Significant outcomes
∙ Mechanisms mediating dipyrone analgesia and hypothermia are not totally understood. The nociceptin/orphanin FQ system is implicated in many physiological processes, including nociception and thermoregulation.
∙ Antagonising N/OFQ peptide receptors attenuates dipyrone-induced anti-nociception, but does not reverse hypothermia.
Limitations
∙ The site of N/OFQ peptide (NOP) receptor antagonist application can differentially modulate the nociceptin/orphanin FQ system; NOP antagonist may also be tested intra-thecally and intra-cerebroventicularly.
∙ Motor activity may also be tested to observe whether NOP receptor antagonist elicits any effect on motor function.
Introduction
Dipyrone (metamizol) is an analgesic antipyretic drug that is being used worldwide to treat mild-to-moderate pain and to reduce elevated body temperature. Differently from the classical non-steroidal anti-inflammatory drugs (NSAIDs), it exerts little anti-inflammatory but strong analgesic and hypothermic actions. These effects of dipyrone have long been suspected to be centrally mediated (Reference Akman, Aksu and Gultekin1,Reference Carlsson and Jurna2); however, despite intensive investigations, the precise mechanisms mediating dipyrone analgesia and hypothermia are not fully elucidated. The periaqueductal grey matter (PAG), rostral ventromedial medulla (RVM) and the spinal cord appear to be the central targets of dipyrone, even when administered systemically (Reference Vazquez, Hernandez, Escobar and Vanegas3,Reference Vanegas and Tortorici4). Descending inhibitory pathways and endogenous opioids seem to play roles in dipyrone actions (Reference Carlsson and Jurna2–Reference Hernandez-Delgadillo and Cruz5). Moreover, recent research indicates that endogenous cannabinoid system may also participate in some of the pharmacological effects of dipyrone (Reference Ulugol6–Reference Rogosch, Sinning and Podlewski8).
The endogenous ligand nociceptin/orphanin FQ (N/OFQ) and its receptor (N/OFQ peptide receptor, NOP) comprise a peptide receptor system expressed in brain areas implicated in many physiological processes, including nociception and thermoregulation. Although contradictory findings are obtained from studies on the effects of NOP receptor system on modulation of pain response, it appears that N/OFQ more often than not possesses supra-spinal hyperalgesic rather than spinal analgesic effects (Reference Heinricher9,Reference Mika, Obara and Przewlocka10). In case of thermoregulation, it is known that administering N/OFQ causes hypothermia, whereas NOP receptor knockout results in hyperthermia (Reference Rawls, Schroeder, Ding, Rodriguez and Zaveri11,Reference Uezu, Sei and Sano12).
Aims of the study
Like dipyrone, evidence also suggests a central component to the mechanism of action of paracetamol (Reference Mallet, Daulhac and Bonnefont13,Reference Dogrul, Seyrek, Akgul, Cayci, Kahraman and Bolay14). N/OFQ administration has been shown to prevent the anti-nociceptive action of paracetamol on the rat hot-plate test (Reference Sandrini, Vitale, Pini, Lopetuso, Romualdi and Candeletti15). On the other hand, NOP receptors have been indicated not to mediate paracetamol-induced hypothermia in rats (Reference Corley and Rawls16). Taking into account the similarities between dipyrone and paracetamol, the purpose of this work was to determine whether NOP receptors are involved in the anti-nociceptive and hypothermic effect of dipyrone.
Material and methods
Animals and ethics
Adult male Balb/c albino mice (Center of the Laboratory Animals, Trakya University) weighing 20–30 g were used. Animals were housed in groups of 10, and were maintained under controlled light (12 : 12 h light : dark cycles) and temperature (21±2°C) conditions with water and food available ad libitum. This study was conducted according to the guidelines of the Ethical Committee of the International Association for the Study of Pain after approval by the ‘Animal Care Ethics Committee’ at Trakya University.
Hot-plate test
A conventional hot-/cold-plate analgesia meter (Ugo Basile, Comerio, Italy) was used to perform the hot-plate test. In brief, the animals were placed on an electrically heated plate that was kept constant at a temperature of 55±0.1°C. Response latencies either to jump or a hind-paw lick were measured using an electronic timer. A cut-off time of 25 s was set in order to minimise tissue damage. Test latencies were converted to the percentage of the maximal possible effect (%MPE) according to the following equation: %MPE=[(post-drug latency−baseline latency)/cut-off time−baseline latency]×100.
Tail-flick test
To perform the tail-flick test, a standardised tail-flick apparatus (Commat, Ankara, Turkey) was used. In brief, radiant heat was focussed on the dorsal surface of the mouse tail, and response latencies to a tail-flick were recorded using an electronic timer. Baseline tail-flick latency for each mouse ranged from 2 to 3 s. A cut-off time of 10 s was set in order to minimise tissue damage. Test latencies were converted to the %MPE according to the following equation: %MPE=[(post-drug latency−baseline latency)/cut-off time−baseline latency]×100.
Measurement of rectal temperature
To measure rectal temperature of mice to the nearest 0.1°C by means of an Ellab thermometer, a 2-mm-diameter probe was inserted 2.5 cm into the rectum of mice and left there until steady readings were achieved.
Study design and drugs
Animals were randomly divided into control and treatment groups and habituated to the environment 3 days before the testing period. Subsequently, the effects of different doses of dipyrone (150, 300, 600 mg/kg, i.p.) and JTC-801 (1, 3, 10 mg/kg, i.p.), a NOP receptor antagonist, on nociception and rectal temperature were tested. Next, by administering the ineffective dose of JTC-801 (3 mg/kg) 30 min before dipyrone, the effect of NOP receptor antagonism on the anti-nociceptive and hypothermic effects of dipyrone were observed. Different cohorts of animals were used for JTC-801 administrations. Hot-plate, tail-flick and hypothermia tests were performed in all animals and each of them were used only once. For standardisation, the tests were conducted in the same order, tail-flick being the first and hypothermia the last.
Drug doses and treatment times were selected from previous studies (Reference Gunduz, Karadag and Ulugol17–Reference Yilmaz and Ulugol19). Dipyrone was purchased from Santa Cruz, and JTC-801 from Sigma Chemical Co. Dipyrone was dissolved in saline and JTC-801 was dissolved in 20% DMSO, 5% Tween 80, 5% ethanol and 70% saline; both were administered i.p. in a volume of 0.1 ml/10 g body weight.
Statistical analysis
Analysis of variance followed by the Bonferroni t test were used to compare the data of the groups. Values of p<0.05 were considered to be statistically significant. All data are expressed as mean±SEM.
Results
Dipyrone (150–600 mg/kg, i.p.) administration exerted dose-dependent anti-nociceptive and hypothermic effects (Fig. 1). The NOP receptor antagonist, JTC-801 (1, 3, 10 mg/kg, i.p.), did not affect either nociception or body temperature (data not shown). JTC-801 (3 mg/kg), at a dose that elicited no action on its own, partially abolished the anti-nociceptive action of dipyrone in both hot-plate and tail-flick tests, but did not reverse dipyrone-induced hypothermia (Fig. 2). JTC-801 augmented the hypothermic effect of 300 mg/kg dose of dipyrone (Fig. 2).
Fig.1 Time course of the anti antinociceptive and hypothermic effects of dipyrone (150, 300, 600 mg/kg). (Compared to control: *p<0.05, +p<0.01, p<0.005, &p<0.001; n=10 for each group; MPE=maximal possible effect).
Fig. 2 Effects of the NOP receptor antagonist, JTC-801 (3 mg/kg), on the antinociceptive and hypothermic effects of dipyrone. JTC-801 was injected 30 min before, and tests were performed 30 min after dipyrone administration. (Compared to control: +p<0.01, #p<0.05, &p<0.001; Compared to dipyrone: *p<0.05, **p<0.01, ***p<0.005; n=10 for each group; MPE=maximal possible effect).
Discussion
Far from being typical NSAIDs, dipyrone and paracetamol are two similar non-opioid analgesics possessing very little anti-inflammatory action. Their central activity appears to mediate their analgesic and hypothermic effects in addition to their well-known peripheral effects. NOP receptor activation antagonised paracetamol-induced anti-nociception, whereas inhibition of these receptors was ineffective in preventing paracetamol-induced hypothermia (Reference Sandrini, Vitale, Pini, Lopetuso, Romualdi and Candeletti15,Reference Corley and Rawls16). Here, unlike paracetamol, NOP receptor inhibition attenuated the anti-nociceptive action of dipyrone. On the other hand, similar to results obtained with paracetamol, we did not find any evidence that NOP receptor activation is involved in the hypothermic effect of dipyrone.
Pain-related central nervous system structures, such as the PAG, RVM and spinal cord, are known to mediate the anti-nociceptive effects of dipyrone not only when the drug is administered spinally or supra-spinally but also when administered systemically, pointing to the clinical relevance of this effect (Reference Vazquez, Hernandez, Escobar and Vanegas3). Accordingly, facilitation of endogenous opioidergic circuits and activation of descending pain-control system, which inhibits pain transmission at the level of the spinal cord, are involved in this effect of dipyrone (Reference Vazquez, Hernandez, Escobar and Vanegas3–Reference Hernandez-Delgadillo and Cruz5). Dipyrone is also proposed to be involved in interaction with the L-arginine-nitric oxide pathway, the glutamatergic system and the endogenous cannabinoid system, as well as its well-characterised effect of cyclooxygenase inhibition (Reference Rogosch, Sinning and Podlewski8–Reference Siebel, Beirith and Calixto23). Here, we observed whether NOP receptors participate in dipyrone-induced anti-nociception. Contrary to the results showing that NOP receptor activation prevents the anti-nociceptive action of paracetamol, our findings suggest that blockade of NOP receptors attenuates anti-nociception induced by dipyrone. Our results may be due to the complex role of the N/OFQ system in pain modulation, because whether N/OFQ has pro-nociceptive or anti-nociceptive properties and/or bidirectional-modulating actions still remain unknown (Reference Heinricher9,Reference Mika, Obara and Przewlocka10). Supporting this complex modulation of pain by the N/OFQ system, both NOP receptor agonists and antagonists demonstrate potential utility in treating pain states (Reference Tamai, Sawamura, Takeda, Orii and Hanaoka24,Reference Khroyan, Polgar and Orduna25). Therefore, obtaining contradictory results by administration of paracetamol and dipyrone, especially when the N/OFQ system is modulated, seems not to be unusual. Nevertheless, our findings indicating attenuation of dipyrone anti-nociception by the NOP antagonist are plausible, when it is taken into consideration that activation of NOP receptors has been associated with pain enhancement at some brain sites (Reference Mogil, Grisel, Reinscheid, Civelli, Belknap and Grandy26,Reference Gear, Bogen, Ferrari, Green and Levine27).
Regarding thermoregulation, we observed that NOP receptor antagonism did not reverse dipyrone-induced hypothermia. This effect is similar to that of paracetamol (Reference Corley and Rawls16), whose hypothermic action has been studied more intensively than dipyrone. Paracetamol-induced hypothermia has been shown to be independent of the transient receptor vanilloid potential−1 system and opioid, cannabinoid and NOP receptors (Reference Corley and Rawls16–Reference Ayoub, Pryce, Seed, Bolton, Flower and Baker28). A cyclooxygenase-1 variant and its antioxidant and anti-glutamatergic properties are among the possible hypothermic mechanisms of paracetamol (Reference Ayoub, Botting, Goorha, Colville-Nash, Willoughby and Ballou29–Reference Huang, Wang and Lin31). In case of dipyrone, very little is known; it exerts hypothermia at high doses and its antipyretic mechanism is suggested not to involve PGE2 synthesis inhibition (Reference Schlosburg, Radanova, Di Marzo, Imming and Lichtman32,Reference Malvar, Soares and Fabricio33). As described in the introduction, N/OFQ administration results in hypothermia, whereas NOP receptor knockout exerts hyperthermia (Reference Rawls, Schroeder, Ding, Rodriguez and Zaveri11,Reference Uezu, Sei and Sano12). In contrast to these reports, we have observed that JTC-801 increased dipyrone-induced hypothermia at the 300 mg/kg dose of dipyrone. This unexpected finding may have resulted from differences in experimental procedures, species and strains of animals and other methodologies. Despite this discrepancy, our results indicate that, similar to its effect on paracetamol (Reference Corley and Rawls16), NOP receptor antagonism seems not to reverse dipyrone-induced hypothermia.
In summary, we report that antagonising NOP receptors attenuates dipyrone-induced anti-nociception, but does not reverse dipyrone-induced hypothermia. The mechanisms through which dipyrone exerts its anti-nociceptive and hypothermic effects await further exploration, although it seems that they are mediated by dissimilar mechanisms.
Acknowledgement
Ismet Hande Ertin, MSc, contributed to the acquisition and interpretation of data, drafting the article and final approval of the version to be published. Associate Professor Dr. Ozgur Gunduz contributed to the acquisition, analysis and interpretation of data, drafting the article and final approval of the version to be published. Professor Dr. Ahmet Ulugol contributed to the conception, design, analysis and interpretation of data, drafting and revising the article for important intellectual content and final approval of the version to be published.
Financial Support
This work was supported by a grant from Trakya University Research Council (TUBAP-2013/32).
Conflicts of Interest
None.
Ethical Standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
