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.

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.

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.

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.

The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration.

Tachykinin peptides such as substance P and neurokinin B have been widely studied as mediators of pain transmission. The expression of neurokinin-1 and neurokinin-3 receptor messenger RNAs in the spinal cord is increased following intense nociception. The opiate ligands morphine and naltrexone alter behavioral responses to formalin-induced pain and alter evoked substance P release. This study investigated whether these opiates similarly alter the expression of substance P-, neurokinin B-, neurokinin-1 receptor- and neurokinin-3 receptor-encoding messenger RNAs in spinal systems following formalin-induced nociception. Expression levels of various messenger RNAs were quantitated using solution hybridization-nuclease protection assays. Six hours after hindpaw treatment, the levels of substance P-encoding preprotachykinin messenger RNA in the lumbar dorsal root ganglia and neurokinin B, neurokinin-1 receptor and neurokinin-3 receptor messenger RNAs in the lumbar dorsal horn were increased by approximately two-fold as compared to sham-treated controls. Pretreatment with naltrexone resulted in a further increase in the nociception-induced substance P messenger RNA expression in the dorsal root ganglia; preprotachykinin messenger RNA expression was not affected by morphine. Nociception-induced neurokinin-1 receptor messenger RNA expression in the dorsal horn was blocked by morphine, but was not affected by naltrexone. Both morphine and naltrexone blocked the formalin-induced increases in neurokinin B and neurokinin-3 receptor messenger RNA levels. Increased neurokinin B messenger RNA expression may reflect increased neurokinin B turnover in spinal interneurons activated by nociception. Neurokinin-3 receptor messenger RNA expression levels varied closely with, and thus may be regulated by, the levels of neurokinin B messenger RNA in the same regions. The results of this study indicate that pretreatment with opiate ligands modulates the expression of tachykinin peptide and neurokinin receptor encoding mRNAs in spinal systems following a peripheral chemogenic inflammatory stimulus. Thus, endogenous opioid systems may be involved in activity-induced changes in the expression of spinal tachykinin peptides and neurokinin receptors.

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.

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.

Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat.

Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin injected into the hindpaw as a nociceptive stimulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord in vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull cannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-like immunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechanical pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100 microliters of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 min and greater than 60 min after formalin injection. The application of a noxious pinch following formalin injection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the hindpaw activates segmental antinociceptive systems which block SP release and limit nociceptive transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Kainic acid induced seizures cause a marked increase in the expression of neurokinin-3 receptor mRNA in the rat cerebellum.

Marked changes in the expression of the tachykinin peptide neurokinin B (NKB) have been recently observed in animal models of epilepsy. In this study we investigated mRNA levels encoding the receptor for NKB, the neurokinin-3 receptor (NK-3R), after limbic seizures induced by kainic acid (KA) in the rat. NK-3R mRNA levels were determined by nuclease protection assay at various time intervals after i.p. injection of KA in the rat. Increases of more than 200% were observed in NK-3R mRNA in the cerebellum after 7 and 30 days. In the hippocampus a moderate, reversible increase (of 70%, 1 day after KA) was seen. In the frontal cortex a reduction of NK-3R mRNA (2 days after KA) was found. In the amygdala, levels of the transcript were decreased (by 50% and more) at all intervals investigated. The decreases in mRNA levels in the amygdala are consistent with the severe damage observed in this brain area. The increases in NK-3R mRNA in the cerebellum point to the development of receptor supersensitivity and suggest a functional role of NKB in this animal model of epilepsy.

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.

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.

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.