Jill C. Fehrenbacher

Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C.

Gabapentin and pregabalin are amino acid derivatives of &ggr;‐amino butyric acid that have anticonvulsant, analgesic, and anxiolytic‐like properties in animal models. The mechanisms of these effects, however, are not well understood. To ascertain whether these drugs have effects on sensory neurons, we studied their actions on capsaicin‐evoked release of the sensory neuropeptides, substance P and calcitonin gene‐related peptide from rat spinal cord slices in vitro. Although release of immunoreactive peptides from non‐inflamed animals was not altered by either drug, prior in vivo treatment by intraplantar injection of complete Freunds adjuvant enhanced release from spinal tissues in vitro, which was attenuated by gabapentin and pregabalin. These drugs also reduced release of immunoreactive neuropeptides in spinal tissues pretreated in vitro with the protein kinase C activator, phorbol 12,13‐dibutyrate. Our results suggest that gabapentin and pregabalin modulate the release of sensory neuropeptides, but only under conditions corresponding to significant inflammation‐induced sensitization of the spinal cord.

Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

A-Kinase Anchoring Protein Mediates TRPV1 Thermal Hyperalgesia through PKA Phosphorylation of TRPV1

&NA; Certain phosphorylation events are tightly controlled by scaffolding proteins such as A‐kinase anchoring protein (AKAP). On nociceptive terminals, phosphorylation of transient receptor potential channel type 1 (TRPV1) results in the sensitization to many different stimuli, contributing to the development of hyperalgesia. In this study, we investigated the functional involvement of AKAP150 in mediating sensitization of TRPV1, and found that AKAP150 is co‐expressed in trigeminal ganglia (TG) neurons from rat and associates with TRPV1. Furthermore, siRNA‐mediated knock‐down of AKAP150 expression led to a significant reduction in PKA phosphorylation of TRPV1 in cultured TG neurons. In CHO cells, the PKA RII binding site on AKAP was necessary for PKA enhancement of TRPV1‐mediated Ca2+‐accumulation. In addition, AKAP150 knock‐down in cultured TG neurons attenuated PKA sensitization of TRPV1 activity and in vivo administration of an AKAP antagonist significantly reduced prostaglandin E2 sensitization to thermal stimuli. These data suggest that AKAP150 functionally regulates PKA‐mediated phosphorylation/sensitization of the TRPV1 receptor.

PAR-2 agonists activate trigeminal nociceptors and induce functional competence in the delta opioid receptor

&NA; The role of protease activated receptor‐2 (PAR‐2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR‐2 activate the capsaicin‐sensitive subclass of trigeminal nociceptors in a PLC–PKC‐dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR‐2 agonist (SL‐NH2) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch‐clamp. The specificity of SL‐NH2 and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR‐2. The in vivo role of PAR‐2 activation was determined measuring SL‐NH2 and trypsin‐evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL‐NH2/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL‐NH2 (100 &mgr;M) and trypsin (1–600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 &mgr;M). In cultures treated with siRNA against PAR‐2, both SL‐NH2 and trypsin responses were significantly diminished. Both SL‐NH2 and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL‐NH2 rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR‐2 can also induce functional competence in DOR.

Tumor necrosis factor α and interleukin-1β stimulate the expression of cyclooxygenase II but do not alter prostaglandin E2 receptor mRNA levels in cultured dorsal root ganglia cells

&NA; Tumor necrosis factor α (TNFα) and interleukin 1β (IL‐1β) are pro‐inflammatory cytokines capable of altering the sensitivity of sensory neurons. Because sensitization elicited by IL‐1β and TNFα is blocked by inhibition of the inducible enzyme, cyclooxygenase‐II (COX‐2), we examined whether these cytokines could increase COX‐2 expression in dorsal root ganglion (DRG) cultures. Treatment of cell cultures with either IL‐1β or TNFα increases immunoreactive COX‐2, as measured by immunoblotting, in a time‐ and concentration‐dependent manner. A 24‐h pretreatment with 10 ng/ml IL‐1β or 50 ng/ml TNFα augmented COX‐2 expression 50‐ and 8‐fold over basal levels, respectively. Immunohistochemistry established the presence of COX‐2‐like immunoreactivity in both neuronal and non‐neuronal cells in culture. The addition of IL‐1 receptor antagonist blocked the induction of COX‐2 expression by IL‐1β, but did not alter TNFα‐stimulated increases in COX‐2, indicating that the mechanism of TNFα is not limited to increasing the expression of IL‐1β. The basal and TNFα‐induced expression of COX‐2 was not dependent on the presence of NGF in the growth media. IL‐1β and TNFα treatment for 24 h enhanced prostaglandin E2 (PGE2) production 2–4‐fold, which was blocked by pretreatment with the COX‐2 inhibitor, NS‐398. Exposing cultures to PGE2, IL‐1β, or TNFα for 24 h did not alter PGE2 receptor (EP) mRNA levels. These results indicate that TNFα and IL‐1β induce the functional expression of COX‐2 but not EP receptors in DRG cells in culture and suggest that cytokine‐induced sensitization of sensory neurons is secondary to prostaglandin production and not alterations in EP receptors.

Rapid Pain Modulation with Nuclear Receptor Ligands

We discuss and present new data regarding the physiological and molecular mechanisms of nuclear receptor activation in pain control, with a particular emphasis on non-genomic effects of ligands at peroxisome proliferator-activated receptor (PPAR), GPR30, and classical estrogen receptors. PPARalpha agonists rapidly reduce both acute and chronic pain in a number of pain assays. These effects precede transcriptional anti-inflammatory actions, and are mediated in part by IK(ca) and BK(ca) channels on DRG neurons. In contrast to the peripheral site of action of PPARalpha ligands, the dorsal horn supports the expression of PPARgamma. Intrathecal administration of PPARgamma ligands rapidly (< or =5 min) attenuated mechanical and thermal hypersensitivity associated with nerve injury in a dose-dependent manner that could be blocked with PPARgamma antagonists. By contrast, a PPARgamma antagonist itself rapidly increased the mechanical allodynia associated with nerve injury. These data suggest that ligand-dependent, non-genomic activation of spinal PPARgamma decreases behavioral signs of inflammatory and neuropathic pain. We also report that the GPR30 is expressed on cultured sensory neurons, that activation of the receptor elicits signaling to increase calcium accumulation. This signaling may contribute to increased neuronal sensitivity as treatment with the GPR30 agonist induces hyperalgesia. Finally, application of the membrane-impermeable 17beta-E(2)-BSA rapidly (within 15 min) enhanced BK-stimulated inositol phosphate (IP) accumulation and PGE(2)-mediated cAMP accumulation in trigeminal ganglion cultures. We conclude that nuclear receptor ligands may operate through rapid, non-genomic mechanisms to modulate inflammatory and neuropathic pain.

Capsaicin-evoked iCGRP release from human dental pulp: a model system for the study of peripheral neuropeptide secretion in normal healthy tissue.

ABSTRACT The mechanisms underlying trigeminal pain conditions are incompletely understood. In vitro animal studies have elucidated various targets for pharmacological intervention; however, a lack of clinical models that allow evaluation of viable innervated human tissue has impeded successful translation of many preclinical findings into clinical therapeutics. Therefore, we developed and characterized an in vitro method that evaluates the responsiveness of isolated human nociceptors by measuring basal and stimulated release of neuropeptides from collected dental pulp biopsies. Informed consent was obtained from patients presenting for extraction of normal wisdom teeth. Patients were anesthetized using nerve block injection, teeth were extracted and bisected, and pulp was removed and superfused in vitro. Basal and capsaicin‐evoked peripheral release of immunoreactive calcitonin gene‐related peptide (iCGRP) was analyzed by enzyme immunoassay. The presence of nociceptive markers within neurons of the dental pulp was characterized using confocal microscopy. Capsaicin increased the release of iCGRP from dental pulp biopsies in a concentration‐dependent manner. Stimulated release was dependent on extracellular calcium, reversed by a TRPV1 receptor antagonist, and desensitized acutely (tachyphylaxis) and pharmacologically by pretreatment with capsaicin. Superfusion with phorbol 12‐myristate 13‐acetate (PMA) increased basal and stimulated release, whereas PGE2 augmented only basal release. Compared with vehicle treatment, pretreatment with PGE2 induced competence for DAMGO to inhibit capsaicin‐stimulated iCGRP release, similar to observations in animal models where inflammatory mediators induce competence for opioid inhibition. These results indicate that the release of iCGRP from human dental pulp provides a novel tool to determine the effects of pharmacological compounds on human nociceptor sensitivity.

Models of Inflammation: Carrageenan‐ or Complete Freund's Adjuvant (CFA)–Induced Edema and Hypersensitivity in the Rat

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the anti‐inflammatory properties of agents such as nonsteroidal anti‐inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. This unit details methods to elicit and measure carrageenan‐ and complete Freunds adjuvant (CFA)–induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections of either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) and by the trigeminal ganglion neurons (vibrissal pad). Curr. Protoc. Pharmacol. 56:5.4.1‐5.4.7.

Chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is common in patients receiving anticancer treatment and can affect survivability and long-term quality of life of the patient following treatment. The symptoms of CIPN primarily include abnormal sensory discrimination of touch, vibration, thermal information, and pain. There is currently a paucity of pharmacological agents to prevent or treat CIPN. The lack of efficacious therapeutics is due, at least in part, to an incomplete understanding of the mechanisms by which chemotherapies alter the sensitivity of sensory neurons. Although the clinical presentation of CIPN can be similar with the various classes of chemotherapeutic agents, there are subtle differences, suggesting that each class of drugs might induce neuropathy via different mechanisms. Multiple mechanisms have been proposed to underlie the development and maintenance of neuropathy; however, most pharmacological agents generated from preclinical experiments have failed to alleviate the symptoms of CIPN in the clinic. Further research is necessary to identify the specific mechanisms by which each class of chemotherapeutics induces neuropathy.

Models of Inflammation: Carrageenan Air Pouch

The subcutaneous air pouch is an in vivo model that can be used to study acute and chronic inflammation, the resolution of the inflammatory response, and the oxidative stress response. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti‐inflammatory drugs. Curr. Protoc. Pharmacol. 56:5.6.1‐5.6.8.