Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons.

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu(31), Pro(34)]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu(31), Pro(34)]-NPY after intraplantar injection of complete Freund's adjuvant (CFA) in rat. We found that [Leu(31), Pro(34)]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [(35)S]GTPγS binding simulated by [Leu(31), Pro(34)]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception.

Constitutive µ-opioid receptor activity leads to long-term endogenous analgesia and dependence.

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced µ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Oestrogen increases nociception through ERK activation in the trigeminal ganglion: evidence for a peripheral mechanism of allodynia.

The mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), is activated in experimental models of chronic pain, and is also activated by oestrogen. We used an established model of inflammatory trigeminal pain, injection of Complete Freund's Adjuvant (CFA) into the masseter muscle, to determine whether ERK activation may play a role in hormone-related trigeminal pain disorders. We measured withdrawal responses to stimulation of the masseter (V3, primary allodynia) and whisker pad (V2, secondary allodynia) using graded monofilaments. Oestrogen treatment in the presence of inflammation increased withdrawal response to stimulation of both masseter and whisker pad compared with inflammation alone, indicating an additive effect of inflammation and oestrogen on both primary and secondary allodynia. We examined ERK activation in trigeminal ganglia from each treatment group using western blot and immunohistochemistry. Both masseter inflammation and oestrogen treatment increased ERK activation, and combined treatment had an additive effect. Both masseter inflammation and oestrogen increased the percentage of pERK immunoreactive neurons in divisions 1 and 2 (V1/2), and combined treatment increased pERK immunoreactivity in V1/2 compared with inflammation alone. We stereotactically administered ERK antagonist U0126, or inactive control U0124, to the trigeminal ganglion of CFA+E2-treated rats. U0126 decreased withdrawal responses to mechanical stimulation of the whisker pad compared with U0124-treated rats. Because the secondary allodynia in V2 after inflammation in V3 was reduced by antagonizing ERK activation in the periphery, these data suggest a peripheral component to secondary allodynia mediated through ERK activation.

Role of the oestrogen receptors GPR30 and ERalpha in peripheral sensitization: relevance to trigeminal pain disorders in women.

Oestrogen increases facial allodynia through its actions on activation of the MAPK extracellular-signal regulated kinase (ERK) in trigeminal ganglion neurons. This goal of study was to determine which oestrogen receptor is required for behavioural sensitization. Immunohistochemical studies demonstrated the presence of oestrogen receptor alpha (ERalpha) in nuclei of larger neurons and cytoplasm of smaller neurons, and the novel oestrogen receptor G-protein coupled receptor 30 (GPR30) in small diameter neurons that also contained peripherin, a marker of unmyelinated C-fibres. Specific agonists for ERalpha (PPT) and GPR30 (G-1), but not ERbeta (DPN), activated ERK in trigeminal ganglion neurons in vitro. Both G-1 and PPT treatment increased allodynia after CFA injections into the masseter of ovariectomized Sprague-Dawley rats. Treatment with oestrogen increased expression of ERalpha but not GPR30, while masseter inflammation increased GRP30 but not ERalpha. Differential modulation of these ERK-coupled receptors by oestrogen and inflammation may play a role in painful episodes of temporomandibular disorder and migraine.

Hippocampal neurokinin-1 receptor and brain-derived neurotrophic factor gene expression is decreased in rat models of pain and stress.

Acute or chronic stress can alter hippocampal structure, cause neuronal damage, and decrease hippocampal levels of the neurotrophin brain-derived neurotrophic factor (BDNF). The tachykinin substance P and its neurokinin-1 (NK-1) receptor may play a critical role in neuronal systems that process nociceptive stimuli; their importance in stress-activated systems has recently been demonstrated by the antidepressant-like actions of NK-1 receptor antagonists. However, the functional similarities between neurokinin receptors in the hippocampus and those in sensory systems are poorly understood, as is the significance of hippocampal NK-1 receptor in the context of chronic pain. Therefore, we investigated the effects of immobilization stress or inflammatory stimuli on NK-1 receptor and BDNF gene expression in the rat hippocampus. Rats received an acute or chronic immobilization stress, or an acute (formalin) or chronic (complete Freund's adjuvant) inflammatory stimulus to the right hind paw. Subsequently hippocampal volume and specific gravity were measured and NK-1 receptor and BDNF mRNA levels quantified using ribonuclease protection assays. Results showed that either stress or pain down-regulates expression of both NK-1 receptor and BDNF genes in the hippocampus. Hippocampal volume was increased by either pain or stress; this may be due to edema (decreased specific gravity). Thus, BDNF and NK-1 receptor gene plasticity may reflect sensory activation or responses to neuronal injury. These data may provide useful markers of hippocampal activation during chronic pain, and suggest similarities in the mechanisms underlying chronic pain and depression.

Differential regulation of GABA B receptor subunit expression and function.

The GABA(B) receptor is a G protein-coupled heterodimer composed of GABA(B1) and GABA(B2) subunits. In the present study, experiments were undertaken to examine the relationship between GABA(B) receptor function and subunit expression in the rat lumbar spinal cord following pharmacological and physiological manipulation of this receptor system. Although formalin-induced hind paw inflammation increases the production of GABA(B1) and GABA(B2) protein in the spinal cord within 24 h, there is no change in receptor function, as measured by the baclofen-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay. Conversely, although chronic (7 days) administration of baclofen, a GABA(B) receptor agonist, abolishes baclofen-stimulated [(35)S]GTPgammaS binding in the spinal cord tissue, causes tolerance to the sedative and antinociceptive effects of the drug, increases the number of formalin-induced hind paw flinches, and induces mechanical hyperalgesia, this treatment had no effect on the levels of GABA(B1) or GABA(B2) mRNAs in the lumbar spinal cord. The results indicate a lack of concordance between expression of GABA(B1) and GABA(B2) subunits and GABA(B) receptor function, suggesting these subunit proteins may serve multiple functions in the cells. Moreover, these findings indicate that nongenomic mechanisms are primarily responsible for the GABA(B) receptor desensitization that occurs during prolonged exposure to receptor agonist.

Sensory nerves and neuropeptides in uterine cervical ripening.

At the time of parturition (fetal delivery) the uterine cervix must "ripen," becoming soft, pliable, and dilated to accommodate the fetus' delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.

Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

Developmental regulation of GABA(B) receptor function in rat spinal cord.

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.

D(3) dopamine receptors in rat spinal cord: implications for sensory and motor function.

Quantitative autoradiography was used to determine the distribution of D(3) receptors in rat spinal cord and compare it with the distribution of D(1)-like and D(2) (and D(4)) receptors. [(3)H]PD 128907-labeled D(3) sites were observed in roughly 6-fold lower density than [(3)H]spiperone-labeled D(2) (D(4)) sites and 60-fold lower density than [(3)H]SCH 23390-labeled D(1)-like sites. Highest densities of D(3) binding were observed in the superficial layers of the dorsal horn at cervical and lumbar levels followed by the pars centralis and dorsal horn. Lowest densities of D(3) sites were detected in the ventral horn. These observations suggest that spinal D(3) receptors may play a role in sensory and/or motor function or contribute to the pharmacological effects of dopaminergic drugs.

Time-dependent changes in neurokinin(3) receptors and tachykinins during adjuvant-induced peripheral inflammation in the rat.

Although considerable evidence exists that spinal neurokinin(1) receptors are involved in central sensitization of nociception, recent evidence from knockout studies indicates that other neurokinin receptors in the spinal cord may mediate a portion of the hyperalgesia caused by substance P and neurokinin A. The present study determined whether the second most abundant class of neurokinin receptors, neurokinin(3) receptors, are regulated during persistent peripheral inflammation. Inflammation in the hind paw of the rat was induced by intraplantar injection of complete Freund's adjuvant. Receptor autoradiography revealed specific binding of [125I]-MePhe(7)-NKB, a selective ligand for neurokinin(3) receptors, in the superficial dorsal horn of the spinal cord. Specific binding of [125I]-MePhe(7)-NKB in the medial dorsal horn was reduced bilaterally two days after unilateral injection of complete Freund's adjuvant. Binding returned to basal levels four days after injection of complete Freund's adjuvant. Neurokinin(3) receptor messenger RNA levels doubled in the dorsal spinal cord at 12h and remained elevated for at least four days. The change in neurokinin(3) receptor binding and messenger RNA during adjuvant-induced inflammation may be a consequence of activation of the receptor. Spinal levels of potential endogenous ligands for spinal neurokinin(3) receptors were measured by radioimmunohistochemistry. Immunoreactive substance P but not neurokinin B peptide 2, a marker for neurokinin B, was reduced bilaterally during adjuvant-induced inflammation.Collectively, these data indicate that spinal neurokinin(3) receptors may play a role in spinal neurotransmission of injured rats and require consideration of other tachykinins as physiologically relevant ligands to spinal neurokinin(3) receptors.

Identifying pain genes: bottom-up and top-down approaches.

A major goal of pain research at the present time is the identification of pain genes. Such genes have been informally defined in a number of ways, including the deletion or transcriptional inhibition of which produces alterations in behavioral responses on nociceptive assays; those the transcription of which is selective to pain-relevant anatomic loci (eg, small-diameter cells of the dorsal root ganglion); those the transcription of which is enhanced in animals experiencing tonic nociception or hypersensitivity states; and, finally, those existing in polymorphic forms relevant to interindividual variability. The purpose of this review is to compare the utility of various bottom-up and top-down approaches in defining, identifying, and studying pain genes. We will focus on 4 major techniques: transgenic knockouts, antisense knockdowns, gene expression assays (including DNA microarray-based expression profiling), and linkage mapping.

Nociceptive regulation of GABA(B) receptor gene expression in rat spinal cord.

Activation of gamma-aminobutyric acid (GABA) neurotransmission evokes antinociceptive responses in laboratory animals. The recent cloning of GABA(B) receptor gene products makes it possible to examine the regulation of this receptor system as it relates to the mediation of pain-related sensory information. Inasmuch as acute and chronic pain alter the expression of a number of nociception-related receptors, and because such changes are important components in the regulation of pain, the present study was undertaken to determine whether GABA(B) receptor gene expression is altered in sensory systems following a peripheral nociceptive stimulus. Solution hybridization-nuclease protection assays conducted 24 h after formalin injection into the right hindpaw of the rat revealed a significant bilateral increase in GABA(B) R1 and R2 receptor expression in the dorsal lumbar spinal cord, and a significant increase in GABA(B) R1 receptor mRNA in the ipsilateral lumbar dorsal root ganglion. These findings indicate an activity-dependent, differential regulation of GABA(B) R1 and R2 receptor gene expression in spinal sensory systems in response to chemogenic nociceptive activation, suggesting that GABA(B) receptor plasticity may play an important role in regulating the mediation, and perception, of chronic pain.

Central and peripheral expression of neurokinin-1 and neurokinin-3 receptor and substance P-encoding messenger RNAs: peripheral regulation during formalin-induced inflammation and lack of neurokinin receptor expression in primary afferent sensory neurons.

The neurokinin-1 receptor and its tachykinin neuropeptide ligand substance P are associated with the mediation of nociception. Substance P released from primary afferent sensory neurons activates neurokinin receptors on both central and peripheral targets that mediate specific aspects of central sensitization and inflammatory function; however, an autoreceptor function for the neurokinin-1 receptor remains highly controversial. Activation of the neurokinin-1 receptor by substance P during chronic nociception increases neurokinin-1 receptor gene expression in the spinal cord. Similarly, neurokinin-3 receptors on peripheral or target tissues or neurons could play an important role in the sensitization of sensory neurons. Therefore, this study (i) mapped the steady-state levels of substance P-encoding preprotachykinin, neurokinin-1 and neurokinin-3 receptor messenger RNAs in central and peripheral tissues including sensory ganglia, and (ii) investigated whether formalin-evoked nociception altered the quantity or location of neurokinin-1 or neurokinin-3 receptor messenger RNAs in the sensory ganglia or inflamed peripheral targets for substance P. Solution hybridization-nuclease protection assays quantified neurokinin receptor messenger RNA levels in central and peripheral tissues from normal and formalin-inflamed rats. High concentrations of the neurokinin-1 receptor were found in whole brain, spinal cord, and peripheral target organs innervated by substance P-containing neurons. Measurable levels of neurokinin-3 receptor messenger RNA were found only in brain, spinal cord and urinary bladder. Results also show that neither neurokinin-1 nor neurokinin-3 receptor messenger RNAs were detectable in primary afferent sensory neurons in the dorsal root ganglia of normal or formalin-inflamed rats. Neurokinin-1 receptor messenger RNA levels were, however, significantly increased in hindpaw tissues inflamed by formalin for 6 h. These results indicate that the plasticity of neurokinin-1 receptor gene expression in non-neuronal peripheral cells could regulate sensitivity to substance P in a manner similar to that in the spinal cord dorsal horn. Altered neurokinin-1 receptor gene expression provides a useful marker of long-term nociceptive activation and may mediate peripheral mechanisms of hyperalgesia and cellular sensitization during inflammation. Importantly, inflammation does not induce a phenotypic change in afferent sensory neurons providing neurokinin receptor targets for the direct sensitization of these neurons by substance P.

Cyclic AMP regulates the expression of neurokinin1 receptors by neonatal rat spinal neurons in culture.

Neurokinin1 (NK1) receptors are up-regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)-responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl-cAMP (dbcAMP) resulted in a time-dependent increase in 125I-Bolton-Hunter-substance P (BHSP) binding in the cultures; treatment with dibutyryl-cyclic GMP did not. Treatment with forskolin plus 3-isobutyl-1-methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP-induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP-induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)-induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.

Effect of factor XIII on endothelial barrier function.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 +/- 7% (basal value of 5.9 +/- 0.4 x 10(-6) cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 +/- 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

Chronic non-peptide neurokinin receptor antagonist treatment alters striatal tachykinin peptide and receptor gene expression in the rat.

The neurokinin-1 receptor (NK-1R) and the tachykinin peptide substance P (SP) are found throughout the central nervous system (CNS) and are involved in the regulation of sensory, cardiovascular, and inflammatory function. Selective antagonists for the NK-1R such as CP-122,721 block NK-1R-mediated responses both in vitro and in vivo. This study investigated the effects of long-term daily CP-122,721 treatment on gene expression of SP and the NK-1R in the striatum and hindbrain of the rat. The striatum and hindbrain of rats receiving CP122,721 (5, 30, or 150 mg/kg) once-daily for 30 days were assayed for SP- and NK-1R-encoding mRNAs using solution hybridization-nuclease protection assays. Results show that treatment with CP-122,721 significantly increased SP-encoding mRNA and NK-1R mRNA levels in the striatum, but not in the hindbrain. The ability of CP-122,721 to alter SP and NK-1R gene expression may provide a use for non-peptide neurokinin receptor antagonists in the modulation of systems regulated by NK-1R function.

Regulation of neurokinin-1 receptor expression by GABA(B) receptor agonists.

Activation of GABA(B) receptors produces analgesia in acute and chronic pain models. Data indicate that a possible mechanism for this effect is a GABA(B) receptor-induced blockade of neurokinin-1 (NK-1) receptor gene expression in the spinal cord. While much more potent GABA(B) receptor agonists (CGP 44532) have been developed, there is no information on their antinociceptive properties or their ability to influence NK-1 receptors. To address these issues, rats were treated with baclofen or CGP 44532 and tested for sedation, ataxia, and pain-related behaviors in a chronic pain model (formalin hindpaw injection). In a separate group of experiments the analgesic response to a single dose of CGP 44532 was tested prior, and subsequent to, its chronic administration. The results indicate that CGP 44532 is a substantially more potent analgesic than baclofen. In addition, after chronic administration baclofen was no longer capable of inducing analgesia or of inhibiting the increased expression of NK-1R mRNA and CGP 44532 was still fully effective in both regards. The results suggest that GABA(B) agonists could be clinically useful analgesics.

The neurokinin-1 receptor antagonist LY306,740 blocks nociception-induced increases in dorsal horn neurokinin-1 receptor gene expression.

Dilute formalin injected into the rat hindpaw as a nociceptive stimulus increases neurokinin-1 receptor (NK-1R) mRNA levels in the dorsal horn of the spinal cord. Increased NK-1R mRNA levels could result from increased mRNA stability or an increased rate of NK-1R mRNA transcription. In this study, RNA samples prepared from the spinal cords of rats receiving an injection of formalin into the right hindpaw were assayed for NK-1R mRNA with the use of solution hybridization-nuclease protection assays. The mature and incompletely spliced NK-1R-encoding RNAs protected by endogenous rat RNAs were characterized by the use of NK-1R plasmid constructs containing intron sequences. NK-1R pre-mRNA species were enriched in the nuclear fractions of spinal cord samples, and in the steady state, total molar amounts of NK-1R pre-mRNAs were 2-fold greater than those of mature NK-1R mRNA. In formalin-treated animals, the temporal pattern of peak pre-mRNA levels compared with mature mRNA indicated that chemogenic nociception either activates transcription of the NK-1R gene or decreases the rate of pre-mRNA splicing. In the ipsilateral lumbar dorsal hom, NK-1R mRNA levels were significantly increased at 2 and 6 hr after formalin injection. Levels of the NK-1R pre-mRNA containing both introns A and B were significantly increased 1 hr after formalin treatment, and levels of NK-1R pre-mRNAs containing intron A were significantly elevated at 2 hr after formalin treatment. Pretreatment of rats with the selective NK-1R antagonist LY306,740 was used to determine the role of NK-1R activation in the regulation of nociception-induced NK-1R mRNA levels. Rats were pretreated with either LY306,740 or LY307,679 (an inactive enantiomer) before injection of formalin into the right hindpaw. Pretreatment with LY306,740 (but not LY307,679) completely blocked the formalin-induced increase in dorsal horn NK-1R mRNA levels and significantly reduced formalin-induced behavioral activity. Thus, activation of the NK-1R during nociception increases dorsal horn NK-1R mRNA levels through activation of transcriptional or splicing mechanisms. The stimulation of NK-1R gene expression by activation of the NK-1R provides a homologous mechanism for altering the sensitivity of dorsal horn cells to substance P, potentially via the actions of second messengers, which presumably results in the maintenance of proper sensory information processing during long term nociception.

Nerve growth factor induces mechanical allodynia associated with novel A fibre-evoked spinal reflex activity and enhanced neurokinin-1 receptor activation in the rat.

A single dose of nerve growth factor (NGF, 1 microgram/g, i.p.) administered to rats aged between postnatal days (PND) 12 and 14 resulted in a behavioural hypersensitivity of the hindlimb flexion withdrawal reflex to mechanical stimuli which developed 2 h after NGF and remained significant for 24 h. Heat hyperalgesia occurred some 4 h following NGF injection and lasted for 24 h. Isolated spinal cords were prepared from animals treated with NGF and were maintained in vitro for physiological and pharmacological analysis of lumbar spinal reflex activity. Repetitive, low-frequency group I/II A beta-fibre stimulation evoked a novel wind-up response after NGF injection similar to that produced by C-fiber group III/IV stimulation in normal animals. The neurokinin-1 (NK1) receptor antagonist RP67580 reduced the C fiber-evoked responses following NGF treatment but not in naive preparations. The novel A beta fiber-evoked wind-up response was also reduced by RP67580. The NGF-induced changes in NK1 receptor responses occurred in the absence of any detectable changes in either spinal cord NK1 receptor dose-response relationships or NK1 receptor mRNA levels. These findings are likely to be related to the behavioural allodynia observed in the present study and to central excitability changes observed after chronic inflammation where NGF levels are increased.