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.

Visualization of spinal afferent innervation in the mouse colon by AAV8-mediated GFP expression.

BACKGROUND: Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. METHODS: Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. KEY RESULTS: Analysis of colon from rAAV8-GFP-treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. In addition, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. CONCLUSIONS & INFERENCES: These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents.

BACKGROUND AND PURPOSE: Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein-protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain. EXPERIMENTAL APPROACH: We generated two molecules that interfered with the nNOS-PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1-299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation. KEY RESULTS: IC87201 and tat-nNOS (1-299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3',5'-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve. CONCLUSIONS AND IMPLICATIONS: nNOS-PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS-PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.

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.

Transient changes in the synthesis of nitric oxide result in long-term as well as short-term changes in acetic acid-induced writhing in mice.

A single injection of nitric oxide (NO) synthase (NOS) inhibitors prevents the development of persistent hyperalgesia induced by various manipulations, suggesting that NO precipitates long-term changes in nociception. We examined the possibility that inhibition of NOS may also be sufficient to produce long-term decreases in nociceptive assays, such as writhing, that are known to be sensitive to the short-term effects of NOS inhibitors. We characterized short- and long-term effects of NOS inhibitors, N(omega)-nitro-L-arginine (L-NAME) or 7-nitro indazole (7-NI) injected intrathecally (i.t.) in mice on acetic acid-induced writhing. Doses of L-NAME that had no effect on hot plate or tail flick latencies inhibited writhing (0. 01-30 nmol) as well as spinal nNOS activity (5 and 100 nmol) when injected i.t. 60-90 min before testing. Anti-nociception was not mimicked by D-NAME but was prevented by co-administration with the NO precursor, L-arginine. Injection i.t. of 7-NI (30 min), a selective inhibitor of neuronal NOS (nNOS), inhibited NOS activity in the spinal cord and produced anti-nociception, confirming that writhing is sensitive to inhibition of nNOS. Although the acute action of both NOS inhibitors dissipated completely by 3-6 h, a delayed and prolonged inhibition of writhing was again observed 24 h after L-NAME (5-100 nmol), a time when spinal NOS activity was no longer inhibited by L-NAME (5 and 100 nmol) or 7-NI (25 nmol). This novel effect appears to be initiated by the transient inhibition of nNOS as delayed anti-nociception was mimicked by 7-NI at doses (10-100 nmol) that no longer inhibited spinal nNOS (25 nmol) at 24 h. Co-administration with L-arginine prevented the delayed (24 h) anti-nociceptive effects of L-NAME (30 nmol). L-Arginine (30 and 100 nmol) was without effect on nociception when administered alone 60 min or 24 h prior to testing. Together these data indicate that brief changes in the activity of nNOS induce both long- as well as short-term changes in nociception.

Parenterally administered kainic acid induces a persistent hyperalgesia in the mouse and rat.

Nociceptive primary afferent C-fibers express a subset of glutamate receptors that are sensitive to kainic acid. Thus, we tested the possibility that activation of these receptors alters nociception. Intraperitoneal (i.p.) injection of kainic acid induced a persistent thermal hyperalgesia, when tested using the hot plate (mice) and tail flick (mice and rats) assays, and mechanical hyperalgesia when tested using von Frey monofilaments (rats), but had no effect on acetic acid-induced chemical nociception (mice). When administered i. p., 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an (R, S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid HBr/kainate (AMPA/KA) antagonist, completely blocked hyperalgesia. When injected intrathecally (i.t.), kainic acid itself failed to induce hyperalgesia and AMPA/KA antagonists given i.t. also failed to attenuate the hyperalgesic effect of kainic acid administered i.p. , indicating that the spinal cord is not the primary site of action. Kainic acid injected subcutaneously in the back of mice decreased response latencies in the hot plate and tail flick assays, indicating that hyperalgesia is achieved by a variety of parenteral routes of injection. Histological evaluation of rat spinal cord and dorsal root ganglia revealed no neurodegenerative changes 24 h after kainic acid. Together these data suggest that a persistent hyperalgesia results from the transient activation of AMPA/KA receptors that are located outside the spinal cord, perhaps on the distal projections of primary afferent fibers.

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.

Chelation of zinc in the extracellular area of the spinal cord, using ethylenediaminetetraacetic acid disodium-calcium salt or dipicolinic acid, inhibits the antinociceptive effect of capsaicin in adult mice.

Capsaicin depolarizes primary afferent C-fibers releasing substance P (SP) whose N-terminal metabolites appear to play a role in the development of antinociception. Because some effects of SP(1-7) are similar to those of zinc, we tested the hypothesis that zinc in the extracellular area plays a role in capsaicin-induced antinociception, as measured using the abdominal stretch (writhing) assay. Decreases in zinc were achieved by intrathecal (i.t.) injection of membrane-impermeable compounds: ethylenediaminetetraacetic acid disodium-calcium salt (Ca++ EDTA), a calcium-saturated chelator of divalent cations, or dipicolinic acid, a zinc chelator. Ten nanomoles of Ca++ EDTA had no effect on writhing at either 90 min or 24 h after injection, yet pretreatment with Ca++ EDTA prevented the development of antinociception 24 h after i.t. injection of either 2. 8 nmol of capsaicin or 10 nmol of SP(1-7). One nanomole of dipicolinic acid injected i.t. also blocked capsaicin- and SP(1-7)-induced antinociception. When injected 24 h after SP(1-7), Ca++ EDTA failed to reverse antinociception. Acute antinociception produced 30 min after injection of SP(1-7) was also blocked when Ca++ EDTA was injected 24 h, but not 60 min, before SP(1-7). Thus, the optimal time of Ca++ EDTA-induced hyperalgesia (90 min), described previously, did not correspond to that of its inhibitory effect on antinociception (24 h). In contrast, we found that the previously described antinociception after an i.t. injection of zinc (90 min) is greatly attenuated by 24 h. Thus, zinc appears to be necessary, but may not be sufficient, for the long-term antinociceptive effect of capsaicin, acting downstream from the action of substance P N-terminal metabolites.

Manipulations of zinc in the spinal cord, by intrathecal injection of zinc chloride, disodium-calcium-EDTA, or dipicolinic acid, alter nociceptive activity in mice.

Zinc is concentrated in the dorsal horn of the spinal cord and has been proposed to alter excitability of primary afferent C-fibers, structures believed to be important in nociceptive transmission. Based on the inhibitory effect of zinc on the activity of various other neurotransmitters that play a role in nociception, we tested the hypothesis that zinc modulates pain transmission. To test this, we examined the effect of exogenous zinc, administered intrathecally (i.t.), on nociception in the mouse. We also assessed the impact of decreased concentrations of endogenously occurring zinc in the extracellular fluid brought about by an i.t. injection of either ethylenediaminetetraacetic acid disodium-calcium salt (Ca++EDTA), a calcium-saturated, membrane-impermeable chelator of divalent cations, or of dipicolinic acid, a zinc chelator. Injection of zinc produced a dose-related antinociceptive effect, optimal at 90 min in the writhing assay, but had no effect on tail-flick response latencies. In contrast, injection of either Ca++EDTA or dipicolinic acid produced a dose-related hyperalgesia in the tail-flick assay at 90 min after injection. Responses induced in the writhing assay were unaffected by Ca++EDTA. Although zinc had no effect on thermal nociception, the hyperalgesic effect of Ca++EDTA was antagonized by coadministration of Ca++EDTA with zinc. Similarly, the antinociceptive effect of zinc on writhing responses was attenuated when coadministered with Ca++EDTA. Zinc also inhibited primary afferent C-fiber activity because 10 ng of zinc i.t. inhibited the behavioral response induced by injection i.t. of 1 nmol of capsaicin. Neither zinc nor Ca++EDTA altered writhing or tail-flick latencies, respectively, when injected intracerebroventricularly. These findings support the hypothesis that endogenous zinc, localized in the dorsal horn of the spinal cord, plays a role in the regulation of pain.

Mutual antagonism between nerve growth factor and substance P N-terminal activity on nociceptive activity in mice.

Nerve growth factor (NGF) induces a relatively long-term hyperalgesia in rats, whereas substance P (SP) N-terminal fragments, like SP(1-7), produce a long-lasting antinociception in mice. We used various nociceptive assays to compare the effects of these compounds on pain transmission when injected intrathecally (i.t.) in mice, and to determine whether either compound affects the action of the other. NGF produced thermal hyperalgesia when injected i.t. in mice 24 and 48 hr before testing by the tail-flick assay. During this same interval, NGF elicited no effect on the response to von Frey fibers or on chemically induced nociception measured by the writhing assay. In contrast to NGF, SP(1-7) had no effect on tail-flick latencies but induced antinociception in the writhing assay 24 hr after injection. When administered 2 hr before NGF, SP(1-7) antagonized the thermal hyperalgesic effect of NGF in a dose-related fashion, despite the inability of SP(1-7) to alter tail-flick latency when administered alone. NGF, in turn, antagonized the antinociceptive effects of SP(1-7) in the writhing assay. The D-amino acid-substituted analog, D-SP(1-7), failed to mimic the antinociceptive effect of SP(1-7) or to alter the hyperalgesic effect of NGF, which indicated a stereoselective action of SP(1-7). D-SP(1-7), that inhibits SP(1-7) binding, did reverse the ability of SP(1-7) to antagonize NGF-induced hyperalgesia, consistent with its action as a SP N-terminal antagonist. Mutual antagonism between NGF and SP may reflect modulatory roles of these endogenously occurring peptides during chronic pain when N-terminal metabolites of SP may accumulate.

The alpha2a adrenergic receptor subtype mediates spinal analgesia evoked by alpha2 agonists and is necessary for spinal adrenergic-opioid synergy.

Agonists acting at alpha2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the alpha2a adrenergic receptor (alpha2aAR) to investigate the role of the alpha2aAR in alpha2 agonist-evoked analgesia and adrenergic-opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective alpha2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased alpha2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the delta-opioid agonist deltorphin II and the micro-opioid agonist [D-ALA2,N-Me-Phe4, Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the alpha2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the alpha2aAR subtype is the primary mediator of alpha2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the alpha2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

CP-96,345, which inhibits [3H] substance P binding, selectively inhibits the behavioral response to intrathecally administered N-methyl-D-aspartate, but not substance P, in the mouse.

((2S,3S)-[cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-az abicyclo[2.2.2]octan-3-amine]) (CP-96,345) noncompetitively inhibits substance P (SP) binding at the neurokinin-1 (NK-1) site and has been widely used to determine the extent of NK-1 activity in nociception. To test the selectivity of this compound in vivo regarding other putative nociceptive transmitters, such as excitatory amino acids, we compared the actions of CP-96,345 to those of ((2R,3R)-[cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-az abicyclo[2.2.2]octan-3-amine]), a less active isomer, on behavioral responses induced by SP, N-methyl-D-aspartate (NMDA) and kainic acid (KA) injected intrathecally in mice. When injected intrathecally, SP, NMDA or KA produce a caudally directed biting and scratching behavior that lasted for approximately 60 to 90 sec. At a dose as high as 2 nmol, CP-96,345 had no effect on responses induced by a single injection of 22.5 pmol of SP. In contrast, NMDA-induced behaviors were inhibited by CP-96,345 in a dose-related fashion beginning at a dose as low as 0.02 nmol. There was also an inhibitory effect of CP-96,345 on KA-induced activity that was not dose related. The more potent inhibitor of [3H] SP binding, (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994), was approximately 10 times more potent in inhibiting NMDA-induced activity than CP-96,345. CP-99,994 also inhibited NMDA-induced activity at doses that failed to inhibit SP-induced behavior. Also attenuated by CP-96,345 was the development of sensitization to the behavioral effects produced by repeated injections of KA and desensitization to repeated injections of SP, phenomena linked to an action of the N-terminus of SP. NMDA-induced behaviors and sensitization to KA were found to be sensitive to verapamil, consistent with their mediation by calcium. These results indicate that either CP-96,345 and CP-99,994 do not inhibit NK-1-induced activity in the mouse spinal cord, or that exogenously administered SP does not induce behavioral responses by an interaction with NK-1 receptors. Whether CP-96,345 acts by a mechanism that involves inhibition of calcium channels and/or SP N-terminal activity requires further testing.

Evidence that the NH2-terminus of substance P modulates N-methyl-D-aspartate-induced activity by an action involving sigma receptors.

Behaviors induced in mice by intrathecal injections of either N-methyl-D-aspartate (NMDA) or kainic acid are modulated by NH2-terminal fragments of substance P, such as substance P-(1-7). The action of substance P-(1-7) on kainic acid depends on sigma receptor activity. The present study was designed to test the hypothesis that sigma receptor activity is also necessary for modulation of NMDA by substance P-(1-7). Intrathecal injection of mice with NMDA results in a brief burst of biting and scratching behaviors which decrease in intensity when NMDA is injected repeatedly at 2 min intervals. Pretreatment with 1,3-di-O-tolylguanidine (DTG), a ligand at both sigma 1 and sigma 2 sites, converted NMDA-induced desensitization to sensitization, thereby enhancing tonic NMDA receptor activity. Although haloperidol (30 min) alone was without effect, the potentiation of NMDA-induced activity by DTG was abolished by haloperidol but unaffected by an equimolar dose of either spiperone or thiothixine, two dopamine receptor antagonists. When mice received substance P-(1-7), NMDA-induced behaviors were initially inhibited but then potentiated. Pretreatment with haloperidol prevented both inhibitory and potentiative effects of substance P-(1-7) whereas thiothixine did not, suggesting inhibitory as well as potentiative modulation of NMDA by sigma receptor activity. Endogenous sigma 1 receptor activity may enhance NMDA receptor activity as a treatment regimen that down-regulates sigma 1 binding also inhibited responses to NMDA. In contrast, pretreatment with haloperidol just 5 min prior to challenge, which blocks both sigma 1 and sigma 2 receptor activity, increased responses to NMDA suggesting an inhibitory effect of sigma 2 receptor activity. In summary, modulation of NMDA by substance P-(1-7) appears to depend on activity at sigma sites as substance P-(1-7) mimicked the potentiative effects of DTG, while haloperidol inhibited the effects of both DTG and substance P-(1-7).

Sensitization to the behavioral effect of kainic acid in the mouse is mediated by nitric oxide.

Kainic acid (KA)-sensitive receptors are located on primary afferent C-fibers. Behavioral sensitization to each of four repeated injections of KA appears to involve activation of primary afferent C-fibers based on its susceptibility to capsaicin pretreatment. Hyperalgesia, thought to involve transmission along C-fibers, is sensitive to pharmacologic manipulation of nitric oxide (NO). We tested the hypothesis that KA activates C-fibers, either directly or indirectly, by a mechanism that involves NO. Pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, inhibited KA sensitization whereas D-NAME, the inactive isomer, failed to mimic this action. D-Arginine also inhibited sensitization to KA, whereas L-arginine, a NO precursor, was inactive when administered alone but reversed the inhibitory effect of L-NAME. Methylene blue, which inhibits guanylyl cyclase and NO synthase, attenuated KA sensitization, suggesting that cyclic GMP synthesis may also be involved in this phenomenon. Reduced hemoglobin, which sequesters NO in the extracellular space, attenuated KA sensitization, indicating that the effect of NO is brought about in structures adjacent to cells in which it is synthesized. This convergence of data is consistent with the mediation of behavioral sensitization to KA by NO. KA sensitization has been shown to involve an action of the NH2 terminus of substance P (SP) and NO may thus mobilize SP. Consistent with this, in the presence of SP(1-7), methylene blue was no longer able to inhibit sensitization to KA, suggesting that NO evokes, rather than results from, mobilization of SP.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P N-terminal metabolites and nitric oxide mediate capsaicin-induced antinociception in the adult mouse.

The present investigation describes the antinociceptive effect of capsaicin in the acetic acid-induced abdominal stretch assay and its mediation by substance P(1-7) fragment [SP(1-7)] and nitric oxide (NO). When injected intrathecally 24 hr before testing, SP(1-7) produced a dose-related decrease in the number of abdominal stretches induced by an i.p. injection of acetic acid. The antinociceptive effect of SP(1-7) (10 nmol) persisted for 62 hr after its injection, a time course that was similar to that produced by a dose of capsaicin (2.6 nmol) that produced an effect of similar magnitude. Antinociception induced by 10 nmol of SP(1-7) was completely reversed by coadministration of 10 nmol of D-SP(1-7); the equivalent antinociception produced by capsaicin was reversed by as small a dose as 1 nmol of D-SP(1-7). The guanylate cyclase inhibitor, methylene blue, at a dose of 10 nmol, prevented both SP(1-7)- and capsaicin-induced antinociception. Capsaicin-induced, but not SP(1-7)-induced, antinociception was prevented by Nw-nitro-L-arginine methyl ester, an NO synthase inhibitor. This inhibition of capsaicin was reversed by coadministration of 120 nmol of L-arginine. Reduced hemoglobin did not prevent capsaicin-induced antinociception. These findings suggest NO is produced and acts within capsaicin-sensitive primary afferent fibers in the dorsal spinal cord to mobilize substance P, resulting in N-terminal induced-antinociception.

Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse.

Intrathecal injection of N-methyl-D-aspartate (NMDA) induces a short duration hyperalgesia in mice. An inhibitor of nitric oxide synthase (NOS), N omega-nitro-L-arginine methyl ester (L-NAME), administered either systemically or intrathecally, blocked the NMDA-induced hyperalgesia. This effect was partially reversed by the NOS substrate, L-arginine. Intrathecal hemoglobin mimicked the effects of L-NAME. Intrathecal injection of the NO-donating compounds, sodium nitroprusside (SNP) and hydroxylamine, resulted in a hyperalgesia that lasted 3 h and was reduced by coadministration of hemoglobin. Thus, nitric oxide production appears to mediate NMDA-induced hypersensitivity and may contribute to other forms of centrally induced hyperalgesia.

Spinal interactions between opioid and noradrenergic agonists in mice: multiplicativity involves delta and alpha-2 receptors.

The nature of the interaction between spinally administered opioid and alpha-2 agonists was investigated using the substance P behavioral test in mice. Morphine and agonists which more selectively activate mu or delta opioid receptors were co-administered intrathecally with direct and indirect acting adrenergic agonists norepinephrine, cocaine or clonidine and the behavioral responses to intrathecally coadministered substance P were evaluated. The ED50 values for agonists administered separately and concurrently were computed and drug interactions were evaluated using isobolographic analyses. After separate administration, all the opioid and adrenergic agonists inhibited the substance P-induced behavioral responses. Upon coadministration of opioid and adrenergic agonists, a multiplicative interaction was observed between morphine or the delta agonist D-Pen2-D-Pen-5-enkephalin and the adrenergic agonists. Additive or antagonistic interactions were found between the mu agonist Tyr-D-Ala-NMe-Phe-Gly(ol) and the same adrenergic agonists. The opioid antagonist naloxone and the alpha-2 adrenergic antagonist idazoxan were given as intrathecal pretreatments at doses chosen to shift the dose-response curves of their corresponding agonist (given alone) 4- to 10-fold to the right; this always resulted in a smaller, but significant (2- to 4-fold) shift in the dose-response curve of the other agonist given alone. Intrathecal pretreatment with naloxone or idazoxan altered some interactions between the opioids and clonidine. Although naloxone blocked completely the multiplicative interaction between morphine and clonidine, idazoxan did not. Both naloxone and idazoxan changed the antagonistic interaction between Tyr-D-Ala-NMe-Phe-Gly(ol) and clonidine to a multiplicative interaction. Neither antagonist blocked the multiplicative interaction between D-Pen2-D-Pen5-enkephalin and clonidine. These results suggest that: 1) interactions between opioid and adrenergic agonists in mouse spinal cord are mediated by delta and alpha-2 receptor subtypes; 2) the synergistic interaction between morphine and alpha-2 adrenergic agonists may involve action at delta opioid receptors; and 3) antagonist action on these drug interactions is complex.