Potentiation of formalin-evoked adenosine release by an adenosine kinase inhibitor and an adenosine deaminase inhibitor in the rat hind paw: a microdialysis study

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Abstract

The present study examined the effects of local subcutaneous administration of formalin on adenosine release from the rat hind paw, and the effects of inhibitors of adenosine metabolism on such release. Microdialysis probes were inserted into the subcutaneous tissue of the plantar surface of rat hind paws. Samples were collected every 10 min at a perfusion rate of 2 μl/min and high performance liquid chromatography was used to measure adenosine levels. At lower concentrations of formalin (0.5–2.5%), a significant increase in adenosine levels was observed in the first 10 min after formalin injection, while at the highest concentration of formalin (5%), the increase in adenosine release was observed over 60 min. Co-administration of the adenosine kinase inhibitor 5′-amino-5′-deoxyadenosine (100 nmol) with formalin, significantly increased adenosine release evoked by 0.5–1.5% formalin, but did not produce a further enhancement of release evoked by 5% formalin. The adenosine deaminase inhibitor 2′-deoxycoformycin (100 nmol) significantly increased adenosine levels at 5% formalin but had no effect at lower concentrations of formalin. In confirmation of previous studies, subcutaneous injection of formalin (0.5–5%) produced a characteristic biphasic concentration-related expression of nociceptive behaviours and an increase in paw volume. This study directly demonstrates that formalin can evoke a concentration-dependent local release of adenosine from the rat hind paw. The ability of an adenosine kinase inhibitor and an adenosine deaminase inhibitor to modulate this release is dependent on substrate adenosine concentrations.

Introduction

Both clinical and animal studies demonstrate that adenosine, an endogenous neuromodulator, has an important role in pain modulation Segerdahl and Sollevi, 1998, Sawynok, 1998. Studies in humans show that low-dose adenosine infusion increases pain thresholds (Ekblom et al., 1995), reduces post-operative analgesic requirements (Segerdahl et al., 1997) and reduces pain evoked by both nerve injury Belfrage et al., 1995, Belfrage et al., 1999, Segerdahl et al., 1995, Sollevi et al., 1995 and inflammatory injury (Sjölund et al., 1999). A decrease in adenosine concentrations in plasma and cerebrospinal fluid in patients with neuropathic pain has even been suggested to contribute to the pathophysiology of neuropathic pain (Guieu et al., 1996). In rodent studies, tonic antinociceptive influences of endogenous adenosine mediated by adenosine A1 receptors have been demonstrated, both peripherally (Doak and Sawynok, 1995) and centrally Sawynok et al., 1986, Keil and DeLander, 1996.

Besides, its effects on pain, adenosine also has been suggested to be an autocoid modulating inflammation, primarily acting on adenosine A2A receptors Cronstein, 1994, Sullivan and Linden, 1998. The anti-inflammatory effects of certain agents used clinically, such as methotrexate and sulfasalazine, are mediated by an increase in extracellular adenosine concentrations Cronstein et al., 1993, Gadangi et al., 1996, Morabito et al., 1998.

There are at least four key factors that are important in adenosine metabolism. Adenosine is formed from ATP via 5′-nucleotidase, metabolised by adenosine kinase and adenosine deaminase to 5′-AMP and inosine, respectively, and transported bidirectionally by adenosine transporters on the cell surface (Geiger et al., 1997). The net effect of formation, degradation and transportation determines intracellular and extracellular levels of adenosine. Modulation of extracellular adenosine concentrations by inhibitors of adenosine metabolism can produce antinociceptive and anti-inflammatory effects. Thus, behavioural studies demonstrate that adenosine kinase inhibitors produce a peripherally mediated antinociceptive effect in the formalin-induced persistent pain model (Sawynok et al., 1998), and an anti-inflammatory effect in the carrageenan-induced inflammation model (Poon and Sawynok, 1999). These effects can be blocked by adenosine receptor antagonists, indicating an activation of adenosine receptors.

The formalin test has been widely used to study persistent pain with inflammation (Tjølsen et al., 1992). Behavioural studies suggest that there is a tonic antinociceptive effect of endogenous adenosine (Doak and Sawynok, 1995) and that this effect can be modulated by an adenosine kinase inhibitor (Sawynok et al., 1998), but there has been no direct evidence indicating that adenosine levels are increased by formalin. Microdialysis provides a method to continuously measure the extracellular level of substances in vivo. The aim of the present study was to use the microdialysis technique to examine (1) whether adenosine is released subcutaneously after formalin injection, (2) whether such release can be modulated by an adenosine kinase inhibitor and an adenosine deaminase inhibitor and (3) whether the released adenosine and the modulation of adenosine by enzyme inhibitors correlates with previously reported antinociceptive effects and anti-inflammatory effects produced by adenosine indirectly acting agents.

Section snippets

Materials and methods

Male Sprague–Dawley rats (Charles River, Quebec, Canada) 120–160 g (formalin test) or 250–300 g (microdialysis study) were used. Rats were housed in pairs and allowed free access to food and water on a 12/12 h light/dark cycle at 21±1°C. Rats were used only once. Procedures were approved by the University Committee on Laboratory Animals.

Basal adenosine levels in the subcutaneous space of the rat hind paw

Fig. 2A shows the time course of changes in adenosine levels in the subcutaneous space after implantation of the microdialysis probe. Samples were taken with a flow rate of 2 μl/min from the start of perfusion and collected at 10-min intervals. Adenosine concentrations were initially high, but fell to a steady state level within 80–100 min after probe implantation. Basal adenosine levels were thus measured 120 min after probe implantation. The basal subcutaneous adenosine level was 1.50±0.10

Discussion

Microdialysis has previously been used to measure purine release in both brain Porkka-Heiskanen et al., 1997, Bell et al., 1998, Britton et al., 1999 and peripheral tissues Lönnroth et al., 1989, Blay et al., 1997, MacLean et al., 1998, Manthei et al., 1998. In the present study, using this technique, we demonstrate that the basal adenosine concentration in the subcutaneous area of the rat hind paw was about 0.23±0.02 μM. This is comparable to basal adenosine concentrations in subcutaneous

Conclusion

In summary, this study demonstrates that subcutaneous formalin evokes adenosine in the rat hind paw. The pattern of adenosine release is dependent on formalin concentration, which suggests that at different levels of inflammation, different mechanisms mediate this release. The ability of inhibitors of adenosine kinase and adenosine deaminase to modulate this release accords with the kinetics of the two enzymes.

Acknowledgements

The present study was supported by the Medical Research Council of Canada. X.J. Liu is a recipient of the Izaak Walton Killam Memorial Scholarship. We thank Allison Reid for technical assistance in performing the behavioural and paw volume measurements. We thank Parke-Davis Pharmaceuticals for the provision of 2′-deoxycoformycin.

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