systems, Warner Instruments, Holliston, MA, USA) and cells perfused for additional 10 min. This procedure was repeated in the presence of cannabinoid receptor agonists (WIN55212-2, CP55940, or methanandamide) in KHB containing low or high eCa²⁺. Cells were pre-incubated for 15 min for the treatments with antagonists, and the corresponding antagonists were added to the low and high eCa²⁺ solutions. Only one treatment was carried out on each coverslip used.

2.3. Drugs

The ratiometric fluorescent dye Fura-2 was purchased from Molecular Probes (Eugene, OR, USA), dissolved in dimethylsulfoxide at 1 mM, and further diluted in KHB containing 0.25 mM Ca²⁺ to a working concentration of 5 μM as described [17]. The aminoalkylindole agonist of CB_1/2 cannabinoid receptors WIN55212-2 (5 μM) [6], the prototype bicyclic non-selective CB_1/2R agonist CP55940 (5 μM) [6], and the stable chiral analog of anandamide, a CB₁R partial agonist, methanandamide (5 μM) [6], were from Cayman Chemical Co, (Ann Arbor, MI, USA). Receptor antagonists used include the blocker of CaSR, NPS2314 (3 μM) [18], the selective store-operated calcium (SOC) channel inhibitor N-propargyl-nitrendipine (MRS1845, 10 μM) [19], the CB₁R antagonist SR141716 (1 μM) [6], and the non CB₁/CB₂ receptor blocker O-1918 (10 μM) [20], all from Cayman Chemical Co, (Ann Arbor, MI, USA). The cannabinoid compounds were stored at −20 °C as 10 mM stock solutions in ethanol. Immediately before use, an aliquot of drug stocks was air-dried and re-suspended in 0.25 mM Ca²⁺ KHB. All other chemical reagents were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

2.4. Data Analysis

Average changes in F₃₄₀ and F₃₈₀ were recorded continuously, and [Ca²⁺]_i responses were determined using the ratiometric method (ratio between F₃₄₀ and F₃₈₀) [17]. Imaging measurements were repeated 5 to 7 times with a total of 200 to 300 cells analyzed per each condition, and the background was subtracted automatically. Results were expressed as the difference between baseline and peak response (ΔF_340/380), with data expressed as mean ±SEM (n = 5–7). Statistical analyses were performed by One-Way Analysis of Variance (ANOVA) and Newman-Keuls multiple comparisons test for data obtained in 0.25 or 2.5 mM Ca²⁺ using GraphPad Prism v6 (GraphPad Software Inc, La Jolla, CA, USA). A p < 0.05 was accepted as an indication of statistical significance.

3. Results

3.1. WIN55212-2 Increased [Ca²⁺]_i in N18TG2 Cells at Both Low eCa²⁺ and during a High-eCa²⁺ Stimulus

After resting at 0.25 mM extracellular Ca²⁺, perfusion of N18TG2 cells with 0.25 mM eCa²⁺ increased [Ca²⁺]_i transiently by 11%. When eCa²⁺ was changed to 2.5 mM, [Ca²⁺]_i increased by 304% over basal (ΔF_340/380 0.11 ± 0.04 vs. 0.334 ± 0.06 p < 0.05, Figure 1A,B). We tested the Ca²⁺ mobilization response to the non-selective CB_1/2R aminoalkylindole agonist WIN55212-2 at concentrations that have previously been demonstrated to stimulate Ca²⁺ mobilization in HEK293 cells [6]. In the presence of WIN55212-2 (5 μM), [Ca²⁺]_i increased by 700% over basal in 0.25 mM eCa²⁺ (ΔF_340/380 0.11 ± 0.04 vs. 0.84 ± 0.12 p < 0.05) and by 350% over basal in 2.5 mM eCa²⁺ (ΔF_340/380 0.334 ± 0.06 vs. 1.29 ± 0.13 p < 0.05, Figure 1B–D).

3.2. CaSR Mediates Increases in eCa²⁺-Induced [Ca²⁺]_i in N18TG2 Cells

NPS2143 is a CaSR negative allosteric modulator that binds to the 7-transmembrane domain of the CaSR to inhibit the Ca²⁺ mobilization signaling pathway [21,22]. We used this calcilytic agent at concentrations previously shown to block increases in [Ca²⁺]_i promoted by activation of the Ca²⁺ receptor in HEK293 cells expressing the human Ca²⁺ receptor [18]. High eCa²⁺-induced [Ca²⁺]_i increase was effectively antagonized with NPS2143 (3 μM) (ΔF_340/380 0.36 ± 0.06 vs. 0.16 ± 0.02, p < 0.05, 56% of reduction, Figure 2A), demonstrating the functional activity of the CaSR evident at supra-physiological eCa²⁺. The WIN55212-2-induced elevations in [Ca²⁺]_i in low eCa²⁺ conditions were also attenuated by simultaneous perfusion with the NPS2143 (3 μM) (ΔF_340/380 0.84 ± 0.12 vs. 0.54 ± 0.05, p < 0.05, 36% of reduction, Figure 2B). Interestingly, in the presence of high eCa²⁺, the WIN55212-2-induced [Ca²⁺]_i increase was not inhibited by NPS2143 (ΔF_340/380 1.29 ± 0.13 vs. 1.25 ± 0.14, p > 0.05). These findings might suggest that the WIN55212-2 can influence [Ca²⁺]_i under “basal” CaSR conditions, but the WIN55212-2 stimulus was not influenced by NPS2143-inhibited CaSR. An alternative interpretation is that WIN55212-2 provided a mechanism to protect the CaSR from inhibition by the negative allosteric modulator.

3.3. Aminoalkylindole-Specific Potentiation of the eCa²⁺-Mediated Increase in [Ca²⁺]_i

To check the selectivity of WIN55212-2-induced increase in [Ca²⁺]_i in neuronal cells, the non-classical cannabinoid full agonist, CP55940, and endocannabinoid partial agonist, methanandamide (Me-AEA) were used at concentrations previously shown to inhibit cAMP accumulation [23]. Both compounds at 5 μM failed to significantly increase [Ca²⁺]_i relative to basal values at either 0.25 mM (ΔF_340/380 CP 0.29 ± 0.08, Me-AEA 0.05 ± 0.01, p > 0.05) or at 2.5 mM eCa²⁺ (ΔF_340/380 CP 0.44 ± 0.03, Me-AEA 0.75 ± 0.17, p > 0.05, Figure 3). The responses to either CP55940 or Me-AEA on [Ca²⁺]_i at both levels of eCa²⁺ were significantly lower than the response to WIN55212-2 (p < 0.05). These findings support the WIN55212-2 selectivity, implicating a non-CB₁ and non-CB₂ mechanism for this response.

3.4. WIN55212-2-Stimulated Increases in [Ca²⁺]_i Require Operational Store Operated Calcium Entry (SOCE)

Ca²⁺ mobilization by GPCR-mediated production of inositol triphosphate (IP₃) promotes Ca²⁺ release from ER stores, which requires continuous repletion via store operated Ca²⁺ entry (SOCE) mechanisms [24,25]. The most effective SOCE mechanism is based upon the ER [Ca²⁺] sensor stromal interacting molecule (STIM) and its association and activation of Ca²⁺ release-activated Ca²⁺ channels (CRAC) comprised of Orai1, Orai2, and Orai3 proteins. Cells also utilize non-selective cation channels as store-operated channels (SOCs), comprised of both Orai1 and transient receptor potential canonical channel 1 (TRPC1) channel subunits. Current reviews describe these processes in detail [25,26,27,28].

To evaluate the role of SOCE in WIN55212-2-stimulated increases in [Ca²⁺]_i, we employed the Orai1 inhibitor N-propargyl-nitrendipine (MRS1845), which has a reported IC₅₀ = 1.7 μM to block capacitative Ca²⁺ influx in HL60 cells [19], and also inhibits the ER Ca²⁺ replacement via TRPC1 at higher concentrations [29]. The WIN55212-2-stimulated increases in [Ca²⁺]_i, in both eCa²⁺ conditions (0.25 mM or 2.5 mM Ca²⁺) were attenuated by incubation with MRS1845 (10 μM) (ΔF_340/380 MRS 0.33 ± 0.07, n = 4, 61% reduction at 0.25 mM; ΔF_340/380 MRS 0.7 ± 0.14, n = 7, 46% reduction at 2.5 mM, Figure 4, p < 0.05). These results are consistent with a requirement for continuous refilling of the intracellular Ca²⁺ stores in the ER as the source of the mobilized Ca²⁺.

3.5. WIN55212-2-Dependent Increases in [Ca²⁺]_i Are Mediated by either CB₁R or a nonCB₁/CB₂ Receptor as a Function of the eCa²⁺ Stimulus

The N18TG2 neuronal cell expresses CB₁R but fails to express CB₂R [30,31,32], and thus, cellular signaling via cannabinoid receptors is expected to be inhibited by a CB₁R competitive antagonist/inverse agonist such as SR141716 in this model [33]. Several non-CB₁, non-CB₂ GPCRs have been promoted as “Cannabinoid Related” receptors based on their ability to be orthosterically stimulated/inhibited or allosterically modified by phytocannabinoid or endocannabinoid-like compounds (see [34,35,36] for review). The cannabinoid related GPCRs GPR18 and GPR55 both signal through Ca²⁺ mobilization, and both interact with endocannabinoid-like N-arachidonoylglycine and N-arachidonoylserine, phytocannabinoid CBD, and CBD analogs abnormal-cannabidiol (abn-CBD), O-1602 and O-1918 [37]. For this reason, we chose to test O-1918 for its potential as an inhibitor of Ca²⁺ mobilization in these studies, and we selected a concentration of O-1918 (10 μM) that has been shown to block cannabinoid-dependent effects that are independent of CB₁R or CB₂R [20]. The WIN55212-2-induced increase in [Ca²⁺]_i in 0.25 mM eCa²⁺ was partially blocked by simultaneous perfusion with the CB₁R antagonist SR141716 (1 μM) (ΔF_340/380 SR 0.45 ± 0.07, n = 6, p < 0.05, 46% reduction). Under conditions of 2.5 mM eCa²⁺, SR141716 does not affect the WIN55212-2-dependent increase in [Ca²⁺]_i (ΔF_340/380 SR 1.57 ± 0.11, n = 6, p > 0.05). Simultaneous perfusion with the nonCB₁/CB₂ receptor antagonist O-1918 (10 μM), attenuated WIN55212-2-promoted increases in [Ca²⁺]_i at both eCa²⁺ levels (ΔF_340/380 O-1918 0.28 ± 0.07, n = 5, p < 0.05, 67% reduction at 0.25 mM; ΔF_340/380 O-1918 0.72 ± 0.05, n = 5, p < 0.05, 44% reduction at 2.5 mM, Figure 5B). These findings implicate the role of the CB₁R in WIN55212-2-promoted [Ca²⁺]_i increases in the absence of a CaSR stimulus. On the other hand, a prominent influence of a nonCB₁/CB₂ stimulus appears under conditions of activation of CaSR by supra-physiological eCa²⁺.

4. Discussion

The aminoalkylindole WIN55212-2 can modulate [Ca²⁺]_i in neuroblastoma cells via at least two mechanisms. At low eCa²⁺, the WIN55212-2 induced potentiation of [Ca²⁺]_i partially depends on CB₁R defined by its sensitivity to inhibition by SR141716. At supra-physiologic eCa²⁺, which activates the CaSR, the effect of WIN55212-2 is CB₁R-independent. At both eCa²⁺ levels, the release of Ca²⁺ is from intracellular stores filled by store-operated Ca²⁺ channels.

We observed that the effect of WIN55212-2 on [Ca²⁺]_i depends on the eCa²⁺ level. At low eCa²⁺, WIN55212-2 increases [Ca²⁺]_i acting via CB₁R and nonCB₁/CB₂ receptors, probably acting on different intracellular transduction pathways. Actions of CB₁R through pertussis toxin-sensitive G_i/o proteins leading to inhibition of cAMP production were first demonstrated in N18TG2 neuroblastoma cells [38]. CB₁R acting through Gα_i could serve a modulatory role, as has also been proposed for Gα_q signaling, in mediating [Ca²⁺]_i increases in these cells [12]. The requirement for Gα_q on increasing [Ca²⁺]_i after CB₁R activation was demonstrated in HEK293 cells and hippocampal neurons [6].

Our results of an eCa²⁺-dependent elevation in [Ca²⁺]_i confirm a role for CaSR in the modulation of [Ca²⁺]_i in N18TG2 neuroblastoma cells, as previously demonstrated [12]. The effects of WIN55212-2 we report at low and high eCa²⁺ were attenuated by SOCE blockade, suggesting that WIN55212-2 promotes the release of Ca²⁺ from intracellular stores. The main transduction pathway associated with this particular increase in [Ca²⁺]_i is described as dependent on Gα_q, phospholipase C (PLC) activation, synthesis of diacylglycerol (DAG), and inositol triphosphate (IP₃) with further activation of ER IP₃ receptors promoting Ca²⁺ release [11]. PLC can be activated by either Gα_q or G_i/o βγ subunits, with these two effectors interacting with distinct regions of PLCs; Gα_q binds to the C-terminal and Gβγ binds to the catalytic domain [39]. Gα_q and G_i/o βγ can cooperate synergistically, increasing [Ca²⁺]_i after GPCR activation [40]. Recently a role for G_i/o βγ subunits as modulators of Gα_q activation of PLC, forming a Gα_q-PLC-G_i/o βγ complex, and depending on the affinity for the plasma membrane of the γ subunits has been proposed [41]. Regarding potential interactions between CaSR- and cannabinoid-dependent pathways modulating [Ca²⁺]_i, it is conceivable that WIN55212–2-dependent CB₁R activation increases [Ca²⁺]_i by augmenting CaSR-Gα_q-dependent activation of PLC with the participation of CB₁R-mediated G_i/o βγ release in neuroblastoma cells.

Two conundrums remain. One is that only the WIN55212-2 but no other cannabinoid or endocannabinoid agonist family representatives could stimulate the Ca²⁺ mobilization. The other is that we observed a role for CaSR in WIN55212-2/CB₁R-dependent increases in [Ca²⁺]_i at low eCa²⁺ and not at high eCa²⁺. These results suggest the possibility of different Gα_q-PLC-G_i/o βγ complexes being formed depending on eCa²⁺ and the cellular proximity of the receptors and the G proteins to which they are pre-coupled (Figure 6). For example, our earlier studies demonstrated that WIN55212-2 behaves as an agonist for all three Gi subtypes, whereas the THC analog desacetyllevonantradol behaves as an agonist for Gi₁ and Gi₂ but an inverse agonist for Gi₃; and methanandamide behaves as an agonist at Gi₃ but an inverse agonist for Gi₁ and Gi₂ [42].

In the current study, we also showed a role for nonCB₁/CB₂ receptors in controlling [Ca²⁺]_i. Among the candidates for these receptors is GPR55, which can be activated by CB₁R antagonist/inverse agonist AM251 (but not SR141716), and the lysophospholipid, lysophosphatidylinositol (LPI). GPR55 utilizes Gα_q or Gα_12/13 for signal transduction [43] and can promote Ca²⁺ mobilization and mitogen-activated protein kinase (MAPK) phosphorylation [44]. GPR55 modulates neurotransmitter release through modulation of neuronal [Ca²⁺]_i [45]. Activation of GPR55 in dorsal root ganglia neurons by various cannabinoids, including Δ⁹-THC and methanandamide, increases [Ca²⁺]_i through a mechanism involving Gα_q, PLC, and IP₃ receptors [7].

Our results with O-1918, could implicate a role for GPR55. However, since WIN55212-2 fails to activate [Ca²⁺]_i by GPR55 [7] the effects of O-1918 on WIN55212-2-dependent responses we observed may be explained by the CB₁-induced activation of phospholipase A and synthesis of LPI, which in turn would activate GPR55. This possibility has been suggested to explain the observed actions of CB₁R antagonists/inverse agonists on GPR55-mediated actions [46].

Another possibility for the nonCB₁/CB₂ receptor is GPR18, which originally was characterized by its activation by abnormal cannabidiol (abn-CBD) and inhibition by CBD and O-1918, but now de-orphanized as a GPCR activated by the endogenous anandamide metabolite, N-arachidonoyl-glycine (see review for original references [34,37]). In exogenous expression models, GPR18 responds to N-arachidoyl-glycine by Ca²⁺ mobilization [47]. GPR18 also responds to the inflammation pro-resolving polyunsaturated, hydroxylated 22-C lipid, resolvin D2 (RvD2). In monocytes, macrophages, microglia, and BV2 microglia, and polymorphonuclear neutrophils, GPR18 couples to G_i/o, G_q/11, and G_s to signal by increasing cAMP and protein kinase A (PKA), and phosphorylation of signal transducer and activator of transcription 3 (STAT3) (for review and original references, see [37,48]). However, WIN55212-2 has not been demonstrated to elicit any signaling responses by GPR18 (see summary tables and text for original references [37,48]).

A final possibility is that WIN55212-2 might activate a putative Alkyl Indole receptor as described for microglia and astrocyte cellular signaling in response to WIN55212-2 and analogs [49,50]. Such receptors may be those identified as [³H]WIN55212-2 binding sites in neuroblastoma-glioma hybrid NG108-15 cells [51]. The potential for a specific WIN55212-2 “receptor” in brain membranes was suggested by studies of the C57Bl/6 CB₁ knock-out mouse which showed that anandamide and WIN55212-2 could promote G protein activation ([³⁵S]GTPγS binding) [52]. The properties of a putative Alkyl Indole receptor have yet to be fully characterized.

In summary, the aminoalkylindole WIN55212-2 can modulate [Ca²⁺]_i in N18TG2 cells via CB₁R-dependent and independent mechanisms. We observed functional interactions between the CB₁R and CaSR activation in regulating [Ca²⁺]_i, and these interactions are dependent on eCa²⁺ with the participation of nonCB₁/CB₂ receptors in neuroblastoma cells. Future studies should address the effects of LPI, abn-CBD, CBD and RvD2 to determine the role of GPR55 and GPR18 on Ca²⁺ mobilization influenced by the CaSR. Additionally, studies should address whether aminoalkylindole analogs that act on the putative WIN55212-2 Alkyl Indole “receptor” are involved in the regulation of [Ca²⁺]_i by CaSR. Details regarding the mechanism by which the CaSR interfaces with Class A GPCRs to regulate intracellular calcium stores could be analyzed using thapsigargin-dependent control of [Ca²⁺]_i [6] and other current methods of structural and functional analysis.