Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores

Neuropharmacology 48 (2005) 1086e1096 www.elsevier.com/locate/neuropharm

Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores Shou-Yuan Zhuang, Daniel Bridges, Elena Grigorenko, Stephen McCloud, Andrew Boon, Robert E. Hampson, Sam A. Deadwyler* Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA Received 7 September 2004; received in revised form 20 December 2004; accepted 6 January 2005

Abstract Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 mM). WIN 55,212-2 was also shown to inhibit the NMDAinduced increase in intracellular calcium concentration ([Ca2C]i) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2C]i occurred with similar concentrations (25e100 nM) and in the same time-dependent manner (preexposure 1e15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2C]i was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositoltrisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2C]i via a cAMP/PKA-dependent process during the neurotoxic event. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Neuroprotection; Cannabinoids; NMDA-excitotoxicity; Calcium imaging; FURA-2; Intracellular calcium; Ryanodine receptor; Phosphorylation; Protein kinase A

1. Introduction Cannabinoids have been shown to protect against neurotoxicity in a number of different cellular, animal, and human experimental paradigms (Davies et al., 2002; Fride and Shohami, 2002; Hampson et al., 2000a; Mechoulam et al., 2002; Pryce et al., 2003; van der Stelt et al., 2002). We have previously demonstrated that * Corresponding author. Tel.: C1 336 716 8541; fax: C1 336 716 8501. E-mail address: [email protected] (S.A. Deadwyler). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.01.005

cultured rat hippocampal neurons were protected from excitotoxic insult by pretreatment with either THC or WIN 55,212-2 and that these compounds were effective in preventing cell death even if administered prior to the neurotoxin exposure (Hampson et al., 1998; Zhuang et al., 1999). Other models have demonstrated cannabinoid protection in vivo with respect to neurodegeneration resulting from experimental ischemia (Leker et al., 2003; Martinez-Orgado et al., 2003; Nagayama et al., 1999). Although it is now apparent that cannabinoids have neuroprotective properties, many of the mechanisms involved in the process have yet to be characterized

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(Baker et al., 2003; Mechoulam et al., 2002; van der Stelt et al., 2002). In a brief report we showed that cannabinoid receptor mediated neuroprotection is sensitive to intracellular calcium levels (Zhuang et al., 2001). Here we expand those findings with a detailed analysis of how cannabinoids act to reduce or block release of intracellular calcium [Ca2C]i under neurotoxic conditions (Zhuang et al., 2002). In addition, we demonstrate that such neuroprotection is based on cannabinoid CB1 receptor mediated decreases in cAMP-dependent protein kinase (PKA), an effect that alters the sensitivity of particular intracellular calcium channels. Several possible alternative signaling pathways were also investigated and systematically ruled out on the basis that they did not block the NMDA provoked increase in [Ca2C]i in the same manner as CB1 receptor activation. It is shown that the time course for the following was similar: (1) the neuroprotective effects on cultured neurons, (2) the blockade of intracellular calcium release, and (3) the inhibition of PKA. Common factors underlying these correlated changes were (1) the alteration in sensitivity of Type-II ryanodine receptor (RyR) coupled intracellular calcium channels and (2) the decrease in cAMP due to cannabinoid inhibition of adenylyl cyclase, as originally demonstrated by Howlett and co-workers (Bidaut-Russell et al., 1990; Howlett et al., 1990). Consequently, one process whereby cannabinoids can exert neuroprotective action appears to be a reduction in excitotoxic [Ca2C]i levels directly related to the RyR receptor coupled calcium channel.

2. Methods 2.1. Hippocampal cell cultures Preparation of primary cultures of hippocampal neurons was similar to that described in several previous reports (Deadwyler et al., 1993, 1995; Grigorenko et al., 2002; Mu et al., 1999). Hippocampi from fetal (E-18) rats (Zivic-Miller) were incubated with neutral protease (dispase 1, Boehringer Mannheim Biochemica, 2 U/ml) for 40e50 min at 37  C. After stopping the enzymatic reaction with 1.0 mM NaEDTA, cells were dissociated by gentle trituration via two polished Pasteur glass pipettes and plated at a density of 3e4 ! 105 cells per 35 mm dish. The plating medium consisted of 59% Dulbecco’s Modified Eagle Medium (DMEM, 1!), 19.5% F-12 nutrient mixture (HAM, 1!), 10% fetal bovine serum (FBS), 10% horse serum (HS), 1% L-glutamine (200 mM), all from GIBCO BRL (Gaithersburg, MD). Cultures were grown at 37  C in a humidified 5% CO2 incubator. After 48 h, half of the medium was replaced with ‘‘feeding’’ medium, which consisted of 98% NeurobasalÔ Medium, 2% B-27

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supplement, 0.25% L-glutamine (200 mM), 0.1% 2-mercaptoethanol and 25 mM KCl. At 72 h after plating, half the medium was again replaced with feeding medium, and cultures were treated with 0.75 mM cytosine b-D-arabinofuranoside (Ara-C, Sigma) to prevent glial proliferation. The culture medium was changed every 3 days for the remainder of the experiment. All experiments were performed on cultured cells at days 7e10. 2.2. Drug preparation The cannabinoid CB1 receptor agonists CP 55,940 and WIN 55,212-2, and the CB1 receptor antagonist SR141716A were provided by the National Institute on Drug Abuse (NIDA), Research Triangle Institute (Research Triangle Park), NC and RBI (Boston, MA). Pluronic F-68 acid (Sigma; St. Louis, MO) was dissolved in pure ethanol containing a concentration of 10 mM of CP 55,940. The ethanol was evaporated under a stream of nitrogen gas and CP 55,940 was diluted to 1 mM in saline and stored at 4  C. The final concentrations of CP 55,940 employed ranged from 10 to 500 nM. WIN 55,212-2 and SR141716A were both dissolved in 12 mg/ml DMSO (0.15% of final volume) and added to the culture medium at concentrations of 10e500 nM. All solutions were prepared fresh prior to addition to the culture medium and in all cases exposure to appropriate vehicles was included. Inhibitors were prepared from concentrated stocks in purified water, and diluted to final concentrations in bathing medium (with NMDA and/or cannabinoids as appropriate). Concentrations of SpcAMPS (20 mM), Rp-cAMPS (20 mM), and okadaic acid (1e10 mM) are from prior reports by this laboratory (Deadwyler et al., 1995; Hampson et al., 1995; Mu et al., 2000). Concentrations for thapsigargin (1 mM), xestospongin (1 mM), ryanodine (10e50 mM), and dantrolene (10 mM) were derived from existing reports of the effectiveness and specificity of these compounds in similar cell preparations (Lauri et al., 2003; Martin and Buno, 2003; Nakajima et al., 2001). 2.3. Pretreatment with cannabinoids Hippocampal cell cultures were pretreated with CB1 receptor agonists (CP 55,940; WIN 55,212-2) for 1.5, 2, 3, 5, 15 min periods which was then removed from the medium at various time intervals ranging from 1 to 75 min prior to NMDA exposure (see below) and returned to the incubator for the duration of the preexposure period. Treatment with the CB1 receptor antagonist (SR141716A) was at equal concentrations and preceded addition of agonists by 5 min, whereupon media containing both the antagonist and agonist was administered for the remainder of the 1e15 min pretreatment period. Cultures were gently agitated following addition of all solutions.

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2.4. Treatment with NMDA At the termination of the prescribed cannabinoid treatment period cells were taken from the incubator, washed three times with 500 ml of serum free artificial CSF (ACSF), which was replaced with media containing NMDA (15 mM), and then the cells were returned to the incubator for 5 h (NMDA). The neurotoxic NMDA media was then removed and cells washed three times with 500 ml of CSF, and returned in normal (non NMDA) culture medium to the incubator for an additional 48 h prior to assessment of cell survival. Assessments of cell survival were made 48 h following removal from the 5 h NMDA exposure period.

2.5. Determination of cell survival The LIVE/DEAD Viability/Cytotoxicity marker kit (Molecular Probes, OR) was used to assess survival ratios in each culture dish. Cells were stained with 320 nM calcein AM and 1.6 mM ethidium bromide in D-PBS media (2.7 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4) at room temperature for 20 min. Live cells were stained with calcein, while dead cells were stained with ethidium bromide. Five randomly chosen fields of 4 mm2 diameter (w150 cells/ field) were counted on each of five plates and served as the basis for comparison. Thus the ratio of dead to live cells could be calculated from the differential staining. Maximal cell death was defined using this cytotoxic marker assay as the ratio of the number of dead cells to total cells (live C dead). The condition which produced maximal neuron loss in all cases was exposure to NMDA, thus the cell death ratio for other conditions was plotted relative to this maximum (100%) NMDA ratio. Each experiment also contained a vehicle (i.e. nondrug) control condition from which baseline cell death could be determined and compared across different conditions and manipulations. All values were analyzed using repeated measure ANOVAs comparing % cell death in treated vs. untreated conditions, with orthogonal linear contrasts to compare specific treatment conditions. The results obtained with the cytotoxicity marker kit were validated by comparison with assays of LDH activity (Koh and Choi, 1987) performed on media from the same culture dish. LDH content in cultured cell media was analyzed for oxidation of NADH (340 nm) after the addition of 0.5 mM pyruvate and 0.21 mM NADH to the culture medium (Beckman Instruments). Cultures exposed to NMDA (15 mM) were tested for LDH activity and assessed for the number of live/dead cells detected by the cytotoxic cell marker assay. There was a strong correlation (r2 Z 0.96) between LDH activity and the percentage of cell death estimated by the cytotoxic cell marker assay. The

cytotoxic cell marker measure was, therefore, utilized for assessments of cannabinoid treatments.

2.6. Calcium imaging Primary cultures of hippocampal neurons prepared for calcium imaging were similar to those used for cell survival measures with the exception that the dissociated cells were grown on glass coverslips. After dispersion and plating, cells were incubated in DMEM medium for 7e10 days before testing. Prior to testing, cover slips were removed from the incubator and the cells loaded with Fura-2 AM for calcium imaging purposes via immersion in medium. Cells were pretreated with cannabinoids prior to exposure to NMDA via a 250 ms pulsed pressure application (NMDA 15 mM) through a glass pipette visually placed in the immediate vicinity of a selected cell to be imaged. Calcium influx into the cell was imaged using a double shutter system (Connor and Cormier, 2000; Malinow et al., 1994). Changes in intracellular calcium concentration were indicated by changes in the ratio of Fura-2 fluorescence at 340 and 365 nm excitations. Actual intracellular calcium concentrations could not be calculated in this study; however, relative increases in the Fura-2 fluorescence ratio indicated increased intracellular free calcium [Ca2C]i, while a decrease in the ratio indicated a decrease in [Ca2C]i concentration within the cell. Following NMDA delivery, this fluorescence ratio increased to a maximum within 200 ms, and rapidly returned to baseline within 600 ms of initial NMDA exposure. Fluorescence ratios obtained by measuring 100e200 s after NMDA were normalized to the resting baseline (no NMDA) then averaged across 8e15 different cells for comparison between treatment conditions. Typical baseline ratios of 300 (corresponding to 3-to-1 ratio of 340e365 nm Fura-2 fluorescence) were indicative of nanomolar concentrations of [Ca2C]i, with ratios O500 corresponding to micromolar [Ca2C]i concentrations. The calculated ratios were subjected to ANOVAs to compare baseline and NMDA conditions with cannabinoid pretreatment and/or effects of other modulators of intracellular signaling pathways.

3. Results The neuroprotective consequences of cannabinoid pretreatment are shown in Fig. 1. Cultured hippocampal neurons exposed to NMDA (15 mM) showed a significant increase in % cell death over vehicle baseline (F(1,179) Z 21.6, p ! 0.001). However, if pretreated with different concentrations of WIN 55,212-2 or CP 55,940

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Fig. 1. Cannabinoid concentration and time dependence of neuroprotection in hippocampal cell cultures. (A) Effects of different concentrations (10e 500 mM) of cannabinoid CB1 receptor agonists WIN 55,212-2 (WIN) and CP 55,940 (CP) on % cell death following exposure to NMDA. Cultured fetal (E-18) hippocampal cells were exposed to NMDA (15 mM, black bar) in the medium for 5 h, removed and incubated in normal media then assessed for cell death via cytotoxicity markers at 48 h post NMDA exposure. The ratio of dead cells to total (live C dead) cells was calculated for each treatment condition. This ratio following NMDA treatment for each condition was plotted as 100% with other conditions as a percentile of the NMDA value. White bar indicates treatment with ACSF vehicle. All values represent mean G SEM from 4 to 12 experiments each employing five 35 mm dishes of 7e10 day cultured hippocampal cells. **p ! 0.001 relative to NMDA for all concentrations O10 mM. (B) Cultures in which CB1 receptor antagonist SR141716A (500 nM) was added for 5 min preceding exposure to WIN and CP at the same concentrations as in (A). (C) Time dependence of cannabinoid pretreatment protective effect. WIN (500 nM) applied for the indicated time intervals (1.5e5 min) immediately before (dark gray bars), or 15 min before (light gray bars) NMDA exposure. Also shown is effect of WIN pretreatment for 15 min duration, 30 and 60 min prior to NMDA exposure (striped bars). Zero minute Z vehicle only control. **p ! 0.001 relative to NMDA for all durations. (D) Comparison of WIN (100 nM) and CP (100 nM) pretreatment 0, 45 and 75 min prior to NMDA exposure. NMDA (45 min delay) indicates exposure to vehicle prior to NMDA (15 mM). *p ! 0.01, **p ! 0.001 all compared to NMDA.

(20e500 nM) there was a significant systematic decrease in % cell death (F(4,179) Z 12.2, p ! 0.001, vs. NMDA, Fig. 1A). This neuroprotective effect was blocked (F(1,179) Z 1.2, N.S.) if cultures were pretreated with equal concentrations of the receptor antagonist SR141716A (Fig. 1B). The cannabinoid-produced decrease in cell death was also a function of the duration and time between cannabinoid and NMDA exposure as shown in Fig. 1C (asterisks, F(1,179) O 10.1, p ! 0.001). It was found that pretreatment with cannabinoids for 3e15 min (F(1,179) Z 14.6, p ! 0.001) or as much as 75 min prior (F(1,179) Z 7.3, p ! 0.01) to NMDA exposure still produced significant decreases in % cell death (Fig. 1D). Thus CB1 receptor specific neuroprotection was demonstrated with two different cannabinoid agonists, and shown to be concentration, duration and delay dependent. The existence of gradients in all three of these dimensions suggests that there was a linear correspondence between degree of cannabinoid receptor activation and degree of suppression of NMDA-induced neurotoxicity.

This neuroprotective action was investigated by ratiometric imaging of [Ca2C]i in the same cultured neurons filled with the calcium-sensitive fluorophore, Fura-2, under the same cannabinoid pre-exposure conditions as shown in Fig. 1. A pressure pulse of NMDA delivered next to the cell provoked an immediate elevation in [Ca2C]i as exemplified by a large increase in the fluorescence ratio (see Section 3) of Fura2. This is shown in Fig. 2A as a marked increase in mean fluorescence ratio assessed across different cells following exposure to NMDA alone. Pretreatment with WIN 55,212-2, 3 min prior to the NMDA pulse blocked the mean increase in a concentration-dependent manner (25e500 nM) within the same dose range as that for neuroprotection (Fig. 1A). Even at low concentrations (25 nM), pretreatment with WIN 55,212-2 significantly (F(1,842) Z 9.2, p ! 0.01) suppressed the NMDA-induced increase in [Ca2C]i over the entire 10 min post treatment testing period. In addition, Fig. 2B shows that pretreatment with WIN 55,212-2 at different times prior to NMDA exposure produced a similar (all

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F(1,842) O 8.6, p ! 0.01) temporal gradient for suppression of [Ca2C]i as demonstrated for neuroprotection (Fig. 1C and D), with %15 min as the most effective interval (F(1,842) Z 12.2e18.1, all p ! 0.001). Several potential pathways could be involved in the CB1 receptor mediated reduction of NMDAincreased [Ca2C]i (Berridge, 1998; Berridge et al., 2000; Bezprozvanny et al., 1991; Fagni et al., 2000). We investigated a candidate mechanism in which the release of [Ca2C]i from the endoplasmic reticulum (ER) via NMDA stimulation could be regulated by cannabinoids via ryanodine-sensitive intracellular calcium channels (Friel and Tsien, 1992; Marx et al., 2001; Westhoff et al.,

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2003). We tested this by exposing cells to ryanodine (50 mM) prior to NMDA treatment. Fig. 2C shows that ryanodine successfully reduced the NMDA-induced increase in [Ca2C]i (F(1,842) Z 17.3, p ! 0.001). In addition, WIN 55,212-2 and ryanodine were additive in suppressing the increase in [Ca2C]i (F(1,842) Z 15.5, p ! 0.001) when combined at submaximal concentrations (WIN 25 nM C ryanodine 10 mM in Fig. 2C). Alternatively, the ryanodine receptor ligand dantrolene (10 mM) blocked suppression of the [Ca2C]i increase by ryanodine (F(1,842) Z 1.4, N.S.), while pretreatment with the CB1 receptor antagonist (SR141716A) had no effect on ryanodine action. Because of potential interactions between cannabinoid receptor activation and other signaling pathways (Felder et al., 1998; Hampson et al., 2000b; Twitchell et al., 1997) it was necessary to examine other agents that alter [Ca2C]i. Fig. 3A shows that selective blockers of N, P, and Q-type voltage controlled calcium channels (VCCs) did not prevent the increase in NMDA-elicited [Ca2C]i (all F(1,842) ! 3.2, N.S.). In addition, Fig. 3B shows that WIN 55,212-2 also remained effective in the presence of either the phospholipase C (PLC) inhibitor U73122 or the less active enantiomer U73343 (F(1,842) Z 14.6, p ! 0.001, F(1,842) Z 17.9, p ! 0.001, respectively). Thapsigargin, a potent intracellular [Ca2C]i uptake blocker, also did not block the cannabinoid stimulated reduction in NMDA-increased [Ca2C]i (F(1,842) Z 13.1, p ! 0.001). Finally, xestospongin, which alters inositol-1,4,5-trisphosphate (IP3) activity, did not significantly reduce the action of WIN 55,212-2 (F(1,842) Z 16.2, p ! 0.001, Fig. 3B). Thus the above signaling pathways, each of which has been shown to alter [Ca2C]i, did not interfere with the suppression of NMDA-induced increase in [Ca2C]i by WIN 55,212-2. To verify that changes in [Ca2C]i were also produced by NMDA perfusion as in the neuroprotection experiments shown in Fig. 1, imaging of cells after Fig. 2. Ratiometric imaging of Fura-2 in single hippocampal neurons shows cannabinoid reduction of NMDA-induced increase of [Ca2C]i. (A) Pressure pipette application of NMDA (15 mM, 1 s) increased neuronal [Ca2C]i in imaged neurons as indicated by an increase in the mean fluorescence ratio (ratio of 340e365 nm Fura-2 fluorescence) measured at its maximum 100e200 ms after delivery, then averaged across 8e15 cells per condition. Pretreatment with WIN for 5 min at concentrations of 10e500 nM followed by 5 min ACSF wash proportionally reduced the increase in [Ca2C]i induced by NMDA (decrease in mean fluorescence ratio). Cannabinoid effects were blocked by SR141716A (100 nM). *p ! 0.01; **p ! 0.001 relative to NMDA. (B) Effect of delay between WIN (100 nM, 5 min) pretreatment and NMDA exposure. (C) Pretreatment with ryanodine (Ryano., 50 mM, 5 min) blocked NMDA-elicited increase in [Ca2C]i, similar to effect of WIN. WIN and ryanodine were administered for 5 min in submaximal concentrations (25 and 10 mM, respectively), individually and together (WIN C Ryano.). Effects of both WIN (100 nM) and ryanodine (50 mM) were blocked by 5 min pretreatment with dantrolene (Dantr., 10 mM). *p ! 0.01; **p ! 0.001.

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Fig. 3. Examination of other signaling pathway effects on [Ca2C]i. (A) Voltage-dependent cell membrane Q, N and P-type Ca2C channel blockers (all 1 mM) failed to alter the NMDA-induced increase in [Ca2C]i. Ryanodine (50 mM) significantly (**p ! 0.001) blocked NMDA-induced increase in [Ca2C]i in the same cells. (B) Compounds that alter phospholipase C (PLC) activity e U73122, U73343; or inhibit inositol-1,4,5-trisphosphate (IP3) e xestospongin, did not significantly reduce the effect of cannabinoid pretreatment when coadministered with WIN (100 nM). Thapsigargin, which blocks Ca2C reuptake into the endoplasmic reticulum, was also ineffective. **p ! 0.001 relative to NMDA alone.

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additive (F(1,842) Z 18.6, p ! 0.001) if administered at submaximal concentrations (WIN 25 nM and RpcAMPS 10 mM, F(1,842) Z 11.4, p ! 0.001, data not shown). In contrast, WIN 55,212-2 (100 nM) was unable to significantly alter (F(1,842) Z 1.3, N.S.) the influence of Sp-cAMPS which increased [Ca2C]i in a manner similar to NMDA (Fig. 4A). This suggests that the CB1 receptor-activated adenylyl cyclase, and subsequent reduction in cAMP and PKA levels (Bidaut-Russell and Howlett, 1991; Felder et al., 1993; Hampson et al., 1995), were critical for WIN 55,212-2 blockade of NMDA-induced increase in [Ca2C]i. This was further supported by showing (Fig. 4B) that direct manipulation of PKA-meditated protein phosphorylation via bath perfusion of the phosphatase inhibitor okadaic acid, alone or in combination with PKA (activated by coapplication of Sp-cAMPS), increased resting levels of [Ca2C]i in the same manner as NMDA. These effects were also blocked by prior perfusion of WIN 55,212-2 in the same paradigm that reduced cell death shown in Fig. 1. The time course of cannabinoid-reduced PKA stimulant of [Ca2C]i release was investigated by monitoring the duration of the WIN 55,212-2 triggered positive shift in voltage-dependent inactivation of potassium A-current in the same cultured neurons (Deadwyler et al., 1993). Previously it was shown that such a shift resulted from dephosphorylation of Acurrent channel proteins in these same cultured hippocampal neurons (Hampson et al., 2000b; Mu et al., 2000). The duration of altered voltage dependence could, therefore, be utilized as a marker for determining the time course of cAMP inhibition following a 3.0 min pretreatment and subsequent of WIN 55,212-2 (100 nM). Fig. 4C illustrates the time course of the shift in A-current voltage dependence which is compatible with the effective pretreatment time course for both (1) neuroprotection (Fig. 1D) and (2) suppression of NMDA-mediated increases in [Ca2C]i (Fig. 2B).

4. Discussion addition of NMDA to the perfusion medium was performed. Assessments were made 5 min following application of compounds, which reflected the steadystate [Ca2C]i levels in imaged neurons. Fig. 4A shows that the perfused NMDA increased [Ca2C]i and that this was significantly reduced by prior exposure to WIN 55,212 (100 nM). Significantly, however, application of Rp-cAMPS (20 mM, F(1,842) Z 15.2, p ! 0.001), an inhibitor of cAMP-dependent protein kinase (PKA) maximally reduced the NMDA-induced increase in [Ca2C]i, which was slightly enhanced by addition of an effective concentration of WIN 55,212-2 (F(1,842) Z 2.8, N.S). However, WIN 55,212-2 and Rp-cAMPS were

The neuroprotective actions of cannabinoids have been examined in several different experimental contexts, including in vitro (Grigorenko et al., 2002; Hampson et al., 1998; Iuvone et al., 2004; Khaspekov et al., 2004; Mechoulam et al., 2002; Nagayama et al., 1999) as well as in vivo (Filbert et al., 1999; Lastres-Becker et al., 2003; Martinez-Orgado et al., 2003; Mechoulam et al., 2002; Pryce et al., 2003; Veldhuis et al., 2003) paradigms. In many studies the critical basis for the protective actions of cannabinoids was not completely identified. There is, however, increasing evidence that excitotoxic elevation of intracellular calcium [Ca2C]i produces apoptosis through

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Fig. 4. (A) Inhibition of protein kinase A (PKA) by Rp-cAMPS (20 mM) blocks NMDA-elicited increase in [Ca2C]i, and occludes the effect of 100 nM WIN (C). Activation of PKA with Sp-cAMPS (20 mM) alone was similar to NMDA-elicited increase in [Ca2C]i, and blocked the effect of WIN (C). (B) Phosphatase inhibitor, okadaic acid, was perfused (1, 5 and 10 mM) over same time course as NMDA. Concentrations of okadaic acid (alone) R5 mM increased [Ca2C]i, and the effect at 10 mM was not significantly different from NMDA. Concomitant stimulation of receptor protein phosphorylation via Sp-cAMPS (20 mM), in addition to okadaic acid, also elevated [Ca2C]i to levels not significantly different from NMDA. *p ! 0.01; **p ! 0.001 relative to NMDA alone. (C) Time course of decrease in PKA effect following exposure to WIN (100 nM) in neuroprotection paradigm (see Section 2). Change in PKA activity was determined by monitoring the duration of altered voltage-dependent inactivation of potassium A-current subsequent to 3 min pretreatment with WIN (100 nM), followed by washout in ACSF. Duration of altered Acurrent (mean % G SEM) reflects time course of reduced cAMP and subsequent inhibition of PKA.

direct stimulation of calcineurin which activates caspase via Bcl-2 proteins and Cytochrome c (Polster and Fiskum, 2004), providing a plausible basis for cannabinoid effects on [Ca2C]i mediating the neuroprotection demonstrated here (Fig. 1). The present results are consistent with several other studies showing that cannabinoids mediate alterations in [Ca2C]i levels via the same intracellular second messenger pathway (Guo and Ikeda, 2004; Hajos and Freund, 2002; Twitchell et al., 1997). The current study systematically eliminated several potential alternative sources of CB1 receptor mediated suppression of NMDA-induced increases in [Ca2C]i, including IP3 and PLC pathways (Fig. 3B). Perhaps the

most striking aspect of the above cannabinoid action was the fact that it could be essentially eliminated or greatly diluted in the medium following pretreatment, and still provide protection (Figs. 1 and 4) over an extended period of time (Zhuang et al., 1999; Zhuang et al., 2001). Confirmation that intracellular cAMP levels reflected the same duration of extended action was shown by tracking the PKA-dependent shift in voltage dependence of A-current (Fig. 4C). That cannabinoid treatment did in fact produce a change in PKAmediated phosphorylation of RyR-sensitive intracellular calcium channels (Bultynck et al., 2003; Nozaki et al., 1999; Yoshida et al., 1992) was suggested by a similar reduction in NMDA-increased [Ca2C]i by agents that

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Proposed Cannabinoid Neuroprotection Pathway

A.

Cannab.

NMDA

CB1

- AC

GP U73122

Gi

PLC

-

Sp-cAMPS

IP3

-

Rp-cAMPS

Xesto. Ca2+

+ PKA -

Ca2+

Dantrolene

Ca2+

+

-

+

-

Ryanodine

Ca2+

IP3R Red: Inhibition

Ca2+

Ca2+

+

Thaps.

Ca2+

VCC

Ca2+

RyR (ER: Intracellular Calcium Stores)

Blue: Activation

B. Ca2+

PKA

Phosphatase

+ P i -

Ca2+

Dantrolene

+ + -

Ryanodine

RyanodineReceptor

Fig. 5. Diagram of neuroprotection via reduced release of [Ca2C]i by pre-exposure to cannabinoids. (A) NMDA binds to its receptor and under appropriate conditions triggers Ca2C entry into cell (blue receptor, NMDA); producing increased calcium binding to the intracellular ryanodine receptor-coupled (RyR) Ca2C channel (purple) and subsequent increase in Ca2C release from the endoplasmic reticulum (ER). Manipulations of PLC/IP3 pathways (red and green stippled receptors) did not alter cannabinoid reduction of NMDA-elicited increases in [Ca2C]i. Manipulations of cAMP-dependent protein kinase (pink, PKA) activity via indicated compounds modulated effects of WIN on NMDA-increased [Ca2C]i. WIN inhibitory effect on PKA mediated via CB1/G-protein coupled inhibition of adenylyl cyclase (light blue, AC). (B) PKA and RyR channel interaction: NMDA-induced increase in binding of Ca2C to the RyR-sensitive calcium channel produces positive feedback to increase release of [Ca2C]i. Ryanodine renders the channel less sensitive to Ca2C and prevents the NMDA-elicited increase in [Ca2C]i; however, the effect is blocked by dantrolene. The binding of Ca2C to its site on this channel is also dependent on PKA-regulated phosphorylation of channel proteins. Sp-cAMPS, which activates the catalytic subunit of PKA (PKAc), and the phosphatase inhibitor okadaic acid both blocked the capacity for WIN to reduce the NMDA-induced increased [Ca2C]i. VCC e voltage-activated calcium channel; PLC e phospholipase C; IP3 e inositol-1,4,5-trisphosphate; IP3R e inositol-1,4,5-trisphosphate receptor; AC e adenylyl cyclase; PKA e cAMP-dependent protein kinase; CB1 e cannabinoid receptor; Gi e inhibitory G-protein; ER e endoplasmic reticulum.

act more directly in this pathway than CB1 receptor ligands (e.g., Rp-cAMPS and Sp-cAMPS, Fig. 4B). A conflicting report by Netzeband et al. (1999) showed that similar cannabinoid manipulations produced increases in [Ca2C]i in cultured neurons; however, those experiments were conducted in low Mg2C medium, a condition that we determined provokes enhanced NMDA elevation in [Ca2C]i, and decreases cannabinoid receptor mediated inhibition of cAMP. The studies reported here utilized normal levels of Mg2C in the medium (see Section 2).

Fig. 5A is a diagram of the proposed pathway mediating cannabinoid neuroprotection via reduced [Ca2C]i release. In the proposed process, NMDA receptor-gated Ca2C enters the cell under appropriate Mg2C conditions. Apparently, constitutive levels of PKA are sufficient to provoke increased Ca2C binding to a site on the RyR coupled intracellular calcium channel under these conditions which allows calcium stimulated [Ca2C]i release from the ER (Bultynck et al., 2003; Fagni et al., 2000). Blockade of constitutively active PKA either directly by Rp-cAMPS (Fig. 4B) or

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via CB1 initiated reduction in cAMP, reduces Ca2C flux from the RyR channel (Fig. 4A). In addition, sensitivity of [Ca2C]i to okadaic acid (Fig. 4B) suggests that PKA-mediated phosphorylation is critical for NMDAincreased [Ca2C]i (PKA, Fig. 5). In contrast, manipulations of the PLC and IP3 pathways (Hoesch et al., 2002; Ronde et al., 2000; Seymour-Laurent and Barish, 1995) did not alter the reduction of [Ca2C]i by WIN 55,212 (Fig. 5). Activation of the RyR calcium channel, however, produced effects similar to, and additive with, WIN 55,212-2 (Fig. 2C). Fig. 5B illustrates this interaction between PKA and ryanodine sensitivities: binding of NMDA-triggered Ca2C to a site on the RyR calcium channel produces a positive feedback to elevate and maintain increased Ca2C release from the ER (Kuba, 1994; Verkhratsky, 2002). This feedback can be blocked or reduced by ryanodine and renders the channel less sensitive to NMDA-triggered Ca2C (Ayar and Scott, 1999; Emptage et al., 2001; Sharma and Vijayaraghavan, 2003). However, ryanodine is ineffective in the presence of dantrolene (Krause et al., 2004; Power and Sah, 2002; Xu et al., 1998), showing specificity of the NMDA increase to RyR. The binding of Ca2C to the RyR channel is apparently dependent on its phosphorylation status as has been shown in both neurons (Nozaki et al., 1999; Sabatini et al., 1997; Yoshida et al., 1992) and muscle cells (Hain et al., 1994; Lehnart et al., 2003; Reiken et al., 2003). The significant feature of the above mechanism is that once triggered by relatively low concentrations of cannabinoids acting at the CB1 receptor, neuroprotection from elevated [Ca2C]i persists for relatively prolonged time periods due to the extended recovery time of PKA to constitutive levels. Since the cannabinoid pretreatment effects were concentration, duration and time dependent with respect to the magnitude and time of occurrence of excitotoxic insult, there exists considerable potential for adjustment of these parameters with respect to the possible therapeutic application of potent synthetic cannabinoid compounds (Croxford, 2003; Drysdale and Platt, 2003; Mechoulam and Hanus, 2001; Piomelli, 2003). The involvement of endocannabinoids in the demonstrated CB1 receptor mediated neuroprotective action demonstrated here, remains unclear. There was no indication that endogenous cannabinoids were being released spontaneously by cells in these cultures as evidenced by the lack of significant effects of SR141617A on resting or vehicle [Ca2C]i levels (Fig. 2A). However, it is quite possible that endocannabinoids could be released to perform these neuroprotective actions once cellular depolarization levels exceeded thresholds for calcium entry through NMDA receptors (Malinow et al., 1994; Nakamura et al., 2002). Finally, it is unlikely that the demonstrated benefit of this neuroprotective action would be limited to hippocampus, since CB1 receptors

have been demonstrated on cells in a number of different brain regions (Hoffman and Lupica, 2000; Tsou et al., 1998; Varma et al., 2002; Wilson et al., 2001).

Acknowledgements The authors thank Lucy Fasano for technical assistance. Support was contributed by NIDA grants DA00119, DA07625, DA03502 to S.A.D. and DA08549 to R.E.H.

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