The involvement of a cyclooxygenase 1 gene-derived protein in the antinociceptive action of paracetamol in mice
Introduction
Three isoforms of the prostaglandin synthesising enzyme, cyclooxygenase (COX), have been characterised, COX-1, COX-2 and COX-3 (DeWitt and Smith, 1988, Xie et al., 1991, Chandrasekharan et al., 2002). COX-1, generally expressed constitutively, is responsible for the synthesis of prostaglandins involved in the regulation of physiological functions (Crofford, 1997). Thus, COX-1 is regarded as a ‘house-keeping’ enzyme, unlike COX-2, which is induced by various pro-inflammatory stimuli (Jones et al., 1993) and in pathological conditions such as inflammation, pain and fever (Vane et al., 1994, Yamamoto and Nozaki-Taguchi, 1996, Cao et al., 1996). COX-3 is a splice-variant of COX-1 that retains the intron-1 gene sequence at the mRNA level which encodes a 30 aa sequence inserted into the N-terminal hydrophobic signal peptide of the enzyme protein. In the dog, COX-3 protein was expressed predominantly in the central nervous system (CNS) and in the heart (Chandrasekharan et al., 2002). Paracetamol selectively inhibited this COX-3 protein in preference to COX-1 and COX-2. In the canine, COX-3 mRNA intron-1 is within frame, but in humans and rodents there is a frameshift mutation in intron-1 of the COX-3 transcript. The mechanism for conversion of COX-3 mRNA to active enzyme protein in mice is therefore not clear as yet. However, in rat tissues, an approximately 64 kDa protein was detected using antibody against the intron-1 moiety of a COX-1 variant protein (Snipes et al., 2005). Also, an intron-retained COX-1 protein was identified in human tissues (Qin et al., 2005). Although this human COX-1 variant (named COX-1b2) synthesised prostaglandin F2α from arachidonic acid, its activity was not selectively inhibited by paracetamol (Qin et al., 2005).
Prostaglandin E2 (PGE2), is a potent hyperalgesic mediator at the site of injury (Ferreira et al., 1973), in the spinal cord (Yaksh and Malmberg, 1993, Malmberg and Yaksh, 1994) and in the brain (Hori et al., 2000, Abe et al., 2001). An increased release of PGE2 into the splenic vein of an anaesthetised dog was detected after a nociceptive injection of bradykinin into the spleen (Ferreira et al., 1973). Subsequently, PGE2 release from spinal cord slices in vitro was reported after stimulation with capsaicin or K +(60 mM) (Malmberg and Yaksh, 1994, Dirig et al., 1997). PGE2 was also collected by microdialysis with a probe implanted in the spinal cord of rats in vivo. The concentrations of PGE2 in the dialysate increased after an injection of formalin into the hind paw of the rat (Malmberg and Yaksh, 1995, Muth-Selbach et al., 1999, Tegeder et al., 2001) or after an intraspinal injection of substance P (Yaksh et al., 2001). A hyperalgesic, systemic injection of bacterial lipopolysaccharide (Abe et al., 2001) raised the levels of PGE2 in the preoptic area of the rat hypothalamus and PGE2 concentrations in mouse brains fell after systemic administration of cyclooxygenase inhibitors (Ferrari et al., 1990).
Paracetamol is a widely used analgesic and antipyretic with weak anti-inflammatory effects (Clissold, 1986). Although its mode of pharmacological action has not been fully elucidated, paracetamol is generally believed to act centrally rather than by a peripheral action (Clissold, 1986). The initial report of Flower and Vane (1972), describing an inhibitory effect of paracetamol on COX activity in the CNS is now supported by a number of other studies (Malmberg and Yaksh, 1994, Muth-Selbach et al., 1999, Ferrari et al., 1990). For example, in mice, antinociceptive doses of paracetamol attenuated the ex vivo synthesis of brain PGE2 in a dose-related manner (Ferrari et al., 1990) and in human volunteers, paracetamol raised the threshold to painful electrical stimulation of the sural nerve (Piletta et al., 1991). In addition, in the formalin test in the rat, antinociceptive doses of paracetamol reduced flinching behaviour and release of PGE2 from the spinal cord (Muth-Selbach et al., 1999). In spite of its potent inhibition of the biosynthesis of prostaglandins in the CNS in vivo (Muth-Selbach et al., 1999, Ferrari et al., 1990, Flower and Vane, 1972, Grèen et al., 1989), paracetamol was shown to be a weak inhibitor of both COX-1 and COX-2 in vitro (Warner et al., 1999). It has been suggested that the recently discovered COX-1 variant, namely COX-3, may be the target for the antinociceptive action of paracetamol, which inhibits the activity of COX-3 at therapeutic concentrations in vitro (Chandrasekharan et al., 2002).
Section snippets
Animals
Female C57Bl/6 mice (Harlan-Olac Ltd, UK) were maintained under 12 h/12 h light/dark cycle at 22 ± 1 °C. Food and water were provided ad libitum. The animals were acclimatized to the experimental room prior to testing. Experimental procedures were conducted in accordance with the United Kingdom Home Office Guidelines. The C57Bl/6 strain of mice is moderately sensitive to acetic acid-induced writhing and has a greater sensitivity to paracetamol than to most other NSAIDs (Lariviere et al., 2001).
Comparison of the effect of paracetamol and diclofenac on nociception and on peripheral and central prostaglandin biosynthesis
Paracetamol (100–400 mg/kg) and diclofenac (10–100 mg/kg) dose-dependently reduced acetic acid-induced writhing (Fig. 1A and B). The ID50 values calculated for paracetamol and diclofenac were 172.0 (CI: 158.0–188.0) mg/kg and 16.0 (CI: 8.0–32.0) mg/kg, respectively. Paracetamol (100–250 mg/kg) reduced iloprost-induced writhing (Fig. 2A), but there was no significant reduction of iloprost-induced writhing after diclofenac up to a dose of 100 mg/kg (Fig. 2B). The ID50 value calculated for
Discussion
In this study, the mechanism of the antinociceptive action of paracetamol was investigated using the mouse writhing model of acute nociception (Collier et al., 1968). This model is sensitive to the antinociceptive action of non-steroid antiinflammatoty drugs (NSAIDs) including paracetamol, which reduce writhing responses to i.p. injection of acetic acid (Collier et al., 1968) or of iloprost (Akarsu et al., 1989). It has also been used to study the different components of pain pathways (Gyires
Acknowledgements
This study was funded through the Case PhD Studentship to S.S. Ayoub from the Biotechnological and Biological Sciences Research Council (BBSRC) and GlaxoSmith Kline (GSK). The authors would like to thank Dr Michael Seed for expert consultation. The authors acknowledge the assistance of Ms. Priscilla Sawmynaden with some of the writhing measurements.
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Professor Derek A. Willoughby died on 13 March 2004.