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Abstract

The endogenous cannabinoid R(+)-methanandamide (mAEA) exerts differential anti- and pronociceptive effects by activating both cannabinoid (CB1) and vanilloid (TRPV1) receptors of nociceptive primary afferents. The significance of these effects in meningeal nociception was evaluated by modulation of calcitonin gene-related peptide (CGRP) release from meningeal afferents measured in an in vitro preparation of the hemisected rat skull. Temperature steps to 39°C and 45°C caused heat-dependent increases in CGRP release. One micromolar mAEA inhibited CGRP release at 32°C but facilitated it at 45°C. This effect was abolished in the presence of the TRPV1 receptor antagonist capsazepine. Lower doses of mAEA had no effect on basal or heat-evoked release. In the presence of the CB1 receptor antagonist SR141716 (0.2 μM) heat-stimulated increase in CGRP release was facilitated. CGRP release in the presence of SR141716 (0.2 μM) was further increased by adding mAEA at a concentration which had no effect on its own. These results confirm an opposing functional role for anandamide at CB1 and TRPV1 receptors on meningeal afferents.

Keywords

Anandamide endocannabinoids headache meningeal nociception trigeminal

Introduction

Two cannabinoid receptors, CB1 and CB2, have been described to be linked to antinociceptive effects. CB1 activation inhibits neurotransmitter release (1) and decreases nociceptive neurotransmission (2). The endogenous cannabinoid anandamide [arachydonylethanolamide (AEA)] and its more stable analogue R(+)-methanandamide [R(+)-arachidonyl-1′-hydroxy-2′-propylamide (mAEA)] are agonists at the CB1 receptor at nanomolar doses. Recent data indicate also a role for CB2 receptors in antinociception but their functional relevance is not yet clear (3). On the other hand, mAEA is an agonist at the vanilloid receptor TRPV1 (4, 5), a ligand-gated ion channel that is activated by a variety of noxious stimuli including capsaicin, heat and protons (6, 7). Both CB1 and TRPV1 receptors are found on central and peripheral terminals of small dorsal root ganglion cells (8, 9), most of which are polymodal nociceptors (10). The two receptor types are major players in nociception with opposite effects (11). Low methanandamide concentrations, which preferably activate CB1 receptors, inhibit nociceptive afferents, while higher concentrations, which preferentially activate TRPV1 receptor channels, sensitize nociceptive afferents and facilitate nociceptive transmission (12–14). Such opposing effects of cannabinoid agonists have been described in several tissues innervated by spinal and trigeminal primary afferents (15). It is not yet known, however, if the same opposing mechanisms of cannabinoids can be attributed to intracranial trigeminal afferents. The afferent innervation of the meninges and intracerebral blood vessels is regarded as exclusively nociceptive (16, 17) and the activation of intracranial afferents is believed to be closely linked to the generation of headaches. The neuropeptide calcitonin gene-related peptide (CGRP) is contained in a major proportion of these trigeminal afferents (18) and is relevant in the pathophysiology of migraine (19).

Methods

The experiments were performed in accordance with the ethical guidelines of the International Association for the Study of Pain (20). The protocol for in vivo experiments was reviewed by an ethics committee and approved by the local district government. For all experiments adult male Wistar rats were used. Animals were housed in the Institute and supplied with water and pet food ad libitum.

Neuropeptide release from hemisected skull

Methods are described in detail in previous publications (21, 22). Briefly, male Wistar rats were killed in a CO₂ atmosphere. The head was separated from the body at the atlanto-occipital joint. The skin and the galea were cut along the midline and retracted from the skull. The skull was divided into two equal halves along the sagittal suture. The cerebral hemispheres were removed without lesioning the dura mater encephali that remained attached to the skull. The skull cavities with the dura were washed for 30 min at room temperature with carbogen-gassed synthetic interstitial fluid (SIF, pH 7.4) containing (mm): 108 NaC1, 3.48 KC1, 3.5 MgSO₄, 26 NaHCO₃, 11.7 NaH₂PO₄, 1.5 CaC1₂, 9.6 sodium gluconate, 5.55 glucose and 7.6 sucrose. For the following procedures SIF was buffered by nitrogen gassed HEPES because of the oxygen sensitivity of methanandamide. The skull halves were mounted in a water bath that formed a closed humid chamber with a baseline temperature of 32°C. During the experiments the skull halves were repetitively filled with 400 µl SIF that completely covered the supratentorial dura but not the posterior fossa. Five consecutive samples were collected at intervals of 5 min by evacuating the fluid from the skull halves with a pipette without touching the dura.

Experimental protocols

The skull cavities were filled with preheated SIF at temperatures of 32, 32, 39, 45 and 32°C each for periods of 5 min. The temperatures, which represented baseline temperature, non-noxious and noxious heat, were held throughout the 5-min incubation periods by a newly developed feedback thermocontroller which is technically based on the vortex principle (23). This software-controlled vortex device quickly achieves and maintains target temperatures, compensating for the high thermal capacity of the skull half in relation to the small volume of the eluation fluid. The temperature achieved with the vortex thermocontroller without preheated solutions was recorded in a pilot experiment (see Fig. 1).

Figure 1

Release of immunoreactive calcitonin gene-related peptide (iCGRP) measured with an ELISA in the hemisected rat skull preparation. CGRP content after incubation periods of 5 min at the given temperatures, normalized to the value of the first incubation period (at 5 min). ▪, Control experiments; open symbols show modulation of iCGRP release by different R(+)-methanandamide (mAEA) concentrations, added in the second and the subsequent samples. ∗Significant differences between mAEA 1 µm and control. Inset: Modulation of iCGRP release by mAEA in the second incubation period at basal temperature. Lower panel: Temperature measured on the surface of the dura without preheated solutions in a pilot experiment.

Control experiments without any chemical stimulation (n = 9) were performed to measure heat-evoked immunoreactive CGRP (iCGRP) release from meningeal afferents. To study the modulation of iCGRP release by TRPV1 and CB1 receptor activation at the three temperatures, agonists and antagonists at these receptors were added in subsequent experiments to the HEPES-SIF in the second through fifth 5-min incubation periods. Methanandamide (mAEA) was used at concentrations of 10 nm, 100 nm and 1 µm. The 100-nm and 1-µm mAEA solutions were additionally applied in the presence of the TRPV1 antagonist capsazepine (10 µm) and the 100-nm mAEA solution was also combined with iodo-resiniferatoxin (200 nm). The CB1 receptor antagonist SR141716 (200 nm) was applied either alone or combined with mAEA (100 nm) in separate experiments. SR141716 was added throughout the whole experiment and a preceding 5-min period without SR141716 served as a control.

Enzyme-immunoassay for iCGRP

The method for detecting CGRP in the eluate has been previously published in detail (24). Briefly, incubation fluids were processed immediately after the experiment using commercial iCGRP enzyme-linked immunoassay (EIA) buffers and kits (Cayman, distributed by SPIbio, Paris, France). Peptide degradation was prevented by an EIA buffer that contains peptidase inhibitors. The antibody in the iCGRP-EIA kit is directed against human CGRP α/β but has 100% cross-reactivity with rat and mouse CGRP. The iCGRP detection level in this assay is about 2 pg/ml. All iCGRP values measured in the present experiments were at least 10 times above the detection limit. The released iCGRP concentration is given in pg/ml of fluid evacuated from the skull cavity.

Substances

mAEA was obtained from Biotrend (Cologne, Germany) and diluted in saline from a 10-mm stock solution in pure dimethylsulphoxide (DMSO), and iodo-resiniferatoxin and capsazepine (Sigma-Aldrich, Schnelldorf, Germany) were dissolved in pure DMSO and ethanol, respectively. In the final solutions the primary solvents were below one per thousand. SR141716 (25) was courtesy of Sanofi-Synthelabo.

Data analysis

In all experiments iCGRP concentrations were normalized to the value of the first incubation period (basal release). Subsequent measurements within the same groups of experiments were compared using repeated measures analysis of variance (anova) followed by a Bonferroni corrected post hoc Wilcoxon matched pairs test. Differences in iCGRP release caused by experimental substances were analysed using two-factorial anova with repeated measures followed by Fisher's least significant difference (LSD) test. All tests were performed by the Statistica^® 7 software package (StatSoft, College Station, TX, USA). All values are given as mean ± standard error of the mean unless stated otherwise. Differences were considered significant at P < 0.05 and are marked by asterisks in the figures.

Results

In total, 106 experiments were performed using skull halves of 53 male Wistar rats (296 ± 24 g). The left and the right skull half from one and the same animal were used for parallel experimental groups. The grand mean of iCGRP release in the first incubation period (basal release) was 41 ± 3 pg/ml.

Basal and heat-induced iCGRP release (control experiments, Fig. 1)

The stimulated iCGRP release was temperature dependent (P < 0.001, n = 9, d.f. = 4, Friedmann anova). Wilcoxon post hoc tests over two sampling periods revealed a stable CGRP release at a temperature of 32°C (P = 0.26, first sample 43 ± 4 pg/ml, second sample 45 ± 4 pg/ml). At a temperature of 39°C, iCGRP release increased to 174 ± 16% of the basal value (P = 0.008). At 45°C, iCGRP release further increased to 529 ± 64% (P = 0.008), which was a 3.1-fold increase compared with 39°C (P = 0.008).

Modulation of iCGRP release by methanandamide (Fig. 2)

mAEA at 10 and 100 nm (n = 9 and n = 10) did not alter the temperature-dependent iCGRP release (repeated measures anova). Although there was a tendency towards a decrease in iCGRP release at 32°C and 39°C, there was no significant difference at any temperature when compared with the control experiments. At the concentration of 1 µm, however, mAEA (n = 7) modulated iCGRP release significantly (P = 0.022, repeated measures anova): at 32°C iCGRP release decreased to 77.5% of the basal release (P = 0.012, Fisher's LSD post hoc test). At 39°C neither an inhibitory nor an excitatory effect of mAEA could be detected. Finally, at 45°C mAEA caused a 62.5% increase in iCGRP release compared with the control experiments (P = 0.011, Fisher's LSD post hoc test).

Figure 2

(a) Changes in immunoreactive calcitonin gene-related peptide (iCGRP) release displayed normalized to control experiments (dotted line) at the temperatures shown. □, Effects of R(+)-methanandamide (mAEA) (100 nm) on iCGRP release. The cannabinoid antagonist SR141716 (200 nm) facilitated iCGRP release (hatched bars), further increased by the addition of mAEA 100 nm. ∗P < 0.05 vs. control. (b) Changes in iCGRP release displayed in relation to control experiments (dotted line) at the temperatures shown. □, Effects of mAEA (1 µm). At both temperatures the addition of capsazepine (10 µm) (hatched bars) reduced iCGRP release. Results are given as mean ± SEM. P < 0.05 between experimental groups(†) or in comparison with control (∗), respectively.

Modulation of iCGRP release by CB1 antagonist and methanandamide (Fig. 2a)

At 32°C the iCGRP release was not changed by the cannabinoid receptor (CB1) antagonist SR141716 at 200 nm concentration. During heat stimulation (39°C; 45°C), iCGRP release was higher in the presence of SR141716 (n = 8) than in the control experiments (P = 0.009, two-factorial anova, Fisher's LSD post hoc test). The combination of mAEA (100 nm) with SR141716 (200 nm; n = 12) tended to increase further the heat-induced iCGRP release compared with SR14176 alone (P = 0.056, two-factorial anova, Fisher's LSD post hoc test). Data are shown normalized to iCGRP levels of controls obtained at the same temperatures (Fig. 2a).

Modulation of iCGRP release by TRPV1 antagonists and methanandamide (Fig. 2b)

The effect of mAEA (100 nm and 1 µm) on heat-induced iCGRP release was studied in the presence of the TRPV1 receptor antagonist capsazepine. Since capsazepine has recently been reported to exert partial agonistic effects through TRPV1 receptors (26), capsazepine (10 µm) was applied at 32°C, but did not significantly change iCGRP release (P = 0.31, U-test, data not shown). In contrast, the TRPV1 receptor antagonist iodo-resiniferatoxin increased iCGRP release at 32°C to 140% of the basal value in pilot experiments (P = 0.005, U-test). Due to this partial agonistic effect on TRPV1 receptors, iodo-resiniferatoxin was no longer used. In the presence of 10 µM capsazepine, 100 nm mAEA had no effect on heat-induced iCGRP release (n = 10, data not shown). However, at 39°C and 45°C the increase in iCGRP release caused by 1 µm mAEA was significantly lower in the presence (n = 7) of capsazepine (P < 0.001, two-factorial anova, Fisher's LSD post hoc test), namely it was 43% less at 39°C and 52% less at 45°C (P = 0.019 and P = 0.010, Fisher's LSD post hoc test).

Discussion

In the hemisected rat skull preperation, CGRP release from the cranial dura was used as an index to assess the activation of meningeal primary afferents. The stimulated CGRP release is known to depend on the activity of primary peptidergic afferents and the presence of extracellular Ca²⁺ (unpublished data from our laboratory). In our experiments CGRP was released in a temperature-dependent manner, showing a small but significant increase in release when the temperature was changed from the basal level to mild heat (39°C) and a marked increase when it was changed to noxious heat (45°C). In the present experiments administration of the cannabinoid mAEA (1 µm) reduced CGRP release at the basal temperature of 32°C and facilitated CGRP release at the noxious temperature of 45°C, which suggests the presence of two opposite mechanisms induced by mAEA, as discussed below.

Functional significance of CB1 and TRPV1 receptors in meningeal afferents

CB1 receptors have been found immunohistochemically on peripheral endings (27), cell bodies (10, 28) and central endings of primary afferents (27, 29, 30). CB1 receptors have additionally been localized in second-order neurons in the spinal cord (27, 31) and the trigeminal nucleus caudalis (32). TRPV1 receptors, particularly located on peripheral endings, have been identified in a large majority of C-fibre nociceptive neurons (33). Up to 75% of mainly small dorsal root ganglion cells have been shown to coexpress CB1 and TRPV1 receptors (8, 10).

AEA has been shown to activate both CB1 receptors (34–36) and TRPV1 receptors (4, 5, 36). In trigeminal neuron cultures, 70% of the neurons responded to the TRPV1 receptor agonist capsaicin (1 µm) with an increase in free intracellular calcium concentration (38), which is generally believed to be a precondition for neuropeptide release. There is no direct evidence yet that TRPV1 activation by cannabinoids induces the same intracellular effects as does capsaicin; however, labelled AEA is displaced by capsaicin from TRPV1 receptors, suggesting that the binding sites of both agonists are identical (39).

Opposing effects of methanandamide

The CGRP release measurements showed differential and partly opposing effects of the cannabinoid on meningeal afferents. In general, apart from species differences, lower concentrations of AEA activate preferably CB1 receptors, while higher concentrations activate TRPV1 pathways. In cultured primary sensory neurons, AEA reduced CGRP release at nanomolar concentrations but increased CGRP release at low micromolar concentrations. In such cultures the inhibitory effect of AEA on CGRP release was abolished by the CB1 receptor antagonist SR141716, while the stimulating effect of AEA was blocked by capsazepine (12). This reversal of AEA effects was also seen in other preparations, albeit with different effective concentrations. In an isolated rat trachea preparation, 1 µm AEA had no effect, 10 µm AEA was inhibitory, while 50 and 100 µm AEA facilitated neuropeptide release (14).

Stimulus-dependent formation of endogenous cannabinoids

In the presence of the CB1 receptor antagonist SR141716, iCGRP levels were significantly increased during heat stimulation, but not at basal temperature. This may be explained by a stimulus-induced formation of endogenous AEA from arachidonic acid in the cell membrane. Stimulated endogenous AEA formation may explain why inhibition of the CB1 receptors facilitated the 45°C-induced neuropeptide release (53% increase), whereas basal iCGRP release at 32°C was unchanged. We assume that without SR141716 this facilitation of CGRP release caused by the endogenous cannabinoid may be balanced by an inhibitory effect resulting from CB1 receptor activation in meningeal nerve endings (Fig. 2a, 45°C, open bar).

Effective concentrations of endogenous cannabinoids

Neuronal AEA formation depends on an intracellular Ca²⁺ increase (40), which may reach effective concentrations only at noxious temperatures. Under non-noxious conditions endogenous AEA concentration may be too low to exert any significant effect that could be detected by CB1 receptor inhibition in our experiments. With unstimulated primary sensory neurons in culture, AEA concentrations have been reported to be below the detection level of 0.05 µm, while capsaicin increased the AEA concentration to about 2 µm in the supernatant (41). The EC₅₀ of AEA at TRPV1 receptors of primary sensory neurons is in the range of 3–10 µm (42). AEA levels measured in the skin are obviously high enough to activate cannabinoid receptors (43), but it is unknown whether these concentrations also activate TRPV1 receptors.

In our preparation, mAEA at concentrations <1 µm did not change iCGRP release. We therefore assumed that at these low doses the inhibitory and stimulating effects of mAEA may have been balanced. To test this assumption, pharmacological tools were used to unmask either one of the opposing effects of exogenous mAEA. When 100 nm mAEA was added in the presence of SR141716, which blocked the CB1 receptors, stimulated iCGRP release tended to be greater than with SR141716 alone (Fig. 2a, black vs. hatched bars). We assume that inhibition of CB1 receptors unmasked the pronociceptive effect of mAEA, namely iCGRP release through TRPV1 activation.

Using intravital microscopy, it has been demonstrated in vivo that vasodilation of dural blood vessels, which is known to be primarily a function of CGRP release (44, 45), can be induced through activation of TRPV1 receptors by anandamide (46, 47). In our experiments, capsazepine reduced iCGRP release induced by 1 µm mAEA at 39°C and 45°C, therefore we conclude that TRPV1 receptors mediated the facilitation of heat-induced iCGRP release by mAEA.

Role of the endocannabinoid system in meningeal nociception

Analogues of anandamide are of potential interest for the treatment of patients. It is of note that cannabinoids may regulate neuronal activity not only at the peripheral but also at the central terminals of primary trigeminal afferents. There is clear evidence that functional cannabinoid receptors exist also at these central terminals. In a rat brainstem preparation, a presynaptic inhibitory effect on signal transmission has been shown using the cannabinoid agonist WIN 55,212–2 (48). The functional presence of CB1 and TRPV1 receptors at the first synapse in the afferent signal transduction has also been found for the rat spinal dorsal horn. CB1 receptors mediate presynaptic inhibition, thereby reducing capsaicin-induced transmission (36). Further studies are necessary to clarify the functional relevance of both peripheral and central sites of cannabinoid action.

The opposing effects of the endocannabinoids indicate that CB1 receptors may be part of a peripheral regulatory system that contributes to the balance of pro- and antinociceptive mechanisms at the peripheral terminal. To evaluate systemic cannabinoid actions in this respect, it will be of interest to investigate the effects of intravenous application of anandamide or analogues on trigeminal afferent signalling in the brainstem, thalamus and cortical pain-processing areas.

Acknowlegments

We acknowledge Dr Lothar Kohlloeffel for constructing the vortex thermode, Dr Peter Reeh for reading the manuscript and Iwona Izydorczyk and Annette Kuhn for technical assistance. Supported by BMBT (German Headache Consortium).

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Cannabinoid and Vanilloid Effects of R(+)-Methanandamide in the Hemisected Meningeal Preparation

Abstract

Keywords

Introduction

Methods

Neuropeptide release from hemisected skull

Experimental protocols

Enzyme-immunoassay for iCGRP

Substances

Data analysis

Results

Basal and heat-induced iCGRP release (control experiments, Fig. 1)

Modulation of iCGRP release by methanandamide (Fig. 2)

Modulation of iCGRP release by CB1 antagonist and methanandamide (Fig. 2a)

Modulation of iCGRP release by TRPV1 antagonists and methanandamide (Fig. 2b)

Discussion

Functional significance of CB1 and TRPV1 receptors in meningeal afferents

Opposing effects of methanandamide

Stimulus-dependent formation of endogenous cannabinoids

Effective concentrations of endogenous cannabinoids

Role of the endocannabinoid system in meningeal nociception

Acknowlegments

References