Both natural and synthetic cannabinoids (CBs) acting on G protein–coupled CB1 and CB2 receptors can suppress responses to acute and persistent noxious stimulation.1 CB1 receptors are expressed mainly in the central nervous system and in peripheral tissues.2,3 CB2 receptors occur predominantly peripherally in immune cells, but recently they have also been found in the brain, spinal cord, and in the dorsal root ganglia (DRG), mainly on glial cells.1,4 – 7 A major limitation to the use of CB for therapeutic purposes is the profile of side effects (such as dysphoria) and potential abuse. An alternative approach, which may prevent such side effects, is to influence the endogenous CB system.
Hemopressin (HP), a nonapeptide (H-PVNFKFLSH-OH), is a product of the hemoglobin α chain, discovered in rat brain and so named because it can cause small decreases in arterial blood pressure.8,9 A number of in vitro studies show that HP acts as a CB1 receptor inverse agonist, and it can act on both peripheral and central pain pathways in vivo.10 – 12 These studies showed that HP pretreatment caused antinociceptive effects at systemic, local, and spinal levels. The authors proposed that after CB1 blockade by the inverse agonist HP, the released endocannabinoids might induce antinociception by interfering with other pain transmission mechanisms.12 Our primary goal was to synthesize this peptide and to determine the effects of HP posttreatment on the mechanical pain threshold in a joint inflammation model at the spinal level.
The family of endocannabinoids comprises several polyunsaturated fatty acid derivates, such as N-arachidonoylethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG).13,14 Anandamide was characterized as an endogenous eicosanoid with moderate affinity for the CB1 and CB2 receptors.13 However, anandamide activates other receptors as well, including the capsaicin-sensitive transient receptor potential vanilloid 1 channels (TRPV1), and some of its effects (such as antinociception) may be at least partially attributed to TRPV1 activation.15 – 23 As for the lipid derivative 2-AG, it is a full agonist of CB1 and CB2 receptors with no direct binding to the TRPV1 receptor.14 Some studies have investigated its antinociceptive potency at systemic and peripheral levels.14,24 – 27 However, there are no data either about the effect of 2-AG after intrathecal administration, or about the effect of anandamide on mechanical allodynia at the spinal level. Therefore, our secondary goal was to determine the antinociceptive potency of 2-AG and anandamide in the above-mentioned circumstances. Finally, we also sought to determine the consequences of the administration of synthetic CB1 and CB2 antagonists and HP on the effects of 2-AG.
Amino acid derivatives and resins were purchased from Bachem AG (Bubendorf, Switzerland) and Sigma (St. Louis, MO); coupling agents were from Calbiochem–Novabiochem AG (Läufelingen, Switzerland). All other chemicals and solvents were of analytical grade from commercial sources.
The following drugs were administered in the in vivo experiments: λ-carrageenan (Sigma-Aldrich Ltd., Budapest, Hungary), 2-AG (Tocris Bioscience, Bristol, UK), anandamide (Sigma-Aldrich Ltd.), AM 251 (CB1 receptor antagonist; Tocris Bioscience), and SSR144528-2 (SSR) (CB2 receptor antagonist; a generous gift from Sanofi Aventis, Paris, France).
Carrageenan and HP were dissolved in physiological saline. Anandamide and 2-AG were dissolved in ethanol: Tween = 2:1, respectively. Stock solutions were diluted with saline to a final ethanol concentration of 10%. AM 251 and SSR were dissolved in dimethyl sulfoxide (Sigma-Aldrich, Ltd.) and ethanol and it was further diluted with distilled water. The concentration of dimethyl sulfoxide and ethanol was 15% and 9%, respectively. Intrathecally administered drugs were injected over 120 seconds in a volume of 10 μL, followed by a 10-μL flush of physiological saline.
In Vitro Experiments: Synthesis of HP
HP (H-Pro-Val-Asn-Phe-Lys-Phe-Leu-Ser-His-OH) was prepared by in situ neutralization solid-phase peptide synthesis on Boc-His(Tos)-PAM (0.46 mmol/g loading) resin using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide (TBTU) as coupling agent. Completion of the couplings was tested by the Kaiser test. Removal of the orthogonal protecting groups and cleavage of the peptide from the resin were achieved by anhydrous hydrogen fluoride (10 mL/g peptide-resin) in the presence of 10% (v/v) anisole and 10% (v/v) dimethyl sulfide at 0°C. The crude peptide was precipitated with diethyl ether from the trifluoroacetic acid (TFA) solution and then purified by reversed phase high-performance liquid chromatography (RP-HPLC) on a Vydac 218TP1010 semipreparative column (250 × 10 mm, 12 μm; Grace, Deerfield, IL) with a gradient of acetonitrile (0.08% TFA) in water (0.1% TFA). The purity of the peptide was examined by analytical RP-HPLC and its molecular weight was confirmed by electrospray ionization mass spectrometry.
In Vivo Experiments
The animal surgery and testing procedures were approved by the Institutional Animal Care Committee of the University of Szeged, Faculty of Medicine. Male Wistar rats (weight, 232 ± 2.0 g) were anesthetized with a mixture of ketamine hydrochloride and xylazine (72 and 8 mg/kg intraperitoneally, respectively). An intrathecal catheter (PE-10 tubing, inside diameter 0.28 mm; outside diameter 0.61 mm; Intramedic, Clay Adams, Becton Dickinson, Parsippany, NJ) was inserted through the cisterna magna and passed 8.5 cm caudally into the subarachnoid space28 to place the catheter tip between the vertebrae T12 and L2, corresponding to the spinal segments that innervate the hindpaws.29 After the surgery, the rats were housed separately, and they had free access to food and water. Rats exhibiting postoperative neurologic deficits (approximately 10%) or those that did not show paralysis of one of the hindpaws after 100 μg lidocaine were excluded.29 The rats were allowed to recover for at least 4 days before the testing and they were assigned randomly to the treatment groups (6–15 rats per group). The observer was blinded to the treatment administered in all cases.
Inflammation was elicited by injecting carrageenan (300 μg/30 μL) into one of the tibiotarsal joints (on the paralyzed side during lidocaine administration).29,30 Carrageenan was given to gently restrained conscious animals, using a 27-gauge needle, without anesthesia, so as to exclude any drug interaction. These injections did not elicit any sign of major distress. This way hyperalgesia was induced, peaking at 2 to 3 hours after the injection. To determine the changes in the size of the inflamed joint, we measured the anteroposterior and mediolateral diameter of the paw at the level of the ankle joint with a digital caliper. The cross-sectional area was calculated with the formula a × b × π, where a and b signify the radius in the 2 aspects.
Behavioral Nociceptive Testing
The threshold of withdrawal from mechanical stimulation to the plantar aspect of the hindpaws was assessed using the Dynamic Aesthesiometer apparatus (mod-37450; Ugo Basile, Comerio, Italy), which consists of an elevated wire mesh platform to allow access to the ventral surface of the hindpaws. Before baseline testing, each rat was habituated to the testing box for at least 20 minutes. A steel rod (diameter 0.5 mm) was pushed against the hindpaw with ascending force. The force ranged from 0 to 50 g over an 8-second period. When the animal withdrew the hindpaw, the mechanical stimulus was automatically stopped, and the force at which the animal withdrew the paw was recorded at 0.1-g correctness.
After baseline determination of joint diameter and mechanical paw withdrawal threshold (precarrageenan baseline value at −180 minutes), carrageenan was injected. These measurements were performed again 3 hours after carrageenan injection (postcarrageenan baseline values at 0 minutes). After postcarrageenan baseline determination, HP (0.3–30 μg), 2-AG (1–200 μg), or anandamide (10–200 μg) was given intrathecally, and mechanical sensitivity was defined at 10, 20, 30, 45, 60, 75, 90, and 105 minutes postadministration. The control group received physiological saline (vehicle of HP) or vehicle of 2-AG/anandamide (see Methods). Because vehicle-treated groups did not differ from the saline-treated, we merged the data of these animals.
To determine the involvement of CB1 and CB2 receptors in the effects of 2-AG, separate groups of animals were pretreated with AM 251 (antagonist of CB1 receptors, 10 μg) or SSR (antagonist of CB2 receptors, 15 μg) 20 minutes before 200-μg 2-AG injection. The control group was injected with vehicles of 2-AG and CB antagonists. To investigate the potential antagonistic effects of HP on the 2-AG–induced antinociception, we coadministered 3 or 30 μg HP with 200 μg 2-AG.
At the end of the experiment, the joint diameters were measured again. As for the behavioral changes, we did not observe any sign of altered behavior (i.e., immobility, flaccidity, excitation, or motor weakness), except for when 100 or 200 μg of anandamide was administered. Anandamide in these high doses caused temporary (during the injection) vocalization and excitation, suggesting a pain-inducing potential of anandamide.23,25 Animal suffering and the number of animals per group were kept at a minimum.
Data are presented as means ± SEM. Data sets were examined by repeated measures analysis of variance (ANOVA). Post hoc comparisons were performed with the Fisher LSD test. A P value <0.05 was considered significant. Data analyses were performed with the STATISTICA for Windows software (Statistica Inc., Tulsa, OK).
In Vitro Experiments: Synthesis
HP was prepared by manual solid-phase peptide synthesis using in situ neutralization Boc chemistry. The peptide was purified to homogeneity by semipreparative RP-HPLC and its molecular weight was confirmed by mass spectrometry. To investigate the hydrolytic stability of the peptide, it was dissolved in phosphate-buffered saline and the resulting solutions were incubated at ambient temperature for 10 hours. During the incubation period, samples were taken and analyzed by RP-HPLC. HP was found to be stable, because there were no impurities or hydrolytic fragments detected by RP-HPLC.
In Vivo Experiments
Three hours after the injection of carrageenan into the ankle, there was a significant (P < 0.01) increase in joint cross-sectional area compared with preinjection control levels (from 36 ± 0.1 mm2 to 73 ± 0.5 mm2). This conspicuous increase in joint size was a result of edema formation, confirming that carrageenan treatment resulted in an inflammatory reaction. None of the treatments influenced the degree of edema; the cross-section of the ankle was 72 ± 0.5 mm2 at the end of the experiments, which did not differ from the postcarrageenan baseline value (to 73 ± 0.5 mm2).
The basal mechanical withdrawal threshold was 45 ± 0.4 g, and carrageenan did cause a significant decrease in paw withdrawal threshold on the inflamed side (10 ± 0.3 g), but it did not have a significant influence on the noninflamed side. None of the treatments changed the mechanosensitivity on the normal side; therefore, results were analyzed only on the inflamed paws.
HP caused neither a significant antiallodynic effect compared with the control group, nor were any motor impairments observed in this wide dose range (0.3–30 μg) (Fig. 1A).
2-AG by itself produced a dose-dependent antiallodynic effect, which developed gradually, and reached a maximum between 45 and 60 minutes (Fig. 1B). ANOVA with repeated measures showed significant effects of treatment (F 4,48 = 4.7, P < 0.005) and time (F 9432 = 94.3, P < 0.001). Thus, 1 μg 2-AG was ineffective, whereas 200 μg caused a prolonged antinociceptive effect.
Anandamide elicited a dose-dependent antinociceptive effect, which reached maximum at approximately 20 minutes after administration (Fig. 1C). ANOVA with repeated measures showed significant effects of treatment (F 4,47 = 5.2, P < 0.005), time (F 9423 = 68.5, P < 0.001), and interaction (F 36,423 = 1.9, P < 0.005). Thus, 10 μg anandamide was ineffective, whereas 200 μg caused a prolonged effect.
Regarding the effects of antagonists AM 251 and SSR at CB1 and CB2 receptors, respectively, none of these substances alone influenced the pain threshold (Fig. 2, A and B). AM 251 pretreatment antagonized the antiallodynic effect of 2-AG (200 μg), whereas SSR did not influence it (Fig. 2, A and B). Cotreatment of 3 μg or 30 μg HP with 200 μg 2-AG significantly decreased the antinociceptive effect of 2-AG (Fig. 2, C and D).
Our results showed that HP is a stable peptide, and its intrathecal administration after induction of joint inflammation does not influence mechanical allodynia in a wide dose range, but it does inhibit the antinociceptive effects of 2-AG.
Only a few studies investigated the in vivo and in vitro characteristics of HP. Conformation-state sensitive antibodies were used for the investigation of binding characteristics of HP to different opioid, CB, adrenergic, bradykinin, and angiotensin receptors in cell lines and striatum.12 It has been found that HP is an inverse agonist of CB1 receptors, thus HP is able to block the constitutive activity of CB1 but not CB2 receptors.12 A recent study demonstrated that HP can antagonize CB1 agonist-induced internalization of the CB1 receptors in vitro.10 As for the few earlier in vivo results, it was observed that HP causes hypotension by activation of nitric oxide release,8,9,31 and it induces hypophagia only in mice with functional CB1 receptors.10
Regarding the antinociceptive potency of HP, Dale et al.11 found that intraplantarly administered HP (0.1–20 μg) did not affect the paw pressure threshold in the noninflamed paws, but cotreatment with carrageenan or bradykinin significantly decreased the development of mechanical allodynia, as measured with the paw pressure test, and the effect was not inhibited by an opioid antagonist. Because the contralaterally administered HP was also effective in this respect, the data suggest systemic effects of the ligand. Orally (50 or 100 μg/kg) or intrathecally (0.5 or 5 μg) administered HP pretreatments were also effective in the same test.12 Intraperitoneally administered HP (50 or 500 μg/kg) exhibited marked antinociceptive potency in the acetic acid–induced visceral nociception model. This high dose of HP did not impair motor activity or alter pentobarbital-induced sleeping time, indicating the absence of unwanted sedative or motor side effects. Unfortunately, we did not observe similar antinociceptive effects in our model. It is possible that the controversial results might be attributable to differences in the timing of administration. That is, we applied HP after the mechanical allodynia had been established (posttreatment), whereas earlier studies prevented the development of the hyperalgesia (pretreatment). Furthermore, there were differences regarding either the applied pain test (paw pressure versus von Frey) or the site of administration of carrageenan (intraplantar versus intrajoint administration). In agreement with our results, the latest evidence suggests inefficacy of HP at the spinal level in an acute heat pain test and in a neuropathic pain model.32,33 As for the HP pretreatment before formalin administration, a low dose of HP (3 μg) decreased, but a higher dose (10 μg) enhanced the formalin-induced nocifensive behavior. The authors observed the inefficacy of HP as an antagonist after CB1 receptor activation. This is in contrast with our results, because HP, similarly to the synthetic CB1 antagonist, antagonized the antinociceptive effect of 2-AG in our study. We suppose that the differences in the pain models and the applied CB ligand (WIN 55,212-2 versus 2-AG) might be the explanation for the different results.
Spinally administered anandamide and 2-AG significantly decreased mechanical inflammatory pain sensitivity. The use of CB for the management of a wide range of painful disorders has been well documented at spinal, supraspinal, and peripheral levels,1,4,34 whereas endogenous ligand data are scarce, especially at the spinal level. Earlier studies showed that intrathecal anandamide decreased acute heat pain sensitivity (in hotplate and tail-flick tests) and carrageenan-induced thermal hyperalgesia in rodents, and that both the CB1 and TRPV1 receptors have a role in these effects.23,35,36 To our knowledge, we are the first to offer evidence to suggest that anandamide inhibits mechanical allodynia at the spinal level as well. Because several systems may be influenced by anandamide (e.g., CB, TRPV1, glycine, and serotonin-3 receptors), their net effect may be observed under these circumstances.15,16,37 – 40 Because the high dose of anandamide caused temporary pain, the desensitization of TRPV1 receptors can also be involved in its antinociceptive effect, as suggested earlier.18,23 Therefore, it is possible that alterations in the release of excitatory and inhibitory transmitters can modify the activation of projection neurons, either presynaptically from primary sensory neurons or postsynaptically from interneurons, or both.
2-AG, similarly to anandamide, reduced allodynia in the carrageenan-induced arthritis model, and its antinociceptive effect was inhibited by a CB1 antagonist, whereas it was not influenced by a CB2 antagonist. This is the most abundant endogenous CB, and its concentration in the brain is 50 to 500 times higher than that of anandamide. It has also been identified peripherally.2,41 2-AG is a full agonist for CB1 and CB2 receptors with no direct binding to the TRPV1 receptor.14 It is also a substrate for cyclooxygenase-2, and 2-AG is capable of suppressing an increase of cyclooxygenase-2 expression by activating the CB1 receptors.42,43 There is only little evidence to support the antinociceptive potency of 2-AG. Endogenous 2-AG has been implicated as a major transmitter involved in endocannabinoid-mediated stress-induced analgesia.44,45 Thus 2-AG, but not anandamide, is mobilized in the lumbar spinal cord after exposure to footshock stress, and spinal 2-AG levels show marked correlation with stress-induced antinociception.45,46 Additionally, intrathecal administration of an inhibitor of the 2-AG hydrolyzing enzyme, monoacylglycerol lipase, enhances stress-induced antinociception in a CB1-dependent manner.45 In systemic administration to mice, 2-AG (50% effective dose = 12.5 mg/kg) caused antinociception in acute pain tests, immobility, reduction of spontaneous activity, and decrease of rectal temperature.14,24 Topical administration of 2-AG also decreased the nocifensive behavior in a formalin test, decreased mechanical allodynia and thermal hyperalgesia in a neuropathic pain model, and it was also effective in the alleviation of inflammatory joint pain.25 – 27 The local antinociceptive effects of 2-AG were prevented by CB1 and/or CB2 antagonists.25 – 27 As far as the spinal level is concerned, we are the first to show its antinociceptive potency, and that the effect is reversed by a CB1 antagonist drug (but not by a CB2 antagonist), suggesting that the antiallodynic effect of 2-AG is mainly attributable to the activation of CB1 receptors at the spinal level. CB1 receptors, the molecular targets of 2-AG, are located on primary afferent fiber endings and/or on intrinsic interneurons in the dorsal horn of the spinal cord47,48; therefore, their activation could have led to the observed antinociception.
It is important to consider that these ligands can influence the activity of neurons in DRG too, because the CB receptors can be found on DRG neurons,49,50 and it has been shown that intrathecal injection of sodium fluorescein results in massive staining in the DRG both in the cellular and fiber portions.51 As for the ineffectivity of CB1 and CB2 antagonists alone on inflamed and noninflamed sides, a number of scenarios may be suggested. First, it might be supposed that the mechanical pain threshold after carrageenan administration (approximately 10–15 g) is a very low value, which could not be further decreased by an antagonist. However, the threshold on the normal side did not change either; therefore, this is not likely. Another possibility is that the endogenously released CBs have no significant inhibitory effect on the mechanical threshold in inflammatory circumstances, either on the normal or on the inflamed side. Similar results were found in a bone cancer–induced pain model52; however, other studies have shown that intrathecal injection of CB1 receptor antagonists can evoke nociceptive responses.53,54 It is assumed that the differences in the pain models can lead to these controversial findings. However, the level of the released endogenous CBs was not determined in our study; therefore, it cannot be decided whether this is attributable to lack of production or lack of effect of endogenous CB agonists.
In conclusion, we found that HP was not capable of influencing the established mechanical allodynia in a model of arthritic pain, but inhibited the antinociceptive effects of 2-AG at the spinal level. Furthermore, these findings are the first to demonstrate the antinociceptive potency of 2-AG at the spinal level, and the effect of anandamide on mechanical allodynia in an arthritic pain model.
Name: Zita Petrovszki, MSc.
Contribution: This author helped conduct the study, analyze the data, and write the manuscript.
Attestation: Zita Petrovszki has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Gyula Kovacs, MD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Gyula Kovacs has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Csaba Tömböly, MSc, PhD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Csaba Tömböly has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: György Benedek, MD, DSc.
Contribution: This author helped design the study and write the manuscript.
Attestation: György Benedek has seen the original study data and approved the final manuscript.
Name: Gyongyi Horvath, MD, DSc.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Gyongyi Horvath has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Quinn Hogan, MD.
The authors thank Agnes Tandari for her technical assistance.
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