Research report 741
Interaction of the adenosine A1 receptor agonist N6-cyclopentyladenosine and κ-opioid receptors in rat spinal cord nociceptive reflexes Guillermo A. Ramos-Zepeda, Carlos Herrero-Zorita and Juan F. Herrero Antinociception induced by the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) is linked to opioid receptors. We studied the subtype of receptors to which CPA action is related, as well as a possible enhancement of antinociception when CPA is coadministered with opioid receptor agonists. Spinal cord neuronal nociceptive responses of male Wistar rats with inflammation were recorded using the single motor unit technique. CPA antinociception was challenged with naloxone or norbinaltorphimine. The antinociceptive activity of fentanyl and U-50488H was studied alone and combined with CPA. Reversal of CPA antinociception was observed with norbinaltorphimine (82.9 ± 13% of control) but not with low doses of naloxone (27 ± 8% of control), indicating an involvement of κ-opioid but not µ-opioid receptors. Low doses of CPA did not modify fentanyl antinociception. However, a significant enhancement of the duration of antinociception was seen when U-50488H was coadministered with CPA. We conclude that antinociception
Introduction Adenosine A1 receptor agonists such as N6-cyclopentyladenosine (CPA) are effective antinociceptive agents, with an action mainly associated with the spinal cord processing of nociceptive information (Karlsten et al., 1991; Reeve and Dickenson, 1995; Lee and Yaksh, 1996; Sawynok, 1998; Sawynok and Liu, 2003; Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2005). We have previously reported that CPA antinociception is reversed by high doses of the nonselective opioid receptor antagonist naloxone in animals with paw inflammation (Ramos-Zepeda and Herrero, 2013). This implies an interaction between adenosine and opioid systems that might be useful in the treatment of inflammatory pain. The dose of naloxone used in our previous study, however, although clearly indicating an interaction with opioid receptors, was too high to discriminate the subtype of the opioid receptor involved in that phenomenon (Herrero and Headley, 1991). We therefore wondered whether the interaction between CPA and opioid receptors involves µ-opioid or κ-opioid receptors, or even both, as these are the main opioid receptors associated with spinal cord antinociception. The main objective of the present study was to challenge the antinociceptive effect of CPA with low doses of naloxone (selective for µ-opioid receptors in similar experiments; Herrero and Headley, 1991) and with the κ-opioid receptor selective antagonist norbinaltorphimine (nor-BNI; Portoghese et al., 1994). 0955-8810 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
mediated by CPA in the spinal cord is associated with activation of κ-opioid but not µ-opioid receptors in inflammation. In addition, coadministration of CPA and κ-opioid receptor agonists is followed by significantly longer antinociception, opening new perspectives in the treatment of chronic inflammatory pain. Behavioural Pharmacology 25:741–749 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Behavioural Pharmacology 2014, 25:741–749 Keywords: adenosine, inflammation, opioids, pain, rat, single motor unit, spinal cord, wind-up Department of System Biology, Unit of Physiology, Faculty of Medicine, University of Alcalá, Madrid, Spain Correspondenceto Juan F. Herrero, MD, PhD, Department of System Biology, Unidad de Fisiologia, Campus Universitario, Universidad de Alcala, Alcala de Henares, 28871 Madrid, Spain E-mail: [email protected] Received 27 May 2014 Accepted as revised 20 August 2014
In addition, numerous experiments using a similar protocol as that used in the present experiments have shown that the combination of opioids with other drugs may induce supraadditive, or even synergic, antinociception (Gaitán and Herrero, 2002, 2005; Gaitán et al., 2003, 2005). In fact, systemic administration of adenosine or its analogs enhances the analgesia produced by morphine (Ahlijanian and Takemori, 1985; Contreras et al., 1990; Malec and Michalska, 1990). We therefore wondered whether the administration of CPA together with opioid receptor agonists might result in an enhancement of the potency or duration of antinociception. We attempted to address this question, in the second part of our study, by comparing the antinociception observed after the administration of the µ-opioid receptor agonist fentanyl with that after the administration of the κ-opioid receptor agonist U-55488H, alone and in the presence of low doses of CPA, following the same protocol as that adopted in previous studies using the same technique (Curros-Criado and Herrero, 2009; Molina and Herrero, 2011).
Methods Subjects
All the experiments were carried out on adult male Wistar rats, weighing between 250 and 340 g, with carrageenaninduced soft-tissue inflammation. Inflammation was induced by intraplantar injection of 100 μl carrageenan λ (10 mg/ml in distilled water; Sigma-Aldrich, Madrid, Spain) into the right DOI: 10.1097/FBP.0000000000000091
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742 Behavioural Pharmacology 2014, Vol 25 No 8
hind paw 16 h before the experiment under brief halothane anesthesia (5% in oxygen for induction and 2% for maintenance). The effectiveness of carrageenan was studied by measuring the volume of the paw before the induction of inflammation and after the experiment using a plethysmometer (Letica Scientific Instruments, Barcelona, Spain).
review). The mean intensity of electrical stimulation used was 4.7 ± 0.7 mA. Assessment of nociception
All the experimental procedures conformed to the institutional, national, and European guidelines for the use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals were killed with an overdose of sodium pentobarbital at the end of the experiment (Euta-Lender, Normon).
A computer-controlled pincher device (Cibertec, Madrid, Spain) was used to apply noxious mechanical stimuli and to determine the threshold force required to trigger the withdrawal response using constantly increasing pressure ramp stimulation. The intensity of the stimulation used in the experiments was 200 mN above the threshold for each SMU. The mean force used in the experimental groups was always very similar, with an average of 1.23 ± 0.1 N.
Preparatory surgery
Behavioral experiments
The electrophysiological recording procedures have been described in detail previously (Herrero and Headley, 1991; Solano and Herrero, 1997). Preparatory surgery was always performed under halothane anesthesia (5% in oxygen for induction and 2% for maintenance) and included cannulation of the trachea (to keep airways open and ensure correct ventilation), two superficial branches of the jugular veins (for the administration of anesthesia and drugs), and one carotid artery (for monitoring the arterial blood pressure). The core body temperature was maintained at 37 ± 0.5°C using a homeothermic blanket throughout the surgery and the experiment. The animal was transferred after the surgery to a standard frame for electrophysiological recordings. The right hind limb was fixed with plaster onto a Perspex block, halothane was discontinued, and anesthesia was maintained with αchloralose (50 mg/kg for induction and 20 mg/kg/h through a perfusion pump for maintenance; Sigma). The preparation was left to rest for at least 1 h before the experiment started. Blood pressure was monitored constantly, and in all cases the systolic blood pressure was above 100 mmHg before the administration of the drugs.
In the first series of experiments, antinociception observed after the administration of increasing doses of CPA (cumulative log2 doses every 6 min, 10 to 320 μg/kg; Ramos-Zepeda and Herrero, 2013) was challenged with either 1 mg/kg naloxone (n = 7), 100 μg/kg naloxone (n = 9), or 2.5 mg/kg nor-BNI (n = 9). The CPA vehicle was tested in control experiments (n = 3), using the same protocol as that described for the administration of CPA. The effect of 100 μg/kg naloxone on fentanyl-mediated antinociception was tested in a separate experimental group (n = 5). In these experiments, the effect of a single bolus of 16 μg/kg fentanyl was studied in the presence of 100 μg/kg naloxone injected 18 min before bolus administration (see also Herrero and Headley, 1996).
Stimulus presentation and recording systems
Bipolar tungsten electrodes, inserted percutaneously, were used to isolate and record single motor units (SMUs) in muscles of the right hind limb involved in the withdrawal reflex. The units were isolated by moving the electrode with a micromanipulator while a mild pressure was applied to the paw. A window discriminator was also used to ascertain the recording of an SMU. The units were activated in cycles of 3-min duration, each cycle consisting of 10 s of noxious mechanical stimulation (0.2 N above the threshold over an area of 14 mm2) and 16 electrical stimuli applied to the most sensitive area of the cutaneous receptive field of the SMU (20 ms width, 1 Hz, twice the threshold intensity for the recruitment of long latency responses; Herrero and Cervero, 1996a, 1996b; Fig. 1). Original recordings and the protocol of stimulation are illustrated in Fig. 1. Electrical stimulation was used to study the effect of the drugs on the phenomenon of wind-up (see Herrero et al., 2000 for a
In a second series of experiments, a possible enhancement of opioid-mediated antinociception by CPA was studied following the same protocol as above. The effect of cumulative doses of fentanyl (2–32 μg/kg) on SMU nociceptive responses was studied either alone (n = 9) or in the presence of subeffective doses of CPA (10 μg/kg; n = 7), injected intravenously 6 min before fentanyl administration. Similar experiments to study the antinociception induced by cumulative doses of U-50488H (1–16 mg/kg) alone (n = 9) or in the presence of 10 μg/kg CPA (n = 9) were carried out. The doses of all drugs were chosen according to results observed in preliminary experiments and previous reports following the same technique and experimental protocol (Hartell and Headley, 1991; Herrero and Headley, 1991, 1993; Thorn et al., 1994; Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2005, 2013). Drugs
All the drugs used in this study were prepared fresh on a daily basis, before their administration to the rats, and injected intravenously in a total volume of 0.3 ml. CPA (Sigma) was dissolved in 0.5 μg/μl dimethyl sulfoxide (Sigma) and diluted in saline. The administration of each dose of CPA was very slow, over a minimum of 3 min, so as to minimize its effect on blood pressure. The µ-opioid receptor antagonist naloxone, the µ-opioid receptor selective agonist fentanyl, the κ-opioid receptor selective antagonist nor-BNI, and the κ-opioid
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 743
Fig. 1
(a)
CPA 320 μg/kg
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Elec. stimuli
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CPA 320 μg/kg
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20 spikes 50 s Original recordings of two units showing the antinociceptive effect of CPA and its reversal by naloxone or nor-BNI. The figure shows the protocol followed in the experiments, alternating noxious mechanical and electrical stimulation in 3-min cycles of stimulation. The responses of the units are represented as frequency histograms. (a) The cumulative dose of 320 μg/kg CPA induced inhibition of SMU nociceptive responses that was not reversed by a low dose of the opioid receptor antagonist naloxone. (b) CPA antinociception, however, was fully reversed by the κ-opioid receptor selective antagonist nor-BNI. CPA, N6-cyclopentyladenosine; Elec., electrical; nor-BNI, norbinaltorphimine; SMU, single motor unit.
receptor selective agonist U-50488H (all from Sigma) were dissolved and diluted in saline. Data analysis
Data are expressed as a percentage of control (± SEM), where control is the mean of the three responses before the administration of a drug. The quantitative analysis was based on counts of spikes evoked during the two cycles of stimulation between each dose. The data from electrical stimulation were analyzed by counting the number of spikes evoked between 150 and 650 ms after each stimulus (C-fiber responses, Herrero and Cervero, 1996a, 1996b). The procedure of stimulation and collection of data was performed using a computer with commercial software (Spike 2; CED, Cambridge, UK). Statistical comparisons were made with raw data, also using commercial software (GraphPad-Prism and GraphPad-Instat for Windows, San Diego, CA, USA). One-way analysis of variance with post-hoc Dunnett’s test was used for the comparison between mechanical stimulation and control, as well as for the analysis of wind-up curves.
Results Reversal of the effect of CPA by nor-BNI in response to noxious mechanical stimulation
Paw volume increased from an average of 1.4 ± 0.1 to 2.5 ± 0.1 ml (P < 0.001) after plantar injection of carrageenan.
This increment was not modified (2.6 ± 0.1 ml) by the administration of CPA, at the doses tested, which indicates that, under the present experimental conditions, CPA is devoid of anti-inflammatory effects and, therefore, any depression of nociceptive responses is not secondary to a reduction in paw swelling. As in previous reports (Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2013), systemic administration of CPA virtually abolished SMU responses to noxious mechanical stimulation. The minimum effective dose was 80 μg/kg (P < 0.05) in two experimental groups (naloxone 1 mg/kg and nor-BNI) and 160 μg/kg (P < 0.01) in a third group (naloxone 100 μg/kg). The residual activity observed was between 8.2 ± 3 and 21 ± 8% of the control response (Figs 1 and 2a), depending on the group of units studied (P < 0.001 vs. control in all cases). Control experiments, using equivalent doses of vehicle, did not modify SMU activity to noxious mechanical stimulation with the highest dose studied (103 ± 11%; data not shown in figures). The inhibitory action of CPA lasted for a minimum of 53 min (9.5 ± 6%, P < 0.001), studied in those experiments in which the depression of responses was not challenged with an antagonist. As previously reported, the administration of the nonselective opioid receptor antagonist naloxone, at a high
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744 Behavioural Pharmacology 2014, Vol 25 No 8
dose of 1 mg/kg, fully reversed CPA-mediated inhibition of SMU nociceptive responses (95 ± 18%, P < 0.01 vs. CPA effect; Fig. 2a). However, as illustrated in Fig. 1, the reversal effect was not observed after the administration of a low dose of 100 μg/kg naloxone (27 ± 8%; Fig. 2a). In contrast, the administration of the selective κ-opioid receptor antagonist nor-BNI induced full reversal of CPA antinociception (82.9 ± 13%, P < 0.01 vs. CPA effect; Figs 1 and 2a). The reversal effects of nor-BNI and 1 mg/kg naloxone, although starting immediately after their administration, were significant 18 min after their administration (74 ± 9% in the experiments with naloxone, P < 0.01 vs. CPA effect; 72 ± 9% with nor-BNI, P < 0.01 vs. CPA effect). To be sure that the lack of effect of 100 μg/kg naloxone was not due to loss of activity of the antagonist at that time, a single dose of fentanyl (16 μg/kg, intravenously) was injected 18 min after the same dose of naloxone in a different experimental group. The experiments showed no reduction in SMU responses after fentanyl administration (102 ± 9%; Fig. 2a), indicating effective naloxone antagonism at the time tested. Reversal of the effect of CPA by nor-BNI in response to noxious electrical stimulation
Similar to the observed effects on responses to noxious mechanical stimulation, intravenous administration of CPA dose-dependently reduced wind-up (P < 0.01 vs. control, with a CPA dose of 320 μg/kg; Figs 1 and 2b). The administration of equivalent doses of vehicle did not cause any significant change in wind-up (data not shown). As reported previously, the administration of the nonselective opioid receptor antagonist naloxone induced complete reversal of the CPA effect when injected at a dose of 1 mg/kg (data not shown), but no reversal was observed at a dose of 100 μg/kg (Fig. 1). The administration of nor-BNI was followed by full recovery of CPAmediated depression of wind-up in some experiments (Fig. 1), but only a partial recovery, although significant, was seen in others. The average results are shown in Fig. 2b (P < 0.05 vs. CPA effect). Antinociceptive effect of the μ-opioid selective receptor agonist fentanyl alone and in the presence of low doses of CPA
As the first series of experiments indicates a link between CPA and κ-opioid receptors, but not µ-opioid receptors, the possible enhancement of opioid antinociceptive actions in combination with CPA was studied in the following set of experiments. The administration of cumulative doses of fentanyl dose-dependently depressed responses to noxious mechanical stimulation to 13 ± 5% of control (P < 0.01; Fig. 3a) when injected alone [inhibitory dose (ID)50 of 17 ± 1 μg/kg]. When fentanyl was injected together with a subeffective dose of 10 μg/kg CPA, the antinociceptive effect was very similar:
ID50 of 18 ± 2 μg/kg. The dose of CPA studied (10 μg/kg) caused no change on the nociceptive responses when studied alone: 90 ± 5% (Fig. 3a, inset). In all the experiments, the antinociceptive effect observed on administration of fentanyl fully recovered 24 min after the administration of the highest dose studied. The administration of CPA did not modify the duration of the effect of fentanyl. The administration of fentanyl dose-dependently reduced the effect of CPA on wind-up responses, with complete inhibition after the dose of 32 μg/kg (P < 0.01 vs. control; data not shown in figures). The inhibitory action was not different from that seen after the coadministration of fentanyl and 10 μg/kg CPA (data not shown). As in responses to noxious mechanical stimulation, full recovery of the effect was always observed within 24 min after the administration of the drugs in both groups of experiments. Antinociceptive effect of the κ-opioid selective receptor agonist U-50488H alone and in the presence of low doses of CPA
The administration of the κ-opioid receptor selective agonist U-50488H induced dose-dependent inhibition of responses to noxious mechanical stimulation. The maximal effect observed was 2 ± 1% of control (P < 0.001 vs. control; Fig. 3b), with an ID50 of 4 ± 1 mg/kg. A similar depression in responses was observed when U-50488H was studied in the presence of 10 μg/kg CPA: ID50 of 5 ± 1 mg/kg (Fig. 3b). No significant differences were observed between groups. The dose of CPA studied (Fig. 3b, inset) did not reduce nociceptive responses significantly: 89 ± 1.5%. Antinociception induced by U-50488H lasted for a maximum of 45 min when injected alone (88 ± 1% of control; Fig. 4). However, in the presence of CPA the duration of the antinociceptive effect was longer and remained significant 45 min after administration: 40 ± 11% (P < 0.01 vs. control; P < 0.05 vs. U-50488H alone; Fig. 4). Full recovery was never observed 90 min after administration (73.4 ± 5%). Nociceptive responses to high-intensity electrical stimulation were dose-dependently reduced by U-50488H, with complete inhibition after a dose of 16 mg/kg (P < 0.01 vs. control; Fig. 5a). The effect was similar in the presence of CPA (P < 0.01 vs. control; Fig. 5b). However, whereas full recovery was recorded within 45 min after the administration of U-50488H alone, antinociception was still significant 45 min after the administration of the highest studied dose of U-50488H in the presence of CPA (P < 0.01 vs. control; Fig. 5b).
Discussion Two main observations have been made in the present study: (i) the interaction between the adenosine A1
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 745
Fig. 2
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Reversal of the effect of CPA by naloxone and nor-BNI. (a) The figure shows the antinociceptive effect of CPA after the administration of the highest dose (320 μg/kg) in responses to noxious mechanical stimulation. A high dose of naloxone (1 mg/kg) induced full reversal of CPA-mediated antinociception. However, the effect was not observed with a low dose of naloxone (100 μg/kg). The administration of the κ-opioid receptor antagonist nor-BNI was followed by full reversal of CPA antinociception. The reversal effect of naloxone and BNI, although started immediately after administration, was significant 18 min after administration. The effect of a single dose of 16 μg/kg fentanyl was prevented by a low dose of naloxone (100 μg/kg, injected intravenously 18 min before), showing that naloxone was active at the dose and time tested. (b) Wind-up activity to electrical stimulation was also fully depressed by CPA, although in this case the administration of nor-BNI only partially, although significantly, reversed the inhibition of responses. The effect of antagonists and the effect of CPA were statistically compared using the one-way ANOVA, with the post-hoc Dunnett’s test (*P < 0.05; **P < 0.01). ANOVA, analysis of variance; CPA, N6-cyclopentyladenosine; FENT, fentanyl; NAL, naloxone; nor-BNI, norbinaltorphimine.
receptor agonist CPA and κ-opioid receptors, and (ii) enhancement of the duration of antinociceptive activity of the κ-opioid receptor agonist U-50488H when injected together with small doses of CPA. We have previously
shown that CPA is a very effective antinociceptive drug either in in-vitro preparations (Ramos-Zepeda et al., 2004) or in single units recorded in anaesthetized rats (Ramos-Zepeda and Herrero, 2013), using the same techniques as in present
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746 Behavioural Pharmacology 2014, Vol 25 No 8
Fig. 3
(a) 120 100 80
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Antinociceptive effect of (a) fentanyl and (b) U-50488H, in the absence and in the presence of a low dose of CPA. The administration of fentanyl and U-50488H dose-dependently reduced responses to noxious mechanical stimulation. The injection of 10 μg/kg CPA 6 min before did not modify the antinociceptive activity of fentanyl or U-50488H. The inset shows the lack of an effect of CPA at the dose administered. Statistical analysis as in Fig. 2 (*P < 0.05; **P < 0.01; ***P < 0.001, compared with control response). CPA, N6-cyclopentyladenosine.
experiments. In the latter study, we observed that the CPAmediated antinociceptive effect was fully reversed by a high dose (1 mg/kg) of the opioid receptor antagonist naloxone, indicating an interaction of CPA with opioid receptors. Such an interaction is supported by some studies: for example,
adenosine and its analogs potentiated morphine-induced analgesia, tolerance and dependence in tail flick and acetic acid writhing assays (Ahlijanian and Takemori, 1985). In addition, morphine led to the release of endogenous adenosine from the spinal cord in an in-vivo preparation
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 747
discussion). However, there is evidence that CPA and other analogs are highly selective agonists of adenosine receptors (Jacobson et al., 1992; Ralevic and Burnstock, 1998), indicating that a direct interaction between CPA and opioid receptors is not likely. This is supported by other studies that showed a lack of interaction between opioid receptor antagonists and CPA or other adenosine A1 receptor agonists (DeLander and Hopkins, 1986; Aley and Levine, 1997; Borghi et al., 2002, see below). This controversial observation might reflect divergences in the techniques utilized by different groups or, more probably, in the usage of different agonists or antagonists of adenosine and opioid receptors. In fact, our previous experiments (Ramos-Zepeda and Herrero, 2013) showed that CPA-mediated antinociception was reversed or prevented by the µ-opioid receptor antagonist naloxone at a high dose of 1 mg/kg. Some of those experiments were repeated for the present study, giving the same results and confirming this interaction. The dose of naloxone used, however, has been shown to be nonselective for opioid receptors in experiments using the same technique (Hartell and Headley, 1991; Herrero and Headley, 1991). Therefore, the CPA interaction might be related to µ-opioid, κ-opioid, or both populations of receptors in the spinal cord. The present study helps in clarifying this issue as the administration of a low dose of naloxone (100 μg/kg), which has been shown to be selective for µ-opioid receptors under the present experimental conditions (Herrero and Headley, 1991, 1993; Thorn et al., 1994), did not modify CPA-mediated antinociception. This is supported by the lack of effect observed in previous studies when opioid receptor antagonists were injected systemically before, or in conjunction with, CPA
Fig. 4
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Recovery of the antinociceptive effect of U-50488H in the absence and in the presence of CPA. The inhibition of responses to noxious mechanical stimulation always recovered fully between 30 and 45 min after administration of U-50488H. However, the effect was still significant 45 min after the administration of U-50488H in the presence of CPA. Statistical analysis and layout as in Fig. 3. CPA, N6-cyclopentyladenosine.
(Sweeney et al., 1987a, 1987b; Sawynok et al., 1989), and morphine-induced antinociception was increased by pretreatment with adenosine agonists in mouse in-vivo tests (Contreras et al., 1990). In addition, the effect of morphine and other opiates was attenuated dose-dependently by intrathecal pretreatment with the adenosine receptor antagonist caffeine (Cahill et al., 1995; see also Sawynok, 1998 and Sawynok and Liu, 2003 for further references and
Fig. 5
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Recovery of the wind-up depression induced by U-50488H in the absence (a) and in the presence of CPA (b). As in responses to noxious mechanical stimulation, wind-up inhibition observed by the administration of 16 mg/kg of U-50488H fully recovered in all cases within 45 min after the last dose (a; U-50488H 16 mg/kg 45 min). However, a significant depression of wind-up responses was still observed 45 min after the administration of U-50488H, when injected in the presence of CPA (b; CPA+U-50488H 16 mg/kg 45 min). Statistical analysis and layout as for Fig. 2b. CPA, N6-cyclopentyladenosine.
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748 Behavioural Pharmacology 2014, Vol 25 No 8
or other adenosine A1 agonists (DeLander and Hopkins, 1986; Aley and Levine, 1997; Borghi et al., 2002). However, the administration of nor-BNI, a selective κ-opioid receptor antagonist (Portoghese et al., 1994), fully reversed the antinociceptive effects of CPA. It therefore seems clear that the inhibition of nociceptive spinal cord reflexes observed following the administration of CPA results from an interaction between CPA and κ-opioid, but not µ-opioid, receptors under the present experimental conditions. The mechanism of the CPA–κ-opioid receptor interaction does not seem to be related to a direct effect of CPA on opioid receptors, as discussed above and in a previous study (Ramos-Zepeda and Herrero, 2013), as CPA is a very selective agonist of the adenosine A1 receptor (Jacobson et al., 1992; Ralevic and Burnstock, 1998). It seems more likely that the effect was due to secondary activation of opioid receptors, initiated by the interaction of CPA with adenosine receptors (Ramos-Zepeda and Herrero, 2013). Further and different experimental approaches are needed to clarify this point, but this would explain the lack of full recovery of CPA antinociception on challenge with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine in previous experiments (see Ramos-Zepeda and Herrero, 2005, 2013 for further discussion on this point). Our results provide in-vivo evidence that κ-opioid receptors and adenosine A1 receptors are somehow connected in the generation of antinociception, most likely through a common intracellular mechanism of action. It is not possible to deduce the nature of this link from the results obtained in the present experiments. However, as the release of adenosine induced by opioids, through the activation of protein kinase C, has been suggested to explain the relationship between the two systems, a similar connection might explain some of the observations made here (see Sawynok and Liu, 2003 for further discussion on this subject). In contrast, the adenosine A1 receptor has been proposed to exist as a part of a μ-opioid and α2-adrenergic multireceptor complex in peripheral nerve endings (Aley and Levine, 1997; Sawynok, 1998). This type of interaction with κ-opioid receptors might also explain some of our observations. Finally, although the experimental support is not too evident, a pharmacokinetic interaction between CPA and U-50488H should also be considered. It is difficult to comment on whether the effect is fully localized to the spinal cord or whether there is also a peripheral component to the action, presumably localized to inflammation-sensitized nociceptors. Depression of wind-up by CPA, to a level similar to that observed in responses to noxious mechanical stimulation, is in agreement with a spinal cord-mediated action, as windup is a progressive and frequency-dependent facilitation of the responses of spinal cord neurons (see Herrero et al., 2000 for further discussion). However, although full reversal of the CPA effect was observed in responses to
noxious mechanical stimulation, submaximal reversal of wind-up responses was seen in some, but not all, experiments. A partial action localized to the periphery should therefore be considered. Previous observations from experiments similar to those carried out in the present study have shown that subanalgesic doses of drugs enhance antinociception induced by opioids (Gaitán et al., 2003, 2005; Gaitán and Herrero, 2005). We therefore considered that the interaction observed between CPA and opioid receptors might also result in the enhancement of opioid-mediated antinociception. The administration of subanalgesic doses of CPA did not modify the activity of the µ-opioid receptor agonist fentanyl, nor its duration. This is in agreement with our observation of the lack of a relationship between CPA and µ-opioid receptors. It is also possible that the dose of CPA studied was not sufficient to induce a visible effect in our experiments; however, it was the maximum dose possible without a reduction of spinal cord neuronal activity, as observed in the present and previous experiments (Ramos-Zepeda et al., 2004). Coadministration of CPA and the κ-opioid receptor agonist U-50488H did not modify the potency of the antinociceptive activity induced by the latter, as shown by the comparison of ID50 values in the presence and absence of CPA. However, the duration of U-50488Hmediated antinociception was significantly longer in the presence of CPA. In this case, nociceptive activity evoked by noxious mechanical stimulation was only 40 ± 11% of the control response 45 min after the administration of the drug, whereas full recovery was always observed at that time in the absence of CPA. Likewise, the depression of wind-up activity observed after the administration of U-50488H was not potentiated by its coadministration with low doses of CPA. Full recovery of the effect was observed 45 min after the administration of U-50488H alone, in parallel to the recovery of mechanically evoked responses. However, as for responses to noxious mechanical stimulation, only partial recovery of wind-up was observed 45 min after the administration of U-50488H on coadministration with CPA. These results again support an interaction between CPA and κ-opioid, but not µ-opioid, receptors. Enhancement of the duration of antinociception induced by κ-opioid receptor agonists might open new perspectives in the treatment of chronic inflammatory pain. Conclusion
The antinociceptive activity mediated by the adenosine A1 receptor agonist CPA in the spinal cord is linked to a secondary activation of κ-opioid, but not µ-opioid, receptors in adult rats with inflammation. In addition, antinociception mediated by κ-opioid receptor agonists has a significantly longer duration when the agonists are coadministered with low doses of the adenosine A1 receptor agonist CPA.
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 749
Acknowledgements This work was supported by the Spanish Ministry of Science and Technology (grant SAF2001-1048-C03-03). The authors thank Dr E.J. Taylor for the English and scientific revisions of the manuscript. Conflicts of interest
There are no conflicts of interest.
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Interaction of the adenosine A1 receptor agonist N6-cyclopentyladenosine and κ-opioid receptors in rat spinal cord nociceptive reflexes Guillermo A. Ramos-Zepeda, Carlos Herrero-Zorita and Juan F. Herrero Antinociception induced by the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) is linked to opioid receptors. We studied the subtype of receptors to which CPA action is related, as well as a possible enhancement of antinociception when CPA is coadministered with opioid receptor agonists. Spinal cord neuronal nociceptive responses of male Wistar rats with inflammation were recorded using the single motor unit technique. CPA antinociception was challenged with naloxone or norbinaltorphimine. The antinociceptive activity of fentanyl and U-50488H was studied alone and combined with CPA. Reversal of CPA antinociception was observed with norbinaltorphimine (82.9 ± 13% of control) but not with low doses of naloxone (27 ± 8% of control), indicating an involvement of κ-opioid but not µ-opioid receptors. Low doses of CPA did not modify fentanyl antinociception. However, a significant enhancement of the duration of antinociception was seen when U-50488H was coadministered with CPA. We conclude that antinociception
Introduction Adenosine A1 receptor agonists such as N6-cyclopentyladenosine (CPA) are effective antinociceptive agents, with an action mainly associated with the spinal cord processing of nociceptive information (Karlsten et al., 1991; Reeve and Dickenson, 1995; Lee and Yaksh, 1996; Sawynok, 1998; Sawynok and Liu, 2003; Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2005). We have previously reported that CPA antinociception is reversed by high doses of the nonselective opioid receptor antagonist naloxone in animals with paw inflammation (Ramos-Zepeda and Herrero, 2013). This implies an interaction between adenosine and opioid systems that might be useful in the treatment of inflammatory pain. The dose of naloxone used in our previous study, however, although clearly indicating an interaction with opioid receptors, was too high to discriminate the subtype of the opioid receptor involved in that phenomenon (Herrero and Headley, 1991). We therefore wondered whether the interaction between CPA and opioid receptors involves µ-opioid or κ-opioid receptors, or even both, as these are the main opioid receptors associated with spinal cord antinociception. The main objective of the present study was to challenge the antinociceptive effect of CPA with low doses of naloxone (selective for µ-opioid receptors in similar experiments; Herrero and Headley, 1991) and with the κ-opioid receptor selective antagonist norbinaltorphimine (nor-BNI; Portoghese et al., 1994). 0955-8810 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
mediated by CPA in the spinal cord is associated with activation of κ-opioid but not µ-opioid receptors in inflammation. In addition, coadministration of CPA and κ-opioid receptor agonists is followed by significantly longer antinociception, opening new perspectives in the treatment of chronic inflammatory pain. Behavioural Pharmacology 25:741–749 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Behavioural Pharmacology 2014, 25:741–749 Keywords: adenosine, inflammation, opioids, pain, rat, single motor unit, spinal cord, wind-up Department of System Biology, Unit of Physiology, Faculty of Medicine, University of Alcalá, Madrid, Spain Correspondenceto Juan F. Herrero, MD, PhD, Department of System Biology, Unidad de Fisiologia, Campus Universitario, Universidad de Alcala, Alcala de Henares, 28871 Madrid, Spain E-mail: [email protected] Received 27 May 2014 Accepted as revised 20 August 2014
In addition, numerous experiments using a similar protocol as that used in the present experiments have shown that the combination of opioids with other drugs may induce supraadditive, or even synergic, antinociception (Gaitán and Herrero, 2002, 2005; Gaitán et al., 2003, 2005). In fact, systemic administration of adenosine or its analogs enhances the analgesia produced by morphine (Ahlijanian and Takemori, 1985; Contreras et al., 1990; Malec and Michalska, 1990). We therefore wondered whether the administration of CPA together with opioid receptor agonists might result in an enhancement of the potency or duration of antinociception. We attempted to address this question, in the second part of our study, by comparing the antinociception observed after the administration of the µ-opioid receptor agonist fentanyl with that after the administration of the κ-opioid receptor agonist U-55488H, alone and in the presence of low doses of CPA, following the same protocol as that adopted in previous studies using the same technique (Curros-Criado and Herrero, 2009; Molina and Herrero, 2011).
Methods Subjects
All the experiments were carried out on adult male Wistar rats, weighing between 250 and 340 g, with carrageenaninduced soft-tissue inflammation. Inflammation was induced by intraplantar injection of 100 μl carrageenan λ (10 mg/ml in distilled water; Sigma-Aldrich, Madrid, Spain) into the right DOI: 10.1097/FBP.0000000000000091
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742 Behavioural Pharmacology 2014, Vol 25 No 8
hind paw 16 h before the experiment under brief halothane anesthesia (5% in oxygen for induction and 2% for maintenance). The effectiveness of carrageenan was studied by measuring the volume of the paw before the induction of inflammation and after the experiment using a plethysmometer (Letica Scientific Instruments, Barcelona, Spain).
review). The mean intensity of electrical stimulation used was 4.7 ± 0.7 mA. Assessment of nociception
All the experimental procedures conformed to the institutional, national, and European guidelines for the use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals were killed with an overdose of sodium pentobarbital at the end of the experiment (Euta-Lender, Normon).
A computer-controlled pincher device (Cibertec, Madrid, Spain) was used to apply noxious mechanical stimuli and to determine the threshold force required to trigger the withdrawal response using constantly increasing pressure ramp stimulation. The intensity of the stimulation used in the experiments was 200 mN above the threshold for each SMU. The mean force used in the experimental groups was always very similar, with an average of 1.23 ± 0.1 N.
Preparatory surgery
Behavioral experiments
The electrophysiological recording procedures have been described in detail previously (Herrero and Headley, 1991; Solano and Herrero, 1997). Preparatory surgery was always performed under halothane anesthesia (5% in oxygen for induction and 2% for maintenance) and included cannulation of the trachea (to keep airways open and ensure correct ventilation), two superficial branches of the jugular veins (for the administration of anesthesia and drugs), and one carotid artery (for monitoring the arterial blood pressure). The core body temperature was maintained at 37 ± 0.5°C using a homeothermic blanket throughout the surgery and the experiment. The animal was transferred after the surgery to a standard frame for electrophysiological recordings. The right hind limb was fixed with plaster onto a Perspex block, halothane was discontinued, and anesthesia was maintained with αchloralose (50 mg/kg for induction and 20 mg/kg/h through a perfusion pump for maintenance; Sigma). The preparation was left to rest for at least 1 h before the experiment started. Blood pressure was monitored constantly, and in all cases the systolic blood pressure was above 100 mmHg before the administration of the drugs.
In the first series of experiments, antinociception observed after the administration of increasing doses of CPA (cumulative log2 doses every 6 min, 10 to 320 μg/kg; Ramos-Zepeda and Herrero, 2013) was challenged with either 1 mg/kg naloxone (n = 7), 100 μg/kg naloxone (n = 9), or 2.5 mg/kg nor-BNI (n = 9). The CPA vehicle was tested in control experiments (n = 3), using the same protocol as that described for the administration of CPA. The effect of 100 μg/kg naloxone on fentanyl-mediated antinociception was tested in a separate experimental group (n = 5). In these experiments, the effect of a single bolus of 16 μg/kg fentanyl was studied in the presence of 100 μg/kg naloxone injected 18 min before bolus administration (see also Herrero and Headley, 1996).
Stimulus presentation and recording systems
Bipolar tungsten electrodes, inserted percutaneously, were used to isolate and record single motor units (SMUs) in muscles of the right hind limb involved in the withdrawal reflex. The units were isolated by moving the electrode with a micromanipulator while a mild pressure was applied to the paw. A window discriminator was also used to ascertain the recording of an SMU. The units were activated in cycles of 3-min duration, each cycle consisting of 10 s of noxious mechanical stimulation (0.2 N above the threshold over an area of 14 mm2) and 16 electrical stimuli applied to the most sensitive area of the cutaneous receptive field of the SMU (20 ms width, 1 Hz, twice the threshold intensity for the recruitment of long latency responses; Herrero and Cervero, 1996a, 1996b; Fig. 1). Original recordings and the protocol of stimulation are illustrated in Fig. 1. Electrical stimulation was used to study the effect of the drugs on the phenomenon of wind-up (see Herrero et al., 2000 for a
In a second series of experiments, a possible enhancement of opioid-mediated antinociception by CPA was studied following the same protocol as above. The effect of cumulative doses of fentanyl (2–32 μg/kg) on SMU nociceptive responses was studied either alone (n = 9) or in the presence of subeffective doses of CPA (10 μg/kg; n = 7), injected intravenously 6 min before fentanyl administration. Similar experiments to study the antinociception induced by cumulative doses of U-50488H (1–16 mg/kg) alone (n = 9) or in the presence of 10 μg/kg CPA (n = 9) were carried out. The doses of all drugs were chosen according to results observed in preliminary experiments and previous reports following the same technique and experimental protocol (Hartell and Headley, 1991; Herrero and Headley, 1991, 1993; Thorn et al., 1994; Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2005, 2013). Drugs
All the drugs used in this study were prepared fresh on a daily basis, before their administration to the rats, and injected intravenously in a total volume of 0.3 ml. CPA (Sigma) was dissolved in 0.5 μg/μl dimethyl sulfoxide (Sigma) and diluted in saline. The administration of each dose of CPA was very slow, over a minimum of 3 min, so as to minimize its effect on blood pressure. The µ-opioid receptor antagonist naloxone, the µ-opioid receptor selective agonist fentanyl, the κ-opioid receptor selective antagonist nor-BNI, and the κ-opioid
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 743
Fig. 1
(a)
CPA 320 μg/kg
Control
Elec. stimuli
36 min
Naloxone 100 μg/kg
24 min
0.5 N
20 spikes
(b)
CPA 320 μg/kg
Control Elec. stimuli 0.5 N
36 min
Nor-BNI 2.5 mg/kg
24 min
20 spikes 50 s Original recordings of two units showing the antinociceptive effect of CPA and its reversal by naloxone or nor-BNI. The figure shows the protocol followed in the experiments, alternating noxious mechanical and electrical stimulation in 3-min cycles of stimulation. The responses of the units are represented as frequency histograms. (a) The cumulative dose of 320 μg/kg CPA induced inhibition of SMU nociceptive responses that was not reversed by a low dose of the opioid receptor antagonist naloxone. (b) CPA antinociception, however, was fully reversed by the κ-opioid receptor selective antagonist nor-BNI. CPA, N6-cyclopentyladenosine; Elec., electrical; nor-BNI, norbinaltorphimine; SMU, single motor unit.
receptor selective agonist U-50488H (all from Sigma) were dissolved and diluted in saline. Data analysis
Data are expressed as a percentage of control (± SEM), where control is the mean of the three responses before the administration of a drug. The quantitative analysis was based on counts of spikes evoked during the two cycles of stimulation between each dose. The data from electrical stimulation were analyzed by counting the number of spikes evoked between 150 and 650 ms after each stimulus (C-fiber responses, Herrero and Cervero, 1996a, 1996b). The procedure of stimulation and collection of data was performed using a computer with commercial software (Spike 2; CED, Cambridge, UK). Statistical comparisons were made with raw data, also using commercial software (GraphPad-Prism and GraphPad-Instat for Windows, San Diego, CA, USA). One-way analysis of variance with post-hoc Dunnett’s test was used for the comparison between mechanical stimulation and control, as well as for the analysis of wind-up curves.
Results Reversal of the effect of CPA by nor-BNI in response to noxious mechanical stimulation
Paw volume increased from an average of 1.4 ± 0.1 to 2.5 ± 0.1 ml (P < 0.001) after plantar injection of carrageenan.
This increment was not modified (2.6 ± 0.1 ml) by the administration of CPA, at the doses tested, which indicates that, under the present experimental conditions, CPA is devoid of anti-inflammatory effects and, therefore, any depression of nociceptive responses is not secondary to a reduction in paw swelling. As in previous reports (Ramos-Zepeda et al., 2004; Ramos-Zepeda and Herrero, 2013), systemic administration of CPA virtually abolished SMU responses to noxious mechanical stimulation. The minimum effective dose was 80 μg/kg (P < 0.05) in two experimental groups (naloxone 1 mg/kg and nor-BNI) and 160 μg/kg (P < 0.01) in a third group (naloxone 100 μg/kg). The residual activity observed was between 8.2 ± 3 and 21 ± 8% of the control response (Figs 1 and 2a), depending on the group of units studied (P < 0.001 vs. control in all cases). Control experiments, using equivalent doses of vehicle, did not modify SMU activity to noxious mechanical stimulation with the highest dose studied (103 ± 11%; data not shown in figures). The inhibitory action of CPA lasted for a minimum of 53 min (9.5 ± 6%, P < 0.001), studied in those experiments in which the depression of responses was not challenged with an antagonist. As previously reported, the administration of the nonselective opioid receptor antagonist naloxone, at a high
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dose of 1 mg/kg, fully reversed CPA-mediated inhibition of SMU nociceptive responses (95 ± 18%, P < 0.01 vs. CPA effect; Fig. 2a). However, as illustrated in Fig. 1, the reversal effect was not observed after the administration of a low dose of 100 μg/kg naloxone (27 ± 8%; Fig. 2a). In contrast, the administration of the selective κ-opioid receptor antagonist nor-BNI induced full reversal of CPA antinociception (82.9 ± 13%, P < 0.01 vs. CPA effect; Figs 1 and 2a). The reversal effects of nor-BNI and 1 mg/kg naloxone, although starting immediately after their administration, were significant 18 min after their administration (74 ± 9% in the experiments with naloxone, P < 0.01 vs. CPA effect; 72 ± 9% with nor-BNI, P < 0.01 vs. CPA effect). To be sure that the lack of effect of 100 μg/kg naloxone was not due to loss of activity of the antagonist at that time, a single dose of fentanyl (16 μg/kg, intravenously) was injected 18 min after the same dose of naloxone in a different experimental group. The experiments showed no reduction in SMU responses after fentanyl administration (102 ± 9%; Fig. 2a), indicating effective naloxone antagonism at the time tested. Reversal of the effect of CPA by nor-BNI in response to noxious electrical stimulation
Similar to the observed effects on responses to noxious mechanical stimulation, intravenous administration of CPA dose-dependently reduced wind-up (P < 0.01 vs. control, with a CPA dose of 320 μg/kg; Figs 1 and 2b). The administration of equivalent doses of vehicle did not cause any significant change in wind-up (data not shown). As reported previously, the administration of the nonselective opioid receptor antagonist naloxone induced complete reversal of the CPA effect when injected at a dose of 1 mg/kg (data not shown), but no reversal was observed at a dose of 100 μg/kg (Fig. 1). The administration of nor-BNI was followed by full recovery of CPAmediated depression of wind-up in some experiments (Fig. 1), but only a partial recovery, although significant, was seen in others. The average results are shown in Fig. 2b (P < 0.05 vs. CPA effect). Antinociceptive effect of the μ-opioid selective receptor agonist fentanyl alone and in the presence of low doses of CPA
As the first series of experiments indicates a link between CPA and κ-opioid receptors, but not µ-opioid receptors, the possible enhancement of opioid antinociceptive actions in combination with CPA was studied in the following set of experiments. The administration of cumulative doses of fentanyl dose-dependently depressed responses to noxious mechanical stimulation to 13 ± 5% of control (P < 0.01; Fig. 3a) when injected alone [inhibitory dose (ID)50 of 17 ± 1 μg/kg]. When fentanyl was injected together with a subeffective dose of 10 μg/kg CPA, the antinociceptive effect was very similar:
ID50 of 18 ± 2 μg/kg. The dose of CPA studied (10 μg/kg) caused no change on the nociceptive responses when studied alone: 90 ± 5% (Fig. 3a, inset). In all the experiments, the antinociceptive effect observed on administration of fentanyl fully recovered 24 min after the administration of the highest dose studied. The administration of CPA did not modify the duration of the effect of fentanyl. The administration of fentanyl dose-dependently reduced the effect of CPA on wind-up responses, with complete inhibition after the dose of 32 μg/kg (P < 0.01 vs. control; data not shown in figures). The inhibitory action was not different from that seen after the coadministration of fentanyl and 10 μg/kg CPA (data not shown). As in responses to noxious mechanical stimulation, full recovery of the effect was always observed within 24 min after the administration of the drugs in both groups of experiments. Antinociceptive effect of the κ-opioid selective receptor agonist U-50488H alone and in the presence of low doses of CPA
The administration of the κ-opioid receptor selective agonist U-50488H induced dose-dependent inhibition of responses to noxious mechanical stimulation. The maximal effect observed was 2 ± 1% of control (P < 0.001 vs. control; Fig. 3b), with an ID50 of 4 ± 1 mg/kg. A similar depression in responses was observed when U-50488H was studied in the presence of 10 μg/kg CPA: ID50 of 5 ± 1 mg/kg (Fig. 3b). No significant differences were observed between groups. The dose of CPA studied (Fig. 3b, inset) did not reduce nociceptive responses significantly: 89 ± 1.5%. Antinociception induced by U-50488H lasted for a maximum of 45 min when injected alone (88 ± 1% of control; Fig. 4). However, in the presence of CPA the duration of the antinociceptive effect was longer and remained significant 45 min after administration: 40 ± 11% (P < 0.01 vs. control; P < 0.05 vs. U-50488H alone; Fig. 4). Full recovery was never observed 90 min after administration (73.4 ± 5%). Nociceptive responses to high-intensity electrical stimulation were dose-dependently reduced by U-50488H, with complete inhibition after a dose of 16 mg/kg (P < 0.01 vs. control; Fig. 5a). The effect was similar in the presence of CPA (P < 0.01 vs. control; Fig. 5b). However, whereas full recovery was recorded within 45 min after the administration of U-50488H alone, antinociception was still significant 45 min after the administration of the highest studied dose of U-50488H in the presence of CPA (P < 0.01 vs. control; Fig. 5b).
Discussion Two main observations have been made in the present study: (i) the interaction between the adenosine A1
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 745
Fig. 2
(a)
∗∗ ∗∗
100
% of control
80
60
40
20
0
CPA NAL 320 1 μg/kg mg/kg
CPA 320 μg/kg
NAL 100 μg/kg
CPA Nor-BNI 320 2.5 μg/kg mg/kg
NAL 100 μg/kg
FENT 16 μg/kg
(b) 20 18 Mean number of responses
16
∗
14 12
∗∗
10
∗
8 6 4 2 0 0
2
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6
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Stimulus number Control
CPA 320 μg/kg
Nor-BNI 2.5 mg/kg
Reversal of the effect of CPA by naloxone and nor-BNI. (a) The figure shows the antinociceptive effect of CPA after the administration of the highest dose (320 μg/kg) in responses to noxious mechanical stimulation. A high dose of naloxone (1 mg/kg) induced full reversal of CPA-mediated antinociception. However, the effect was not observed with a low dose of naloxone (100 μg/kg). The administration of the κ-opioid receptor antagonist nor-BNI was followed by full reversal of CPA antinociception. The reversal effect of naloxone and BNI, although started immediately after administration, was significant 18 min after administration. The effect of a single dose of 16 μg/kg fentanyl was prevented by a low dose of naloxone (100 μg/kg, injected intravenously 18 min before), showing that naloxone was active at the dose and time tested. (b) Wind-up activity to electrical stimulation was also fully depressed by CPA, although in this case the administration of nor-BNI only partially, although significantly, reversed the inhibition of responses. The effect of antagonists and the effect of CPA were statistically compared using the one-way ANOVA, with the post-hoc Dunnett’s test (*P < 0.05; **P < 0.01). ANOVA, analysis of variance; CPA, N6-cyclopentyladenosine; FENT, fentanyl; NAL, naloxone; nor-BNI, norbinaltorphimine.
receptor agonist CPA and κ-opioid receptors, and (ii) enhancement of the duration of antinociceptive activity of the κ-opioid receptor agonist U-50488H when injected together with small doses of CPA. We have previously
shown that CPA is a very effective antinociceptive drug either in in-vitro preparations (Ramos-Zepeda et al., 2004) or in single units recorded in anaesthetized rats (Ramos-Zepeda and Herrero, 2013), using the same techniques as in present
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746 Behavioural Pharmacology 2014, Vol 25 No 8
Fig. 3
(a) 120 100 80
% of control
100
% of control
80
60 40 20
∗∗
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0
CPA 10 μg/kg
60
40 ∗∗
20
∗∗∗ 0
2
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32
Dose (μg/kg) CPA+fentanyl
Fentanyl (b) 100
100
% of control
80
80 ∗ % of control
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60 40 20
60 0
CPA 10 μg/kg
∗∗∗
40 ∗∗∗ 20
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1
2
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16
Dose (mg/kg) U-50488H
CPA+U-50488H
Antinociceptive effect of (a) fentanyl and (b) U-50488H, in the absence and in the presence of a low dose of CPA. The administration of fentanyl and U-50488H dose-dependently reduced responses to noxious mechanical stimulation. The injection of 10 μg/kg CPA 6 min before did not modify the antinociceptive activity of fentanyl or U-50488H. The inset shows the lack of an effect of CPA at the dose administered. Statistical analysis as in Fig. 2 (*P < 0.05; **P < 0.01; ***P < 0.001, compared with control response). CPA, N6-cyclopentyladenosine.
experiments. In the latter study, we observed that the CPAmediated antinociceptive effect was fully reversed by a high dose (1 mg/kg) of the opioid receptor antagonist naloxone, indicating an interaction of CPA with opioid receptors. Such an interaction is supported by some studies: for example,
adenosine and its analogs potentiated morphine-induced analgesia, tolerance and dependence in tail flick and acetic acid writhing assays (Ahlijanian and Takemori, 1985). In addition, morphine led to the release of endogenous adenosine from the spinal cord in an in-vivo preparation
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 747
discussion). However, there is evidence that CPA and other analogs are highly selective agonists of adenosine receptors (Jacobson et al., 1992; Ralevic and Burnstock, 1998), indicating that a direct interaction between CPA and opioid receptors is not likely. This is supported by other studies that showed a lack of interaction between opioid receptor antagonists and CPA or other adenosine A1 receptor agonists (DeLander and Hopkins, 1986; Aley and Levine, 1997; Borghi et al., 2002, see below). This controversial observation might reflect divergences in the techniques utilized by different groups or, more probably, in the usage of different agonists or antagonists of adenosine and opioid receptors. In fact, our previous experiments (Ramos-Zepeda and Herrero, 2013) showed that CPA-mediated antinociception was reversed or prevented by the µ-opioid receptor antagonist naloxone at a high dose of 1 mg/kg. Some of those experiments were repeated for the present study, giving the same results and confirming this interaction. The dose of naloxone used, however, has been shown to be nonselective for opioid receptors in experiments using the same technique (Hartell and Headley, 1991; Herrero and Headley, 1991). Therefore, the CPA interaction might be related to µ-opioid, κ-opioid, or both populations of receptors in the spinal cord. The present study helps in clarifying this issue as the administration of a low dose of naloxone (100 μg/kg), which has been shown to be selective for µ-opioid receptors under the present experimental conditions (Herrero and Headley, 1991, 1993; Thorn et al., 1994), did not modify CPA-mediated antinociception. This is supported by the lack of effect observed in previous studies when opioid receptor antagonists were injected systemically before, or in conjunction with, CPA
Fig. 4
100
∗
U-50488H
% of control
80
CPA+U-50488H
60
∗∗
40
∗∗∗
∗∗∗
20 ∗∗∗ 0
∗∗∗
16 mg/kg
24 min
45 min
Recovery of the antinociceptive effect of U-50488H in the absence and in the presence of CPA. The inhibition of responses to noxious mechanical stimulation always recovered fully between 30 and 45 min after administration of U-50488H. However, the effect was still significant 45 min after the administration of U-50488H in the presence of CPA. Statistical analysis and layout as in Fig. 3. CPA, N6-cyclopentyladenosine.
(Sweeney et al., 1987a, 1987b; Sawynok et al., 1989), and morphine-induced antinociception was increased by pretreatment with adenosine agonists in mouse in-vivo tests (Contreras et al., 1990). In addition, the effect of morphine and other opiates was attenuated dose-dependently by intrathecal pretreatment with the adenosine receptor antagonist caffeine (Cahill et al., 1995; see also Sawynok, 1998 and Sawynok and Liu, 2003 for further references and
Fig. 5
(a)
(b) CPA+U-50488H
18
18
16
16
Mean number of responses
Mean number of responses
U-50488H
14 12 10 8 6 4 2
∗∗
0
14 12 10 8
∗∗
6 4 ∗∗
2 0
0
2
4
6
8
10 12 14 16
Stimulus number Control U-50488H 16 mg/kg U-50488H 16 mg/kg 45 min
0
2
4
6 8 10 12 14 16 Stimulus number
CPA+U-50488H 16 mg/kg Control CPA+U-50488H 16 mg/kg 45 min
Recovery of the wind-up depression induced by U-50488H in the absence (a) and in the presence of CPA (b). As in responses to noxious mechanical stimulation, wind-up inhibition observed by the administration of 16 mg/kg of U-50488H fully recovered in all cases within 45 min after the last dose (a; U-50488H 16 mg/kg 45 min). However, a significant depression of wind-up responses was still observed 45 min after the administration of U-50488H, when injected in the presence of CPA (b; CPA+U-50488H 16 mg/kg 45 min). Statistical analysis and layout as for Fig. 2b. CPA, N6-cyclopentyladenosine.
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748 Behavioural Pharmacology 2014, Vol 25 No 8
or other adenosine A1 agonists (DeLander and Hopkins, 1986; Aley and Levine, 1997; Borghi et al., 2002). However, the administration of nor-BNI, a selective κ-opioid receptor antagonist (Portoghese et al., 1994), fully reversed the antinociceptive effects of CPA. It therefore seems clear that the inhibition of nociceptive spinal cord reflexes observed following the administration of CPA results from an interaction between CPA and κ-opioid, but not µ-opioid, receptors under the present experimental conditions. The mechanism of the CPA–κ-opioid receptor interaction does not seem to be related to a direct effect of CPA on opioid receptors, as discussed above and in a previous study (Ramos-Zepeda and Herrero, 2013), as CPA is a very selective agonist of the adenosine A1 receptor (Jacobson et al., 1992; Ralevic and Burnstock, 1998). It seems more likely that the effect was due to secondary activation of opioid receptors, initiated by the interaction of CPA with adenosine receptors (Ramos-Zepeda and Herrero, 2013). Further and different experimental approaches are needed to clarify this point, but this would explain the lack of full recovery of CPA antinociception on challenge with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine in previous experiments (see Ramos-Zepeda and Herrero, 2005, 2013 for further discussion on this point). Our results provide in-vivo evidence that κ-opioid receptors and adenosine A1 receptors are somehow connected in the generation of antinociception, most likely through a common intracellular mechanism of action. It is not possible to deduce the nature of this link from the results obtained in the present experiments. However, as the release of adenosine induced by opioids, through the activation of protein kinase C, has been suggested to explain the relationship between the two systems, a similar connection might explain some of the observations made here (see Sawynok and Liu, 2003 for further discussion on this subject). In contrast, the adenosine A1 receptor has been proposed to exist as a part of a μ-opioid and α2-adrenergic multireceptor complex in peripheral nerve endings (Aley and Levine, 1997; Sawynok, 1998). This type of interaction with κ-opioid receptors might also explain some of our observations. Finally, although the experimental support is not too evident, a pharmacokinetic interaction between CPA and U-50488H should also be considered. It is difficult to comment on whether the effect is fully localized to the spinal cord or whether there is also a peripheral component to the action, presumably localized to inflammation-sensitized nociceptors. Depression of wind-up by CPA, to a level similar to that observed in responses to noxious mechanical stimulation, is in agreement with a spinal cord-mediated action, as windup is a progressive and frequency-dependent facilitation of the responses of spinal cord neurons (see Herrero et al., 2000 for further discussion). However, although full reversal of the CPA effect was observed in responses to
noxious mechanical stimulation, submaximal reversal of wind-up responses was seen in some, but not all, experiments. A partial action localized to the periphery should therefore be considered. Previous observations from experiments similar to those carried out in the present study have shown that subanalgesic doses of drugs enhance antinociception induced by opioids (Gaitán et al., 2003, 2005; Gaitán and Herrero, 2005). We therefore considered that the interaction observed between CPA and opioid receptors might also result in the enhancement of opioid-mediated antinociception. The administration of subanalgesic doses of CPA did not modify the activity of the µ-opioid receptor agonist fentanyl, nor its duration. This is in agreement with our observation of the lack of a relationship between CPA and µ-opioid receptors. It is also possible that the dose of CPA studied was not sufficient to induce a visible effect in our experiments; however, it was the maximum dose possible without a reduction of spinal cord neuronal activity, as observed in the present and previous experiments (Ramos-Zepeda et al., 2004). Coadministration of CPA and the κ-opioid receptor agonist U-50488H did not modify the potency of the antinociceptive activity induced by the latter, as shown by the comparison of ID50 values in the presence and absence of CPA. However, the duration of U-50488Hmediated antinociception was significantly longer in the presence of CPA. In this case, nociceptive activity evoked by noxious mechanical stimulation was only 40 ± 11% of the control response 45 min after the administration of the drug, whereas full recovery was always observed at that time in the absence of CPA. Likewise, the depression of wind-up activity observed after the administration of U-50488H was not potentiated by its coadministration with low doses of CPA. Full recovery of the effect was observed 45 min after the administration of U-50488H alone, in parallel to the recovery of mechanically evoked responses. However, as for responses to noxious mechanical stimulation, only partial recovery of wind-up was observed 45 min after the administration of U-50488H on coadministration with CPA. These results again support an interaction between CPA and κ-opioid, but not µ-opioid, receptors. Enhancement of the duration of antinociception induced by κ-opioid receptor agonists might open new perspectives in the treatment of chronic inflammatory pain. Conclusion
The antinociceptive activity mediated by the adenosine A1 receptor agonist CPA in the spinal cord is linked to a secondary activation of κ-opioid, but not µ-opioid, receptors in adult rats with inflammation. In addition, antinociception mediated by κ-opioid receptor agonists has a significantly longer duration when the agonists are coadministered with low doses of the adenosine A1 receptor agonist CPA.
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Interaction of CPA and κ-opioids Ramos-Zepeda et al. 749
Acknowledgements This work was supported by the Spanish Ministry of Science and Technology (grant SAF2001-1048-C03-03). The authors thank Dr E.J. Taylor for the English and scientific revisions of the manuscript. Conflicts of interest
There are no conflicts of interest.
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