Andrea G. Nackley

Human Catechol-O-Methyltransferase Haplotypes Modulate Protein Expression by Altering mRNA Secondary Structure

Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

Catechol-O-methyltransferase gene polymorphisms are associated with multiple pain-evoking stimuli

&NA; Variations in the gene encoding catechol‐O‐methyltransferase (COMT) are linked to individual differences in pain sensitivity. A single nucleotide polymorphism (SNP) in codon 158 (val158met), which affects COMT protein stability, has been associated with the human experience of pain. We recently demonstrated that three common COMT haplotypes, which affect the efficiency of COMT translation, are strongly associated with a global measure of pain sensitivity derived from individuals’ responses to noxious thermal, ischemic, and pressure stimuli. Specific haplotypes were associated with low (LPS), average (APS), or high (HPS) pain sensitivity. Although these haplotypes included the val158met SNP, a significant association with val158met variants was not observed. In the present study, we examined the association between COMT genotype and specific pain‐evoking stimuli. Threshold and tolerance to thermal, ischemic, and mechanical stimuli, as well as temporal summation to heat pain, were determined. LPS/LPS homozygotes had the least, APS/APS homozygotes had average, and APS/HPS heterozygotes had the greatest pain responsiveness. Associations were strongest for measures of thermal pain. However, the rate of temporal summation of heat pain did not differ between haplotype combinations. In contrast, the val158met genotype was associated with the rate of temporal summation of heat pain, but not with the other pain measures. This suggests that the val158met SNP plays a primary role in variation in temporal summation of pain, but that other SNPs of the COMT haplotype exert a greater influence on resting nociceptive sensitivity. Here, we propose a mechanism whereby these two genetic polymorphisms differentially affect pain perception.

Idiopathic pain disorders--pathways of vulnerability.

Idiopathic pain disorders (IPDs) consist of such conditions as temporomandibular joint disorders (TMJD), fibromyalgia syndrome (FMS), irritable bowel syndrome (IBS), chronic headaches, interstitial cystitis, chronic pelvic pain, chronic tinnitus, whiplash-associated disorders, and vulvar vestibulitis (VVS). IPDs commonly aggregate as ‘‘comorbid’’ conditions that are characterized by a complaint of pain as well as a mosaic of abnormalities in motor function, autonomic balance, neuroendocrine function, and sleep. Although the mechanisms that underlie the majority of these conditions are poorly understood, IPDs have been associated with a state of pain amplification and psychological distress (McBeth et al., 2001; Bradley and McKendree-Smith, 2002; Verne and Price, 2002; Gracely et al., 2004).

Catechol-O-methyltransferase inhibition increases pain sensitivity through activation of both β2- and β3-adrenergic receptors

Abstract Catechol‐O‐methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Our group demonstrated that human genetic variants of COMT are predictive for the development of Temporomandibular Joint Disorder (TMJD) and are associated with heightened experimental pain sensitivity [Diatchenko, L, Slade, GD, Nackley, AG, Bhalang, K, Sigurdsson, A, Belfer, I, et al., Genetic basis for individual variations in pain perception and the development of a chronic pain condition, Hum Mol Genet 2005;14:135–43.]. Variants associated with heightened pain sensitivity produce lower COMT activity. Here we report the mechanisms underlying COMT‐dependent pain sensitivity. To characterize the means whereby elevated catecholamine levels, resulting from reduced COMT activity, modulate heightened pain sensitivity, we administered a COMT inhibitor to rats and measured behavioral responsiveness to mechanical and thermal stimuli. We show that depressed COMT activity results in enhanced mechanical and thermal pain sensitivity. This phenomenon is completely blocked by the nonselective β‐adrenergic antagonist propranolol or by the combined administration of selective β2‐ and β3‐adrenergic antagonists, while administration of β1‐adrenergic, α‐adrenergic, or dopaminergic receptor antagonists fail to alter COMT‐dependent pain sensitivity. These data provide the first direct evidence that low COMT activity leads to increased pain sensitivity via a β2/3‐adrenergic mechanism. These findings are of considerable clinical importance, suggesting that pain conditions resulting from low COMT activity and/or elevated catecholamine levels can be treated with pharmacological agents that block both β2‐ and β3‐adrenergic receptors.

Selective activation of cannabinoid CB2 receptors suppresses spinal fos protein expression and pain behavior in a rat model of inflammation

Activation of cannabinoid CB(2) receptors attenuates thermal nociception in untreated animals while failing to produce centrally mediated effects such as hypothermia and catalepsy [Pain 93 (2001) 239]. The present study was conducted to test the hypothesis that activation of CB(2) in the periphery suppresses the development of inflammatory pain as well as inflammation-evoked neuronal activity at the level of the CNS. The CB(2)-selective cannabinoid agonist AM1241 (100, 330 micrograms/kg i.p.) suppressed the development of carrageenan-evoked thermal and mechanical hyperalgesia and allodynia. The AM1241-induced suppression of carrageenan-evoked behavioral sensitization was blocked by the CB(2) antagonist SR144528 but not by the CB(1) antagonist SR141716A. Intraplantar (ipl) administration of AM1241 (33 micrograms/kg ipl) suppressed hyperalgesia and allodynia following administration to the carrageenan-injected paw but was inactive following administration in the contralateral (noninflamed) paw, consistent with a local site of action. In immunocytochemical studies, AM1241 suppressed spinal Fos protein expression, a marker of neuronal activity, in the carrageenan model of inflammation. AM1241 suppressed carrageenan-evoked Fos protein expression in the superficial and neck region of the dorsal horn but not in the nucleus proprius or the ventral horn. The suppression of carrageenan-evoked Fos protein expression induced by AM1241 was blocked by coadministration of SR144528 in all spinal laminae. These data provide evidence that actions at cannabinoid CB(2) receptors are sufficient to suppress inflammation-evoked neuronal activity at rostral levels of processing in the spinal dorsal horn, consistent with the ability of AM1241 to normalize nociceptive thresholds and produce antinociception in inflammatory pain states.

A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation.

The present studies were conducted to test the hypothesis that systemically inactive doses of cannabinoids suppress inflammation-evoked neuronal activity in vivo via a peripheral mechanism. We examined peripheral cannabinoid modulation of spinal Fos protein expression, a marker of neuronal activity, in a rat model of inflammation. Rats received unilateral intraplantar injections of carrageenan (3%). In behavioral studies, carrageenan induced allodynia and mechanical hyperalgesia in response to stimulation with von Frey monofilaments. The cannabinoid agonist WIN55,212-2 (30 microg intraplantarly), administered concurrently with carrageenan, attenuated carrageenan-evoked allodynia and hyperalgesia relative to control conditions. In immunocytochemical studies, WIN55,212-2 suppressed the development of carrageenan-evoked Fos protein expression in the lumbar dorsal horn of the spinal cord relative to vehicle treatment. The same dose administered systemically or to the noninflamed contralateral paw failed to alter either carrageenan-evoked allodynia and hyperalgesia or carrageenan-evoked Fos protein expression, consistent with a peripheral site of action. The suppressive effects of WIN55,212-2 (30 microg intraplantarly) on carrageenan-evoked Fos protein expression and pain behavior were blocked by local administration of either the CB(2) antagonist SR144528 (30 microg intraplantarly) or the CB(1) antagonist SR141716A (100 microg intraplantarly). WIN55,212-3, the enantiomer of the active compound, also failed to suppress carrageenan-evoked Fos protein expression. These data provide direct evidence that a peripheral cannabinoid mechanism suppresses the development of inflammation-evoked neuronal activity at the level of the spinal dorsal horn and implicate a role for CB(2) and CB(1) in peripheral cannabinoid modulation of inflammatory nociception.

Quantification of condylar resorption in temporomandibular joint osteoarthritis

OBJECTIVE This study was performed to determine the condylar morphologic variation of osteoarthritic (OA) and asymptomatic temporomandibular joints (TMJs) and to determine its correlation with pain intensity and duration. STUDY DESIGN Three-dimensional surface models of mandibular condyles were constructed from cone-beam computerized tomography images of 29 female patients with TMJ OA (Research Diagnostic Criteria for Temporomandibular Disorders group III) and 36 female asymptomatic subjects. Shape correspondence was used to localize and quantify the condylar morphology. Statistical analysis was performed with multivariate analysis of covariance analysis, using Hotelling T(2) metric based on covariance matrices, and Pearson correlation. RESULTS The OA condylar morphology was statistically significantly different from the asymptomatic condyles (P < .05). Three-dimensional morphologic variation of the OA condyles was significantly correlated with pain intensity and duration. CONCLUSION Three-dimensional quantification of condylar morphology revealed profound differences between OA and asymptomatic condyles, and the extent of the resorptive changes paralleled pain severity and duration.

Cytokine biomarkers and chronic pain: association of genes, transcription, and circulating proteins with temporomandibular disorders and widespread palpation tenderness.

Summary Women with widespread vs localized chronic pain exhibit distinctive alterations in molecular‐genetic profiles of the inflammatory mediators MCP‐1, IL‐1ra, IL‐8, and TGFβ1. ABSTRACT For reasons unknown, temporomandibular disorder (TMD) can manifest as localized pain or in conjunction with widespread pain. We evaluated relationships between cytokines and TMD without or with widespread palpation tenderness (TMD−WPT or TMD+WPT, respectively) at protein, transcription factory activity, and gene levels. Additionally, we evaluated the relationship between cytokines and intermediate phenotypes characteristic of TMD and WPT. In a case‐control study of 344 females, blood samples were analyzed for levels of 22 cytokines and activity of 48 transcription factors. Intermediate phenotypes were measured by quantitative sensory testing and questionnaires asking about pain, health, and psychological status. Single nucleotide polymorphisms (SNPs) coding cytokines and transcription factors were genotyped. TMD−WPT cases had elevated protein levels of proinflammatory cytokine monocyte chemotactic protein (MCP‐1) and antiinflammatory cytokine interleukin (IL)‐1ra, whereas TMD+WPT cases had elevated levels of proinflammatory cytokine IL‐8. MCP‐1, IL‐1ra, and IL‐8 were differentially associated with experimental pain, self‐rated pain, self‐rated health, and psychological phenotypes. TMD−WPT and TMD+WPT cases had inhibited transcription activity of the antiinflammatory cytokine transforming growth factor β1 (TGFβ1). Interactions were observed between TGFβ1 and IL‐8 SNPs: an additional copy of the TGFβ1 rs2241719 minor T allele was associated with twice the odds of TMD+WPT among individuals homozygous for the IL‐8 rs4073 major A allele, and half the odds of TMD+WPT among individuals heterozygous for rs4073. These results demonstrate how pro‐ and antiinflammatory cytokines contribute to the pathophysiology of TMD and WPT in genetically susceptible people. Furthermore, they identify MCP‐1, IL‐1ra, IL‐8, and TGFβ1 as potential diagnostic markers and therapeutic targets for pain in patients with TMD.

A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism.

BackgroundOpioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to μ-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood.ResultsWe recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o complex, MOR1K couples to the stimulatory Gαs complex.ConclusionThe major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.

Low Enzymatic Activity Haplotypes of the Human Catechol-O-Methyltransferase Gene: Enrichment for Marker SNPs

Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val 158 met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.