Analgesic synergy between opioid and α2 -adrenoceptors.

UNLABELLED: Opioid and α2 -adrenoceptor agonists are potent analgesic drugs and their analgesic effects can synergize when co-administered. These supra-additive interactions are potentially beneficial clinically; by increasing efficacy and/or reducing the total drug required to produce sufficient pain relief, undesired side effects can be minimized. However, combination therapies of opioids and α2 -adrenoceptor agonists remain underutilized clinically, in spite of a large body of preclinical evidence describing their synergistic interaction. One possible obstacle to the translation of preclinical findings to clinical applications is a lack of understanding of the mechanisms underlying the synergistic interactions between these two drug classes. In this review, we provide a detailed overview of the interactions between different opioid and α2 -adrenoceptor agonist combinations in preclinical studies. These studies have identified the spinal cord as an important site of action of synergistic interactions, provided insights into which receptors mediate these interactions and explored downstream signalling events enabling synergy. It is now well documented that the activation of both µ and δ opioid receptors can produce synergy with α2 -adrenoceptor agonists and that α2 -adrenoceptor agonists can mediate synergy through either the α2A or the α2C adrenoceptor subtypes. Current hypotheses surrounding the cellular mechanisms mediating opioid-adrenoceptor synergy, including PKC signalling and receptor oligomerization, and the evidence supporting them are presented. Finally, the implications of these findings for clinical applications and drug discovery are discussed. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Ligand requirements for involvement of PKCε in synergistic analgesic interactions between spinal µ and δ opioid receptors.

BACKGROUND AND PURPOSE: We recently found that PKCε was required for spinal analgesic synergy between two GPCRs, δ opioid receptors and α2 A adrenoceptors, co-located in the same cellular subpopulation. We sought to determine if co-delivery of µ and δ opioid receptor agonists would similarly result in synergy requiring PKCε. EXPERIMENTAL APPROACH: Combinations of µ and δ opioid receptor agonists were co-administered intrathecally by direct lumbar puncture to PKCε-wild-type (PKCε-WT) and -knockout (PKCε-KO) mice. Antinociception was assessed using the hot-water tail-flick assay. Drug interactions were evaluated by isobolographic analysis. KEY RESULTS: All agonists produced comparable antinociception in both PKCε-WT and PKCε-KO mice. Of 19 agonist combinations that produced analgesic synergy, only 3 required PKCε for a synergistic interaction. In these three combinations, one of the agonists was morphine, although not all combinations involving morphine required PKCε. Morphine + deltorphin II and morphine + deltorphin I required PKCε for synergy, whereas a similar combination, morphine + deltorphin, did not. Additionally, morphine + oxymorphindole required PKCε for synergy, whereas a similar combination, morphine + oxycodindole, did not. CONCLUSIONS AND IMPLICATIONS: We discovered biased agonism for a specific signalling pathway at the level of spinally co-delivered opioid agonists. As the bias is only revealed by an appropriate ligand combination and cannot be accounted for by a single drug, it is likely that the receptors these agonists act on are interacting with each other. Our results support the existence of µ and δ opioid receptor heteromers at the spinal level in vivo. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Nociceptive characteristics of tumor necrosis factor-alpha in naive and tumor-bearing mice.

A nociceptive role for tumor necrosis factor-alpha (TNF-alpha) in naive mice and in mice with fibrosarcoma tumor-induced primary hyperalgesia was investigated. The presence of TNF-alpha mRNA was confirmed in tumor site homogenates by reverse transcription-polymerase chain reaction (RT-PCR), and examination of TNF-alpha protein levels in tumor-bearing mice indicated a significantly higher concentration of this cytokine in tumor microperfusates and tumor site homogenates compared with that obtained from a similar site on the contralateral limb or in naive mice. Intraplantar injection of TNF-alpha into naive or fibrosarcoma tumor-bearing mice induced mechanical hypersensitivity, as measured by withdrawal responses evoked by von Frey monofilaments. This hypersensitivity suggests that TNF-alpha can excite or sensitize primary afferent fibers to mechanical stimulation in both naive and tumor-bearing mice. In addition, the hyperalgesia produced by TNF-alpha was completely eliminated when the injected TNF-alpha was pre-incubated with the soluble receptor antagonist TNFR:Fc. Importantly, pre-implantation systemic as well as post-implantation intra-tumor injection of TNFR:Fc partially blocked the mechanical hyperalgesia, indicating that local production of TNF-alpha may contribute to tumor-induced nociception.

Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain.

This paper describes a model of tumor-induced bone destruction and hyperalgesia produced by implantation of fibrosarcoma cells into the mouse calcaneus bone. Histological examination indicates that tumor cells adhere to the bone edge as early as post-implantation day (PID) 3, but osteolysis does not begin until PID 6, correlating with the development of hyperalgesia. C3H/He mice exhibit a reproducible hyperalgesia to mechanical and cold stimuli between PID 6 and 16. These behaviors are present but significantly reduced with subcutaneous implantation that does not involve bone. Systemic administration of morphine (ED(50) 9.0 mg/kg) dose-dependently attenuated the mechanical hyperalgesia. In contrast, bone destruction and hypersensitivity were not evident in mice implanted with melanoma tumors or a paraffin mass of similar size. A novel microperfusion technique was used to identify elevated levels of the putative algogen endothelin (ET) in perfusates collected from the tumor sites of hyperalgesic mice between PID 7 and 12. Increased ET was evident in microperfusates from fibrosarcoma tumor-implanted mice but not from melanoma tumor-implanted mice, which are not hyperalgesic. Intraplantar injection of ET-1 in naive and, to a greater extent, fibrosarcoma tumor-bearing mice produced spontaneous pain behaviors, suggesting that ET-1 activates primary afferent fibers. Intraplantar but not systemic injection of the ET-A receptor antagonist BQ-123 partially blocked tumor-associated mechanical hyperalgesia, indicating that ET-1 contributes to tumor-induced nociception. This model provides a unique approach for quantifying the behavioral, biochemical, and electrophysiological consequences of tumor-nerve interactions.

Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain.

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2-3.4 Hz) in 34% of cutaneous C-fibers adjacent to the tumor (9-17 d after implantation). C-fibers in tumor-bearing mice exhibited a mean decrease in heat threshold of 3.5 +/- 0.10 degrees C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16-24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor-bearing mice. The development of spontaneous activity and sensitization to heat in C-fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C-fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.

Mechanisms of induction of persistent nociception by dynorphin.

The opioid peptide dynorphin has been demonstrated to be both nociceptive and antinociceptive. This article will review the potential mechanisms through which dynorphin contributes to spinally mediated nociception. Specifically, we will examine the interaction of dynorphin with multiple sites on the NMDA receptor complex. Dynorphin-induced opioid activity is generally inhibitory, with a tendency to impede nociceptive signals and serve in a neuroprotective capacity. In contrast, dynorphin's interaction with multiple sites on the NMDA receptor complex produces excitatory responses resulting in nociceptive and even toxic effects. Thus, it is hypothesized that dynorphin has both physiological and pathological roles in acute and chronic pain states.

Functional interactions between tumor and peripheral nerve in a model of cancer pain in the mouse.

Cancer is usually accompanied by pain, which tends to increase in relation to metastatic infiltration and destruction. In the United States, 30% to 40% of newly diagnosed cancer patients and 67% to 90% of patients with advanced cancer report moderate to severe pain. Relief for approximately 90% of patients with cancer-related pain may be provided by the World Health Organization's "analgesic ladder," which involves progressing from non-opioid (e.g., acetaminophen, ibuprofen) to weak opioid (e.g., codeine), to strong opioid (e.g., morphine, fentanyl) intervention for pain relief. The severity of cancer pain is affected by diverse factors. In addition to the obvious factors of tumor size and degree of metastatic destruction, the type of tumor and its location are also important factors that contribute to pain severity. Severe cancer pain is especially associated with tumors involving bone destruction and nerve infiltration. Cancer pain seems to involve diverse mechanisms, including characteristics of both nociceptive and neuropathic pain. Unfortunately, even opioid analgesics often produce poor pain relief against neuropathic pain derived from peripheral nerve or root damage common to cancers involving bone metastases and nerve infiltration. In addition, these drugs may induce adverse side effects since they affect various physiological functions, including hormone secretion, neurotransmitter release, feeding, gastrointestinal motility, and respiratory activity. Currently, drug therapies utilizing antidepressants and anticonvulsants are being used to relieve neuropathic pain whereas cancer pain is treated largely with opiods in cancer patients.

Agmatine improves locomotor function and reduces tissue damage following spinal cord injury.

Clinically effective drug treatments for spinal cord injury (SCI) remain unavailable. Agmatine, an NMDA receptor antagonist and inhibitor of nitric oxide synthase (NOS), is an endogenous neuromodulator found in the brain and spinal cord. Evidence is presented that agmatine significantly improves locomotor function and reduces tissue damage following traumatic SCI in rats. The results suggest the importance of future therapeutic strategies encompassing the use of single drugs with multiple targets for the treatment of acute SCI. The therapeutic targets of agmatine (NMDA receptor and NOS) have been shown to be critically linked to the pathophysiological sequelae of CNS injury and this, combined with the non-toxic profile, lends support to agmatine being considered as a potential candidate for future clinical applications.

Moxonidine, a selective imidazoline-alpha2 -adrenergic receptor agonist, produces spinal synergistic antihyperalgesia with morphine in nerve-injured mice.

BACKGROUND: Moxonidine, a novel imidazoline-alpha2-adrenergic receptor-selective analgesic, was recently identified as antinociceptive but has yet to be evaluated in neuropathic pain models. alpha2-adrenergic receptor-selective analgesics, and high-efficacy opioids, effectively inhibit neuropathic pain behaviors in rodents. In contrast, morphine potency and efficacy decreases in states of neuropathic pain, both in rodents and in humans, but may be restored or enhanced by coadministration of morphine with alpha2-adrenergic receptor-selective analgesics. The current experiments extend the evaluation of opioid-coadjuvant interactions in neuropathic subjects by testing the respective antihyperalgesic interactions of moxonidine and clonidine with morphine in a test of mechanical hyperalgesia. METHODS: Nerve-injured mice (Chung model) were spinally administered moxonidine, clonidine, morphine, and the combinations moxonidine-morphine and clonidine-morphine. Hyperalgesia was detected by von Frey monofilament stimulation (3.3 mN) to the hind paws (plantar surface). The ED50 values were calculated and the interactions tested by isobolographic analysis. RESULTS: In nerve-injured mice, moxonidine, clonidine, and morphine all dose-dependently inhibited mechanical hyperalgesia. Furthermore, the combinations of moxonidine-morphine and clonidine-morphine resulted in substantial leftward shifts in the dose-response curves compared with those of each agonist administered separately. The calculated ED50 values of the dose-response curves of these combinations were significantly lower than their corresponding theoretical additive ED50 values. These results confirmed that both interactions were synergistic. CONCLUSIONS: Moxonidine and clonidine both synergize with morphine to inhibit paw withdrawal from nociceptive mechanical stimuli in nerve-injured mice.

Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury.

Antagonists of glutamate receptors of the N-methyl-d-aspartate subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) prevent nervous system plasticity. Inflammatory and neuropathic pain rely on plasticity, presenting a clinical opportunity for the use of NMDAR antagonists and NOS inhibitors in chronic pain. Agmatine (AG), an endogenous neuromodulator present in brain and spinal cord, has both NMDAR antagonist and NOS inhibitor activities. We report here that AG, exogenously administered to rodents, decreased hyperalgesia accompanying inflammation, normalized the mechanical hypersensitivity (allodynia/hyperalgesia) produced by chemical or mechanical nerve injury, and reduced autotomy-like behavior and lesion size after excitotoxic spinal cord injury. AG produced these effects in the absence of antinociceptive effects in acute pain tests. Endogenous AG also was detected in rodent lumbosacral spinal cord in concentrations similar to those previously detected in brain. The evidence suggests a unique antiplasticity and neuroprotective role for AG in processes underlying persistent pain and neuronal injury.

Spinal plasticity of acute opioid tolerance.

Spinal acute opioid tolerance remains mechanistically undercharacterized. Expanded clinical use of direct spinal administration of opioids and other analgesics indicates that studies to further understand spinal mechanisms of analgesic tolerance are warranted. Rodent models of spinal administration facilitate this objective. Specifically, acute spinal opioid tolerance in mice presents a plasticity-dependent, rapid, and efficient opportunity for evaluation of novel clinical agents. Similarities between the pharmacology of acute and chronic spinal opioid tolerance, neuropathic pain, and learning and memory suggest that this model may serve as a high through-put predictor of bioactivity of novel plasticity-modifying compounds.

Cytokine involvement in dynorphin-induced allodynia.

Dynorphin A is an endogenous opioid peptide, which has previously been shown to produce a long-lasting allodynia and hyperalgesia in mice, behavioral states consistent with signs of clinically observed neuropathic pain. This dynorphin-induced allodynia was used as a pharmacological, central model of neuropathic pain. In this study, we examined the involvement of the cytokine IL-1beta, the transcription factor nuclear factor kappa B (NF-kappaB), and de novo protein synthesis in the development of allodynia induced by intrathecal (i.t.) administration of dynorphin in male ICR mice. Pretreatment with the protein synthesis inhibitor cycloheximide (0. 3-85nmol), the NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC) (0.001-1000pmol), the IL-1 receptor antagonist (IL-1ra) protein (0. 01-100ng), the caspase-1 inhibitor (YVAD) (0.1-300pmol), and the anti-inflammatory cytokine IL-10 (0.1-300ng) all dose-dependently reduced the induction of dynorphin-induced allodynia. Finally, IL-10 administered within the first 24h after the dynorphin insult prevented the development of chronic allodynia. These results demonstrate that the anti-inflammatory cytokines IL-10 and IL-1ra impede the development of dynorphin-induced allodynia. These results also suggest that production of new proteins through NF-kappaB activation is required for the induction of allodynia. We speculate that IL-1ra, IL-10, PDTC and cycloheximide interfere with the central pro-inflammatory cascade. Modulation of cytokine activity in the spinal cord may therefore prove to be an effective therapeutic strategy for the treatment of chronic pain.

Effects of peripheral nerve injury on alpha-2A and alpha-2C adrenergic receptor immunoreactivity in the rat spinal cord.

Neuropathic pain resulting from peripheral nerve injury can often be relieved by administration of alpha-adrenergic receptor antagonists. Tonic activation of alpha-adrenergic receptors may therefore facilitate the hyperalgesia and allodynia associated with neuropathic pain. It is currently unclear whether alpha2A- or alpha2c-adrenergic receptor subtypes are involved in the pro-nociceptive actions of alpha-adrenergic receptors under neuropathic conditions. We therefore investigated the effects of peripheral nerve injury on the expression of these subtypes in rat spinal cord using immunohistochemical techniques. In addition, neuropeptide Y immunoreactivity was examined as an internal control because it has previously been shown to be up-regulated following nerve injury. We observed a decrease in alpha2A-adrenergic receptor immunoreactivity in the spinal cord ipsilateral to three models of neuropathic pain: complete sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. The extent of this down-regulation was significantly correlated with the magnitude of injury-induced changes in mechanical sensitivity. In contrast, alpha2c-adrenergic receptor immunoreactivity was only increased in the spinal nerve ligation model; these increases did not correlate with changes in mechanical sensitivity. Neuropeptide Y immunoreactivity was up-regulated in all models examined. Increased expression of neuropeptide Y correlated with changes in mechanical sensitivity. The decrease in alpha2A-adrenergic receptor immunoreactivity and the lack of consistent changes in alpha2C-adrenergic receptor immunoreactivity suggest that neither of these receptor subtypes is likely to be responsible for the abnormal adrenergic sensitivity observed following nerve injury. On the contrary, the decrease in alpha2A-adrenergic receptor immunoreactivity following nerve injury may result in an attenuation of the influence of descending inhibitory noradrenergic input into the spinal cord resulting in increased excitatory transmitter release following peripheral stimuli.

Moxonidine, a selective alpha2-adrenergic and imidazoline receptor agonist, produces spinal antinociception in mice.

alpha2-Adrenergic receptor (AR)-selective compounds produce antihypertensive and antinociceptive effects. Moxonidine alleviates hypertension in multiple species, including humans. This study demonstrates that intrathecally administered moxonidine produces antinociception in mice. Antinociception was detected via the (52.5 degrees C) tail-flick and Substance P (SP) nociceptive tests. Moxonidine was intrathecally administered to ICR, mixed C57BL/6 x 129/Sv [wild type (WT)], or C57BL/6 x 129/Sv mice with dysfunctional alpha2aARs (D79N-alpha2a). The alpha2AR-selective antagonist SK&F 86466 and the mixed I1/alpha2AR-selective antagonist efaroxan were tested for inhibition of moxonidine-induced antinociception. Moxonidine prolonged tail-flick latencies in ICR (ED50 = 0.5 nmol; 0. 3-0.7), WT (0.17 nmol; 0.09-0.32), and D79N-alpha2a (0.32 nmol; 0. 074-1.6) mice. Moxonidine inhibited SP-elicited behavior in ICR (0. 04 nmol; 0.03-0.07), WT (0.4 nmol; 0.3-0.5), and D79N-alpha2a (1.1 nmol; 0.7-1.7) mice. Clonidine produced antinociception in WT but not D79N-alpha2a mice. SK&F 86466 and efaroxan both antagonized moxonidine-induced inhibition of SP-elicited behavior in all mouse lines. SK&F 86466 antagonism of moxonidine-induced antinociception implicates the participation of alpha2ARs. The comparable moxonidine potency between D79N-alpha2a and WT mice suggests that receptors other than alpha2a mediate moxonidine-induced antinociception. Conversely, absence of clonidine efficacy in D79N-alpha2a mice implies that alpha2aAR activation enables clonidine-induced antinociception. When clinically administered, moxonidine induces fewer side effects relative to clonidine; moxonidine-induced antinociception appears to involve a different alpha2AR subtype than clonidine-induced antinociception. Therefore, moxonidine may prove to be an effective treatment for pain with an improved side effect profile.

Redox manipulation of NMDA receptors in vivo: alteration of acute pain transmission and dynorphin-induced allodynia.

The redox modulatory site of the N-methyl-D-aspartate (NMDA) receptor directly regulates NMDA receptor function. Sulfhydryl reducing agents, such as dithiothreitol (DTT), potentiate NMDA receptor-evoked currents in vitro, whereas oxidizing agents, such as 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), attenuate these currents. In this study, we examined the effect of this redox manipulations on nociceptive spinal cord signaling in mice. Intrathecal (i.t.) administration of DTT (0.1-30 nmol), presumably reducing the NMDA receptor, dose-dependently enhanced NMDA-induced nociceptive behaviors, and this enhancement was blocked by the oxidizing agent, DTNB. Pretreatment with DTT (10 nmol, i.t.) enhanced NMDA-induced tail-flick thermal hyperalgesia and intraplantar formalin-induced nociceptive behaviors. Finally, DTT pretreatment enhanced the long lasting allodynia induced by i.t. administration of dynorphin, whereas post-treatment with DTNB reduced the permanent allodynia induced by dynorphin for 5 days. Potentiation of all four of these NMDA-dependent nociceptive behaviors by DTT suggests that the reduction of the NMDA receptor by endogenous reducing agents may contribute to augmented pain transmission in response to activation by endogenous glutamate. Moreover, blockade of in vivo NMDA receptor reducing agents or oxidation of the NMDA receptor redox site may prove therapeutically useful in the treatment of chronic pain.

Spinal antinociceptive synergism between morphine and clonidine persists in mice made acutely or chronically tolerant to morphine.

Morphine (Mor) tolerance has been attributed to a reduction of opioid-adrenergic antinociceptive synergy at the spinal level. The present experiments tested the interaction of intrathecally (i.t.) administered Mor-clonidine (Clon) combinations in mice made acutely or chronically tolerant to Mor. ICR mice were pretreated with Mor either acutely (40 nmol i.t., 8 h; 100 mg/kg s.c., 4 h) or chronically (3 mg/kg s.c. every 6 h days 1 and 2; 5 mg/kg s.c. every 6 h days 3 and 4). Antinociception was detected via the hot water (52.5 degrees C) tail-flick test. After the tail-flick latencies returned to baseline levels, dose-response curves were generated to Mor, Clon, and Mor-Clon combinations in tolerant and control mice. Development of tolerance was confirmed by significant rightward shifts of the Mor dose-response curves in tolerant mice compared with controls. Isobolographic analysis was conducted; the experimental combined ED50 values were compared statistically against their respective theoretical additive ED50 values. In all Mor-pretreated groups, the combination of Mor and Clon resulted in significant leftward shifts in the dose-response curves compared with those of each agonist administered separately. In all tolerant and control groups, the combination of Mor and Clon produced an ED50 value significantly less than the corresponding theoretical additive ED50 value. Mor and Clon synergized in Mor-tolerant as well as in control mice. Spinally administered adrenergic/opioid synergistic combinations may be effective therapeutic strategies to manage pain in patients apparently tolerant to the analgesic effects of Mor.

Differential distribution of alpha2A and alpha2C adrenergic receptor immunoreactivity in the rat spinal cord.

alpha2-Adrenergic receptors (alpha2-ARs) mediate a number of physiological phenomena, including spinal analgesia. We have developed subtype-selective antisera against the C termini of the alpha2A-AR and alpha2C-AR to investigate the relative distribution and cellular source or sources of these receptor subtypes in the rat spinal cord. Immunoreactivity (IR) for both receptor subtypes was observed in the superficial layers of the dorsal horn of the spinal cord. Our results suggest that the primary localization of the alpha2A-AR in the rat spinal cord is on the terminals of capsaicin-sensitive, substance P (SP)-containing primary afferent fibers. In contrast, the majority of alpha2C-AR-IR was not of primary afferent origin, not strongly colocalized with SP-IR, and not sensitive to neonatal capsaicin treatment. Spinal alpha2C-AR-IR does not appear to colocalize with the neurokinin-1 receptor, nor is it localized on astrocytes, as evidenced by a lack of costaining with the glial marker GFAP. However, some colocalization was observed between alpha2C-AR-IR and enkephalin-IR, suggesting that the alpha2C-AR may be expressed by a subset of spinal interneurons. Interestingly, neither subtype was detected on descending noradrenergic terminals. These results indicate that the alpha2-AR subtypes investigated are likely expressed by different subpopulations of neurons and may therefore subserve different physiological functions in the spinal cord, with the alpha2A-AR being more likely to play a role in the modulation of nociceptive information.

Acute tolerance to spinally administered morphine compares mechanistically with chronically induced morphine tolerance.

The mechanistic similarity between acutely and chronically induced morphine tolerance has been previously proposed but remains largely unexplored. Our experiments examined the modulation of acutely induced tolerance to spinally administered morphine by agonists that affect the N-methyl-D-aspartate receptor and nitric oxide synthase systems. Antinociception was detected via the hot water (52.5 degrees C) tail flick test in mice. Intrathecal pretreatment with morphine (40 nmol) produced a 9.6-fold rightward shift in the morphine dose-response curve. This shift confirmed the induction of acute spinal morphine tolerance. Intrathecal copretreatment with the receptor antagonists (competitive and noncompetitive, respectively) dizolcipine (MK801, 3 nmol) or LY235959 (4 pmol) and morphine [40 nmol, intrathecally (i.t.)] attenuated acute tolerance to morphine measured 8 hr later. A 60-min pretreatment of 7-nitroindazole (6 nmol, i.t.), a selective neuronal NOS inhibitor, followed by administration of morphine (40 nmol, i.t.) blocked the induction of morphine tolerance. Intrathecal copretreatment with morphine (40 nmol, i.t.) and agmatine (4 nmol, i.t.), an imidazoline, receptor agonist and putative nitric oxide synthase inhibitor, almost completely abolished acute spinal morphine tolerance. The results of these experiments agree with previous reports using models of chronically induced morphine tolerance. This evidence supports the proposal that the mechanisms responsible for acute morphine tolerance parallel those underlying chronic morphine tolerance. This study attests to the powerful predictive value of acute induction as a model for morphine tolerance.

Spinal analgesic actions of the new endogenous opioid peptides endomorphin-1 and -2.

Two highly-selective mu-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous mu-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin-1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.