Control of P2X3 channel function by metabotropic P2Y2 utp receptors in primary sensory neurons.

Purinergic signaling contributes significantly to pain mechanisms, and the nociceptor-specific P2X3 ATP receptor channel is considered a target in pain therapeutics. Recent findings suggesting the coexpression of metabotropic P2Y receptors with P2X3 implies that ATP release triggers the activation of both ionotropic and metabotropic purinoceptors, with strong potential for functional interaction. Modulation of native P2X3 function by P2Y receptor activation was investigated in rat dorsal root ganglia (DRG) neurons using whole cell patch-clamp recordings. Application of the selective P2Y receptor agonist UTP decreased peak amplitudes of α,ß-meATP-evoked homomeric P2X3-mediated currents, but had no effect on heteromeric P2X2/3-mediated currents. Treatment with phospholipase C inhibitor U73122 significantly reversed P2X3 current inhibition induced by UTP-sensitive P2Y receptor activation. We previously reported the modulation of P2X receptors by phospholipids in DRG neurons and injection of exogenous phosphatidylinositol-4,5-bisphosphate (PIP(2)) fully reverses UTP-mediated regulation of P2X3 channel activity. Pharmacological as well as functional screening of P2Y receptor subtypes indicates the predominant involvement of P2Y2 receptor in P2X3 inhibition, and immunolocalization confirms a significant cellular coexpression of P2X3 and P2Y2 in rat DRG neurons. In summary, the function of P2X3 ATP receptor can be inhibited by P2Y2-mediated depletion of PIP(2). We propose that expression of P2Y2 purinoceptor in nociceptive sensory neurons provides an homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptor channels.

Neurotrophic factor changes in the rat thick skin following chronic constriction injury of the sciatic nerve.

BACKGROUND: Cutaneous peripheral neuropathies have been associated with changes of the sensory fiber innervation in the dermis and epidermis. These changes are mediated in part by the increase in local expression of trophic factors. Increase in target tissue nerve growth factor has been implicated in the promotion of peptidergic afferent and sympathetic efferent sprouting following nerve injury. The primary source of nerve growth factor is cells found in the target tissue, namely the skin. Recent evidence regarding the release and extracellular maturation of nerve growth factor indicate that it is produced in its precursor form and matured in the extracellular space. It is our hypothesis that the precursor form of nerve growth factor should be detectable in those cell types producing it. To date, limitations in available immunohistochemical tools have restricted efforts in obtaining an accurate distribution of nerve growth factor in the skin of naïve animals and those with neuropathic pain lesions. It is the objective of this study to delineate the distribution of the precursor form of nerve growth factor to those cell types expressing it, as well as to describe its distribution with respect to those nerve fibers responsive to it. RESULTS: We observed a decrease in peptidergic fiber innervation at 1 week after the application of a chronic constriction injury (CCI) to the sciatic nerve, followed by a recovery, correlating with TrkA protein levels. ProNGF expression in CCI animals was significantly higher than in sham-operated controls from 1-4 weeks post-CCI. ProNGF immunoreactivity was increased in mast cells at 1 week post-CCI and, at later time points, in keratinocytes. P75 expression within the dermis and epidermis was significantly higher in CCI-operated animals than in controls and these changes were localized to neuronal and non-neuronal cell populations using specific markers for each. CONCLUSIONS: We describe proNGF expression by non-neuronal cells over time after nerve injury as well as the association of NGF-responsive fibers to proNGF-expressing target tissues. ProNGF expression increases following nerve injury in those cell types previously suggested to express it.

Delayed reinnervation by nonpeptidergic nociceptive afferents of the glabrous skin of the rat hindpaw in a neuropathic pain model.

Painful peripheral neuropathies have been associated with a reorganization of skin innervation. However, the detailed changes in skin innervation by the different afferent fiber types following a neuropathic nerve injury have never been characterized in an animal model of neuropathic pain. Our objective was to thoroughly characterize such changes in the thick skin of the foot in a well-established rat model of neuropathic pain, namely, the chronic constriction injury (CCI) of the sciatic nerve. We used the immunofluorescence detection of calcitonin gene-related peptide (CGRP), purinergic receptor P2X3, and NF200 as markers of peptidergic nociceptive fibers, nonpeptidergic nociceptive C fibers, and myelinated afferents, respectively. We observed that CCI-operated animals developed significant mechanical allodynia and hyperalgesia as well as thermal hyperalgesia. At 3 days following nerve injury, all CCI-operated animals had a significant decrease in the density of fibers immunoreactive (IR) for CGRP, P2X3, and NF200 within the upper dermis. A recovery of CGRP-IR fibers occurred within 4 weeks of nerve injury and sprouting above control levels was observed at 16 weeks. However, the myelinated (NF200-IR) fibers never recovered to control levels within a period of 16 weeks following nerve injury. Interestingly, the P2X3-IR fibers took considerably more time to recover than the CGRP-IR fibers following the initial loss. A loss in P2X3-IR fibers persisted to 16 weeks and recovered to levels above that of control at 1.5 years following nerve injury. Further studies are required to clarify the relevance of these innervation changes for neuropathic pain generation and maintenance.

Distribution of P2X(3)-immunoreactive fibers in hairy and glabrous skin of the rat.

The skin is innervated by two populations of unmyelinated sensory fibers, the peptidergic and nonpeptidergic, which transmit nociceptive information to the central nervous system. The peptidergic population expresses neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) and has both cutaneous and visceral targets. The nonpeptidergic population expresses the purinergic receptor P2X(3), binds the isolectin B4 (IB4), and innervates mainly the epidermis. To date, the peptidergic nociceptor population in cutaneous tissue of the rat has been well characterized, whereas the nonpeptidergic innervation pattern has lacked an adequate description. To this aim, we used light microscopic immunocytochemistry to investigate the pattern of P2X(3)-immunoreactive (-IR) fiber innervation of both hairy and glabrous skin from male Sprague-Dawley rats. Our results show extensive P2X(3)-IR fibers throughout the upper and lower dermis. Thick bundles of P2X(3)-IR fibers were found to run in parallel with the dermal-epidermal junction and projected multiple thin collateral axons that penetrated the epidermal layer, creating a dense network of innervation throughout the entire epidermis. The distribution of P2X(3)-IR fibers in the epidermis was far more extensive than the distribution of CGRP-IR fibers. P2X(3)-IR fibers also innervate hair follicles but were rarely found in close proximity to glands and blood vessels. The present results suggest a primary role for P2X(3)-IR fibers in the detection of noxious stimuli in cutaneous tissue and provide an anatomical basis for future studies examining a possible functionally distinct role of nonpeptidergic nociceptors in the transmission of nociceptive signals.