Heterogeneity of cannabinoid ligand-induced modulations in intracellular Ca²⁺ signals of mouse pancreatic acinar cells in vitro

Abstract

We recently reported that a CB₂R agonist, GW405833 (GW), reduced both the ACh-induced Ca²⁺ oscillations and the L-arginine-induced Ca²⁺ signal enhancement in mouse pancreatic acinar cells, suggesting that GW-induced inhibition may prevent the pathogenesis of acute pancreatitis. In this study, we aim to evaluate the effects of other cannabinoid ligands on Ca²⁺ signaling in acinar cells. Patch-clamp whole-cell recordings were applied to measure ACh-induced intracellular Ca²⁺ oscillations in pancreatic acinar cells acutely dissociated from wild-type (WT), CB₁R knockout (KO), and CB₂R KO mice, and the pharmacological effects of various cannabinoid ligands on the Ca²⁺ oscillations were examined. We found that all the 8 CB₂R agonists tested inhibited ACh-induced Ca²⁺ oscillations. Among them, GW, JWH133, and GP1a caused potent inhibition with IC₅₀ values of 5.0, 6.7, and 1.2 μmol/L, respectively. In CB₂R KO mice or in the presence of a CB₂R antagonist (AM630), the inhibitory effects of these 3 CB₂R agonists were abolished, suggesting that they acted through the CB₂Rs. The CB₁R agonist ACEA also induced inhibition of Ca²⁺ oscillations that existed in CB₁R KO mice and in the presence of a CB₁R antagonist (AM251), suggesting a non-CB₁R effect. In WT, CB₁R KO, and CB₂R KO mice, a nonselective CBR agonist, WIN55,212-2, inhibited Ca²⁺ oscillations, which was not mediated by CB₁Rs or CB₂Rs. The endogenous cannabinoid substance, 2-arachidonoylglycerol (2-AG), did not show an inhibitory effect on Ca²⁺ oscillations. In conclusion, CB₂R agonists play critical roles in modulating Ca²⁺ signals in mouse pancreatic acinar cells, while other cannabinoid ligands modulate Ca²⁺ oscillations in a heterogeneous manner through a CB receptor or non-CB-receptor mechanism.

Introduction

Cannabis is a common cash crop that can be used for textiles, raw materials, and food. It is also a herbal medicine whose extract can be used as an analgesic. It is considered a drug and is regulated in many countries. At least 4% of adults in the world use cannabis each year, making it one of the most commonly used illicit drugs in the world.

Cannabinoid receptors are divided into two categories, named cannabinoid type 1 receptor (CB₁R) and cannabinoid type 2 receptor (CB₂R), which are both G protein-coupled receptors [1]. The traditional view holds that CB₁R is mainly expressed in central neurons and in small amounts in peripheral tissues and cells, while CB₂R is mainly expressed in peripheral immune cells and the hematopoietic system.

In pancreatic tissues, both CB₁R and CB₂R messenger RNAs (mRNA) are expressed, suggesting that cannabinoid receptors are involved in the regulation of pancreatic function [2, 3]. Additionally, a recent study showed that in animal models of pancreatitis, CB₂R mRNA and protein levels are changed in pancreatic acinar cells and CB₂R agonists can reduce the pathological changes in acinar cells [2, 3]. In acutely dissociated mouse pancreatic acinar cells, we reported that the selective CB₂R agonist GW405833 (GW) inhibited both the acetylcholine (ACh)-induced Ca²⁺ oscillations and the L-arginine-induced elevation of intracellular Ca²⁺ oscillations, indicating that CB₂R plays important roles in regulating the physiology and pathophysiology of pancreatic acinar cells [4].

Many synthetic and endocannabinoid substances show a variety of pharmacological effects. Accumulating lines of evidence suggest that the cannabinoid ligands modulate CB signaling in a complex way, including through diverse targets, signaling pathways, and heterogeneous effects, and through CB receptor (CB₁R and CB₂R) and non-CB receptor mechanisms. However, the pharmacological effects of various cannabinoid ligands on intracellular Ca²⁺ signaling in pancreatic acinar cells have not been well studied. The aim of this study was to detect the pharmacological effects of various cannabinoid ligands, including eight commercially available CB₂R agonists, one CB₁R agonist (arachidonyl-2'-chloroethylamide (ACEA)), a nonspecific CB receptor agonist (WIN55,212-2), and an endocannabinoid substance (2-arachidonoylglycerol (2-AG)), on the ACh-induced Ca²⁺ oscillations in acutely isolated pancreatic acinar cells from wild-type (WT), CB₁R knockout (KO), and CB₂R KO mice. The Ca²⁺ oscillations were determined by measuring the Ca²⁺-activated Cl⁻ currents using patch-clamp whole-cell recordings.

Materials and methods

All experimental protocols were approved by and performed in accordance with the guidelines set by the animal care and use and ethical committees at the Barrow Neurological Institute and the First Affiliated Hospital of Zhengzhou University (Zhengzhou, He-nan, China).

Mouse pancreatic acinar cell preparation

The mice used for this study were adult (4–6 months old) male CD₁ mice (Charles River Laboratories International, Inc., Wilmington, MA, USA). Additionally, C57BL/6J WT, CB₁R KO [5], and CB₂R KO mice [6] of similar ages and gender on a C57BL/6J genetic background were used to determine the targets of cannabinoid ligands. Pancreatic cells were acutely isolated as previously described [7,8,9]. In brief, pancreatic glands were taken from isoflurane-anesthetized mice, and fragments of the pancreatic tissue were minced and digested using collagenase (200 U/mL, 25–30 min, 37 °C; Wako Pure Chemicals, Osaka, Japan) in the presence of 1 mM Ca²⁺. After collagenase digestion, the cell suspension was gently pipetted to obtain further separation of the cells and then washed with physiological saline. A 100 μL volume of cell suspension was then poured into extracellular solution in a 2 mL experimental bath solution. The isolated cells usually adhered to the bottom within 15–20 min and were used for recording within 3 h after preparation. All experiments were performed at room temperature (22 ± 1 °C).

Conventional patch-clamp whole-cell recordings

Conventional patch-clamp whole-cell recording was used to record the Ca²⁺-activated Cl⁻ currents for monitoring intracellular Ca²⁺ oscillations, as reported previously [8, 9]. Recording pipettes were made from borosilicate glass capillaries. They had a resistance of 3–5 MΩ when filled with pipette solution. After a GΩ seal was established between the cell membrane and the pipette, a whole-cell configuration was achieved by brief negative suction. Transmembrane currents were recorded with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Sunnyvale, CA, USA) at a holding potential (V_H) of −30 mV. In this study, the series resistance was not compensated.

Solution and chemicals

The standard extracellular solution contained the following (in mM): 140 NaCl, 1.0 CaCl₂, 4.7 KCl, 1.13 MgCl₂, 10 glucose, and 10 HEPES, adjusted to pH 7.2 with NaOH. The pipette solution contained the following (in mM): 140 KCl, 1.13 MgCl₂, 5 Na₂ATP, 0.24 EGTA, 10 glucose, and 10 HEPES, pH 7.2. The drugs used in this study were GW, GP1a, JWH133, SER601, CB65, JWH015, Hu308, L759656 ACEA, WIN55,212-2, and 2-AG (Fig. 1), which were purchased from Tocris Bioscience (Minneapolis, MN, USA). ACh was purchased from Sigma-Aldrich (St Louis, MO, USA). A stream of the standard extracellular solution was continuously perfused over the cell during recording. A computer-controlled U-tube system was used for drug application [10]. For intracellular drug application, the drug was added to the pipette solution, and the establishment of a whole-cell configuration allowed the drug to diffuse into the recorded cell.

Fig. 1

Chemical structures of 11 tested cannabinoid ligands

Statistical analysis

For patch-clamp experiments, the Ca²⁺-activated Cl⁻ current responses in dissociated mouse pancreatic acinar cells (Fig. 2) were presented as the current charge (current area· Cm⁻¹ min⁻¹), and then the drug-induced changes were compared to the baseline level of charge (induced by ACh). All data are presented as the mean ± SEM. When data were obtained from the same recorded cell and the changes in ACh response were compared before and after drug exposure, paired Student’s t-test was used. To analyze the effects of CB ligands on different groups of cells (eg, WT, CB₁R, and CB₂R KO mice), one-way analysis of variance (ANOVA) with Tukey’s post hoc test was used.

Fig. 2

a Photograph of freshly dissociated mouse pancreatic acinar cells. Typical acinar cells have a kidney shape (indicated by red arrow). b Representative trace of ACh-induced Ca²⁺ oscillations by measuring Ca²⁺-dependent Cl⁻ current oscillations in patch-clamp whole-cell recording

Results

Effects of CB₂R agonists GW, JWH133, GP1a on ACh-induced Ca²⁺ oscillations

To determine the effects of CB₂R agonists on intracellular Ca²⁺ signals, we examined the effect of three CB₂R agonists with similar chemical structures (Fig. 1) on ACh-induced Ca²⁺ oscillations. The results showed that the three tested CB₂R agonists (at 10 μM concentration), GW, JWH133, and GP1a, showed marked inhibition. As shown in Fig. 3a–c, 10 μM GW, JWH133, or GP1a significantly inhibited the 10 nM ACh-induced Ca²⁺ oscillations. Statistical analysis showed that after exposure of WT mice to GW, JWH133, and GP1a, the normalized net charges of 10 nM ACh-induced Ca²⁺ oscillations were reduced to 23% ± 4.4% (P < 0.001, n = 7), 26% ± 4.2% (P < 0.001, n = 9), and 4.6% ± 4.2% (P < 0.001, n = 6), respectively (Fig. 3d). In CB₂R KO mice (Fig. 3e) or in the presence of the CB₂R antagonist AM630 (Fig. 3g), the 10 μΜ GW-, JWH133-, and GP1a-induced inhibition of 30 nM ACh-induced Ca²⁺ oscillations was abolished. The concentration–inhibition relationship curves for these three agonists were measured, showing half-maximal inhibitory concentration (IC₅₀) values of 5.0, 6.7, and 1.2 µM for GW-, JWH133-, and GP1a-induced inhibition, respectively (Fig. 3f). These results indicate that the CB₂R agonists GW, GP1a, and JWH133 potently inhibit the intracellular Ca²⁺ signal through CB₂Rs of mouse pancreatic acinar cells.

Fig. 3

Effects of the CB₂R agonists GW, JWH133, and GP1a on the ACh-induced Ca²⁺ oscillations. a–c Representative traces of the effects of 10 μM GW (a), JWH133 (b) and GP1a (c) on 10 nM ACh-induced Ca²⁺ oscillations in pancreatic acinar cells. d The bar graph summarizes the effects of three CB₂R agonists on the ACh-induced Ca²⁺ oscillations. ***P < 0.001. e Similar experiments were done as in (d) but in CB₂R-KO mice and showed that the above three CB₂R agonists did not affect the ACh-induced Ca²⁺ oscillations in CB₂R-KO mice. f Concentration–inhibition relationship curves of GW, JWH133, and GP1a. g The effects of a CB₂R antagonist (0.1 μM AM630) on the GW-, JWH133-, and GP1a-induced inhibition of the ACh-induced Ca²⁺ oscillations. In this and all following figures, the vertical column represents an averaged mean of total charge (current area/Cm per 2 min), and the vertical bars indicate the standard error

Effects of CB₂R agonists SER601, CB65, Hu308, L759656, and JWH015 on ACh-induced Ca²⁺ oscillations

We tested five other CB₂R agonists, SER601, CB65, Hu308, L759656, and JWH015, that possess different chemical structures (Fig. 1). The results showed a mild reduction in Ca²⁺ oscillations for all tested agonists (Fig. 4a–e). Statistical analysis (Fig. 4f) showed that after exposure to 10 μM SER601, CB65, Hu308, L759656, and JWH015, the normalized net charges of 10 nM ACh-induced Ca²⁺ oscillations were reduced (F_30, 6 = 9.59, P < 0.0001). Tukey’s post hoc comparison between baseline and each CB₂R agonist tested showed the effects of SER601 (88.1% ± 5.1%, P < 0.001), CB65 (85.9% ± 5.5%, P < 0.01), Hu308 (88.0% ± 4.0%, P < 0.001), L759656 (96.4% ± 2.09%, P > 0.05), and 89.0% ± 6.4%, P < 0.01). These results demonstrate that four of the five tested CB₂R agonists showed lower affinity/potency of inhibition of intracellular Ca²⁺ signaling in pancreatic acinar cells, while L759656 did not show a significant effect.

Fig. 4

CB₂R agonists SER601, CB65, Hu308, L759656, and JWH015 show weak inhibition of ACh-induced Ca²⁺ oscillations. a–e Representative traces of the effects of 10 μM SER601 (a), CB65 (b), Hu308 (c), L759656 (d), and JWH015 (e) on 10 nM ACh-induced Ca²⁺ oscillations in pancreatic acinar cells of WT mice. f Statistical analysis shows that these five CB₂R agonists with different chemical structures mildly inhibit the ACh-induced Ca²⁺ oscillations. **P < 0.01, ***P < 0.001

Effect of the CB₁R agonist on ACh-induced Ca²⁺ oscillations

It has been reported that both CB₁R and CB₂R are expressed in mouse pancreatic tissue [3], but in our acute dissociated mouse pancreatic acinar cells, the CB₁R mRNA was not detected [4]. Here, we tested the effect of CB₁R agonists on ACh-induced Ca²⁺ oscillations. As shown in Fig. 5a, the CB₁R agonist ACEA (10 μM) reversibly reduced 30 nM ACh-induced Ca²⁺ oscillations. Considering that ACEA was solubilized with ethanol (10 μM ACEA = 7.3 mM ethanol), we examined the effect of 7.3 mM ethanol on ACh-induced Ca²⁺ oscillations and found a similar inhibitory effect on 30 nM ACh-induced Ca²⁺ oscillations (Fig. 5b). Furthermore, we examined the inhibitory effect of ACEA on the ACh-induced Ca²⁺ oscillations in the presence of the CB₁R antagonist AM251 (Fig. 5c) or in the pancreatic acinar cells isolated from either CB₁R KO (Fig. 5d) or CB₂R KO (Fig. 5e) mice and found a similar inhibition. One-way ANOVA showed that there was no significant difference in ACEA-induced inhibition among the five tested groups (P > 0.05, Fig. 5f). These results suggest that the inhibitory effect of ACEA on the ACh-induced Ca²⁺ oscillations is not mediated through either CB₁R or CB₂R.

Fig. 5

Effects of CB₁R agonist (ACEA, 10 μΜ) on ACh-induced Ca²⁺ oscillations. a A typical case of 10 μM ACEA (dissolved by 7.3 mM ethanol)-induced reduction in ACh-induced Ca²⁺ oscillations in WT mice. b A typical trace of the effects of 7.3 mM ethanol (as a control) on the ACh-induced Ca²⁺ oscillations. c A typical trace of the effects of the CB₁R antagonist AM251 (1 μM) on 10 μM ACEA-induced inhibition of the ACh-induced Ca²⁺ oscillations. d A typical trace of the effects of ACEA on the ACh-induced Ca²⁺ oscillations in CB₁R KO mice. e A typical trace of the effects of ACEA on the ACh-induced Ca²⁺ oscillations in CB₂R KO mice. f Statistical analysis shows similar levels of inhibition of the ACh-induced Ca²⁺ oscillations in all tested groups. One-way ANOVA shows no significant difference among the effects of these CB₂R agonists

Effect of the nonselective cannabinoid receptor agonist WIN55,212-2 on the ACh-induced Ca²⁺ oscillations

To further clarify the regulatory role of cannabinoid CB₁ and CB₂ receptors, we tested the effect of a classic synthetic nonselective CBR agonist, WIN55,212-2, since this compound has been widely used to test the effects of the cannabinoid. The results showed that WIN55,212-2 inhibited the ACh-induced Ca²⁺ oscillations in a concentration-dependent manner, with an IC₅₀ value of 1.1 µM (Fig. 6a–d). Statistical analysis (Fig. 6d) showed that the normalized net charge was reduced to 7% ± 3.5% (P < 0.001, n = 5) on 30 nM ACh-induced Ca²⁺ oscillations after the addition of 10 μM WIN55,212-2. Then, we compared the effects of WIN55,212-2 (10 μM) on 30 nM ACh-induced Ca²⁺ oscillations in WT, CB₁R, and CB₂R KO mice. The results showed that WIN55,212-2 (10 μM) inhibited the 30 nM ACh-induced Ca²⁺ oscillations in all WT (Fig. 6e), CB₁R KO (Fig. 6f), and CB₂R KO mice (Fig. 6g). Statistical analysis showed that the rates of inhibition (the currents of 1-drug/baseline) by WIN55,212-2 (10 μM) of the normalized net charge of the ACh-induced Ca²⁺ oscillations were 92.5% ± 3.5% (P < 0.001, n = 6), 92.5% ± 3.1% (P < 0.001, n = 6), and 90.7% ± 4.6% (P < 0.001, n = 8), respectively, after addition of 10 μM WIN to pancreatic acinar cells isolated from WT, CB₁R KO and CB₂R KO mice (Fig. 6h). These results suggest that the inhibition of Ca²⁺ oscillations by WIN55,212-2 is not mediated by CB₁R or CB₂R.

Fig. 6

Effect of a nonselective CBR agonist WIN55,212-2 (10 μΜ) on 30 nM ACh-induced Ca²⁺ oscillations. a–c Typical traces of the WIN55,212-2-induced inhibition of the ACh-induced Ca²⁺ oscillations in a concentration-dependent manner in WT mice, which formed a sigmoidal concentration–inhibition relationship curve (d). In the acinar cells prepared from WT (e), CB₁R KO (f), or CB₂R KO (g) mice, WIN55,212-2 reduced the ACh-induced Ca²⁺ oscillations. h Statistical analysis (one-way ANOVA) indicates that there are no significant differences among the inhibitory effects of WIN55,212-2 on the Ca²⁺ oscillations of the acinar cells prepared from WT, CB₁R KO, and CB₂R KO mice

Effect of the endocannabinoid substance 2-AG on ACh-induced Ca²⁺ oscillations

Lastly, we examined the effects of an endogenous cannabis-like substance, 2-AG, on 30 nM ACh-induced Ca²⁺ oscillations in pancreatic acinar cells dissociated from WT mice. As shown in Fig. 7, 2-AG at either 10 μM (Fig. 7a) or 100 μM (Fig. 7b) failed to affect the ACh-induced Ca²⁺ oscillations (Fig. 7c).

Fig. 7

Effect of the endocannabinoid substance 2-AG on 30 nM ACh-induced Ca²⁺ oscillations. a A typical trace of the effects of 2-AG (10 μM) on 30 nM ACh-induced Ca²⁺ oscillations. b A typical trace of the effects of 2-AG (100 μM) on 30 nM ACh-induced Ca²⁺ oscillations. c The bar graph summarizes the effects of 2-AG on Ca²⁺ oscillations

Discussion

In this study, we have systematically examined the pharmacological effects of cannabinoid ligands on ACh-induced Ca²⁺ oscillations in single pancreatic acinar cells acutely dissociated from WT, CB₁R, and CB₂R KO mice. We found heterogeneous effects of different cannabinoid ligands on Ca²⁺ oscillations through the different targets. First, all tested CB₂R ligands showed inhibitory effects with different efficiencies. Three higher-efficiency agonists, GW, JWH133, and GP1a, showed remarkable inhibition of the ACh-induced Ca²⁺ oscillations. Based on the IC₅₀ values of the concentration–inhibition curves of these three agonists, the order of affinity was GP1a > GW > JWH133 for inhibiting the ACh-induced Ca²⁺ oscillations. The inhibitory effects of these CB₂R ligands were eliminated in the acinar cells dissociated from CB₂R KO mice or in the presence of a CB₂R antagonist (AM630), which suggests that the inhibition is mediated through the CB₂Rs. Then, a selective CB₁R agonist, ACEA, also showed inhibition of the ACh-induced Ca²⁺ oscillations. However, this inhibition was still maintained in the presence of CB₁R antagonists (AM251) or in the acinar cells dissociated from either CB₁R KO or CB₂R KO mice. This suggests that ACEA inhibits ACh-induced Ca²⁺ oscillations through a non-CBR mechanism. Since ACEA was dissolved in ethanol, we tested the effects of 7.3 mM (equal to 10 µM ACEA) ethanol and found a similar inhibition. Furthermore, a commonly used, nonselective CBR ligand, WIN55,212-2, showed significant inhibition of the ACh-induced Ca²⁺ oscillations, with an IC₅₀ value of 1.1 µM. Unexpectedly, the WIN55,212-2-induced inhibition still occurred in the acinar cells dissociated from either CB₁R KO or CB₂R KO mice, suggesting that WIN55,212-2-induced inhibition of ACh-induced Ca²⁺ oscillations is not mediated by CB₁R or CB₂R. Finally, an endogenous cannabis-like substance, 2-AG, did not alter the ACh-induced Ca²⁺ oscillations, even at a concentration of 100 µM. Collectively, we have profiled the heterogeneous nature of the pharmacological properties of different CBR ligands in the intracellular Ca²⁺ signals of mouse pancreatic acinar cells. Our results show that cannabinoid ligands modulate cellular Ca²⁺ signals through heterogeneous pathways, in which CB₂R ligands inhibit the ACh-induced Ca²⁺ oscillations by targeting CB₂Rs, while other ligands, such as ACEA and WIN55,212-2, modulate Ca²⁺ oscillations that are not mediated through the CBRs. This study provides new insights into cannabinoid modulations of the Ca²⁺ signal in mouse pancreatic acinar cells and suggests that carefully selected CB₂R agonists should be considered as novel therapeutic compounds for treating acute pancreatitis.

CB₂R is the major target to mediate the cannabinoid modulations in the ACh-induced Ca²⁺ oscillations in mouse pancreatic acinar cells

Cannabinoid receptors are divided into two subtypes, CB₁R and CB₂R. CB₂R has been called the “peripheral” cannabinoid receptor because it is mainly expressed in peripheral tissues, including immune cells, cardiomyocytes, gastrointestinal cells, and hepatocytes [11,12,13,14,15,16,17]. In pancreatic acinar cells, CB₂R protein expression has been found in rodents using immunohistochemical staining and Western blotting [3, 18]. In mouse pancreatic tissues, CB₁R and CB₂R mRNAs have been identified using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining [3]. Functionally, we examined the roles of CB₂Rs in the modulation of the ACh-induced Ca²⁺ oscillation in mouse pancreatic acinar cells, a well-established cell model for the study of intracellular Ca²⁺ signals [19]. Using patch-clamp whole-cell recording to measure Ca²⁺-activated Cl⁻currents, we found that all eight commercially available CB₂R agonists showed significant inhibition of Ca²⁺ oscillations. Three of them, GW, JWH133, and GP1a, showed dramatic inhibition, and this inhibition was mediated through CB₂R. These results support previous reports that CB₂R agonists play critical roles in the prevention of acute pancreatitis pathology [3] and the reduction of intracellular Ca²⁺ signals [18].

Effects of CB₁R agonists on the ACh-induced Ca²⁺ oscillations in mouse pancreatic acinar cells

Whether mouse pancreatic acinar cells express CB₁Rs is unknown. Both CB₁R and CB₂R mRNA and proteins have been detected in pancreatic tissue [3, 18]. Using real-time RT-PCR, our recent work detected CB₂R but not CB₁R mRNA in freshly isolated mouse pancreatic acinar cells [4]. Therefore, in this study, we examined the effects of a CB₁R agonist on the ACh-induced Ca²⁺ oscillations in isolated single mouse pancreatic acinar cells. Interestingly, we found that a CB₁R selective agonist, ACEA, also inhibited the ACh-induced Ca²⁺ oscillations. To determine whether the effect of ACEA was mediated by CB₁Rs, we tested the effect of a CB₁R antagonist (AM251, 10 µM) on the ACEA-induced inhibition and found that AM251 failed to prevent ACEA's effect. Moreover, we tested the effects of ACEA on the Ca²⁺ oscillations in the acinar cells dissociated from CB₁R KO and WT mice and found similar inhibition by ACEA in these two groups. These results suggest that the ACEA-induced inhibition of ACh-induced Ca²⁺ oscillations is not mediated by CB₁R. ACEA also inhibited Ca²⁺ oscillations in CB₂R KO mice, suggesting that the CB₂R is also not the target for the ACEA. Considering that ACEA was dissolved in ethanol (10 µM ACEA contained 7.3 mM ethanol), we further examined the effects of 7.3 mM ethanol and found a similar inhibition on the ACh-induced Ca²⁺ oscillations, suggesting that ACEA-induced inhibition was likely a solvent effect. Taken together, our data demonstrate that the ACEA-induced inhibition of cellular Ca²⁺ oscillations is achieved through a non-CBR mechanism. This functional study supports our previous work, in which we failed to detect CB₁R mRNA expression in mouse pancreatic acinar cells [4]. The reason for the differences between previous reports and ours may be the use of different preparations. Previous reports that tested CB₁R and CB₂R mRNA expression used pancreatic tissue [3], while we used dissociated pancreatic acinar cells [4]. In pancreatic tissue, there are other types of cells, such as beta-cells, which have been reported to express both CB₁R and CB₂R [3, 18].

Effects of nonspecific CBR ligands on the ACh-induced Ca²⁺ oscillations

In the cannabinoid signaling field, many studies have used nonspecific CBR ligands such as WIN55,212-2 and endocannabinoid substances such as 2-AG. These ligands nonselectively activate both CB₁Rs and CB₂Rs. Thus, we tested the effects of WIN55,212-2 and 2-AG on the ACh-induced Ca²⁺ oscillations. Interestingly, we found that WIN55,212-2 inhibited Ca²⁺ oscillations in a non-CBR way (Fig. 6), while 2-AG did not show a significant effect on the Ca²⁺ oscillations, even at a high concentration (Fig. 7b, c). Although the precise mechanisms of these diverse effects by cannabinoid ligands on the Ca²⁺ oscillations in mouse pancreatic acinar cells are still unclear, our findings suggest the heterogeneous and complex ways that cannabinoid ligands modulate intracellular Ca²⁺ signals. Emerging evidence shows that there are complex relationships between cannabinoid receptors and intracellular Ca²⁺ signals in different types of cells. For example, the activation of CB₁R or CB₂R increases intracellular Ca²⁺ levels in endothelial cells, submandibular acinar cells, canine kidney cells, and bladder cancer cells [20,21,22,23], while the activation of CB₁R or CB₂R reduces glucose-induced intracellular Ca²⁺ oscillations and insulin release in pancreatic beta-cells [24, 25].

Clinical significance of CB₂R agonists in the modulation of Ca²⁺ signals in pancreatic acinar cells

Acute pancreatitis is an inflammatory disease with complex pathogenesis requiring immediate medical treatment [26, 27]. In clinical practice, there are still no effective drugs for the specific treatment of acute pancreatitis [26]. Pancreatic acinar cells are exocrine pancreatic units that synthesize, store, and secrete inactive digestive enzymes into the lumen of acinar cells. The activity of pancreatic acinar cells is strictly regulated by the secretagogues ACh and cholecystokinin (CCK), both of which act on specific membrane receptors (muscarinic and CCK receptors, respectively) and then increase the Ca²⁺ levels in the cytoplasm. If the intracellular Ca²⁺ has been maintained at a high concentration, the intracellular signal transduction is disrupted, harming the cells and resulting in acute pancreatitis. Recent studies have shown that the early pathological changes in acute pancreatitis are caused by an abnormal increase in Ca²⁺ levels in acinar cells. The sustained increase in intracellular Ca²⁺ levels can trigger digestive enzymes, resulting in inflammation and necrosis. Drugs that block the accumulation of calcium store-operated Ca²⁺ channels will prevent the sustained increase in intracellular Ca²⁺ levels and protease activation [28]. In an animal model of acute pancreatitis, activation of CB₂Rs (but not CB₁Rs) in pancreatic acinar cells can prevent pathological changes caused by cerulein or L-arginine [3]. In our previous study, we reported that the CB₂R agonist GW reduced ACh-induced Ca²⁺ oscillations, eliminated L-arginine-induced enhancement of Ca²⁺ oscillations, and prevented the L-arginine-induced increases in serum amylase and lung myeloperoxidase levels in acute pancreatitis [4]. These results suggest that CB₂R agonists rather than CB₁R agonists may be potential novel therapeutic agents for the prevention and treatment of acute pancreatitis.

In conclusion, our study profiles the pharmacological effects of cannabinoid ligands on the intracellular Ca²⁺ signals in mouse pancreatic acinar cells. It confirms that modulation by CBR ligands can be accomplished through CB₂R as well as through non-CBR pathways. In consideration of the heterogeneity of the regulatory process, we should be cautious when selecting CBR ligands as potential drugs for the treatment of acute pancreatitis.