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Previous work indicates that the antianalgesic action of pentobarbital and neurotensin administered intracerebroventricularly in mice arises from activation of a descending system to release cholecystokinin (CCK) in the spinal cord where CCK is known to antagonize morphine analgesia. Spinal dynorphin, like CCK, has an antianalgesic action against intrathecally administered morphine. This dynorphin action is indirect; even though it is initiated in the spinal cord, it requires the involvement of an ascending pathway to the brain and a descending pathway to the spinal cord where an antianalgesic mediator works. The aim of the present investigation was to determine if the antianalgesic action of intrathecal dynorphin A involved spinal CCK. All drugs were administered intrathecally to mice in the tail flick test. Morphine analgesia was inhibited by dynorphin as shown by a rightward shift of the morphine dose-response curve. The effect of dynorphin was eliminated by administration of the CCK receptor antagonists lorglumide and PD135 158. One hour pretreatment with CCK antiserum also eliminated the action of dynorphin. On the other hand, the antianalgesic action of CCK was not affected by dynorphin antiserum. Thus, CCK did not release dynorphin. Both CCK and dynorphin were antianalgesic against DSLET but not DPDPE, delta 2 and delta 1 opioid receptor peptide agonists, respectively. The results suggest that the antianalgesic action of dynorphin occurred through an indirect mechanism ultimately dependent on the action of spinal CCK.

DynorphinA (Dyn) administered intrathecally or released spinally in mice produces antianalgesia, that is, antagonizes morphine analgesia (tail-flick test). Spinal transection eliminates this Dyn antianalgesia. Present results in mice show that intracerebroventricular administration of flumazenil, a benzodiazepine receptor antagonist, also eliminated the antianalgesic action of Dyn; flumazenil in the brain eliminated the suppressant effect of intrathecal Dyn on intrathecal and intracerebroventricular morphine-induced antinociception. Intracerebroventricular clonidine, naloxone, and norbinaltorphimine release spinal Dyn. The latent antinociceptive actions of these compounds were uncovered by intracerebroventricular flumazenil. Thus, Dyn, given intrathecally or released spinally, activates a pathway that is inhibited by intracerebroventricular flumazenil. Dyn antianalgesia is not significantly altered by intracerebroventricular administration of bicuculline and picrotoxin, suggesting that activation of the gamma-aminobutyric acid receptor has little if any involvement in the antianalgesic action of Dyn. The antagonistic effect of Dyn seems to be mimicked by benzodiazepine agonists. Furthermore, administration of a benzodiazepine receptor inverse agonist (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) inhibited Dyn antianalgesia as did flumazenil. Thus, flumazenil, through a benzodiazepine antagonist or inverse agonist action, interrupts, as does spinal transection, the neuronal circuit (cord/brain/cord) necessary for the antianalgesic action of spinal Dyn. Because Dyn antianalgesia is an indirect action, activation of the neuronal circuit must lead to the release of a direct-acting antianalgesic mediator in the spinal cord.

Morphine and heroin act on supraspinal mu-opioid receptors in ICR mice to activate descending noradrenergic and serotonergic systems to inhibit the tail flick response. Antinociception induced by supraspinal [D-Pen2,5]-enkephalin (DPDPE, delta agonist) involves a descending system mediated by spinal gamma-aminobutyric acid, GABAA and GABAB, receptors. Because in Swiss Webster mice the receptor selectivity of heroin changes to delta whereas morphine remains mu, the purpose of the present study was to determine whether this delta action of heroin was mediated spinally by GABAA and GABAB receptors. Bicuculline (GABAA receptor antagonist) and picrotoxin (chloride ion channel blocker) given intrathecally produced rightward shifts in the dose-response curves of DPDPE and heroin given intracerebroventricularly. Thus, spinal GABAA receptors were involved. Intrathecal administration of 2-hydroxysaclofen (GABAB receptor antagonist) also shifted the dose-response curves to the right. Thus, the antinociception produced by heroin, like DPDPE, by activation of delta receptors in the brain of Swiss Webster mice involved both GABAA and the GABAB receptors in the spinal cord.

Heroin activates delta receptors, whereas morphine activates mu receptors, in the brain of Swiss-Webster mice, to produce antinociception. The present study determined the type of opioid receptor activated by 6-monoacetylmorphine (MAM), a metabolite of heroin. Intracerebroventricular MAM-induced inhibition of the tail-flick response was reduced by coadministration of naltrindole (a delta opioid receptor antagonist), suggesting that i.c.v. MAM, like i.c.v. heroin, acted on delta receptors. This delta receptor-mediated response was not affected by intrathecal (i.t.) administration of yohimbine and methysergide. Thus, the descending noradrenergic and serotonergic neuronal pathways, which are activated by i.c.v. morphine, were not involved in MAM antinociception. In the spinal cord, coadministration of naltrindole with MAM, i.t., decreased antinociception suggesting that MAM acted on spinal delta receptors. This finding is in contrast to i.t. heroin which acts on mu receptors in the spinal cord. These receptor selectivities were also demonstrated for systemically administered MAM and heroin. Thus, i.c.v. naltrindole inhibited both MAM- and heroin-induced antinociception, but i.t. naltrindole only inhibited the MAM response. The following results in ICR mice contrast with those above. Antinociception induced by i.c.v. MAM was decreased by coadministration of naloxone, but not naltrindole, suggesting that MAM, like morphine and heroin, acted on supraspinal mu receptors. Also, this MAM response was inhibited by i.t. administration of methysergide which is consistent with supraspinal mu receptor activation. Furthermore, i.t. MAM acted on spinal mu receptors because i.t. administration of naloxone, but not naltrindole, produced inhibition. In conclusion, the ability to ascribe delta receptor selectivity to the action of heroin and MAM in Swiss-Webster mice served to reinforce the concept that heroin and MAM act primarily on their own and not through formation of morphine. Further elucidation of the difference in heroin and MAM receptor selectivities between Swiss-Webster and ICR mice might contribute to a better understanding of opioid receptor mechanisms.

Studies have shown that midazolam acts in the brain to antagonize the antinociception produced by morphine. The purpose of this study was to determine if spinal dynorphin A(1-17) (Dyn) was involved in the antagonistic effects of midazolam. A number of drugs when administered intracerebroventricularly (ICV) to mice release Dyn in the spinal cord to antagonize morphine-induced antinociception. In the present study using the mouse tail-flick test, midazolam administered ICV produced a dose related reduction of the antinociception induced by morphine given intrathecally (IT). The antagonistic action of midazolam against morphine-induced antinociception involved the release of Dyn in the spinal cord, as evidenced by the following results. 1) Administration of naloxone, nor-binaltorphimine and dynorphin antiserum, IT, eliminated the antagonistic effect of midazolam, given ICV, against morphine. Treatment with these opioid antagonists and dynorphin antiserum is known to inhibit the action of spinally released Dyn. 2) Production of desensitization to the effect of spinal Dyn by pretreating with morphine, 10 mg/kg subcutaneously 3 h before the tail-flick test, abolished the antagonistic action of midazolam given ICV. A 3-h pretreatment with midazolam, ICV, also produced desensitization to the antianalgesic action of Dyn given IT. 3) Elimination of the Dyn component of action of midazolam by administration of naloxone, nor-binaltorphimine and dynorphin antiserum, IT, uncovered slight antinociceptive activity of midazolam, given ICV. Coadministration of flumazenil (a benzodiazepine antagonist), bicuculline (a GABA antagonist) and picrotoxin (a chloride ion channel blocker) inhibited the midazolam effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Earlier studies from this laboratory indicated that intracerebroventricular administration of physostigmine and clonidine activated both a spinal descending analgesic and antianalgesic system. It was proposed that the latter was mediated spinally by dynorphin A (1-17), because small intrathecal doses (fmol) of dynorphin A (1-17) antagonized analgesia, while intrathecal administration of naloxone and nor-binaltorphimine (at doses which had no effect on spinal mu and kappa receptors) enhanced analgesia by attenuating the antianalgesic component. In the present studies in mice, using the tail-flick response, intrathecal administration of dynorphin antibody (antiserum to dynorphin) enhanced the analgesic effect of (10 min) physostigmine and clonidine given intraventricularly. Peak effect for the antiserum was at 1 hr. Inhibition of the tail-flick response, induced by DAMGO (Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, a mu agonist), U50, 488 H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate hydrate, a kappa agonist) and morphine was also enhanced by intrathecal administration of dynorphin antiserum. Thus, a variety of analgesic agonists appear to activate a dynorphin-mediated antianalgesic system. Such a system appears not to be activated by intraventricular administration of beta-endorphin and DPDPE (D-Pen2-D-Pen5-enkephalin, a delta agonist) because neither beta-endorphin- nor DPDPE-induced analgesia was enhanced by intrathecal administration of antiserum. The results of the experiments with the antibody provide further evidence to support the role of dynorphin A (1-17), as a putative endogenous opioid, which mediates an antianalgesic descending system in the spinal cord.