Neuropharmacology Research Group Funded Research
Cannabinoid Modulation of Capsaicin-Sensitive Nociceptors – R01 DA019585
Estrogen Modulation of Human Nociceptors – R01 NS058655
Prolactin Modulation of Trigeminal Nociceptors – R01 DE017696
Sodium Channel Expression in Human Teeth – R01 DE015576
The Role for the GPR30 Receptor in Inflammatory Pain – R21 DE018796
Interactions of TRPV1 and TRPA1 and Inflammatory Pain -- R01 DE019311
Cannabinoid Modulation of Capsaicin-Sensitive Nociceptors:
Many traditional analgesics have insufficient efficacy and therefore continued research is of high importance. Recently, we have developed a novel hypothesis for peripheral cannabinoid antihyperalgesia/antiallodynia via ionotropic cannabinoid receptors. We propose to test the central hypothesis that certain cannabinoids inhibit TRPV1 receptor activity by pharmacological desensitization of TRPV1 and that this action contributes to the peripheral antihyperalgesic/antiallodynic effects of cannabinoids. Our original preliminary data demonstrated that application of cannabinoids to sensory neurons leads to: 1) activation of protein phosphatase 2B (i.e., calcineurin); 2) dephosphor- ylation of TRPV1; and 3) inhibition of TRPV1 activity in a fashion reversed by calcineurin antagonists. Since the original submission, we have discovered that anandamide (AEA) and arachidonyl-2-chloroethylamide (ACEA) desensitize TRPV1 in transfected CHO cells, whereas WIN activates TRPA1 in CHO cells, and that AM1241 preferentially activates a heretofore unreported TRPV1/TRPA1 heteromer. Moreover, peripheral cannabinoid antihyperalgesia/antiallodynia in rats is blocked by pretreatment with a calcineurin antagonist, suggesting that peripheral cannabinoid antihyperalgesia/antiallodynia might be mediated, in part, by cannabinoid ionotropic receptors.
Specific Aim #1: Determine whether cannabinoid pretreatment activates specific calcineurin catalytic subunits (Aa, Ab, or Ag) leading to dephosphorylation of TRPV1.
Specific Aim #2: Determine whether cannabinoid-induced pharmacological desensitization of cannabinoid ionotropic receptors evokes a peripheral antihyperalgesic/antiallodynic effect.
Specific Aim #3: Determine whether TRPA1 is required to mediate the peripheral antihyperalgesic/antiallodynic effects of certain cannabinoids.
If the hypothesis of peripheral cannabinoid ionotropic receptors mediating antihyperalgesia/antiallodynia is confirmed, then we believe that it will be an important and conceptually innovative discovery for several reasons: First, this is the first demonstration of a functional implication of ionotropic cannabinoid receptors for peripheral antihyperalgesia/antiallodynia. Second, ionotropic mechanisms may have considerable significance in understanding cannabinoid modulation of other sites in the central nervous and immune systems. Third, identification of cannabinoid modulation of pain via a TRPA1/TRPV1 heteromer provides a rationale for a novel class of analgesics that are devoid of classical cannabinoid side effects.
Estrogen Modulation of Human Nociceptors:
Clinical studies of multiple pain disorders indicate that a patient’s biological sex is a common risk factor for pain, with many studies demonstrating that women are at increased risk for pain in many acute and chronic conditions. The proposed translational clinical study directly tests the hypotheses that estradiol (17b-E2) increases peripheral nociceptor activity in human acute pain patients and that polymorphisms of the estrogen receptors are associated with increased nociceptor activity and post-operative pain. To address this hypothesis, we have developed a method for evaluating estradiol enhancement of the exocytotic activity of peripheral human nociceptors innervating surgical biopsies of healthy tissue (evaluating substance P (SP) release from neuron terminals innervating dental pulp from extracted third molar teeth). Our preliminary studies identified estrogen receptor a (ERa) and ERa in both human trigeminal ganglia neurons and in peripheral neurons in surgical biopsies, and discovered that estradiol significantly increases bradykinin/prostaglandin E2 (BK/PGE2)-evoked iSP from human tissue biopsies collected from women, but not from men. Moreover, in this A1 revision, we report new findings that certain polymorphisms of ERa and ERb are positively correlated with increased intensity of post-operative pain in these patients. Thus, we have developed a working hypothesis that estrogen sensitizes peripheral terminals of human trigeminal nociceptors. To evaluate this hypothesis, we propose the following aims:
Specific Aim 1: Determine whether females differ from males for increased release of SP following stimulation with either BK/PGE2 or with capsaicin.
Specific Aim 2: Determine whether elevated circulating estradiol levels are associated with increased release of SP following stimulation with either BK/PGE2 or with capsaicin
Specific Aim 3: Determine whether specific polymorphisms of the estrogen receptor alpha (ERa) or beta (ERb) are associated with increased BK/PGE2- or capsaicin-evoked release of SP from surgical biopsies exposed to estradiol.
Specific Aim 4: Establish a data and gene bank to facilitate future genetic epidemiological and pharmacogenetic studies. This database will be used for three proposed secondary analyses including an evaluation of the association between ER polymorphisms and post-surgical pain and will be also available to other investigators to foster the NIH policy for data sharing.
These integrated studies will evaluate estrogen modulation of peripheral human nociceptors at the pharmacological, biochemical and genetic levels. Collectively, these studies provide a comprehensive evaluation of the hypothesis that activation of estrogen receptors enhances nociceptor function in women.
Prolactin Modulation of Trigeminal Nociceptors:
The role of gender and pain remains a major health care problem, and in preliminary studies on this topic, we evaluated the long-term effects of estradiol on gene expression in trigeminal neurons. The results constituted an unexpected discovery that estradiol upregulates prolactin (PRL) more than 40 fold in sensory neurons. Follow-up studies demonstrated that PRL and the PRL receptors (PRL-R) are expressed in the capsaicin-sensitive neurons of both female and male rats, and that application of capsaicin evokes PRL release from trigeminal sensory neurons. Furthermore, application of exogenous PRL significantly increases nociceptor responsiveness to capsaicin as measured by inward currents, CGRP exocytosis, accumulation of intracellular calcium levels, and nocifensive behavior. We now provide new studies demonstrating increased PRL levels in CFA inflamed tissue and antihyperalgesic actions of a PRL receptor antagonist. These preliminary data provide strong initial support for a completely new hypothesis of nociceptor regulation by an autocrine/paracrine system containing PRL. Based upon this hypothesis, PRL may serve as a novel hyperalgesic agent in both females and in males. We believe that this discovery has substantial scientific and medical implications, and is highly innovative from a conceptual perspective. Therefore, this project will characterize the mechanisms mediating prolactin effects in female and male rats and will directly test the hypotheses that PRL evokes a rapid increase in the responsiveness sensory neurons to noxious stimuli such as capsaicin. Our specific aims will:
Specific Aim 1: Determine the effects of exogenous PRL on capsaicin- and inflammation-induced hyperalgesia/allodynia.
Specific Aim 2: Determine the mechanisms by which PRL rapidly increases the responsiveness of trigeminal neurons to noxious chemical and thermal stimuli.
Specific Aim 3: Characterize the stimuli that evoke PRL release in trigeminal sensory neurons from in vitro cultures and from acutely isolated and superfused peripheral terminals.
The discovery that trigeminal sensory neurons express both PRL and PRLR, and that application of exogenous PRL significantly and rapidly sensitizes trigeminal nociceptors to noxious stimuli such as capsaicin, provides strong initial support for a completely new and innovative hypothesis of nociceptor regulation by an autocrine/paracrine PRL system, and compounds that block the PRL-R may serve as a novel class of analgesic drugs in gender dependent pain.
Sodium Channel Expression in Human Teeth:
Voltage-gated sodium channels are critical to the initiation and propagation of nerve action potentials and some sodium channel (NaCh) isoforms are preferentially expressed in primary afferent nociceptors. Experimental animal and a few human studies have shown changes in the distribution and expression of certain isoforms within primary afferent neurons associated with peripheral inflammation and following nerve injury. These alterations implicate dynamic NaCh expression as a basic underlying mechanism contributing to inflammatory and neuropathic pain. The tooth pulp is a rich source of pain fibers and represents a valuable model system to study pain mechanisms. Normal wisdom teeth and diseased teeth with a diagnosis of irreversible pulpitis and known pain levels are commonly extracted, providing an ample supply of tissues for analysis. The responses to sensory stimuli can be evaluated prior to extraction, thus allowing a possible correlation of NaCh expression with receptor expression for various stimuli. The overall objective of this study is to correlate changes in NaCh expression with changes in hot and cold thermoreceptors to pain levels and clinical responses to hot and cold stimuli in normal and diseased human extracted teeth. NaCh and thermoreceptor expressions will be quantified in normal and in modality-specific pain groups of diseased teeth. We hypothesize that in painful teeth the expression of NaCh isoforms dynamically changes, and further that these changes will correlate temporally and spatially with the expression of appropriate thermoreceptors in hot or cold sensitive painful teeth when compared to normal teeth. In a more general sense, the extracted tooth represents a powerful model system to evaluate in a quantitative fashion a possible correlation between known pain states and the alterations in diseased human tissues at the molecular level. Findings from this study will further our knowledge regarding both basic pain mechanisms and the increased occurrence of local anesthesia failures in diseased teeth that can be a major deterrent for utilization of dental care.
Specific Aims- The human tooth pulp is a rich source of pain fibers and is a common site of pathology that is often accompanied by spontaneous and stimulus-induced lingering pain. A common treatment modality includes the extraction of the offending tooth diagnosed with irreversible pulpitis. Normal wisdom teeth are also routinely extracted. Extracted teeth represent an abundant source of normal and diseased human nociceptors and the evaluation of these tissues represents a powerful model to study human pain mechanisms since the character of pain, pain levels, and response to stimuli can be documented prior to extraction.
One of the characteristics of inflammatory pain is the presence of hyperalgesia and allodynia, whereas neuropathic pain is especially characterized by spontaneous nerve activity. Interestingly, the pain felt in diseased teeth with reversible pulpal pathology usually includes an initial hypersensitivity to stimuli showing features of hyperalgesia and allodynia, followed at times by a progression to a lingering response to stimuli and/or spontaneous pain in teeth with irreversible pulpitis. This important transition from hypersensitivity to spontaneous and stimulus-induced lingering pain suggests the use of pulpal pain as a model system for understanding not only inflammatory pain, but also as a model for studying neuropathic pain mechanisms.
The activation of voltage-gated sodium channels (NaChs) is a critical factor in the generation of nerve action potentials and recent interest has focused on the changes that occur in the expression, location and distribution of these NaChs following inflammation and nerve injury. These changes may be associated with the development of increased resting and stimulated fiber excitability leading to increased pain perception and the possible maintenance of a neuropathic pain state. A gradual change in the composition and density of NaCh isoforms associated with the progression of pulpal pathology may represent a mechanism contributing to spontaneous and stimulus-induced lingering pain that characterizes irreversible pulpitis. The extracted human tooth also allows a quantitative investigation of a possible relationship between NaCh expression and expression of thermal receptors involved in nociception. Hot and cold thermal stimuli may differentially induce pain in teeth with irreversible pulpitis and the expressions of the vanilloid receptor subtype 1 (VR1; responsible for moderate heat) and the cold-and menthol-sensitive receptor-1 (CMR1; responsible for cold) can be evaluated. The responses to these stimuli that commonly produce lingering pain in diseased teeth can be evaluated prior to extraction and these responses will form modality-specific pain groups. The response profiles of each of these modality-specific pain groups can then be correlated with the appearance of different NaCh isoform(s) and with their colocalization with modality-related transducers.
The overall objective of this study is to correlate changes in the expression of NaChs with clinical responses to hot and cold thermal stimuli, and the expression of the associated receptors/transducers responsible for receiving that stimulus in extracted normal teeth as compared to in teeth with irreversible pulpitis associated with severe and spontaneous pain. The hypothesis is that there will be a dynamic change in the expression of NaChs in diseased and painful teeth that is correlated with an upregulation of appropriate receptors in teeth that are hypersensitive to heat and/or cold. The following specific aims will address this hypothesis:
Aim 1- To evaluate quantitatively the overall NaCh and Nav1.3, 1.6, 1.7, 1.8, 1.9 isoform expressions in nerves of normal teeth as compared to diseased teeth. This aim will include analysis of NaCh expression in coronal versus radicular pulp and in nodal and non-nodal sites (i.e. unmyelinated axons and newly-observed non-nodal clusters in myelinated axons).
Aim 2- To evaluate quantitatively hot/cold VR1 and CMR1 receptor expression in nerves of normal teeth as compared to the nerves in the modality-specific pain groups of diseased teeth. These findings will be correlated with NaCh expression as determined from Aim 1. These experiments will investigate whether there is a spatially coordinate regulation of receptors and NaCh’s in normal and diseased tooth pulp.
Aim 3- To investigate the ultrastructural localization of NaCh isoforms in in different fiber types and at sites that may be involved in pain generation. These observations will determine whether NaCh distributions seen at the light level in painful teeth are similar to those studied in other foci of neuropathic or inflammatory pain (e.g. neuromas, areas of demyelination), and thus likely to be sites of hypersensitivity to hot/cold or spontaneous pain. These studies will employ electron microscopy and immunoperoxidase methods in normal and diseased teeth.
The Role for the GPR30 Receptor in Inflammatory Pain:
Epidemiological studies indicate a higher prevalence of painful disorders in females than in males (Dao and LeResche, 2000; Yunus, 2002; Heitkemper et al., 2003). Although gender-related differences are numerous, considerable evidence implicates estrogens as critical factors in sex-dependent differences in pain, especially in conditions where inflammation is present (see review by Fillingim and Ness, 2000). Although numerous studies have investigated the genomic effects of estradiol on nociceptive responses (Drury and Gold, 1978; Ratka and Simpkins, 1991; Frye et al., 1992; Frye et al., 1993; Martinez-Gomez et al., 1994; Coyle et al., 1996; Cruz et al., 1996; Flake et al., 2005), the discovery that estradiol can activate intracellular signaling pathways through non-genomic mechanisms opens up the possibility that the steroid hormone might also induce posttranslational changes of ion channels or receptors in trigeminal neurons to alter the sensitivity of neurons to thermal, mechanical, or chemical stimuli. Our preliminary data indicate that both estradiol and G-1, a specific agonist for the GPR30, elicit significant increases in inflammation-induced orofacial thermal hyperalgesia in female rats, but have no effect on thermal sensitivity in saline-injected animals. Likewise, acute treatment with estradiol augments the stimulated release of the nociceptive neuropeptide iCGRP from inflamed peripheral tissues, but has no effect on release from noninflamed biopsies. Inflammation induced by injection of the vibrissal pad with CFA augments the local concentration of estradiol, suggesting that this putative inflammatory mediator is present in vivo at relatively high concentrations around the nerve terminals. Finally, we demonstrate that not only are estradiol levels increased with inflammation, but the GPR30 receptor expression within the trigeminal ganglia is also increased subsequent to vibrissal pad inflammation in trigeminal ganglia isolated from female (OVX + E2 replacement) rats. These data suggest that inflammation and estrogen may interact to facilitate nociceptive signaling and contribute to increased pain perception in females. We will examine the expression and localization of the receptor in trigeminal ganglia, infraorbital nerve, and vibrissal pad skin from control and inflamed rats using both mRNA and protein as endpoints. Furthermore, we will ascertain whether the GPR30 has a functional role in modulating nociceptor sensitivity by means of in vivo behavioral studies and in vitro studies examining the sensitivity of trigeminal neurons. Because the pharmacology of the GPR30 distinguishes it from other estrogen receptors, the findings here may provide an opportunity for the development of therapeutics which can selectively reverse GPR30 activity to attenuate inflammatory pain without affecting the actions of estrogens at the classical receptors.
Interactions of TRPV1 and TRPA1 and Inflammatory Pain:
The management of inflammatory pain represents a major scientific and health care challenge and many currently used analgesics provide inadequate pain relief. A mechanistic-based approach to pain management might contribute to the development of valid and novel hypotheses and improve target selection, assist the development and analysis of animal pain models, and eventually inform the design of clinical trials. Inflammatory pain has peripheral and central components. Research into peripheral mechanisms of inflammatory pain is of particular interest since it offers the potential for developing analgesics with minimal CNS side effects. This is an area in which there have been rapid advances over the past decade, although our understanding of peripheral mechanisms of inflammatory pain still remains incomplete.
In this application, we propose to test the primary hypothesis that a functional interaction between TRPA1 and TRPV1 channels plays a key role in the integration of inflammation-induced stimuli by sensory neurons. According to this hypothesis, inflammation-induced activation of the TRPA1 channel is controlled by the TRPV1 channel that fulfills these actions by serving as a modulator to the TRPA1 channel. Furthermore, inflammation-induced activation of TRPA1 is mediated by endogenous ligands, diacylglycerol (DAG) and/or 2-arachydonic glycerol (2-AG), the effects of which are also greatly enhanced via functional interaction with TRPV1. Our specific aims will:
Specific Aim #1: Determine whether TRPA1 and TRPV1 channels are a part of a receptor complex in sensory neurons and whether this complex exhibits a novel phenotype.
Specific Aim #2: Determine whether the functional activities of the TRPA1 channel are controlled by the TRPV1 channel during inflammation.
Specific Aim #3: Determine whether TRPV1 modulates the properties of TRPA1-mediated diacylglycerol and 2-arachydonic glycerol responses in sensory neurons
This conceptually innovative hypothesis which proposes distinct mechanisms of integration of inflammatory stimuli by sensory neurons has strong potential for scientific and medical implications. It suggests a functional interaction between TRP channels in regulating nociception, particularly integration of inflammatory inputs by a TRPA1/TRPV1 complex. The proposed mechanism provides a rationale for an entirely novel strategy in the design of analgesics that reduce inflammatory hyperalgesia by suppressing activities of a complex containing the TRPA1 and TRPV1 channels, but not altering functioning of individual channels. The last notion is especially important, since it may avoid the newly recognized adverse effect of hyperthermia observed with TRPV1 antagonists given to humans.