Originally published online as doi:10.1189/jlb.0505274 on October 21, 2005

Published online before print October 21, 2005

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(Journal of Leukocyte Biology. 2006;79:202-213.)
© 2006 by Society for Leukocyte Biology

Induction of intracellular calcium elevation by ⁹-tetrahydrocannabinol in T cells involves TRPC1 channels

Gautham K. Rao and Norbert E. Kaminski¹

Department of Pharmacology & Toxicology, Center for Integrative Toxicology, and National Food Safety & Toxicology Center, Michigan State University, East Lansing

¹ Correspondence: Department of Pharmacology & Toxicology and Center for Integrative Toxicology, 315 Food Safety Building, Michigan State University, East Lansing, MI 48824-1317. E-mail: kamins11{at}msu.edu

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We have reported previously that ⁹-tetrahydrocannabinol (⁹-THC) treatment of resting human and murine splenic T cells robustly elevated intracellular calcium ([Ca²⁺]_i). The objective of the present investigation was to examine the putative role of [Ca²⁺]_i store depletion and store-operated calcium (SOC) [¹ ] and receptor-operated cation (ROC) channels in the mechanism by which ⁹-THC increases [Ca²⁺]_i in the cannabinoid-2 receptor-expressing human peripheral blood-acute lymphoid leukemia (HPB-ALL) human T cell line. By using the smooth endoplasmic reticulum Ca²⁺-ATPase pump inhibitor, thapsigargin, and the ryanodine receptor antagonist, 8-bromo-cyclic adenosine diphosphate ribose, we demonstrate that the ⁹-THC-mediated elevation in [Ca²⁺]_i occurs independently of [Ca²⁺]_i store depletion. Furthermore, the ROC channel inhibitor, SK&F 96365 was more efficacious at attenuating the ⁹-THC-mediated elevation in [Ca²⁺]_i than SOC channel inhibitors, 2-aminoethoxydiphenyl borate and La³⁺. Recently, several members of the transient receptor potential canonical (TRPC) channel subfamily have been suggested to operate as SOC or ROC channels. In the present studies, treatment of HPB-ALL cells with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeant analog of diacylglycerol (DAG), which gates several members of the TRPC channel subfamily, rapidly elevated [Ca²⁺]_i, as well as prevented a subsequent, additive elevation in [Ca²⁺]_i by ⁹-THC, independent of protein kinase C. Reverse transcriptase-polymerase chain reaction analysis for TRPC1–7 showed that HPB-ALL cells express detectable mRNA levels of only TRPC1. Finally, small interference RNA knockdown of TRPC1 attenuated the ⁹-THC-mediated elevation of [Ca²⁺]_i. Collectively, these results suggest that ⁹-THC-induced elevation in [Ca²⁺]_i is attributable entirely to extracellular calcium influx, which is independent of [Ca²⁺]_i store depletion, and is mediated, at least partially, through the DAG-sensitive TRPC1 channels.

Key Words: Key Words: • cannabinoid • ⁹-THC • TRP channel • SOC channel • store depletion • OAG

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The immunosuppressive properties of the plant-derived cannabinoid ⁹-tetrahydrocannabinol (⁹-THC) have been widely characterized [^{1 2 3} ]. Previous investigations from this laboratory focusing on the mechanism by which cannabinoids modulate interleukin (IL)-2 have demonstrated a strong correlation between cannabinoid-mediated modulation of IL-2 expression and reciprocal changes in DNA binding and reporter gene activity of the transcription factor, nuclear factor of activated T cells (NFAT) [^{4 5 6} ], which is an essential regulator of IL-2 transcription [⁷ ]. The activation and subsequent nuclear translocation of NFAT are under the control of the calcium-dependent phosphatase, calcineurin, whose activity in turn is regulated by a prolonged elevation in intracellular calcium ([Ca²⁺]_i) [⁸ ]. Previous investigations from this laboratory have also shown that plant-derived cannabinoids, ⁹-THC, and cannabinol elevate [Ca²⁺]_i independently in resting murine and human T cells [⁹ , ¹⁰ ]. Moreover, the elevation of [Ca²⁺]_i by ⁹-THC and cannabinol in T cells was sensitive to the cannabinoid-1 (CB1) and CB2 receptor antagonists and was heavily dependent on the presence of extracellular calcium ([Ca²⁺]_e). Apart from regulating the activity of NFAT, calcium is an important second messenger in T cells, which contributes negatively as well as positively to the expression of many genes [⁷ , ⁸ ]. Furthermore, an elevation of [Ca²⁺]_i prior to T cell activation may render the cells anergic [^{11 12 13} ]. Considering the significant role for calcium in gene expression and anergy in T cells, the present studies aimed to elucidate in detail the mechanism by which ⁹-THC elevates [Ca²⁺]_i in the CB2-expressing human peripheral blood-acute lymphoid leukemia (HPB-ALL) human T cell line.

The normal mechanism of the [Ca²⁺]_i elevation in T cells upon engagement of the T cell antigen receptor involves the depletion of intracellular-stored calcium from the endoplasmic reticulum, leading to the opening of a highly calcium-selective variety of store-operated calcium (SOC) channels called the calcium release-activated calcium channels [⁸ , ¹⁴ ]. Despite years of investigation, the actual mechanism of SOC channel regulation as well as its molecular makeup remains elusive. Recent work on the molecular identity of the SOC and receptor-operated cation (ROC) channels has focused on a novel class of proteins called the transient receptor potential (TRP) channels. More specifically, a subfamily of TRP channels, TRP canonical (TRPC) channels, has received attention, as TRPCs may operate as ROC or SOC channels [^{15 16 17 18 19} ]. The expression of multiple members of the TRPC subfamily has been documented in a variety of cells, including human peripheral blood T cells and the Jurkat T cell line [¹⁷ , ²⁰ ]. However, the behavior of individual TRPC subfamily members, as SOC or ROC channels, has varied depending on the experimental conditions, cell type used, and mode of channel activation.

In view of these previous reports regarding the presence of SOC channels and expression of TRPC channels in T cells, the objective of the present study was to investigate the involvement of SOC and ROC channels in the mechanism by which ⁹-THC elevates [Ca²⁺]_i. In particular, the role of intracellular store-operated calcium and the TRPC channels, many of which are diacylglycerol (DAG)-gated [²¹ , ²² ], was assessed in the elevation of [Ca²⁺]_i by ⁹-THC. Using a variety of pharmacological and biochemical techniques, the present investigation demonstrates that the mechanism of [Ca²⁺]_i elevation by ⁹-THC is independent of [Ca²⁺]_i store depletion, is attributable entirely to [Ca²⁺]_e influx, and involves DAG-sensitive TRPC1 channels.

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Compounds
⁹-THC was provided by the National Institute on Drug Abuse (Bethesda, MD). 1-Oleoyl-2-acetyl-sn-glycerol (OAG), 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR), lanthanum chloride (LaCl₃), thapsigargin (TG), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO). 2-Aminoethoxydiphenyl borate (2-APB) was from Calbiochem (San Diego, CA). 1-{ß-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole (SK&F 96365) was from Biomol (Plymouth Meeting, PA).

Cell culture
Dr. Jeffrey A. Ledbetter (Pacific Northwest Research Institute, Seattle, WA) generously provided the HPB-ALL human T cell line. HPB-ALL cells were cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY), supplemented with 100 units penicillin/ml, 100 units streptomycin/ml, 10% bovine calf serum (Hyclone, Logan, UT), 100 mM nonessential amino acids (Gibco BRL), and 1 mM sodium pyruvate (Gibco BRL).

Calcium determination
Cells were washed twice in Ca²⁺-KREB buffer [129 mM NaCl, 5 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1 mM CaCl₂, 5 mM NaHCO₃, 10 mM HEPES, 2.8 mM glucose, 0.2% bovine serum albumin (BSA)]. All experiments, except for those with LaCl₃, were performed in the Ca²⁺-KREB buffer. The experiments with LaCl₃ were performed in modified Hanks’ balanced saline solution (HBSS) buffer (120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO₄, 1.8 mM CaCl₂, 20 mM HEPES, 11.1 mM glucose, 0.2% BSA), which contained a low concentration of anions [²³ ]. [Ca²⁺]_i was determined by measuring the fluorescence of fura-2 dye, which is dually excited at 340 nm and 380 nm. Briefly, cells were incubated with cell-permeant fura-2 AM dye (1 µM, Molecular Research Products, Eugene, OR) for 30 min at 37°C in the dark. Cells were then harvested, washed three times with buffer to remove extracellular fura-2 dye, and readjusted to 5 x 10⁵ cells/ml in the appropriate buffer. Cells were placed in a quartz cuvette with constant stirring. Calcium determinations were performed at room temperature with a Beckman Spex 1681 0.22 m spectrometer with dual excitation at 340 and 380 nm and emission at 510 nm (all slit widths were 1 mm). [Ca²⁺]_i calculations were based on maximum and minimum calcium values, as assessed with use of 0.1% Triton-X and 500 mM EGTA, respectively. The dissociation constant for the fura-2 calcium complex was 1.45 x 10^–7 M. All compounds used in [Ca²⁺]_i determination were screened for autofluorescence using fura-2 sodium salt containing Ca²⁺-KREB buffer. None of the compounds exhibited autofluorescence nor did they interfere with fura-2 fluorescence.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing
Total RNA from HPB-ALL or Jurkat E6-1 cells was isolated using Tri Reagent (Sigma Chemical Co.). Isolated RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of RT. RT-PCR was performed as described previously [⁴ ]. The PCR master mixture consisted of Mg²⁺-free PCR buffer, 2.5 mM MgCl₂, 1.25 units (0.5 µl) Taq DNA polymerase, 120 nM forward and reverse primers, and 400 ng (4.0 µl) cDNA, reverse-transcribed from total RNA, isolated from HPB-ALL and Jurkat E6-1 cells. Samples were heated to 94°C for 4 min and cycled 40 times at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, after which, an additional extension step of 72°C for 5 min was included. The forward and reverse primer sequences, amplicon sizes, and accession numbers for TRPC1–7 are given in Table 1 . PCR products were resolved in a 1.2% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME) and visualized with ethidium bromide staining. The bands for TRPC1 were excised from the agarose gel and purified using Wizard® PCR preps DNA purification system (Promega, Madison, WI). Sequencing of TRPC1 bands was performed with TRPC1 forward or reverse primers using an ABI PRISM® 3100 genetic analyzer at the Michigan State University Macromolecular Structure, Sequencing and Synthesis Facility (East Lansing, MI).

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Table 1. Sequences of Primers Used for RT-PCR Reactions

Small interference RNA (siRNA) knockdown of TRPC1
Chemically synthesized and preannealed siRNA for TRPC1 and a nonsilencing control sequence were custom-ordered from Ambion Inc. (Austin, TX). HPB-ALL cells (2.5x10⁵ cells/ml) were transiently transfected with 20 nM siRNA-specific TRPC1 or a nonsilencing control sequence for 48 h. Briefly, CodeBreaker^TM siRNA transfection reagent (Promega) was diluted in Opti-MEM® medium (Gibco BRL) according to the manufacturer’s instructions. The mixture was incubated at room temperature for 20 min. siRNA, specific for TRPC1 or a nonsilencing control sequence, was diluted in the prepared mixture and allowed to incubate at room temperature for a further 20 min. Transfection complexes were then dropped onto the cells, swirled to mix, and allowed to incubate at 37°C for 48 h. After 48 h, cells were harvested, washed, and used for RNA isolation or calcium determination. Sequences for siRNA oligonucleotides for TRPC1 and nonsilencing control are listed in Table 2 .

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Table 2. Oligonucleotide Sequences for siRNA

Quantitative real-time PCR for TRPC1
Real-time PCR was performed on a PE Applied Biosystems PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA), as described previously [²⁴ ]. Briefly, total RNA was isolated from siRNA-transfected HPB-ALL cells using the SV Total RNA isolation system (Promega) and reverse-transcribed. The PCR reaction contained 100 ng (2.0 µl) cDNA template, 133 nM forward and reverse primers, and the following components of the SYBR® Green PCR core reagents (Applied Biosystems): 0.025 units (0.2 µl) AmpliTaq^TM Gold DNA polymerase, 1 mM deoxy-unspecified nucleoside 5'-triphosphate mix, 3 mM MgCl₂, and 1x SYBR Green PCR buffer. Each plate contained duplicate standards of purified PCR products of known template concentration, which covered at least seven orders of magnitude to interpolate relative template concentrations of the experimental samples from the standard curves of log copy number versus threshold cycle. In addition, no template controls were included on each plate. The relative level of TRPC1 mRNA was standardized to the housekeeping gene, ß-actin, to control for differences in RNA loading, quality, and cDNA synthesis. Comparisons among vehicle (transfection reagent), nonsilencing control, and TRPC1 siRNA-treated groups were analyzed using a two-way ANOVA followed by Dunnett’s post-test. The forward and reverse primer sequences for TRPC1 and ß-actin are given in Table 3 .

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Table 3. Real-Time PCR Primer Sequences for TRPC1 and ß-Actin

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Effect of calcium channel inhibitors on the rise in [Ca²⁺]_i elicited by ⁹-THC and TG
To elucidate whether the increase in [Ca²⁺]_i elicited by ⁹-THC is mediated through the SOC channels, calcium experiments were performed in HPB-ALL cells pretreated with calcium channel inhibitors, SKF or 2-APB, followed by ⁹-THC treatment. A previous publication from this laboratory has demonstrated that the influx in [Ca²⁺]_i, elicited by ⁹-THC in T cells, is concentration-responsive but is modest at concentrations less than 10 µM [¹⁰ ]. Presently, all studies were conducted using 12.5 µM ⁹-THC, a concentration that yielded a robust elevation in [Ca²⁺]_i. In addition, calcium experiments were carried out to examine the effects of the calcium channel inhibitors on the [Ca²⁺]_i elevation elicited by TG (1 µM), a widely described activator of the SOC current by way of calcium store depletion [²⁵ , ²⁶ ]. Pretreatment of HPB-ALL cells with SKF resulted in a concentration-dependent inhibition of the ⁹-THC-mediated elevation in [Ca²⁺]_i (Fig. 1A ), with substantial inhibition at 20–50 µM SKF. Similarly, 2-APB also inhibited the ⁹-THC-mediated elevation in [Ca²⁺]_i in a concentration-responsive manner; however, the effect of 2-APB, even at concentrations as high as 100 µM, was modest in comparison with SKF, as seen in Figure 1B . Conversely, SKF only modestly attenuated the TG-induced [Ca²⁺]_i rise at 50 µM (Fig. 2A ), whereas 2-APB potently inhibited TG-induced [Ca²⁺]_i elevation at a concentration of 25 µM (Fig. 2B) . The contrasting effects of SKF and 2-APB on the ⁹-THC- and TG-induced rise in [Ca²⁺]_i may be explained by the prior reports demonstrating that 2-APB is a SOC channel inhibitor [¹⁹ ], and SKF is a ROC channel inhibitor [²⁷ ]. Additionally, 2-APB may act as an inositol triphosphatase (IP₃) receptor antagonist or target signaling intermediates, such as phospholipase C (PLC) in lymphocytic cells, apart from inhibiting SOC channels [²⁹ ].

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Figure 1. 9-THC-mediated elevation in [Ca2+]i is (A) strongly attenuated upon pretreatment with SK&F 96365 and (B) partially attenuated upon pretreatment with 2-APB. A 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml) was treated with (A) SK&F 96365 (10–50 µM) or (B) 2-APB (50–100 µM) and/or vehicle [VH; double-distilled (dd)H2O for SKF and 0.1% ethanol for 2-APB] before beginning calcium measurements. At 300 s, 9-THC (12.5 µM) or VH (0.1% ethanol) was injected into the cuvette, and the increase in [Ca2+]i was measured for 1600 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent four independent experiments.

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Figure 2. TG-induced elevation in [Ca2+]i is (A) weakly attenuated upon pretreatment with SK&F 96365 and (B) strongly attenuated upon pretreatment with 2-APB. A 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml) was treated with (A) SK&F 96365 (20–50 µM) or (B) 2-APB (25–50 µM) and/or VH (ddH2O for SKF and 0.1% ethanol for 2-APB) before beginning calcium measurements. At 300 s, TG (1 µM) or VH (0.1% ethanol) was injected into the cuvette, and the increase in [Ca2+]i was measured for 1600 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent three (A) or two (B) independent experiments.

To address the discrepancy of the contrasting effects of SKF and 2-APB on the ⁹-THC- and TG-induced elevation of [Ca²⁺]_i, additional studies were conducted with LaCl₃. La³⁺ cation has been reported as a potent inorganic inhibitor of SOC channels at submicromolar levels, and it is effective at inhibiting a TG-induced [Ca²⁺]_i rise [²⁹ ]. In addition, lanthanide compounds have been reported to inhibit TRPC channels but at concentrations in the micromolar range [¹⁶ , ¹⁹ ]. Presently, the effect of LaCl₃ (20–250 µM) was examined on the ⁹-THC- and TG-induced [Ca²⁺]_i rise. Pretreatment of cells with LaCl₃ markedly inhibited the ⁹-THC-mediated elevation of [Ca²⁺]_i at concentrations greater than or equal to 100 µM (Fig. 3A ). In contrast, LaCl₃ was potent at attenuating the TG-induced rise in [Ca²⁺]_i, even at a low concentration of 20 µM (Fig. 3B) .

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Figure 3. LaCl3 is (A) a weak inhibitor of the 9-THC-induced elevation in [Ca2+]i but (B) a potent inhibitor of the TG-induced elevation in [Ca2+]i. A 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml) in modified HBSS buffer (see Materials and Methods) was treated with LaCl3 (20–250 µM) or VH (ddH2O) before beginning calcium measurements. At 300 s, 9-THC (12.5 µM; A), TG (1 µM; B), or VH (0.1% ethanol) was injected into the cuvette, and the increase in [Ca2+]i was measured for 1600 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent two (A) or three (B) independent experiments.

⁹-THC-mediated elevation in [Ca²⁺]_i is not abolished upon TG pretreatment
The experiments outlined above suggested that the ⁹-THC-mediated elevation in [Ca²⁺]_i was dependent on SKF-sensitive channels but was independent of SOC channels. To eliminate the possibility that ⁹-THC was depleting [Ca²⁺]_i pools and to examine the effect of calcium store depletion on the ⁹-THC-mediated [Ca²⁺]_i rise, calcium measurements were performed with sequential addition of TG, followed by ⁹-THC. In these studies, cells were first treated with TG (1 µM) at 300 s and followed by addition of TG (1 µM) or ⁹-THC (12.5 µM). As seen in Figure 4A , an initial addition of TG to the cells led to a rapid and robust increase in [Ca²⁺]_i, indicating an influx of [Ca²⁺]_e following depletion of stored [Ca²⁺]_i, and the subsequent addition of TG did not lead to a further increase in [Ca²⁺]_i. In contrast, if cells were initially treated with TG to deplete stored calcium and then treated with ⁹-THC, the addition of ⁹-THC led to a further elevation of [Ca²⁺]_i over the TG-induced [Ca²⁺]_i rise.

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Figure 4. The 9-THC-induced elevation in [Ca2+]i is independent of [Ca2+]i store depletion. Calcium measurements were performed using a 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml). (A) TG (1 µM) or VH (0.1% ethanol) was injected into the cuvette at 300 s, followed by a second injection with TG (1 µM) or 9-THC (12.5 µM) at 1200 s. The increase in [Ca2+]i was measured for 2500 s. (B) Cells were treated with 8-Br-cADPR (20–75 µM) or VH (ddH2O) before beginning calcium measurements. 9-THC (12.5 µM) or VH (0.1% ethanol) was injected into the cuvette at 300 s, and the increase in [Ca2+]i was measured for 1600 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent three (A) or two (B) independent experiments.

8-Br-cADPR pretreatment did not attenuate the ⁹-THC-mediated elevation in [Ca²⁺]_i
It has previously been reported that there are two [Ca²⁺]_i stores: the IP₃-sensitive and -insensitive calcium pools, which are depleted by TG; and a TG-insensitive ryanodine receptor (RyR)-gated calcium pool, depleted by the endogenous RyR agonist cADPR [³⁰ ]. To ascertain that ⁹-THC did not elicit the increase in [Ca²⁺]_i following depletion of cADPR-sensitive calcium pools, HPB-ALL cells were pretreated with 8-Br-cADPR, a cell-permeant antagonist of the RyR, for 300 s followed by ⁹-THC. The resulting ⁹-THC-mediated [Ca²⁺]_i increase was not attenuated by 8-Br-cADPR treatment, even at concentrations as high as 75 µM (Fig. 4B) .

OAG elevates [Ca²⁺]_i in HPB-ALL cells
In light of the observation that ⁹-THC-mediated elevation in [Ca²⁺]_i was independent of store depletion and was sensitive to high concentrations of LaCl₃, we examined the possibility that ⁹-THC-induced elevation in [Ca²⁺]_i was mediated through ROC channels in the TRPC subfamily. Recently, various groups have conjectured that TRPC channels may operate as ROC or SOC channels [^{15 16 17 18 19} ]. Moreover, analogs of DAG, a product of phosphotidyl inositol bisphosphate hydrolysis by PLC, have been found to activate four members of the TRPC subfamily, namely TRPC1, -3, -6, and -7 [¹⁶ , ^{19 20 21 22} ]. In the present investigation, the presence of DAG-gated channels in HPB-ALL cells was confirmed by treatment with OAG, a cell-permeant analog of DAG. Treatment of HPB-ALL cells with increasing concentrations of OAG (50–300 µM) resulted in a rapid elevation of [Ca²⁺]_i (Fig. 5A ), which was, however, smaller in magnitude when compared with the ⁹-THC-mediated rise of [Ca²⁺]_i.

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Figure 5. Treatment of HPB-ALL cells with OAG induces an elevation in [Ca2+]i and abolishes the 9-THC-elicited rise in [Ca2+]i. Calcium measurements were performed using a 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml). (A) OAG (50–300 µM) or ethanol VH was injected into the cuvette at 300 s without dilution in Ca2+-KREB buffer. The increase in [Ca2+]i was measured for 1600 s. (B) OAG (300 µM) or ethanol VH was injected into the cuvette at 300 s, followed by a second injection with OAG (300 µM) or 9-THC (12.5 µM) at 900 s, and the increase in [Ca2+]i was measured for 1800 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent five (A) or three (B) independent experiments.

⁹-THC-mediated elevation in [Ca²⁺]_i is abolished upon OAG pretreatment
The hypothesis that ⁹-THC and OAG putatively induced [Ca²⁺]_i elevations through the same TRPC channel was examined in HPB-ALL cells, which were sequentially treated with OAG (300 µM) for 600 s, followed by a second addition of OAG (300 µM) or ⁹-THC (12.5 µM). As expected, treatment of cells initially with OAG elevated [Ca²⁺]_i rapidly. However, as can be gleaned from Figure 5B , the subsequent addition of OAG did not further elevate [Ca²⁺]_i. It is interesting that in cells initially treated with OAG, a subsequent addition of ⁹-THC also failed to elicit a further elevation in [Ca²⁺]_i.

The abrogation of ⁹-THC induced [Ca²⁺]_i elevation by OAG is protein kinase C (PKC)-independent
Several recent studies have reported that TRPC channels are negatively regulated by PKC [³¹ , ³² ]. To investigate the possibility that the pretreatment with OAG inhibits a subsequent ⁹-THC-mediated elevation in [Ca²⁺]_i by activating PKC, a further experiment was performed with the PKC activator PMA. It has been demonstrated previously that treatment of lymphocytes with high concentrations of PMA for long periods of time results in the down-regulation of DAG-sensitive PKC isoforms [³³ , ³⁴ ]. Presently, HPB-ALL cells were treated with VH 0.05% dimethyl sulfoxide (DMSO) or PMA (500 nM) for 20 h and then used for calcium determinations. Cells pretreated with VH or PMA were sequentially treated with OAG (300 µM) for 600 s, followed by a second addition of OAG (300 µM) or ⁹-THC (12.5 µM). As can be seen in Figure 6A and 6B , preincubation of cells with PMA did not prevent the abrogation of ⁹-THC-induced [Ca²⁺]_i elevation by OAG. The observation that OAG inhibits a subsequent and additive elevation of [Ca²⁺]_i, even upon PKC down-regulation, is consistent with a prior report, which demonstrated that the activation of TRPC3 by OAG was independent of PKC [³¹ ].

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Figure 6. Abrogation of the 9-THC-induced elevation in [Ca2+]i by OAG is PKC-independent. Calcium measurements were performed using a 3-ml aliquot of fura-2-AM-loaded HPB-ALL cells (5x105 cells/ml), pretreated for 20 h with (A) VH (0.05% DMSO) or (B) PMA (500 nM). OAG (300 µM) or ethanol VH was injected into the cuvette at 300 s, followed by a second injection with OAG (300 µM) or 9-THC (12.5 µM) at 900 s, and the increase in [Ca2+]i was measured for 1800 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The calcium traces represent two independent experiments.

RT-PCR for TRPC1–7
RT-PCR was performed for all members of the human TRPC channel subfamily, with the exception of TRPC2, which is a pseudogene in humans [³⁵ , ³⁶ ]. RT-PCR results demonstrated that whereas Jurkat E6-1 cells, which served as a comparative control, express transcripts for TRPC1, -3, and -6, the HPB-ALL cells express transcripts only for TRPC1 (Fig. 7 ). It is interesting that in addition to the 686 bp-predicted TRPC1 amplicon, RT-PCR results demonstrated the presence of a second, smaller amplicon in the HPB-ALL and Jurkat E6-1 cells. The smaller TRPC1 amplicon was sequenced from HPB-ALL and Jurkat E6-1 cells using TRPC1 forward and reverse primers. Sequencing confirmed that the smaller 532-bp product was an alternative splice variant of TRPC1, which is spliced between nucleotides 309 and 463 within the TRPC1-coding sequence (data not shown). However, splicing of 154 bp from within the TRPC1-coding sequence shifts the open-reading frame of TRPC1, resulting in a premature stop codon at nucleotides 491–493.

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Figure 7. RT-PCR analysis of TRPC1–7 in HPB-ALL and Jurkat E6-1 cells. Total RNA was isolated from HPB-ALL (left) and Jurkat E6-1 (right) cells. Isolated RNA samples were reverse-transcribed and assayed for expression of TRPC1–7 mRNA transcripts by PCR. The RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of RT. The PCR reaction was performed with 40 amplification cycles using specific primers for TRPC1–7 and resolved on a 1.2% agarose gel (conditions described in Materials and Methods). The results are representative of three independent experiments.

Knockdown of TRPC1
Given that HPB-ALL cells expressed steady-state mRNA for only TRPC1, we further investigated the involvement of TRPC1 in the ⁹-THC-mediated rise in [Ca²⁺]_i. HPB-ALL cells were transiently transfected with siRNA (20 nM) directed against TRPC1 or a nonsilencing control sequence. After a 48-h transfection, the cells were harvested, total RNA was isolated, and real-time PCR was performed for TRPC1 and ß-actin as a loading control. Compared with vehicle (transfection reagent) treatment, the siRNA targeting TRPC1 yielded 50% knockdown in the TRPC1 mRNA level, whereas the nonsilencing control siRNA produced no significant change (Fig. 8A ). Finally, calcium determinations were performed in the siRNA-transfected cells to determine whether knockdown of TRPC1 affected the elevation of [Ca²⁺]_i by ⁹-THC. In accord with the real-time PCR results, knockdown of TRPC1 attenuated the ⁹-THC-induced elevation of [Ca²⁺]_i, whereas the nonsilencing control siRNA did not (Fig. 8B) . siRNA directed against TRPC1 typically yielded between 30% and 50% attenuation of the ⁹-THC-induced elevation of [Ca²⁺]_i, as compared with vehicle control.

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Figure 8. siRNA knockdown of TRPC1 and attenuation of [Ca2+]i elevation elicited by 9-THC. HPB-ALL cells were transiently transfected with siRNA (20 nM) directed against TRPC1 or a nonsilencing control sequence or treated with transfection medium only (vehicle) for 48 h. (A) Total RNA was isolated, reverse-transcribed, and assayed for expression of TRPC1 and ß-actin by real-time PCR. The RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of RT. The relative level of TRPC1 mRNA was standardized to the housekeeping gene, ß-actin, and presented as mean ± SE of the percent knockdown of TRPC1 of vehicle control (see Materials and Methods). *, P < 0.05, for paired comparison analysis (Dunnet’s test). (B) Calcium determinations were performed using a 3-ml aliquot of fura-2AM-loaded HPB-ALL cells (5x105 cells/ml), which had undergone a 48-h transient transfection with TRPC1 siRNA, control siRNA, or vehicle. At 300 s, 9-THC (12.5 µM) or VH (0.1% ethanol) was injected into the cuvette, and the increase in [Ca2+]i was measured for 1600 s. [Ca2+]i changes are presented as changes in the ratio of bound-to-free calcium (340 nm/380 nm). The graphs represent three (A) or four (B) independent experiments.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A number of previous publications have reported in various cell types, including primary murine splenic T cells and human T cell lines, that cannabinoid compounds can elevate [Ca²⁺]_i independently of activation stimuli [⁹ , ¹⁰ , ^{37 38 39 40} ]. In T cells, calcium is a critical second messenger and contributes to the negative as well as positive expression of many genes [⁷ ]. The primary mechanism of calcium influx in T cells is through SOC entry, triggered by a rapid release of calcium pools [⁸ ]. Previously, this laboratory has demonstrated that in the absence of [Ca²⁺]_e, the ⁹-THC-induced rise in [Ca²⁺]_i was severely attenuated and delayed [¹⁰ ]. The delay and the magnitude of the elevation in [Ca²⁺]_i, elicited by ⁹-THC in the absence of [Ca²⁺]_e, were uncharacteristic of calcium store depletion in T cells. The critical role for calcium in T cells coupled with the widely established observation that cannabinoid compounds are immunosuppressive prompted additional studies to examine the mechanism by which ⁹-THC elevates [Ca²⁺]_i in the CB2 receptor-expressing HPB-ALL human T cell line.

The present investigation demonstrated that the mechanism of ⁹-THC-mediated elevation in [Ca²⁺]_i was receptor-operated and independent of calcium store depletion, as supported by the following lines of evidence. First, the ⁹-THC-mediated elevation in [Ca²⁺]_i was strongly inhibited by the ROC channel inhibitor SKF. Second, the SOC channel inhibitors LaCl₃ and 2-APB were less efficacious at attenuating the ⁹-THC-induced [Ca²⁺]_i elevation, as compared with the TG-induced [Ca²⁺]_i elevation. Finally, neither the depletion of stored calcium by TG nor antagonism of the RyRs with 8-Br-cADPR prevented ⁹-THC from eliciting a full [Ca²⁺]_i elevation. In spite of the fact that these data suggest that the mechanism of ⁹-THC-induced [Ca²⁺]_i elevation is receptor-operated and occurs in a calcium store depletion-independent manner, it is intriguing that the SOC channel inhibitors LaCl₃ and 2-APB partially attenuated the ⁹-THC-induced [Ca²⁺]_i elevation at higher concentrations. Recent studies investigating the store-dependent and -independent mechanisms of calcium entry in nonexcitable cells have focused primarily on the TRP channel superfamily and specifically, on the TRPC channel subfamily. Various groups have conjectured that TRPC channels may operate as ROC or SOC channels [^{15 16 17 18 19} , ⁴¹ , ⁴² ]. Consistent with the results from the present studies, several reports have demonstrated that TRPC channels are sensitive to high concentrations of lanthanide compounds as well as 2-APB [¹⁶ , ¹⁹ , ⁴³ ]. Along with the aforementioned reports, the results from the current investigation, demonstrating the partial sensitivity of ⁹-THC- and TG-induced [Ca²⁺]_i elevation to SOC and ROC inhibitors, respectively, may suggest that the signaling mechanisms regulating SOC and ROC entry may indeed converge onto the same channel. Nevertheless, it remains to be fully resolved whether the SOC and ROC channels are distinct proteins or the same channels regulated by alternate signaling mechanisms.

The present studies further examined whether a ROC channel, in particular, a DAG-sensitive TRPC channel, was involved in the mechanism of ⁹-THC-mediated [Ca²⁺]_i elevation. Recently, four members of the TRPC subfamily, namely TRPC1, -3, -6, and -7, have been found to be activated by analogs of DAG, a product of phosphotidyl inositol bisphosphate hydrolysis by PLC [¹⁶ , ¹⁹ , ²⁰ , ²² , ⁴⁴ ]. The presence of a DAG-sensitive TRPC channel in HPB-ALL cells is supported by the observation that OAG, a cell-permeant DAG analog, which activates several TRPC channels, increased [Ca²⁺]_i rapidly and in a concentration-responsive manner. Moreover, cells treated with ⁹-THC failed to elicit a [Ca²⁺]_i response following an OAG-induced [Ca²⁺]_i elevation, suggesting that ⁹-THC and OAG likely increase [Ca²⁺]_i through the same TRPC channel. In addition, preincubation of cells with a high concentration of PMA, which is well established to down-regulate PKC, failed to prevent the abrogation of the ⁹-THC-mediated rise in [Ca²⁺]_i by OAG. The failure of PKC down-regulation to prevent OAG-induced inhibition of a subsequent rise in [Ca²⁺]_i by ⁹-THC suggested again that OAG and ⁹-THC likely gate the same TRPC channel. It is somewhat surprising that RT-PCR analysis for TRPC subfamily genes demonstrated that HPB-ALL cells express transcripts only for TRPC1. To our knowledge, endogenous expression of TRPC1 in the absence of other TRPC subfamily members has not been demonstrated previously. To ascertain that TRPC1 was involved in the ⁹-THC-mediated rise in [Ca²⁺]_i, expression of TRPC1 was knocked down using siRNA, and siRNA targeting TRPC1 knocked down the mRNA expression of TRPC1 by 50%. In concordance with the mRNA expression results, knockdown of TRPC1 also attenuated the magnitude of the ⁹-THC-induced elevation of [Ca²⁺]_i, whereas the nonsilencing control siRNA did not. The TRPC1 knockdown studies strongly suggest that the ⁹-THC-induced elevation of [Ca²⁺]_i in the HPB-ALL cells is mediated, at least in part, by TRPC1 functioning as a ROC channel.

Whether functioning as ROC or SOC channels, it is generally postulated that functional TRPC channel complexes are tetrameric [¹⁶ , ⁴⁵ , ⁴⁶ ]. From the findings in the present study, one can glean that in the HPB-ALL cells, which express only TRPC1, the functional DAG-operated ROC channel is putatively a TRPC1 homotetramer. However, the existence of TRPC1 as homotetramers on the plasma membrane remains controversial, in light of the previous demonstration that in transfected human embryonic kidney 293 cells, the localization of TRPC1 to the plasma membrane required coexpression of TRPC4 [⁴⁵ ]. Moreover, Hassock and coworkers [⁴⁷ ] have shown that TRPC1 is mostly located on intracellular membranes in platelets. Clearly, further studies investigating the functional makeup and cellular trafficking of the functional TRPC1 channel are required before the mechanism of its regulation can be understood fully. Although a number of studies have identified TRPC1 as a putative component of SOC channels [¹⁶ , ⁴⁸ , ⁴⁹ ], it remains unclear how the TRPC channels are regulated when endogenously expressed. Putney Jr. and coworkers [¹ 8] have demonstrated that at least for TRPC3, regulation and behavior depend on the level of expression. Alternatively, it has also been suggested that formation of different multimeric TRPC complexes may result in distinct biophysical and regulatory properties [²¹ , ⁴⁵ , ⁵⁰ ].

Although the present studies provide clear evidence that endogenously expressed, DAG-sensitive TRPC1 channels are involved in the ⁹-THC-mediated elevation of [Ca²⁺]_i in T cells, it is noteworthy that this observation may not be extrapolated to all cannabinoid ligands. A previous publication from this laboratory has shown that although ⁹-THC elicited a robust elevation in [Ca²⁺]_i, the high-affinity CB1/CB2 nonselective agonist CP55,940 did not [¹⁰ ]. Furthermore, the ⁹-THC-mediated rise in [Ca²⁺]_i was sensitive to SR141716A and SR144528, the CB1 and CB2 receptor antagonists, respectively [¹¹ ]. More recent studies have revealed that neither the endogenous cannabinoids, anandamide and 2-arachidonoylglycerol, nor the CB2-selective agonist JWH-133, over a wide range of concentrations (0.1–20 µM), elicited a rise in [Ca²⁺]_i in HPB-ALL cells (unpublished observations). Additional studies are currently under way to investigate the signaling mechanism of TRPC1 regulation by ⁹-THC in T cells, as well as the involvement of cannabinoid receptors therein.

The functional implications of elevation of [Ca²⁺]_i by ⁹-THC in T cells are far-reaching, especially with respect to T cell activation. ⁹-THC has been shown previously to alter many aspects of the function of activated T cells, including mitogen-induced cell proliferation, accessory cell function in T cell-dependent antibody responses, and production of cytokines [^{1 2 3} , ⁵¹ ]. Moreover, recent investigations from several laboratories have shown that treatment of immune cells with micromolar concentrations of ⁹-THC can modulate the expression and replication of various pathogenic viruses [⁵² , ⁵³ ]. The specific mechanism responsible for altered immune cell function by cannabinoids, however, remains poorly understood. The current results showing that ⁹-THC elevates [Ca²⁺]_i through TRPC1 channels coupled with the previous reports, demonstrating that an elevation of [Ca²⁺]_i, independent of activation stimuli, may render T cells anergic [^{11 12 13} , ⁵⁴ , ⁵⁵ ], suggest that one critical aspect of the immunomodulatory effects of ⁹-THC may be the elevation of [Ca²⁺]_i. Overall, the present characterization of the [Ca²⁺]_i elevation by ⁹-THC may provide the crucial information toward the development of novel cannabinoid-based central nervous system-inactive therapeutic agents.

 
This work was supported by funds from the National Institute of Drug Abuse Grants DA07908 and DA016828.

Received May 24, 2005; revised August 19, 2005; accepted August 30, 2005.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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