The ADP receptor P2Y1 is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors

Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Molecular Pain (Impact Factor: 3.65). 02/2011; 7(1):13. DOI: 10.1186/1744-8069-7-13
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P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold.
To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice.
These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.

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    • "Accordingly , there is evidence for the presence of P2Y 1 , P2Y 6 and P2Y 11 receptors in mast cells (Feng et al., 2004; Gao et al., 2010, 2013). Furthermore, some electrophysiological studies have suggested that P2Y 1 receptors are present in primary afferent neurons (Jankowski et al., 2012; Malin and Molliver, 2010; Molliver et al., 2011; Yajima et al., 2005) and keratinocytes (Dussor et al., 2009). "
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    ABSTRACT: Metabotropic P2Y receptors subfamily consists of eight functional mammalian receptors. Specifically, P2Y(1), P2Y(6) and P2Y(11) receptors have been described in the sensory nervous system, but their participation, at peripheral level, in behavioral pain models is scarcely understood. This study assessed the role of peripheral P2Y(1), P2Y(6) and P2Y(11) receptors in formalin-induced inflammatory pain. Ipsilateral, but not contralateral peripheral pre-treatment with the endogenous P2Y(1) (ADP, 100-1000 nmol/paw), P2Y(6) (UDP, 180-300 nmol/paw) and P2Y(11) (ATP, 100-1000 nmol/paw), or selective P2Y(1) (MRS2365, 0.1-10 nmol/paw), P2Y(6) (PS130474, 0.1-0.10 pmol/paw) and P2Y(11) (NF546, 0.3-3 nmol/paw) receptor agonists increased 0.5% formalin-induced flinching behavior. Concordantly, peripheral pre-treatment with the selective P2Y(1) (MRS2500, 0.01-10 pmol/paw), P2Y(6) (MRS2578, 3-30 nmol/paw) and P2Y(11) (NF340, 1-10 nmol/paw) receptor antagonists significantly decreased 1% formalin-induced flinching behavior. Furthermore, the pronociceptive effect of ADP (100 nmol/paw) or MRS2365 (10 nmol/paw), UDP (300 nmol/paw) or PSB0474 (10 pmol/paw) and ATP (1000 nmol/paw) or NF546 (3 nmol/paw) was blocked by the selective P2Y(1) (MRS2500, 0.01 nmol/paw), P2Y(6) (MRS2578, 3 nmol/paw), and P2Y(11) (NF340, 1 nmol/paw) receptor antagonists, respectively. Western blot analysis confirmed the presence of P2Y(1) (66 kDa), P2Y(6) (36 kDa) and P2Y(11) (75 kDa) receptors in dorsal root ganglia (DRG) and sciatic nerve. Results suggest that peripheral activation of P2Y(1), P2Y(6) and P2Y(11) receptors plays a pronociceptive role in formalin-induced pain.
    Pharmacology Biochemistry and Behavior 11/2014; 128. DOI:10.1016/j.pbb.2014.11.001 · 2.78 Impact Factor
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    • "[38,45,46]). After P7 inflammation, we found a unique increase in the pH sensor and mechanical modulator ASIC3 [47,48], the heat transducer TRPM3 [49] and the heat threshold regulator P2Y1 [45,50] in the DRGs (Table 3). These specific changes in gene expression correlate well with the observed changes in mechanical and heat responsiveness in addition to the reduction in heat thresholds in “A”-fiber neurons, respectively (Figure 2). "
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    ABSTRACT: Background It is well-documented that neonates can experience pain after injury. However, the contribution of individual populations of sensory neurons to neonatal pain is not clearly understood. Here we characterized the functional response properties and neurochemical phenotypes of single primary afferents after injection of carrageenan into the hairy hindpaw skin using a neonatal ex vivo recording preparation. Results During normal development, we found that individual afferent response properties are generally unaltered. However, at the time period in which some sensory neurons switch their neurotrophic factor responsiveness, we observe a functional switch in slowly conducting, broad spiking fibers (“C”-fiber nociceptors) from mechanically sensitive and thermally insensitive (CM) to polymodal (CPM). Cutaneous inflammation induced prior to this switch (postnatal day 7) specifically altered mechanical and heat responsiveness, and heat thresholds in fast conducting, broad spiking (“A”-fiber) afferents. Furthermore, hairy skin inflammation at P7 transiently delayed the functional shift from CM to CPM. Conversely, induction of cutaneous inflammation after the functional switch (at P14) caused an increase in mechanical and thermal responsiveness exclusively in the CM and CPM neurons. Immunocytochemical analysis showed that inflammation at either time point induced TRPV1 expression in normally non-TRPV1 expressing CPMs. Realtime PCR and western blotting analyses revealed that specific receptors/channels involved in sensory transduction were differentially altered in the DRGs depending on whether inflammation was induced prior to or after the functional changes in afferent prevalence. Conclusion These data suggest that the mechanisms of neonatal pain development may be generated by different afferent subtypes and receptors/channels in an age-related manner.
    Molecular Pain 06/2014; 10(1):34. DOI:10.1186/1744-8069-10-34 · 3.65 Impact Factor
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    • "ATP binds to both the P2X ionotropic and P2Y metabotropic G-protein coupled family of receptors (Dussor et al., 2009; Burnstock, 2007). The importance of nucleotide signaling in the intercellular communication among cells of the keratinocyte-axon complex after injury has been reported in recent studies (Mandadi et al., 2009; Molliver et al., 2011). Our results show that PO does not interfere with DRG responses to the TRPV1 channel activator capsaicin (0.03–5 mM), eliminating this ion channel as an involved player. "
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    ABSTRACT: A commercial resin-based pine oil (PO) derived from Pinus palustris and Pinus elliottii was the major focus of this investigation. Extracts of pine resins, needles, and bark are folk medicines commonly used to treat skin ailments, including burns. The American Burn Association estimates that 500,000 people with burn injuries receive medical treatment each year; one-half of US burn victims are children, most with scald burns. This systematic study was initiated as follow-up to personal anecdotal evidence acquired over more than 10 years by MH Bhattacharyya regarding PO's efficacy for treating burns. The results demonstrate that PO counteracted dermal inflammation in both a mouse ear model of contact irritant-induced dermal inflammation and a second degree scald burn to the mouse paw. Furthermore, PO significantly counteracted the tactile allodynia and soft tissue injury caused by the scald burn. In mouse dorsal root ganglion neuronal cultures, PO added to the medium blocked adenosine triphosphate-activated, but not capsaicin-activated, pain pathways, demonstrating specificity. These results together support the hypothesis that a pine-oil-based treatment can be developed to provide effective in-home care for second degree burns. Copyright © 2013 John Wiley & Sons, Ltd.
    Phytotherapy Research 02/2014; 28(2). DOI:10.1002/ptr.4991 · 2.66 Impact Factor
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