We also noticed that acetic acid

We also noticed that acetic acid by itself (used to induce pain) increased spinal cord levels of KYNA (see Fig. 5). The most rational explanation of this finding is based on the possibility that acetic acid and KYNA share the same transport systems and the finding is therefore not surprising. Although further studies are needed to prove the specificity of action of both zaprinast and l-kynurenine/kynurenic acid in the writhing test in vivo, the results presented here suggest that GPR35 activation is an interesting approach that may be followed for the design of new molecular entities able to reduce inflammatory pain.
Acknowledgements This work was supported by grants from the Institut de Recherche Pierre Fabre, the University of Florence, Ente Cassa di Risparmio and MIUR.
G-protein coupled receptors (GPCRs) constitute one of the largest gene families in the human genome. While the vast majority of genes encoding GPCRs have been identified, ligands have not yet been identified for many of them. Consequently, the expression pattern and function of these ‘orphan’ GPCRs are largely unknown. GPR35 was identified as an orphan GPCR in 1998 . It shares homology with purinergic P2Y receptors, lysophosphatidic acid receptor GPR23, nicotinic acid receptor HM74, and the cannabinoid receptor GPR55. GPR35 is expressed at highest levels in cells of immune system and gastrointestinal tract , but its function has been poorly understood. Recently GPR35 was identified as a receptor for kynurenic acid . Kynurenic acid is a tryptophan metabolite and is an antagonist of several excitatory amino acid receptors, including -methyl--aspartate (NMDA) receptors . It can inhibit neuronal hyperexcitation induced by these receptors and limit associated excitotoxicity. Kynurenic acid is also an antagonist for α7-nicotinic glycerol receptors and thus may modulate both glutamatergic and cholinergic neurotransmission . Zaprinast, an inhibitor of cyclic GMP (cGMP)-specific phosphodiesterase PDE5, is an agonist for GPR35 . Application of zaprinast mobilizes intracellular calcium concentration in cells co-expressing G chimeric G-proteins and either human or rat GPR35, with EC values of 840nM and 16nM, respectively. This finding is especially interesting given the anti-nociceptive effect of zaprinast. Several studies have proposed that PDE5 inhibitors could produce anti-nociceptive effects by promoting accumulation of cGMP , , , as this second messenger has several targets including cGMP-dependent protein kinases (PKG), cGMP-regulated PDEs, and cyclic nucleotide-gated ion channels . However, while activation of the cGMP-PKG-K channel pathway appears most likely to mediate the anti-nociceptive effects of PDE5 inhibition, a recent study demonstrated that the effects of zaprinast in the rat formalin test were unrelated to cGMP signaling. Intrathecal administration of zaprinast exerted anti-nociceptive effects during both the acute and tonic phase of formalin-induced nociceptive pain that were comparable to morphine but were not abolished by attenuating cGMP accumulation with guanylate cyclase inhibitor . Thus, the effects of zaprinast on nociception are mediated by mechanisms distinct from PDE5 inhibition. As zaprinast is also an agonist for GPR35, here we examined the expression of GPR35 in the nervous system and explored the potential effects of GPR35 activation on nociception. GPR35 mRNA is expressed within nociceptive pathways including the dorsal root ganglion (DRG) and spinal cord . Both kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP formation from cultured rat DRG sensory neurons. Thus GPR35 may modulate nociception by influencing the signaling by second messengers other than cGMP. Materials and methods Isolation of rat DRG sensory neurons. All experimental procedures relating to animal care and treatment were conducted according to the guidelines of the Institutional Animal Care and Use Committee at Pfizer, the ILAR Guide for the Care and Use of Laboratory Animals, and all federal and international regulations. DRGs were isolated from embryonic day 18 embryos of IGS rats and digested in 0.25% trypsin–EDTA for 30min at 37°C. Dissociated cells were plated on poly-l-lysine-coated 96-well plates at a density of 20,000 cells per well and grown in Eagle’s MEM supplemented with 10% fetal bovine serum, 5mg/ml glucose, 40ng/ml gentamicin, 200ng/ml nerve growth factor (NGF), and 10μM arabinoside C. After overnight culture, the medium was replaced with Neurobasal medium supplemented with B27, 2mM GlutaMax, 200ng/mL NGF, and 10μM arabinoside C. The medium was exchanged with fresh Neurobasal medium every two days and experiments were performed after seven days in culture.