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Article

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

by
Victor M. Pulgar
1,2,3,*,
Allyn C. Howlett
4 and
Khalil Eldeeb
4,5,6
1
Department of Pharmaceutical and Clinical Sciences, College of Pharmacy and Health Sciences, Campbell University, Buies Creek, NC 27506, USA
2
Biomedical Research and Infrastructure Center, Winston-Salem State University, Winston-Salem, NC 27101, USA
3
Department of Obstetrics & Gynecology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
4
Department of Physiology & Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA
5
Jerry M. Wallace School of Osteopathic Medicine, Campbell University, Buies Creek, NC 27506, USA
6
AL Azhar Faculty of Medicine, New Damietta 34518, Egypt
*
Author to whom correspondence should be addressed.
Cells 2022, 11(19), 2947; https://doi.org/10.3390/cells11192947
Submission received: 15 August 2022 / Revised: 13 September 2022 / Accepted: 16 September 2022 / Published: 21 September 2022

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 (ΔF340/380). 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 CB1/2R 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 Gαq 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, Gαq, and Gβγ, with activation of phospholipase C, production of IP3 through Gαq, 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 Gαi/o and modulated by Gαq [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 Gαs and Gαq, whereas activation by calcimimetics occurs via Gαi [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 CB1/2 cannabinoid receptors WIN55212-2 (5 μM) [6], the prototype bicyclic non-selective CB1/2R 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 (ΔF340/380), 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 (ΔF340/380 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 CB1/2R 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+ (ΔF340/380 0.11 ± 0.04 vs. 0.84 ± 0.12 p < 0.05) and by 350% over basal in 2.5 mM eCa2+ (ΔF340/380 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) (ΔF340/380 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) (ΔF340/380 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 (ΔF340/380 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 (ΔF340/380 CP 0.29 ± 0.08, Me-AEA 0.05 ± 0.01, p > 0.05) or at 2.5 mM eCa2+ (ΔF340/380 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) (ΔF340/380 MRS 0.33 ± 0.07, n = 4, 61% reduction at 0.25 mM; ΔF340/380 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) (ΔF340/380 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 (ΔF340/380 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 (ΔF340/380 O-1918 0.28 ± 0.07, n = 5, p < 0.05, 67% reduction at 0.25 mM; ΔF340/380 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 Gi/o proteins leading to inhibition of cAMP production were first demonstrated in N18TG2 neuroblastoma cells [38]. CB1R acting through Gαi could serve a modulatory role, as has also been proposed for Gαq signaling, in mediating [Ca2+]i increases in these cells [12]. The requirement for Gαq 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 Gαq, 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 Gαq or Gi/o βγ subunits, with these two effectors interacting with distinct regions of PLCs; Gαq binds to the C-terminal and Gβγ binds to the catalytic domain [39]. Gαq and Gi/o βγ can cooperate synergistically, increasing [Ca2+]i after GPCR activation [40]. Recently a role for Gi/o βγ subunits as modulators of Gαq activation of PLC, forming a Gαq-PLC-Gi/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-Gαq-dependent activation of PLC with the participation of CB1R-mediated Gi/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 Gαq-PLC-Gi/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 Gαq or Gα12/13 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 Gαq, 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 Gi/o, Gq/11, and Gs 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.

Author Contributions

Conceptualization, A.C.H., V.M.P. and K.E.; methodology, V.M.P. and K.E.; formal analysis, V.M.P., A.C.H. and K.E.; investigation, V.M.P. and K.E.; resources, V.M.P. and A.C.H.; data curation, V.M.P.; writing—original draft preparation, V.M.P.; writing—review and editing, V.M.P., A.C.H. and K.E.; funding acquisition, A.C.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by NIH grants R01-DA042157, K12-GM102773 and MD00232. This manuscript represents the authors’ results and interpretations and is not influenced by the NIH. The APC was funded by the Department of Pharmaceutical & Clinical Sciences, Campbell University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank Sandra Leone-Kabler for cell culture maintenance. The facilities of the Winston Salem State University Biomedical Research Infrastructure Center are greatly appreciated.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. WIN55212-2 increases [Ca2+]i: effect of eCa2+. (A). Time course of the changes in F340/380 in N18TG2 cells incubated in 0.25 and 2.5 mM eCa2+ in basal conditions (blue line) or the presence of WIN55212-2 5 µM (red line). (B). Relative changes in [Ca2+]i as ΔF340/380 in basal conditions (Basal, □, n = 6) or in the presence of WIN55212-2 5 µM (+WIN, ■, n = 7). * p < 0.05 vs. basal; # p < 0.05 vs. 0.25 mM eCa2+. (C). Fluorescence image from a representative coverslip with N18TG2 cells observed under the imaging system used (see section Imaging in Materials and Methods) in eCa2+ 0.25 mM. 40× amplification. (D). Same cells as in C after 2 min treatment (approximately at peak response) with WIN55212-2 5 µM in eCa2+ 0.25 mM, 40x amplification. Changes in pseudo color from green to red represent the increase in emission after excitation at 340 nm and decrease in emission after excitation at 380 nm of Fura-2 upon binding to Ca2+, the basis of the ratiometric (F340/380) system for determinations of relative changes in [Ca2+]i [17].
Figure 1. WIN55212-2 increases [Ca2+]i: effect of eCa2+. (A). Time course of the changes in F340/380 in N18TG2 cells incubated in 0.25 and 2.5 mM eCa2+ in basal conditions (blue line) or the presence of WIN55212-2 5 µM (red line). (B). Relative changes in [Ca2+]i as ΔF340/380 in basal conditions (Basal, □, n = 6) or in the presence of WIN55212-2 5 µM (+WIN, ■, n = 7). * p < 0.05 vs. basal; # p < 0.05 vs. 0.25 mM eCa2+. (C). Fluorescence image from a representative coverslip with N18TG2 cells observed under the imaging system used (see section Imaging in Materials and Methods) in eCa2+ 0.25 mM. 40× amplification. (D). Same cells as in C after 2 min treatment (approximately at peak response) with WIN55212-2 5 µM in eCa2+ 0.25 mM, 40x amplification. Changes in pseudo color from green to red represent the increase in emission after excitation at 340 nm and decrease in emission after excitation at 380 nm of Fura-2 upon binding to Ca2+, the basis of the ratiometric (F340/380) system for determinations of relative changes in [Ca2+]i [17].
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Figure 2. Increases in [Ca2+]i depend on the CaSR. (A). Effects of the CaSR inhibitor NPS2314 3 µM (+NPS, diagonal stripes upward bars, n = 7) on [Ca2+]i in basal conditions (open bars, n = 6). (B). Effects of the CaSR inhibitor NPS2314 3 µM (+NPS, diagonal stripes upward bars, n = 7) on [Ca2+]i in the presence of WIN55212-2 5 µM (black bars, n = 7). * p < 0.05 vs. basal or WIN55212-2 at the corresponding eCa2+.
Figure 2. Increases in [Ca2+]i depend on the CaSR. (A). Effects of the CaSR inhibitor NPS2314 3 µM (+NPS, diagonal stripes upward bars, n = 7) on [Ca2+]i in basal conditions (open bars, n = 6). (B). Effects of the CaSR inhibitor NPS2314 3 µM (+NPS, diagonal stripes upward bars, n = 7) on [Ca2+]i in the presence of WIN55212-2 5 µM (black bars, n = 7). * p < 0.05 vs. basal or WIN55212-2 at the corresponding eCa2+.
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Figure 3. Aminoalkylindole-specific increase in [Ca2+]i in N18TG2 cells. Increases in [Ca2+]i in conditions of low (0.25 mM) and high (2.5 mM) eCa2+ in the presence of WIN55212-2 5µM (+WIN, n = 7), the bicyclic mimetic of THC CP55940 5 µM (+CP, n = 4), or the arachidonoylethanolamine analog meth-anandamide 5 µM (+Me-AEA, n = 5) * p < 0.05 vs. basal; # p < 0.05 vs. WIN55212-2.
Figure 3. Aminoalkylindole-specific increase in [Ca2+]i in N18TG2 cells. Increases in [Ca2+]i in conditions of low (0.25 mM) and high (2.5 mM) eCa2+ in the presence of WIN55212-2 5µM (+WIN, n = 7), the bicyclic mimetic of THC CP55940 5 µM (+CP, n = 4), or the arachidonoylethanolamine analog meth-anandamide 5 µM (+Me-AEA, n = 5) * p < 0.05 vs. basal; # p < 0.05 vs. WIN55212-2.
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Figure 4. WIN55212-2-mediated increase in [Ca2+]i is dependent on store operated calcium entry. Increases in [Ca2+]i in low (0.25 mM) and high (2.5 mM) eCa2+ in basal conditions (Basal, n = 6), in the presence of WIN55212-2 (5 µM, n = 7) (+WIN), and in the presence of WIN55212-2 plus MRS1845 (+WIN + MRS, n = 4 or n = 7) (10 µM). * p < 0.05 vs. +WIN.
Figure 4. WIN55212-2-mediated increase in [Ca2+]i is dependent on store operated calcium entry. Increases in [Ca2+]i in low (0.25 mM) and high (2.5 mM) eCa2+ in basal conditions (Basal, n = 6), in the presence of WIN55212-2 (5 µM, n = 7) (+WIN), and in the presence of WIN55212-2 plus MRS1845 (+WIN + MRS, n = 4 or n = 7) (10 µM). * p < 0.05 vs. +WIN.
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Figure 5. WIN55212-2-mediated increase in [Ca2+]i is dependent on the CB1 receptor and on nonCB1/CB2 receptors. (A). Time course of the changes in F340/380 in N18TG2 cells incubated in 0.25 and 2.5 mM eCa2+ in the presence of WIN55212-2 5 µM (+WIN, red line), SR141716 1 µM (+WIN + SR, green line) and WIN55212-2 plus O-1918 10 µM (+WIN + O-1918, light blue line). (B). Increases in [Ca2+]i in low (0.25 mM) and high (2.5 mM) eCa2+ in basal conditions (Basal, open bars, n = 6), in the presence of WIN55212-2 (5 µM) (+WIN, n = 7), in the presence of WIN55212-2 plus SR141716 (1 µM) (+WIN + SR, n = 7), and in the presence of WIN55212-2 plus O-1918 (10 µM) (+WIN + O-1918, n = 5). * p < 0.05 vs. +WIN.
Figure 5. WIN55212-2-mediated increase in [Ca2+]i is dependent on the CB1 receptor and on nonCB1/CB2 receptors. (A). Time course of the changes in F340/380 in N18TG2 cells incubated in 0.25 and 2.5 mM eCa2+ in the presence of WIN55212-2 5 µM (+WIN, red line), SR141716 1 µM (+WIN + SR, green line) and WIN55212-2 plus O-1918 10 µM (+WIN + O-1918, light blue line). (B). Increases in [Ca2+]i in low (0.25 mM) and high (2.5 mM) eCa2+ in basal conditions (Basal, open bars, n = 6), in the presence of WIN55212-2 (5 µM) (+WIN, n = 7), in the presence of WIN55212-2 plus SR141716 (1 µM) (+WIN + SR, n = 7), and in the presence of WIN55212-2 plus O-1918 (10 µM) (+WIN + O-1918, n = 5). * p < 0.05 vs. +WIN.
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Figure 6. Diagram illustrating potential functional interactions between cannabinoid receptors and CaSR in neuroblastoma cells in different levels of eCa2+. The formation of different Gαq-PLC-Gi/o βγ complexes depending on eCa2+ and cellular proximity of the GPCRs involved is proposed. CB1: CB1 receptor. CBx: putative ‘WIN55212-2’ receptor. CaSR: calcium sensing receptor. SOC: store operated calcium channels.
Figure 6. Diagram illustrating potential functional interactions between cannabinoid receptors and CaSR in neuroblastoma cells in different levels of eCa2+. The formation of different Gαq-PLC-Gi/o βγ complexes depending on eCa2+ and cellular proximity of the GPCRs involved is proposed. CB1: CB1 receptor. CBx: putative ‘WIN55212-2’ receptor. CaSR: calcium sensing receptor. SOC: store operated calcium channels.
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Pulgar, V.M.; Howlett, A.C.; Eldeeb, K. WIN55212-2 Modulates Intracellular Calcium via CB1 Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells. Cells 2022, 11, 2947. https://doi.org/10.3390/cells11192947

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Pulgar VM, Howlett AC, Eldeeb K. WIN55212-2 Modulates Intracellular Calcium via CB1 Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells. Cells. 2022; 11(19):2947. https://doi.org/10.3390/cells11192947

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Pulgar, Victor M., Allyn C. Howlett, and Khalil Eldeeb. 2022. "WIN55212-2 Modulates Intracellular Calcium via CB1 Receptor-Dependent and Independent Mechanisms in Neuroblastoma Cells" Cells 11, no. 19: 2947. https://doi.org/10.3390/cells11192947

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