Published online before print March 5, 2007
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,1
* Research and Innovation (R&I) Company, Padova, Italy;
Department of Pharmaceutical Sciences, University of Padova, Padova, Italy; and
San Camillo Hospital, IRCCS, Venezia-Lido, Italy
1 Correspondence: Research and Innovation (R&I) Company, Via Svizzera 16, 35127 Padova, Italy. E-mail: albertaleon{at}researchinnovation.com
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9-tetrahydrocannabinol (THC), is opposite to that inhibitory, exerted by the synthetic cannabinoids WIN 55,212-2 and CP 55,940. Moreover, the vanilloid capsaicin, a full agonist of transient receptor potential channel VR1, did not affect [Ca2+]ilevels in the RBL-2H3 cells, thus excluding the involvement of this receptor in the CBD-mediated effects. Together, these results support existence of yet-to-be identified sites of interaction, i.e., receptors and/or ion channels associated with Ca2+ influx of natural cannabinoids such as CBD and THC, the identification of which has the potential to provide for novel strategies and agents of therapeutic interest.
Key Words: ß-hexosaminidase • calcium • ion channels • 9-tetrahydrocannabinol • CB receptors • VR1 receptors • TRP channels
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the main psychoactive and nonpsychoactive components of cannabis, respectively, possess myriad pharmacological properties, many of which are mimicked by their synthetic (e.g., CP 55,940) as well as endogenous counterparts, the endocannabinoids (e.g., arachidonylethanolamide, also known as anandamide). However, although much evidence points to a role of known cannabinoid receptors, namely CB1 and CB2 [1
, 2
], in determining some of the effects produced by most cannabinoids/endocannabinoids, CBD binds with low affinity to these receptors [3
, 4
]. Yet, CBD exerts, in analogy to THC and other CB receptor agonists, immunosuppressive and anti-inflammatory properties, the combination of which has, for example, been shown to culminate in potent, antiarthritic effects in experimental models of chronic joint inflammation [5
]. CBD also reduces edema and hyperalgesia in a rat paw model of carrageenan-induced inflammation as well as serum TNF-
levels in LPS-treated mice [6
, 7
]. Studies directly investigating the effects of CBD on specific immune cell populations include suppression of NO production and cytokine release in macrophages, modulation of cytokine release in PBMC and suppression of chemokine production in a human B cell line [8
9
10
11
]. Notwithstanding this, the mode(s) of action of CBD are still largely obscure. Although mechanisms related with the endocannabinoid system have been proposed in, at least, the antihyperalgesic actions of CBD, other evidence provides for alternative mechanisms by which CBD can induce immunomodulatory and anti-inflammatory effects [3
, 6
, 7
, 12
, 13
], the further elucidation of which has the potential to open new avenues in the pharmacology of cannabinoids. Mast cells, traditionally associated with immediate hypersensitivity and allergic reactions, play significant roles in the defense against bacteria and parasites as well as in the pathogenesis of a variety of disorders (e.g., autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, congestive heart failure, and pain, among others) [14 15 16 ]. These cells, widely distributed throughout connective and mucosal tissues, often adjacent to blood and lymphatic vessels and in close contact to nerves, can in fact be activated by a variety of stimuli to secrete and produce a battery of mediators with proinflammatory and immunoregulatory potential. However, although activated mast cells may, by virtue of their location and mediator expression, be pivotal in the genesis, magnitude, or duration of an inflammatory response in many tissues, the balance of engaging inhibitory and activatory cell-surface receptors on mast cells determines whether the cell becomes active upon a challenge. Moreover, complex molecular, intracellular networks control the extent, persistence, or both of the secretory response, and genetic and environmental factors control their mediator content as well as other aspects of their phenotype. Therefore, elucidating how mast cell function may be modulated has the potential to provide insight into how mast cells might be manipulated to achieve therapeutic ends.
Given the anti-inflammatory properties of CBD together with the potential, although controversial, of cannabinoids/endocannabinoids to modulate mast cell activation [17
18
19
20
21
22
], we questioned here, for the first time, whether CBD affects mast cell function. To this end, we used RBL-2H3 cell cultures, a rat cell line of basophilic-leukemia origin, displaying characteristics of mucosal mast cells, used extensively to study signaling pathways, leading to degranulation and release of inflammatory mediators upon antigen-induced aggregation of the IgE-bound, high-affinity receptors (Fc
RIs) expressed on their surface [23
24
25
]. It is surprising that our results show that exposure of the RBL-2H3 cells to CBD results in their activation. This effect, similar to that exerted by THC, is opposite to that inhibitory of the synthetic CB receptor agonists WIN 55,212-2 and CP 55,940.
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RBL-2H3 cells and immunologic activation
RBL-2H3 cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown in 75 cm2 culturing flasks at 37°C in a humidified atmosphere of 5% CO2, and the medium was refreshed three times a week.
RBL-2H3 were activated by the IgE receptor cross-linking; in brief, RBL-2H3 cells were seeded into 96-well plates at a density of 105 cells per well in 100 µl culture medium in the presence of 0.5 µg/ml antidinitrophenol (anti-DNP) IgE (Sigma Chemical Co., Clone SPE-7) and incubated overnight at 37°C in a humidified atmosphere of 5% CO2 in air. The adherent cells were washed in PBS and incubated with DNP-human serum albumin (HSA) antigen (HSA conjugated to DNP at 0.1 µg/ml final concentration) or vehicle (PBS) in DMEM phenol red-free containing 1 mg/ml BSA and 2 mg/ml glucose. Cannabinoid receptor agonists, with the exception of THC, and antagonists were dissolved in DMSO at a final concentration of 0.1% and added to culture cells 5 min before antigen (DNP-HSA) stimulation. Controls were at all times exposed to the same solvent concentration. After 1 h, activation was measured by assessing the release of ß-hexosaminidase in the culture medium (see below).
Measurement of ß-hexosaminidase release
ß-Hexosaminidase activity was assayed according to the method described by Smith et al. [26
]. Briefly, 50 µl cell supernatant was added to 50 µl substrate (1.4 mM p-nitrophenyl N-acetyl-d-glucosaminide in 0.2 M citrate buffer, pH 4.5) in a 96-well plate and incubated for 3 h at 37°C. The reaction was stopped by addition of 100 µl Tris buffer, pH 9, and the absorbance was read at 405 nm on a Spectra Count plate reader (Packard, Downers Grove, IL, USA). Release of ß-hexosaminidase is presented as optical absorbance of the ß-hexosaminidase-converted product per 50 µl cell culture medium.
RT-PCR
Total RNA was isolated from RBL-2H3 cell cultures by using Trizol (Life Technologies, Gaithersburg, MD, USA), according to the manufacturer’s instruction. First-strand cDNA was prepared by RT of 1 µg total RNA by using oligo(dT) to prime Moloney murine leukemia virus RT (Promega, Madison, WI, USA). The RT-PCR was performed on identical amounts of cDNA for each sample. Primers used were designed from sequences in the GeneBank database using Primer Express software (Perkin-Elmer, Boston, MA, USA). Primer sequences were the following: rat CB1 sense primer: 5'-GGC ATC TCT TTC TCA GTC AC-3', and antisense primer: 5'-ATC AGG TAG GTC TCG TCA AT-3' (890 bp product); and rat CB2 sense primer: 5'-CAA CGA CTA CCT CCT GGG C-3', and antisense: 5'-TCA GCA GTT GGA GCA GCC-3' (523 bp product). For PCR amplification of specific cDNAs, the 50-µl reactions contained the following reagents: 0.1 mM each deoxy (d)CTP, dGTP, dATP, and dTTP, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 9.0), 0.1% Triton X-100, 500 nM each primer, 1 U Taq polymerase, and 40 ng of the cDNA synthesized in the RT reaction. The number of cycles and reaction temperature conditions were optimized to provide a linear relationship between the amount of input template and the amount of PCR product. Amplification programs were the following: 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for CB2 and 35 cycles of 94°C for 1 min, 55°C for 2 min, and 74°C for 1 min for CB1. PCR products were separated by electrophoresis through a 1.4% agarose gel and stained with ethidium bromide. An image of the gel was captured digitally by using PhotoCapt 99.01 software (Bioprofil, Germany). The identities of the amplicones were confirmed with sequencing.
Measurements of intracellular calcium ([Ca2+]i) by flow cytometry
RBL-2H3 cells (106 cells/ml) were loaded with 5 µM Fluo-3/AM in DMEM phenol red-free containing 1.8 mM Ca2+ plus 1 mg/ml BSA and 2 mg/ml glucose for 1 h at room temperature. Subsequently, cells were washed twice in PBS and resuspended (106 cells/ml) in DMEM phenol red-free containing 1.8 mM Ca2+ plus 1 mg/ml BSA and 2 mg/ml glucose, and 500 µl was used for the analysis. Fluo-3 fluorescence was monitored using a FACScan (Becton Dickinson, San Jose, CA, USA) flow cytometer and CellQuest software. A baseline value was obtained for each sample by fluorescence measurement for 10 s before addition of the compounds. Cannabinoids were added to cells 5 min before antigen (DNP-HSA) stimulation. Fluo-3 fluorescence was expressed in arbitrary fluorescence intensity units and plotted as fluorescence-1 versus time. Mean fluorescence intensity (MFI) was expressed as the percent of increase in fluorescence with respect to baseline fluorescence.
Statistical analysis
Results were represented as mean ± SEM. Significance of difference between groups was assessed by nonparametric ANOVA (Kruskal-Wallis test) followed by post-hoc, multiple comparisons test (Dunn).
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Figure 1. Effect of cannabinoids on ß-hexosaminidase release in RBL-2H3 cells, which were sensitized with anti-DNP-IgE (0.5 µg/ml) for 18–20 h and then incubated with vehicle (DMSO, 0.1%) or increasing concentrations of CBD (A) or (+)WIN 55,212-2 [WIN(+)] and (–)WIN 55,212-3 [WIN(–); B] or CP 55,940 (CP) and JWH-133 (JWH; C), with (IgE-DNP) or without (basal) antigen (DNP-HSA, 0.1 µg/ml). Results are expressed as mean ± SEM of values from five to 10 independent experiments, each one conducted in triplicate. °, P < 0.05; °°, P < 0.01; and °°°, P < 0.001, with respect to basal release, and *, P < 0.05; **, P < 0.01; and ***, P < 0.001, with respect to IgE-DNP stimulation.
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Figure 2. Effect of CBD on synthetic cannabinoid modulation of ß-hexosaminidase release. RBL-2H3 cells sensitized with anti-DNP-IgE (0.5 µg/ml) for 18–20 h were pretreated with increasing concentrations of CBD (1–10 µM) or vehicle (DMSO, 0.1%) for 5 min, followed by increasing concentrations of (+)WIN 55,212-2 (A) or CP 55,940 (B) in the presence of antigen (DNP-HSA, 0.1 µg/ml). Results, expressed as percent of IgE-DNP or IgE-DNP-CBD-induced release, are represented as mean ± SEM of values from three independent experiments conducted in triplicate. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, with respect to vehicle-treated.
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RI-induced serotonin release in RBL-2H3 cells [19
, 20
]. Rather, it appears that CB2 ligation may, given the ability of JWH-133 (10 µM) to facilitate mediator release from the IgE-DNP-stimulated RBL-2H3 cells (Fig. 1C)
, result in opposite effects from those induced by CB1 ligation.
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Figure 3. RT-PCR analysis of CB1 and CB2 expression in RBL-2H3 cells. Total RNA extracted from RBL-2H3 cells was retro-transcribed, and cDNA was amplified with specific primers for CB1 and CB2 receptors. Gel image is representative of three independent experiments (A). Effect of cannabinoid receptor antagonists on cannabinoid modulation of ß-hexosaminidase release. RBL-2H3 cells sensitized with anti-DNP-IgE (0.5 µg/ml) for 18–20 h were pretreated with the CB1 receptor antagonist AM281 (0.1 µM) or the CB2 receptor antagonist AM630 (1 µM) or vehicle (vh; DMSO, 0.1%) for 5 min, followed by 10 µM CBD or vehicle (DMSO, 0.1%) in the presence (IgE-DNP) or absence (basal) of antigen (DNP-HSA, 0.1 µg/ml; B). Results are represented as mean ± SEM of values from three to five independent experiments conducted in triplicate. °°, P < 0.01, with respect to CBD-treated; *, P < 0.05; **, P < 0.01; and ***, P < 0.001, with respect to vehicle-treated. RBL-2H3 cells sensitized with anti-DNP-IgE (0.5 µg/ml) for 18–20 h were pretreated with AM281 (0.1 µM) or AM630 (1 µM) for 5 min, followed by addition of 10 µM (+)WIN 55,212-2 (WIN) or vehicle (DMSO, 0.1%) in the presence of antigen (DNP-HSA, 0.1 µg/ml; C). Results are represented as mean ± SEM of values from three to five independent experiments conducted in triplicate. **, P < 0.01, and ***, P < 0.001, with respect to vehicle-treated, and °, P < 0.05, with respect to (+)WIN 55,212-2-treated.
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i/o-coupled CB1 and CB2 receptor isoforms found in the RBL-2H3 cells [20
], thus raising speculation of a contribution of an endogenous ligand in the ability of AM630 to affect ß-hexosaminidase release from the antigen-stimulated cells in the presence or absence of CBD (Fig. 3B)
.
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Figure 4. Effect of PTX on cannabinoid modulation of ß-hexosaminidase release. RBL-2H3 cells were sensitized with anti-DNP-IgE (0.5 µg/ml) in the presence or absence of PTX (100 ng/ml) for 18–20 h, followed by addition of increasing concentrations of CBD (A), (+)WIN 55,212-2 (B), or CP 55,940 (C) or vehicle (DMSO, 0.1%) in the presence (IgE-DNP) or absence (basal) of antigen (DNP-HSA, 0.1 µg/ml). Results are represented as mean ± SEM of values from at least three independent experiments conducted in triplicate. °°, P < 0.01, and °°°, P < 0.001, with respect to basal release, and *, P < 0.05; **, P < 0.01; and ***, P < 0.001, with respect to the IgE-DNP-stimulated cells. ns, Not significant.
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Figure 5. Effect of CBD on [Ca2+]i. RBL-2H3 cells, loaded with Fluo-3/AM (5 µM) and resuspended in phenol red-free DMEM, were incubated with increasing concentrations of CBD (1–10 µM, A) or with CBD (10 µM) in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 2 mM, C). RBL-2H3 cells, sensitized with anti-DNP-IgE for 18–20 h, followed by loading with Fluo-3/AM (5 µM), were incubated with antigen (DNP-HSA, 0.1 µg/ml) or vehicle (PBS) in the presence or absence of CBD (10 µM, B). Fluo-3 fluorescence intensity was monitored in a time-resolved mode by flow cytometry. Results, expressed as percent of Fluo-3 MFI variation with respect to baseline fluorescence intensity of unstimulated cells, are representative of at least three independent experiments.
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Figure 6. Effect of synthetic cannabinoids on [Ca2+]i in the presence or absence of extracellular Ca2+. RBL-2H3 cells, loaded with 5 µM Fluo-3/AM, were resuspended in phenol red-free DMEM, with (B, D) or without (A, C) BAPTA (2 mM). RBL-2H3 cells, sensitized with anti-DNP-IgE and loaded with Fluo-3/AM, were incubated with 10 µM (+)WIN 55,212-2 (A, B) or 10 µM CP 55,940 (C, D) and subsequently stimulated with (IgE-DNP) or without antigen (DNP-HSA, 0.1 µg/ml). Fluo-3 fluorescence intensity was monitored, in a time-resolved mode, by flow cytometry. Results, expressed as percent of Fluo-3 MFI variation with respect to baseline fluorescence intensity of unstimulated cells, are representative of at least three independent experiments.
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Figure 7. Effect of CPS on [Ca2+]i. RBL-2H3 cells loaded with Fluo-3/AM (5 µM) and resuspended in phenol red-free DMEM were incubated with increasing concentrations of CPS (0.1–10 µM, A). Effect of cation channel blockers on the CBD-evoked elevation of [Ca2+]i in RBL-2H3 cells, which when loaded with 5 µM Fluo-3/AM, were treated with 5 µM CBD alone or in the presence of increasing concentrations (3–30 µM) of nitrendipine (Nitr.; B) or clotrimazole (Clotr.; C). Results, expressed as percent of Fluo-3 MFI variation with respect to baseline fluorescence intensity of unstimulated cells, are representative of at least three independent experiments.
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Effects of THC on RBL-2H3 cell activation
In a final series of experiments, we also evaluated, for comparative purposes, whether THC, the major natural psychoactive component of marijuana, affects [Ca2+]i in RBL-2H3 cells in basal or antigen-stimulated conditions. THC has been reported to induce histamine secretion from rat peritoneal mast cells in vitro via CB receptor-independent interactions [21
]. Moreover, THC elevates [Ca2+]i levels in resting T cells in a PTX-insensitive manner and exerts, unlike other cannabinoids, analgesic effects also via non-CB1/CB2 receptor-related mechanisms involving extracellular Ca2+ influx, independent of VR1 receptors [36
, 37
]. Our results, reported in Figure 8A
, show that THC (5–20 µM) induced, similarly to CBD, a concentration-dependent rise of [Ca2+]i in RBL-2H3 cells, an effect antagonized by clotrimazole (30 µM, Fig. 8B
). Moreover, THC (15 µM) caused PTX-insensitive release of ß-hexosaminidase in the culture medium (Table 1
).
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Figure 8. Effect of THC on [Ca2+]i. RBL-2H3 cells loaded with Fluo-3/AM (5 µM) and resuspended in phenol red-free DMEM were incubated with increasing concentrations of THC (5–20 µM, A). Effect of clotrimazole on THC-9-evoked elevation of [Ca2+]i in RBL-2H3 cells, which when loaded with 5 µM Fluo-3/AM, were treated with 20 µM THC alone or in the presence of clotrimazole (30 µM, B). Results, expressed as percent of Fluo-3 MFI variation with respect to baseline fluorescence intensity of unstimulated cells, are representative of at least three independent experiments.
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Table 1. Effect of PTX on 9-THC-Mediated ß-hexosaminidase Release in RBL-2H3 Cells
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RI immunoreceptor stimulation via a mechanism that is not mediated by Gi/Go protein-coupled receptors but rather, is associated with a robust rise in [Ca2+]i levels. Indeed, unlike CBD, CP 55,940 and (+)WIN 55,212-2, but not the inactive stereoisomer (–)WIN 55,212-3 and the selective CB2 agonist JWH-133 [38
], inhibited ß-hexosaminidase release induced by the IgE receptor FcRI cross-linking. Whereas the inhibitory effect of (+)WIN 55,212-2 was counteracted partially by the CB1 but not the CB2 receptor antagonists/inverse agonists (AM281 and AM630, respectively) as well as PTX-sensitive, the activating effect of CBD was neither sensitive to CB1/2 receptor antagonism nor prevented but rather, potentiated by pretreatment with PTX. In addition, unlike CBD, (+)WIN 55,212-2 and CP 55,940 did not affect basal [Ca2+]i but rather, inhibited antigen-induced [Ca2+]i rise, also in the absence of extracellular Ca2+. All this not only confirms previous results but also most importantly, excludes direct or indirect involvement of known CB and other receptors coupled to Gi/o proteins, herein including the abnormal CBD receptor, in the CBD-induced activation of the RBL-2H3 cells [13
, 22
, 25
, 39
].
Recently, by using resting human and murine T cells, it has been postulated that only cannabinoids, which possess a THC-like, three-ring structure, are able to increase [Ca2+]irobustly in a CB receptor-independent manner [40
]. Although our and other findings [41
] with CBD, a bicyclic cannabinoid, do not support such a hypothesis, we found that THC induces, like CBD, PTX-insensitive ß-hexosamidase release from the RBL-2H3 cells, an effect likely consequent to the elevation of [Ca2+]i. THC is, at difference with (+)WIN 55,212-2 and CP 55,940, a low-affinity, partial agonist for CB1 receptors and unlike (+) WIN55,212-2, largely ineffective in coupling CB1 receptors in a conformation that enables Gq signaling [4
, 42
, 43
]. Moreover, THC has, in analogy to our results, been reported to induce in vitro histamine secretion from rat peritoneal mast cells via mechanisms independent of CB1/CB2 receptors and importantly, solely, partly sensitive to PTX [21
]. These and other findings, showing that CB1 and CB2 receptor isoforms are Gi/o coupled in RBL-2H3 cells, exclude a mechanism of THC action, which involves CB receptors [20
]. Rather, it may be postulated that the capability of THC, like CBD, to activate the RBL-2H3 cells may be mediated by specific non-CB receptors. Moreover, the structural difference between the two molecules raises the question as to whether the same or different sites are implicated.
Ca2+ mobilization from intracellular stores and Ca2+ entry from the extracellular space are key features of antigen-induced degranulation of RBL-2H3 cells [44
]. Here, although FcRI clustering was, upon removal or chelation of extracellular Ca2+, found to induce a transient increase in [Ca2+]i, likely reflecting Ca2+ release from intracellular stores, the CBD-induced [Ca2+]i rise was abolished completely. Moreover, pretreatment with SKF96365, a cation channel blocker commonly used to block voltage-independent Ca2+ channels, abolished the CBD-induced [Ca2+]i rise completely (data not shown). These findings, suggesting that the Ca2+ source is mainly attributable to extracellular Ca2+ influx, raise the possibility that ligand-gated or receptor-operated Ca2+ influx channels are implicated, the most promising molecular candidates of which are represented by the TRP gene superfamily of Ca2+-permeable cation channels [45
]. However, although CBD has been described to be capable of interacting with the TRPV1 channel (also termed VR1 receptors), the vanilloid CPS, a full agonist of TRPV1 receptors, did not affect [Ca2+]i in the RBL-2H3 cells [3
, 33
]. Also THC, although able to increase [Ca2+]i in the RBL-2H3 cells, does not induce Ca2+ transients in cells expressing VR1 receptors [37
]. Together, this excludes the possibility that TRPV1 channels mediate the activation of the RBL-2H3 cells induced by CBD or THC. Nevertheless, as TRP channels other than VR1, e.g., ANKTM1 (TRPA1) and TRPC1, have been implicated in CB1/CB2/VR1 receptor-independent effects of THC and cannabinol, another nonpsychoactive component of marijuana, it would not be surprising if a member of the TRP ion channel family other than TRPV1, directly or indirectly via receptor-operated mechanisms, is implicated [46
47
48
49
50
51
52
].
Conversely, irrespective of the Ca2+ influx channels implicated, flow of ions, such as K+, plays an important role in the activation responses of RBL-2H3 cells, as they regulate cell membrane potential and thus serve to sustain the Ca2+ influx necessary for degranulation [53 54 55 56 ]. Among these, RBL-2H3 cells display, upon antigen stimulation, an outwardly rectifying, Ca2+-dependent K+ efflux pathway, recently proposed to involve conductances of the intermediate conductance Ca2+-activated K+ channel (IKCa)-type, the opening of which is expected to increase the electrical driving force for Ca2+ entry [55 , 56 ]. Here, we found that the CBD- and THC-evoked, intracellular Ca2+ rise was inhibited dose-dependently by the cation channel blockers clotrimazole (10–30 µM) and nitrendipine (10–30 µM). It is intriguing that clotrimazole is also a relatively selective blocker of IKCa channels, an effect mimicked by nitrendipine at the concentrations used [57 58 59 ]. Although this raises the possibility of an action of these receptors on IKCa channels, clotrimazole had, in spite of its ability to markedly reduce the [Ca2+]i rise induced by CBD, no effect on the CBD-induced ß-hexosaminidase release from the RBL-2H3 cells (data not shown). Moreover, clotrimazole did not inhibit antigen-mediated secretion of the RBL-2H3 cells, a result in line with previous reports [55 ]. Also, 1-ethyl-2-benzimidazolinone, a specific IKCa opener, reportedly did not elicit histamine release from mast cells on its own, suggesting that IKCa opening alone is insufficient for degranulation [60 , 61 ]. Thus, although it may be speculated that the concerted action between Ca2+ influx channels and IKCa channels on the cell surface may allow the RBL-2H3 cells to sustain elevated [Ca2+]i in response to CBD, other signaling pathways are likely necessary for the effects of CBD on their mediator release. However, given the promiscuous capability of clotrimazole to block several types of channels, further studies are warranted to confirm and expand these speculations.
Whereas activation of mast cells has, at least traditionally, been implicated in the genesis and/or amplification of inflammatory reactions, most studies show that CBD as well as THC exert, in contrast to our in vitro results, anti-inflammatory and immunosuppressive proprieties in vivo [5 ]. Although difficult to reconcile, it is noteworthy that mast cells are tissue-dwelling cells, heterogeneous in phenotype and function, the activation of which results from the transient displacement of a physiological balance between positive and negative signals delivered by activating and inhibitory receptors and as such, profoundly affected by the microenvironment. Moreover, CBD activates, likely via TRPV1-mediated mechanisms, CPS-sensitive nerve fibers in inflamed tissues, and THC causes CB1/CB2-independent release of a calcitonin gene-related peptide from CPS-sensitive nerves in isolated rat and mouse mesenteric arterial rings, even from TRPV1 knockout animals [35 ]. Given the known role of these nociceptors in not only in hyperalgesia but also in development and progression of inflammation, it cannot be excluded that their activity-induced desensitization by CBD and THC may contribute to their anti-inflammatory action in vivo. Other, although not exclusive, possibilities include effects on endocannabinoid production or degradation, as well as effects on adenosine signaling in vivo [3 , 7 , 41 ].
In sum, results from our study lend support to existence of yet-to-be-identified sites, i.e., receptors and/or ion channels, of interaction of natural cannabinoids, such as CBD and THC. Such sites are, at least in RBL-2H3 mast cells, coupled, directly or indirectly, with Ca2+ influx channels, the opening of which triggers and/or participates in their activation. Although future studies are necessary to identify and address the site(s) implicated, our results not only have the potential of opening new avenues in the pharmacology of plant-derived cannabinoids but also raise the possibility that mast cell activation may contribute to the adverse bronchopulmonary consequences of chronic marijuana smoking.
Received December 20, 2006; revised January 26, 2007; accepted February 5, 2007.
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and suppress interleukin-1 in vitro Int. J. Immunopharmacol. 13,1091-1097[Medline]9 Tetrahydrocannabinol and cannabidiol alter cytokine production by human immune cells Immunopharmacology 40,179-185[CrossRef][Medline] RI signaling Immunol. Today 13,195-197[CrossRef][Medline] S binding in the mouse brain Eur. J. Pharmacol. 321,R1-R3[CrossRef][Medline] activity and Ca2+ mobilization in antigen-stimulated mast cells J. Leukoc. Biol. 65,321-329[Abstract](9)-tetrahydrocannabinol in resting T cells J. Leukoc. Biol. 75,884-892 9-Tetrahydrocannabinol and cannabinol activate capsaicin-sensitive sensory nerves via a CB1 and CB2 cannabinoid receptor-independent mechanism J. Neurosci. 22,4720-47279 tetrahydrocannabinol in T cells involves TRPC1 channels J. Leukoc. Biol. 79,202-213This article has been cited by other articles:
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