cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord.

Using neurokinin 1 receptor (NK1R) internalization to measure of substance P release in rat spinal cord slices, we found that it was induced by the adenylyl cyclase (AC) activator forskolin, by the protein kinase A (PKA) activators 6-Bnz-cAMP and 8-Br-cAMP, and by the activator of exchange protein activated by cAMP (Epac) 8-pCPT-2-O-Me-cAMP (CPTOMe-cAMP). Conversely, AC and PKA inhibitors decreased substance P release induced by electrical stimulation of the dorsal root. Therefore, the cAMP signaling pathway mediates substance P release in the dorsal horn. The effects of forskolin and 6-Bnz-cAMP were not additive with NMDA-induced substance P release and were decreased by the NMDA receptor blocker MK-801. In cultured dorsal horn neurons, forskolin increased NMDA-induced Ca2+ entry and the phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Therefore, cAMP-induced substance P release is mediated by the activating phosphorylation by PKA of NMDA receptors. Voltage-gated Ca2+ channels, but not by TRPV1 or TRPA1, also contributed to cAMP-induced substance P release. Activation of PKA was required for the effects of forskolin and the three cAMP analogs. Epac2 contributed to the effects of forskolin and CPTOMe-cAMP, signaling through a Raf - mitogen-activated protein kinase pathway to activate Ca2+ channels. Epac1 inhibitors induced NK1R internalization independently of substance P release. In rats with latent sensitization to pain, the effect of 6-Bnz-cAMP was unchanged, whereas the effect of forskolin was decreased due to the loss of the stimulatory effect of Epac2. Hence, substance P release induced by cAMP decreases during pain hypersensitivity.

Neurokinin 1 receptor activation in the rat spinal cord maintains latent sensitization, a model of inflammatory and neuropathic chronic pain.

Latent sensitization is a model of chronic pain in which a persistent state of pain hypersensitivity is suppressed by opioid receptors, as evidenced by the ability of opioid antagonists to induce a period of mechanical allodynia. Our objective was to determine if substance P and its neurokinin 1 receptor (NK1R) mediate the maintenance of latent sensitization. Latent sensitization was induced by injecting rats in the hindpaw with complete Freund's adjuvant (CFA), or by tibial spared nerve injury (SNI). When responses to von Frey filaments returned to baseline (day 28), the rats were injected intrathecally with saline or the NK1R antagonist RP67580, followed 15 min later by intrathecal naltrexone. In both pain models, the saline-injected rats developed allodynia for 2 h after naltrexone, but not the RP67580-injected rats. Saline or RP67580 were injected daily for two more days. Five days later (day 35), naltrexone was injected intrathecally. Again, the saline-injected rats, but not the RP67580-injected rats, developed allodynia in response to naltrexone. To determine if there is sustained activation of NK1Rs during latent sensitization, NK1R internalization was measured in lamina I neurons in rats injected in the paw with saline or CFA, and then injected intrathecally with saline or naltrexone on day 28. The rats injected with CFA had a small amount of NK1R internalization that was significantly higher than in the saline-injected rats. Naltrexone increased NK1R internalization in the CFA-injected rats but nor in the saline-injected rats. Therefore, sustained activation of NK1Rs maintains pain hypersensitivity during latent sensitization.

A Src family kinase maintains latent sensitization in rats, a model of inflammatory and neuropathic pain.

Latent sensitization is a long-term model of chronic pain in which hyperalgesia is continuously suppressed by opioid receptors, as demonstrated by the induction of mechanical allodynia by opioid antagonists. Different intracellular signals may mediate the initiation, maintenance and expression of latent sensitization. Our criterion for the involvement of a signal in the maintenance of latent sensitization is that inhibitors should permanently eliminate the allodynia produced by an opioid antagonist. We hypothesized that Src family kinases (SFKs) maintain latent sensitization and tested this hypothesis by inducing latent sensitization in rats with complete Freund's adjuvant (CFA) or spared nerve injury. After measures of mechanical allodynia returned to baseline, vehicle or the SFK inhibitor PP2 were injected intrathecally. The opioid antagonist naltrexone injected intrathecally 15 min later produced allodynia in control rats but not in rats injected with PP2. Vehicle or PP2 were injected daily for two more days and naltrexone was injected five days later. Again, naltrexone induced allodynia in the control rats but not in the rats injected with PP2. Results were similar when latent sensitization was induced with CFA or spared nerve injury. We concluded that an SFK, likely Fyn, maintains latent sensitization induced by inflammation or nerve injury.

Neuropeptide Y release in the rat spinal cord measured with Y1 receptor internalization is increased after nerve injury.

Neuropeptide Y (NPY) modulates nociception in the spinal cord, but little is known about its mechanisms of release. We measured NPY release in situ using the internalization of its Y1 receptor in dorsal horn neurons. Y1 receptor immunoreactivity was normally localized to the cell surface, but addition of NPY to spinal cord slices increased the number of neurons with Y1 internalization in a biphasic fashion (EC50s of 1 nM and 1 µM). Depolarization with KCl, capsaicin, or the protein kinase A activator 6-benzoyl-cAMP also induced Y1 receptor internalization, presumably by releasing NPY. NMDA receptor activation in the presence of BVT948, an inhibitor of protein tyrosine phosphatases, also released NPY. Electrical stimulation of the dorsal horn frequency-dependently induced NPY release; and this was decreased by the Y1 antagonist BIBO3304, the Nav channel blocker lidocaine, or the Cav2 channel blocker ω-conotoxin MVIIC. Dorsal root immersion in capsaicin, but not its electrical stimulation, also induced NPY release. This was blocked by CNQX, suggesting that part of the NPY released by capsaicin was from dorsal horn neurons receiving synapses from primary afferents and not from the afferent themselves. Mechanical stimulation in vivo, with rub or clamp of the hindpaw, elicited robust Y1 receptor internalization in rats with spared nerve injury but not sham surgery. In summary, NPY is released from dorsal horn interneurons or primary afferent terminals by electrical stimulation and by activation of TRPV1, PKA or NMDA receptors in. Furthermore, NPY release evoked by noxious and tactile stimuli increases after peripheral nerve injury.

Corticotropin-Releasing Factor in the Brain and Blocking Spinal Descending Signals Induce Hyperalgesia in the Latent Sensitization Model of Chronic Pain.

Latent sensitization is a model of chronic pain in which an injury triggers a period of hyperalgesia followed by an apparent recovery, but in which pain sensitization persists but is suppressed by opioid and adrenergic receptors. One important characteristic of latent sensitization is that hyperalgesia can be triggered by acute stress. To determine whether the effect of stress is mimicked by the activation of corticotropin-releasing factor (CRF) signaling in the brain, rats with latent sensitization induced by injecting complete Freund's adjuvant (CFA, 50 µl) in one hind paw were given an intracerebroventricular (i.c.v.) injection of CRF. The i.c.v. injection of CRF (0.6 µg, 10 µl), but not saline, induced bilateral mechanical hyperalgesia in rats with latent sensitization. In contrast, CRF i.c.v. did not induce hyperalgesia in rats without latent sensitization (injected with saline in the hind paw). To determine whether descending pain inhibition mediates the suppression of hyperalgesia in latent sensitization, rats with CFA-induced latent sensitization received an intrathecal injection of lidocaine (10%, 1 µl) at the cervical-thoracic spinal cord to produce a spinal block. Lidocaine-injected rats, but not rats injected intrathecally with saline, developed bilateral mechanical hyperalgesia. Intrathecal lidocaine did not induce hyperalgesia in rats without latent sensitization (injected with saline in the hind paw). These results show that i.c.v. CRF mimicked the hyperalgesic response triggered by stress during latent sensitization, possibly by blocking inhibitory spinal descending signals that suppress hyperalgesia.

Mu-opioid receptors in nociceptive afferents produce a sustained suppression of hyperalgesia in chronic pain.

The latent sensitization model of chronic pain reveals that recovery from some types of long-term hyperalgesia is an altered state in which nociceptive sensitization persists but is suppressed by the ongoing activity of analgesic receptors such as µ-opioid receptors (MORs). To determine whether these MORs are the ones present in nociceptive afferents, we bred mice expressing Cre-recombinase under the Nav1.8 channel promoter (Nav1.8cre) with MOR-floxed mice (flMOR). These Nav1.8cre/flMOR mice had reduced MOR expression in primary afferents, as revealed by quantitative PCR, in situ hybridization, and immunofluorescence colocalization with the neuropeptide calcitonin gene-related peptide. We then studied the recovery from chronic pain of these mice and their flMOR littermates. When Nav1.8cre/flMOR mice were injected in the paw with complete Freund adjuvant they developed mechanical hyperalgesia that persisted for more than 2 months, whereas the responses of flMOR mice returned to baseline after 3 weeks. We then used the inverse agonist naltrexone to assess ongoing MOR activity. Naltrexone produced a robust reinstatement of hyperalgesia in control flMOR mice, but produced no effect in the Nav1.8/flMOR males and a weak reinstatement of hyperalgesia in Nav1.8/flMOR females. Naltrexone also reinstated swelling of the hind paw in flMOR mice and female Nav1.8cre/flMOR mice, but not male Nav1.8cre/flMOR mice. The MOR agonist DAMGO inhibited substance P release in flMOR mice but not Nav1.8cre/flMOR mice, demonstrating a loss of MOR function at the central terminals of primary afferents. We conclude that MORs in nociceptive afferents mediate an ongoing suppression of hyperalgesia to produce remission from chronic pain.

Mechanisms of µ-opioid receptor inhibition of NMDA receptor-induced substance P release in the rat spinal cord.

The interaction between NMDA receptors and µ-opioid receptors in primary afferent terminals was studied by using NMDA to induce substance P release, measured as neurokinin 1 receptor internalization. In rat spinal cord slices, the µ-opioid receptor agonists morphine, DAMGO and endomorphin-2 inhibited NMDA-induced substance P release, whereas the antagonist CTAP right-shifted the concentration response of DAMGO. In vivo, substance P release induced by intrathecal NMDA after priming with BDNF was inhibited by DAMGO. ω-Conotoxins MVIIC and GVIA inhibited about half of the NMDA-induced substance P release, showing that it was partially mediated by the opening of voltage-gated calcium (Cav) channels. In contrast, DAMGO or ω-conotoxins did not inhibit capsaicin-induced substance P release. In cultured DRG neurons, DAMGO but not ω-conotoxin inhibited NMDA-induced increases in intracellular calcium, indicating that µ-opioid receptors can inhibit NMDA receptor function by mechanisms other than inactivation of Cav channels. Moreover, DAMGO decreased the ω-conotoxin-insensitive component of the substance P release. Potent inhibition by ifenprodil showed that these NMDA receptors have the NR2B subunit. Activators of adenylyl cyclase and protein kinase A (PKA) induced substance P release and this was decreased by the NMDA receptor blocker MK-801 and by DAMGO. Conversely, inhibitors of adenylyl cyclase and PKA, but not of protein kinase C, decreased NMDA-induced substance P release. Hence, these NMDA receptors are positively modulated by the adenylyl cyclase-PKA pathway, which is inhibited by µ-opioid receptors. In conclusion, µ-opioid receptors inhibit NMDA receptor-induced substance P release through Cav channel inactivation and adenylyl cyclase inhibition.

Sustained Suppression of Hyperalgesia during Latent Sensitization by µ-, δ-, and κ-opioid receptors and α2A Adrenergic Receptors: Role of Constitutive Activity.

Many chronic pain disorders alternate between bouts of pain and periods of remission. The latent sensitization model reproduces this in rodents by showing that the apparent recovery ("remission") from inflammatory or neuropathic pain can be reversed by opioid antagonists. Therefore, this remission represents an opioid receptor-mediated suppression of a sustained hyperalgesic state. To identify the receptors involved, we induced latent sensitization in mice and rats by injecting complete Freund's adjuvant (CFA) in the hindpaw. In WT mice, responses to mechanical stimulation returned to baseline 3 weeks after CFA. In µ-opioid receptor (MOR) knock-out (KO) mice, responses did not return to baseline but partially recovered from peak hyperalgesia. Antagonists of α2A-adrenergic and δ-opioid receptors reinstated hyperalgesia in WT mice and abolished the partial recovery from hyperalgesia in MOR KO mice. In rats, antagonists of α2A adrenergic and µ-, δ-, and κ-opioid receptors reinstated hyperalgesia during remission from CFA-induced hyperalgesia. Therefore, these four receptors suppress hyperalgesia in latent sensitization. We further demonstrated that suppression of hyperalgesia by MORs was due to their constitutive activity because of the following: (1) CFA-induced hyperalgesia was reinstated by the MOR inverse agonist naltrexone (NTX), but not by its neutral antagonist 6ß-naltrexol; (2) pro-enkephalin, pro-opiomelanocortin, and pro-dynorphin KO mice showed recovery from hyperalgesia and reinstatement by NTX; (3) there was no MOR internalization during remission; (4) MORs immunoprecipitated from the spinal cord during remission had increased Ser(375) phosphorylation; and (5) electrophysiology recordings from dorsal root ganglion neurons collected during remission showed constitutive MOR inhibition of calcium channels. SIGNIFICANCE STATEMENT: Chronic pain causes extreme suffering to millions of people, but its mechanisms remain to be unraveled. Latent sensitization is a phenomenon studied in rodents that has many key features of chronic pain: it is initiated by a variety of noxious stimuli, has indefinite duration, and pain appears in episodes that can be triggered by stress. Here, we show that, during latent sensitization, there is a sustained state of pain hypersensitivity that is continuously suppressed by the activation of µ-, δ-, and κ-opioid receptors and by adrenergic α2A receptors in the spinal cord. Furthermore, we show that the activation of µ-opioid receptors is not due to the release of endogenous opioids, but rather to its ligand-independent constitutive activity.

Latent sensitization: a model for stress-sensitive chronic pain.

Latent sensitization is a rodent model of chronic pain that reproduces both its episodic nature and its sensitivity to stress. It is triggered by a wide variety of injuries ranging from injection of inflammatory agents to nerve damage. It follows a characteristic time course in which a hyperalgesic phase is followed by a phase of remission. The hyperalgesic phase lasts between a few days to several months, depending on the triggering injury. Injection of µ-opioid receptor inverse agonists (e.g., naloxone or naltrexone) during the remission phase induces reinstatement of hyperalgesia. This indicates that the remission phase does not represent a return to the normal state, but rather an altered state in which hyperalgesia is masked by constitutive activity of opioid receptors. Importantly, stress also triggers reinstatement. Here we describe in detail procedures for inducing and following latent sensitization in its different phases in rats and mice.

BDNF released during neuropathic pain potentiates NMDA receptors in primary afferent terminals.

NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock-down of NMDA receptors in primary afferents decreased NMDA-induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin-related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75(NTR) ), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA-12 but not by the p75(NTR) inhibitor TAT-Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full-length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr(1472) phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch-clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA-induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain.

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by µ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors.

GABA(B) , µ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that µ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the µ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by µ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein ßγ subunits to calcium channels.

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord, measured as neurokinin 1 receptor internalization.

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of mu-opioid and GABA(B) receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.

Enkephalins, dynorphins, and beta-endorphin in the rat dorsal horn: an immunofluorescence colocalization study.

To characterize neuronal pathways that release opioid peptides in the rat dorsal horn, multiple-label immunohistochemistry, confocal microscopy, and computerized co-localization measures were used to characterize opioid-containing terminals and cells. An antibody that selectively recognized beta-endorphin labeled fibers and neurons in the ventral horn as well as fibers in the lateral funiculus and lamina X, but practically no fibers in the dorsal horn. An anti-enkephalin antibody, which recognized Leu-, Met-, and Phe-Arg-Met-enkephalin, labeled the dorsolateral funiculus and numerous puncta in laminae I-III and V of the dorsal horn. An antibody against Phe-Arg-Met-enkephalin, which did not recognize Leu- and Met-enkephalin, labeled the same puncta. Antibodies against dynorphin and prodynorphin labeled puncta and fibers in laminae I, II, and V, as well as some fibers in the rest of the dorsal horn. Dynorphin and prodynorphin immunoreactivities colocalized in some puncta and fibers, but the prodynorphin antibody additionally labeled cell bodies. There was no co-localization of dynorphin (or prodynorphin) with enkephalin (or Phe-Arg-Met-enkephalin). Enkephalin immunoreactivity did not colocalize with the C-fiber markers calcitonin gene-related peptide (CGRP), substance P, and isolectin B4. In contrast, there was some colocalization of dynorphin and prodynorphin with CGRP and substance P, but not with isolectin B4. Both enkephalin and dynorphin partly colocalized with vesicular glutamate transporter 2, a marker of glutamatergic terminals. The prodynorphin-positive neurons in the dorsal horn were distinct from neurons expressing mu-opioid receptors, neurokinin 1 receptors, and protein kinase C-gamma. These results show that enkephalins and dynorphins are present in different populations of dorsal horn neurons. In addition, dynorphin is present in some C-fibers.

Effects of veratridine and high potassium on micro-opioid receptor internalization in the rat spinal cord: stimulation of opioid release versus inhibition of internalization.

Veratridine and high K+-induced micro-opioid receptor (MOR) internalization in rat spinal cord slices by evoking opioid release. Veratridine induced up to 75% MOR internalization but showed an atypical concentration-response: its effect increased steeply from 5 microM to 10 microM, and declined thereafter to disappear at 100 microM. At 100 microM, veratridine also inhibited of MOR internalization induced by exogenous endomorphin-2. This inhibition was caused by Na+ entry, since the Na+ ionophore monensin (50 microM) also inhibited endomorphin-induced MOR internalization. In contrast, veratridine induced neurokinin 1 receptor internalization (by evoking substance P release) without any inhibition at high concentrations. KCl evoked up to 80% MOR internalization, which disappeared in the presence of lidocaine or in the absence of peptidase inhibitors, indicating that it involved neuronal firing and peptide release. Unlike veratridine, KCl did not inhibit MOR internalization at high concentrations. However, both KCl and veratridine evoked more MOR internalization when applied for 2 min than for 20 min because of a direct inhibition of MOR internalization with the longer incubation times. These results show that short incubations with 20 microM veratridine or 30 mM KCl are optimal stimuli to evoke opioid release and MOR internalization in the spinal cord.

Inhibition of opioid release in the rat spinal cord by alpha2C adrenergic receptors.

Neurotransmitter receptors that control the release of opioid peptides in the spinal cord may play an important role in pain modulation. Norepinephrine, released by a descending pathway originating in the brainstem, is a powerful inducer of analgesia in the spinal cord. Adrenergic alpha2C receptors are present in opioid-containing terminals in the dorsal horn, where they could modulate opioid release. The goal of this study was to investigate this possibility. Opioid release was evoked from rat spinal cord slices by incubating them with the sodium channel opener veratridine in the presence of peptidase inhibitors (actinonin, captopril and thiorphan), and was measured in situ through the internalization of mu-opioid receptors in dorsal horn neurons. Veratridine produced internalization in 70% of these neurons. The alpha2 receptor agonists clonidine, guanfacine, medetomidine and UK-14304 inhibited the evoked mu-opioid receptor internalization with IC50s of 1.7 microM, 248 nM, 0.3 nM and 22 nM, respectively. However, inhibition by medetomidine was only partial, and inhibition by UK-14304 reversed itself at concentrations higher than 50 nM. None of these agonists inhibited mu-opioid receptor internalization produced by endomorphin-2, showing that they inhibited opioid release and not the internalization itself. The inhibitions produced by clonidine, guanfacine or UK-14304 were completely reversed by the selective alpha2C antagonist JP-1203. In contrast, inhibition by guanfacine was not prevented by the alpha2A antagonist BRL-44408. These results show that alpha2C receptors inhibit the release of opioids in the dorsal horn. This action may serve to shut down the opioid system when the adrenergic system is active.

Noxious mechanical stimulation evokes the segmental release of opioid peptides that induce mu-opioid receptor internalization in the presence of peptidase inhibitors.

The internalization of mu-opioid receptors (MORs) provides an ideal way to locate areas of opioid peptide release. We used this method to study opioid release in the spinal cord evoked by noxious stimuli in anesthetized rats. Previous studies have shown that opioids released in the spinal cord produce MOR internalization only when they are protected from peptidase degradation. Accordingly, rats were implanted with chronic intrathecal catheters that were used to inject a mixture of peptidase inhibitors (amastatin, captopril and phosphoramidon) onto the lumbar spinal cord. Five minutes later, a noxious stimulus was delivered to the paw. Lumbar spinal segments were double-stained with antibodies against MORs and neurokinin 1 receptors (NK1Rs) using immunofluorescence. Mechanical stimulation of the hindpaw consisted of repeated 10 s clamps with a hemostat for 10 min. In the ipsilateral dorsal horn, the stimulus produced abundant NK1R internalization in segments L3-L6, and a more modest but significant MOR internalization in segments L5 and L6. In the contralateral dorsal horn, NK1R was substantially lower and MOR internalization was negligible. The same mechanical stimulus applied to a forepaw did not produce NK1R or MOR internalization in the lumbar spinal cord. Thermal stimulation consisted of immersing a hindpaw in water at 52 degrees C for 2 min. It produced substantial NK1R internalization ipsilaterally in segment L6, but no MOR internalization. These results show that mechanical stimulation induces segmental opioid release, i.e., in the dorsal horn receiving the noxious signals and not in other spinal segments.

Comparing analgesia and mu-opioid receptor internalization produced by intrathecal enkephalin: requirement for peptidase inhibition.

Opioid receptors in the spinal cord produce strong analgesia, but the mechanisms controlling their activation by endogenous opioids remain unclear. We have previously shown in spinal cord slices that peptidases preclude mu-opioid receptor (MOR) internalization by opioids. Our present goals were to investigate whether enkephalin-induced analgesia is also precluded by peptidases, and whether it is mediated by MORs or delta-opioid receptors (DORs). Tail-flick analgesia and MOR internalization were measured in rats injected intrathecally with Leu-enkephalin and peptidase inhibitors. Without peptidase inhibitors, Leu-enkephalin produced neither analgesia nor MOR internalization at doses up to 100 nmol, whereas with peptidase inhibitors it produced analgesia at 0.3 nmol and MOR internalization at 1 nmol. Leu-enkephalin was 10 times more potent to produce analgesia than to produce MOR internalization, suggesting that DORs were involved. Selective MOR or DOR antagonists completely blocked the analgesia elicited by 0.3 nmol Leu-enkephalin (a dose that produced little MOR internalization), indicating that it involved these two receptors, possibly by an additive or synergistic interaction. The selective MOR agonist endomorphin-2 produced analgesia even in the presence of a DOR antagonist, but at doses substantially higher than Leu-enkephalin. Unlike Leu-enkephalin, endomorphin-2 had the same potencies to induce analgesia and MOR internalization. We concluded that low doses of enkephalins produce analgesia by activating both MORs and DORs. Analgesia can also be produced exclusively by MORs at higher agonist doses. Since peptidases prevent the activation of spinal opioid receptors by enkephalins, the coincident release of opioids and endogenous peptidase inhibitors may be required for analgesia.

Inhibition of opioid release in the rat spinal cord by serotonin 5-HT(1A) receptors.

Neurotransmitter receptors that inhibit the release of opioid peptides in the spinal cord may play an important role in modulating pain. Serotonin is an important neurotransmitter in bulbospinal descending pathways, and 5-HT(1) receptors have been shown to inhibit synaptic transmission. Our goal was to determine whether 5-HT(1A) receptors inhibit opioid release in the spinal cord. Opioid release was evoked from rat spinal cord slices by electrically stimulating one dorsal horn, and measured in situ through the internalization of micro-opioid receptors in dorsal horn neurons. Stimulation with 1000 square pulses at 500 Hz produced internalization in 60% of the mu-opioid receptor neurons in the stimulated dorsal horn, but not in the contralateral one. The selective 5-HT(1A) receptor agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) inhibited the evoked mu-opioid receptor internalization by about 50%, with an approximate IC(50) of 50 nM. The effect of 8-OH-DPAT was attributed to inhibition of opioid release and not of the receptor internalization process, because 8-OH-DPAT did not inhibit the internalization induced by incubating the slices with a micro-opioid receptor agonist (endomorphin-2, 100 nM). The selective 5-HT(1A) receptor antagonist WAY100135 (10 microM) blocked the inhibition produced by 1 microM 8-OH-DPAT. These results show that 5-HT(1A) receptors inhibit opioid release in the spinal dorsal horn, probably from a subpopulation of enkephalin-containing presynaptic terminals. Therefore, 5-HT(1A) receptors likely decrease the analgesia produced by endogenously released opioids.

The role of neurokinin 1 receptors in the maintenance of visceral hyperalgesia induced by repeated stress in rats.

BACKGROUND & AIMS: The neurokinin 1 receptors (NK(1)Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK(1)Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress. METHODS: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK(1)R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK(1)Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia. RESULTS: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK(1)Rs. Immunohistochemistry showed an increased number of NK(1)R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK(1)Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls. CONCLUSIONS: Stress-induced increased NK(1)R expression on spinal neurons and the inhibitory effect of intrathecal NK(1)R antagonist on visceral hyperalgesia support the key contribution of spinal NK(1)Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat.

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.