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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.

The purpose was to determine the effect on the morphine-morphine glucuronide systems of Triton X-100 instilled into the area of the hepatic canalicular membrane by segmented retrograde intrabiliary injection (40 microliters of 0.4% Triton + 31 microliters of saline) in the isolated in situ perfused livers of male Sprague-Dawley rats. In all experiments, [14C]morphine was given by segmented retrograde intrabiliary injection (40 microliters of [14C]morphine + 110 microliters of saline). The control single pass perfusate contained 15.8% [14C]morphine glucuronide (MG) and 6.2% [14C]morphine. With Triton, the major changes observed were an unusual plateau-like pattern of egress of the [14C]MG into the perfusate and a profound decrease in the [14C]MG excretion into bile. In controls, 45 mg of unlabeled morphine sulfate intraportally 5 min before the [14C]morphine reduced the perfusate [14C]MG and increased [14C]morphine as expected by isotope dilution. Also, [14C]MG recovery in bile was accordingly decreased (intact pentobarbital-anesthetized rat); however, the [14C]morphine in bile did not change even though intracellular availability of [14C]morphine must have been increased. In pulse chase experiments, [14C] morphine was followed in 2 min with 45 mg of morphine sulfate intraportally. The perfusate [14C]MG declined and [14C]morphine increased. However, on the canalicular side (intact pentobarbital-anesthetized rat) no isotope dilution was evident: [14C]MG and [14C]morphine content in bile was similar to controls given no morphine sulfate. Triton treatment decreased the amount of [14C] morphine which could be chased into the perfusate. Several different pools of morphine and MG exist in the liver; the canalicular and sinusoidal sites respond very differently to manipulation of these pools.

In the present study, it is proposed that the opioids applied to supraspinal brain sites produced their analgesic effects by the activation of different descending pain inhibitory systems. The blockade of the spinal endorphinergic system by intrathecal naloxone on the production of tail-flick inhibition induced by intraventricular beta-endorphin and morphine was then studied. Intraventricular injection of beta-endorphin and morphine produced an inhibition of the tail-flick response to the heat stimulus in rats. Intrathecal injection of naloxone at doses of 0.4 to 40 micrograms caused a dose-related blockade of the inhibition of the tail-flick response induced by intraventricular injection of beta-endorphin, and a high dose of naloxone (40 micrograms) completely blocked the tail-flick inhibition induced by intraventricular beta-endorphin (16 micrograms). On the other hand, intrathecal naloxone (12-120 micrograms) had only a very weak effect on the tail-flick inhibition induced by intraventricular morphine (40 micrograms). Intraventricular injection of naloxone at doses of 1.2 to 12 micrograms equally antagonized in a dose-dependent manner the tail-flick inhibition induced by intraventricular beta-endorphin and morphine. The results indicate that a spinal naloxone-sensitive endorphinergic system is involved in the production of beta-endorphin but not morphine-induced tail-flick inhibition, and suggest that intraventricular beta-endorphin and morphine elicit their pharmacological actions via the activation of different descending pain inhibitory systems; descending epsilon and mu systems for beta-endorphin and morphine, respectively, are proposed.

Simultaneous action of morphine on supraspinal and spinal sites produces a multiplicative interaction for antinociception which may be important for the analgesia produced by systemically administered morphine. The purpose of this study was to see whether other agonists with more receptor selective opioid actions than morphine would also produce this multiplicative interaction. DAMPGO (Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5), DPDPE (D-Pen2, D-Pen5, enkephalin) and U50-488H, opioid agonists highly selective for mu, delta and kappa receptors, respectively, were administered alone i.c.v. or intrathecally (i.t.) or in combination (i.c.v. plus i.t.) to determine ED50 values for the tail-flick response in mice. These ED50 values were examined isobolographically in relation to the theoretical additive ED50 values by the potency ratio method. First, DAMPGO given i.cv and i.t. was similar to morphine, indicating that simultaneous supraspinal and spinal mu agonist administration produce the multiplicative interaction. Second, concurrent administration of DPDPE or U50,488H, i.c.v. and i.t., as well as cross-over combinations of DPDPE at one and U50,488H at the other site, produced additive interactions only. The multiplicative interaction was a property characteristic of mu but not delta and kappa agonists. Based on the similarity between morphine and DAMPGO, it was postulated that both mu agonists act on redundant descending pain inhibitory pathways to produce multiplication. A second mechanism for multiplicative interaction was based on the difference between DAMPGO and morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Etorphine, a potent opioid agonist, has been reported to bind to both mu and epsilon opioid receptors. The present studies were designed to determine what types of opioid receptors and neurotransmitters for descending pain control systems were involved in antinociception induced by etorphine in mice. Morphine, a typical mu opioid receptor agonist, and beta-endorphin, an epsilon opioid receptor agonist, were used for comparison. Antinociceptive response induced by etorphine (20 ng) given i.c.v was blocked by i.c.v administration of D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-Pen-Thr-NH2 (CTOP, 25 ng) and beta-endorphin-(1-27) [beta-EP-(1-27)] (6 micrograms), but not ICI 174,864 (ICI, 5 micrograms) or norbinaltorphimine (N-BNI, 5 micrograms). The antinociception induced by i.c.v. etorphine was also antagonized by the i.c.v. pretreatment of beta-funaltrexamine (beta-FNA, 50 ng, 24 hr). Intracerebroventricular administration of beta-EP-(1-27) (3 micrograms) caused a further attenuation of the i.c.v. etorphine-induced antinociception in mice pretreated with beta-FNA. The antinociceptive response induced by morphine (2 micrograms) given i.c.v. was blocked by i.c.v. administration of CTOP (25 ng) or beta-FNA (50 ng), but not beta-EP-(1-27) (6 micrograms), ICI (5 micrograms) or N-BNI (5 micrograms). These results indicate that the antinociception induced by etorphine given i.c.v. is mediated by the stimulation of both mu and epsilon opioid receptors whereas the antinociception induced by morphine given i.c.v. is mediated by the stimulation of mu, but not epsilon opioid receptors at supraspinal sites.(ABSTRACT TRUNCATED AT 250 WORDS)

In previous publications we proposed that dynorphin A (1-17) (Dyn) functions as an antianalgesic agent in the spinal cord of mice. Whether endogenously released or administered directly to the spinal cord, this antianalgesic action attenuates the antinociceptive effect of morphine (Mor) in the mouse tail-flick test. Because this action of Dyn in the spinal cord appeared to be congruous with the function of substance P (SP), experiments were designed to compare the actions of the two on Mor-induced antinociception. Inhibition of the tail-flick response induced by i.c.v. administration of Mor was attenuated by intrathecal (i.t.) administration of SP or Dyn. This antianalgesic effect of Dyn (5 fmol) but not SP (74 pmol) was antagonized by naloxone and nor-binaltorphimine administered i.t. Capsaicin (Cap) i.t. at a 0.1-microgram dose, like SP and Dyn, antagonized the antinociceptive effect of Mor given i.c.v. Excellent evidence exists to indicate that, in rats, Cap (30-70 micrograms i.t.) releases SP in the spinal cord and that Mor inhibits this release. Present experiments indicated, however, that i.t. administration of low doses of Cap (0.05-0.5 microgram) in mice preferentially released Dyn and not SP as based on the following results. 1) The antianalgesic action of Cap i.t. against Mor i.c.v. was antagonized by naloxone and nor-binaltorphimine i.t. as was Dyn i.t. (but not SP i.t.). 2) A SP antagonist, (D-Pro2, D-Phe7, D-Trp9)-SP, did not reverse the effect of Cap or Dyn given i.t., even though it antagonized the effect of SP.(ABSTRACT TRUNCATED AT 250 WORDS)

Previously, a number of analgesic agonists, when administered i.c.v. to mice, were shown putatively to activate the release of dynorphin A (1-17) (Dyn A) in the spinal cord. Whether released endogenously or administered i.t., Dyn A produces an antianalgesic action against i.t. administered morphine. In the present study, the opioid antagonists, naloxone and norbinaltorphimine (N-BNI), were shown to activate the Dyn A system. Intracerebro-ventricular administration of both naloxone and N-BNI antagonized the antinociceptive effect of i.t. morphine in the mouse tail-flick test, an effect designated as an antianalgesic action. This antianalgesic action was demonstrated to be mediated by spinal Dyn A in the following ways: 1) the antagonistic effect of i.c.v. naloxone and N-BNI was eliminated by administration of small doses of i.t. naloxone and N-BNI, a unique situation where administration of the opioid antagonists at a second (i.t.) site reversed the antagonistic effect of opioid antagonists administered at the other (i.c.v.) site; 2) i.t. pretreatment with dynorphin antiserum prevented the antianalgesic effect; 3) morphine pretreatment (s.c., 10 mg/kg), which produces desensitization to the effect of spinal Dyn A, eliminated the antianalgesic effect; and 4) pretreatment with i.c.v. naloxone (3 hours) and N-BNI (24 hours) which presumably releases Dyn A produced desensitization to the antagonistic effect of i.c.v. naloxone and N-BNI as well as to the antianalgesic action of i.t. Dyn A. Taken together, the results indicate that both i.c.v. naloxone and N-BNI produced indirect antagonistic actions which were mediated at the spinal cord by the antianalgesic action of Dyn A.(ABSTRACT TRUNCATED AT 250 WORDS)

Concurrent administration of morphine sulfate i.c.v. and i.t. produces a multiplicative interaction for analgesia in the tail flick response in mice. This interaction decreases to an additive interaction in mice which are tolerant to s.c. morphine. To test the responses of opioids selective for mu or delta receptors, the present study examined the interactions between fentanyl citrate (mu agonist) and D-Ala2-D-Leu5 enkephalin (DADLE, a relatively selective delta agonist) administered i.c.v. and i.t. using the tail flick test in control and morphine pellet-implanted mice. A method was developed for assigning statistical significance to the resulting ED50 values when analyzed isobolographically. When fentanyl or DADLE was administered i.c.v. plus i.t., an additive interaction between sites occurred in control animals, which changed to an antagonistic interaction for fentanyl and a multiplicative interaction for DADLE after morphine pellet treatment. When morphine was given i.c.v. along with i.t. fentanyl or DADLE in control animals, multiplicative interactions occurred when equipotent doses were given. Thus, opioids which were more receptor-selective than morphine did not produce multiplicative interactions, but were multiplicative when given with morphine. These results suggest that activation of combinations of receptors (by morphine) was required for the multiplicative interaction. The supraspinal site involved mu receptors (which are not self-sufficient and require an additional component) and the spinal site involved mu or delta receptors. The use of isobolographic analysis required that the drugs, when administered concurrently at two sites, be given in a constant dose ratio.

Differences in antinociceptive (inhibition of tail-flick response) action of morphine and Tyr-D-Ala2-Gly-NMePhe4-ol5 (DAMGO) were demonstrated by intracerebroventricular (i.c.v.) administration of these agonists along with intrathecal (i.t.) administration of a variety of antagonists: yohimbine, methysergide, naloxone and nor-binaltorphimine. Intracerebroventricular morphine analgesia was antagonized by either i.t. yohimbine or methysergide, whereas i.c.v. DAMGO analgesia was only antagonized by i.t. methysergide. Thus, for i.c.v. morphine-induced analgesia, descending spinal noradrenergic and serotonergic systems were involved, whereas for DAMGO analgesia, only the serotonergic system was involved. The dose-response curve for i.c.v. morphine reached a plateau at high doses, whereas i.c.v. DAMGO analgesia peaked at 10 ng and then decreased thereafter, producing a bell-shaped dose-response curve. This decrement in analgesic response could be reversed by low doses of i.t. methysergide and i.t. pindolol. It was concluded that activation of serotonin-1 (5-HT1) receptors plays a role in the decrease in analgesia from high doses of DAMGO. Combinations of i.t. morphine with i.t. 5-HT or i.t. clonidine produced additive or greater analgesic responses. Combinations of i.t. DAMGO with i.t. 5-HT or i.t. clonidine produced less than additive interactions. Part of the latter responses appeared to be due to activation of 5-HT1 receptors; blockade of these receptors by pindolol enhanced i.t. DAMGO-induced analgesia. Morphine and DAMGO differ further because i.c.v. morphine activated a descending antianalgesic pathway mediated by spinal dynorphin A(1-17), whereas i.c.v. DAMGO at a high dose did not. Thus, morphine and DAMGO differ in their modes of antinociceptive action as measured by the tail-flick response.

The opioid receptor types involved in supraspinal and spinal heroin-induced analgesia in Swiss Webster and ICR mice were determined by intracerebroventricular (i.c.v.) and intrathecal (i.t.) administration of opioid agonists and antagonists. Also, comparisons were made with morphine. Antinociception was measured by changes in tail-flick latency. In Swiss Webster mice, i.c.v. heroin like [D-Pen2-D-Pen5]enkephalin, a delta receptor opioid agonist, activated supraspinal delta opioid receptors as evidenced by inhibition of analgesia by coadministration of naltrindole, a delta receptor antagonist. Lack of effect of i.t. yohimbine and methysergide vs. i.c.v. heroin indicated that spinal descending noradrenergic and serotonergic systems were not involved. Heroin and [D-Pen2-D-Pen5]enkephalin were also matched in producing additive interactions with i.t. opioids. Also, i.c.v. heroin like [D-Pen2-D-Pen5]enkephalin did not activate a dynorphin-mediated antianalgesic system. In ICR mice, i.c.v. heroin receptor selectivity matched that of i.c.v. Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, a selective mu receptor opioid agonist. Analgesia was inhibited by pretreatment with i.c.v. beta-funaltrexamine, a nonequilibrium mu receptor antagonist. Intrathecal administration of methysergide inhibited i.c.v. heroin-induced analgesia whereas i.t. yohimbine had no effect, which indicated that a descending serotonergic system but not a noradrenergic system was involved. Low doses of i.t. naloxone and nor-binaltorphimine increased the analgesic effect. This effect was consistent with activation of an antianalgesic system by i.c.v. heroin, which was mediated by dynorphin A in the spinal cord. Desensitization of the antianalgesic system also resulted in increased analgesia. In both Swiss Webster and ICR mice, i.t. heroin-induced analgesia involved spinal mu receptors like those stimulated by Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5. Analgesia was inhibited by i.t. naloxone. In both strains, i.t. heroin, like i.t. Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, produced an additive interaction with i.t. clonidine. In conclusion, the supraspinal receptors activated by heroin are different between Swiss Webster and ICR mice. In both strains, the receptor selectivities assigned to heroin did not match those for morphine. Heroin did not act by being biotransformed to morphine.

The antinociceptive action (tail-flick test) of physostigmine given i.c.v. to mice was enhanced by the administration intrathecally (i.t.) of narcotic antagonists. Doses, i.t., as low as 0.1 fentog of naloxone, 0.25 ng of beta-funaltrexamine and 0.1 ng of nor-binaltorphimine enhanced physostigmine, 2 micrograms i.c.v., analgesia. These doses of opioid antagonists did not inhibit spinal mu receptors or kappa receptor agonist-induced analgesia as assessed by absence of effect on Tyr-D-Ala2-N-MePhe4-Gly-ol5 or U50,488H i.t. analgesia. Enhancing effects of the opioid antagonists were interpreted to indicate that i.c.v. physostigmine-induced analgesia was mediated spinally by an endogenous opioid which had an antagonistic effect. This putative opioid antagonist was postulated to be dynorphin A (1-17). Thus, i.t. administration of small doses of less than 10 pg of dynorphin was shown to antagonize the analgesic action of physostigmine, i.c.v. Furthermore, this effect of dynorphin was attenuated by the doses of naloxone, beta-funaltrexamine or nor-binaltorphimine which were effective in enhancing physostigmine-induced analgesia. We concluded that physostigmine given i.c.v. had two actions, the first produced analgesia and the second activated a system which had an antianalgesic effect. Evidence indicated that the latter effect was mediated by dynorphin A (1-17). This concept of dynorphin action may be the basis for some of the unusual findings of the analgesic action of naloxone in other situations and support the concept for a descending dynorphin A (1-17)-mediated antianalgesic system.

We have reported previously that i.v.t. beta-endorphin increases the release of immunoreactive Met-enkephalin but not Leuenkephalin or dynorphins from the spinal cord. To determine if the effect is specific to beta-endorphin, the present investigation tested i.v.t. beta-endorphin, its analogs and other opiate agonists with different opioid receptor activities for their ability to release Met-enkephalin using an intrathecal perfusion technique. Human beta-endorphin and its analogs, human beta-endorphin-(1-30), -(1-29) and -(1-28) which have an identical amino acid sequence in the NH2-terminus showed reduced stepwise potencies in releasing Met-enkephalin. The results correlated well with their analgesic potencies. Des-Met5-camel beta-endorphin (64 micrograms i.v.t.) which does not have a complete sequence of Met-enkephalin in its NH2-terminus but still retains 20% of camel beta-endorphin analgesic potency caused the spinal release of Met-enkephalin. Morphine (mu opioid receptor agonist, 40 micrograms), D-Ala2-D-Leu5-enkephalin (delta opioid receptor agonist, 80 micrograms) and U-50488H (kappa opioid receptor agonist, 160 micrograms) injected i.v.t. were unable to cause any release of Met-enkephalin. High-performance liquid chromatography after Sephadex G-50 gel chromatography indicated that the immunoreactive Met-enkephalin in the spinal perfusate released by i.v.t. beta-endorphin had a retention time identical to authentic Met-enkephalin. Intraventricular injection of Met-enkephalin, 4 nmol (2.3 micrograms), caused little increase of Met-enkephalin immunoreactivity in the spinal perfusate, whereas 4 nmol of i.v.t. beta-endorphin caused a marked increase of Met-enkephalin in the spinal perfusate. Inhibition of peptidase by i.v.t. aprotinin and bacitracin does not prevent the spinal release of Met-enkephalin induced by i.v.t. beta-endorphin. It is concluded that the release of Met-enkephalin was specific to beta-endorphin and the results were not due to cross-immunoreactivity of beta-endorphin or its metabolites.

The intraperitoneal injection of cadmium (Cd) in mice produces a characteristic abdominal stretch response similar to that observed with acetic acid. Morphine, clonidine, and tripelennamine ED50 values were the same for inhibition of the response to Cd and to acetic acid. The ED50 values for aspirin and indomethacin were lower against the Cd than the acetic acid response. On repeated injections, Cd did but acetic acid did not produce tolerance to itself. Furthermore, the mice made tolerant to Cd were not cross-tolerant to acetic acid, but the acetic-acid-treated mice were cross-tolerant to Cd. These results suggest that Cd and acetic acid may produce the stretch response through different mechanisms, and the Cd-induced response might serve as a screening test for aspirin-like agents.

Following a 10-min near-field exposure to sham radiation of 2.45 GHz microwave (MW) radiation at a power density of 10 mW/cm2, male Swiss-Cox mice were injected with sodium phenobarbital (PB) (150 mg/kg, iv) and the onset and duration of PB-induced sleep were recorded. MW-pretreated mice exhibited a decreased PB sleep onset relative to sham when PB was administered 1 to 45 min following irradiation; however, no effect was seen by 24 h. The duration of PB-induced sleep was increased; however MW pretreatment did not affect the duration of hexobarbital- or pento-barbital-induced sleep. At the time of onset of PB-induced sleep, PB concentrations in brain of MW-irradiated and control groups were the same despite a twofold difference in the time to sleep onset between these groups. But, when measured 5 min following PB injection, the PB concentrations in brain were higher in MW-pretreated mice, suggesting that a dispositional mechanism accounted for the observed MW effects on PB-induced sleep. Mice heated for 10 min by an incandescent light bulb served as thermal controls, and under certain conditions exhibited an increase in duration of PB sleep and enhanced brain disposition of PB. The effect of MW exposure on PB sleep onset was also influenced by ambient temperature. The data suggest that MW exposure affects PB-induced sleep by enhancing the disposition of PB to the brain and that these effects result in part from thermal effects.

Cold water swim stress for 3 min at 5 degrees C produces antinociception in the tail-flick test in mice by activation of delta opioid receptors in the brain. Also, the inhibition of the tail-flick reflex produced by i.c.v. administration of delta opioid receptor agonists is known to be mediated by spinal gamma-aminobutyric acid (GABA) receptors. The purpose of this investigation was to determine if the cold water swim stress-induced antinociceptive response is mediated by GABA receptors in the spinal cord. First, i.c.v. administration of the delta-2 receptor antagonist, naltriben, but not the delta-1 receptor antagonist, 7-benzylidenenaltrexone, antagonized the cold water swim stress-induced antinociception in ICR mice and confirmed the role of delta-2 receptors in this response. Next, the involvement of spinal GABAA receptors was shown through intrathecal administration of GABAA receptor antagonists, picrotoxin and bicuculline, which inhibited the cold water swim stress-induced antinociceptive response. Thus, the antinociception produced through activation of the delta-2 receptor in the brain by cold water swim stress involved a descending pathway mediated by spinal GABAA receptors. This descending pathway appeared to be the same as that activated by i.c.v. administration of delta-2 opioid agonists in the brain.

Morphine administered concurrently by i.c.v. plus intrathecal (i.t.) injection produces a multiplicative (synergistic) interaction for antinociception in the tail-flick test. Inasmuch as i.c.v. administered beta-endorphin has been proposed to produce antinociception by activating a descending pain inhibitory system different from that activated by morphine, the present experiments compared the two systems in mice. The responses to i.c.v., i.t. and combinations of i.c.v. plus i.t. administration of morphine and beta-endorphin were evaluated by determination of ED50 values which were plotted as isobolograms and compared to calculated theoretical additive ED50 values. The following combinations gave additive interactions: i.c.v. plus i.t. beta-endorphin, i.c.v. beta-endorphin plus i.t. morphine and i.t. morphine plus i.t. beta-endorphin. These results were consistent with the hypothesis that i.c.v. beta-endorphin stimulates supraspinal epsilon receptors which activate a descending pathway involving enkephalinergic neuronal mediation and spinal postsynaptic mu receptors. Stimulation of these mu receptors by i.t. morphine or i.t. beta-endorphin together with the supraspinal effect of beta-endorphin resulted in an additive interaction. Multiplicative interactions were obtained for the following combinations: i.c.v. morphine plus i.t. morphine, i.c.v. morphine plus i.t. beta-endorphin and i.c.v. morphine plus i.c.v. beta-endorphin. Morphine administered i.c.v. stimulated supraspinal mu receptors to activate a descending pain inhibitory pathway which is mediated spinally by monoamines. The i.t. agonists in this case activated the spinal mu receptor which is presumed to be part of the beta-endorphin descending pathway described above. Thus, when both pathways were activated simultaneously the interaction was multiplicative.(ABSTRACT TRUNCATED AT 250 WORDS)

In the rat and mouse when morphine is coadministered intracerebroventricularly (i.c.v.) in the brain and intrathecally (i.t.) in the spinal cord, a multiplicative (synergistic) interaction occurs for the antinociceptive response. In the male Swiss Cox mouse, with the development of a high degree of systemic tolerance to morphine, this multiplicative interaction decreases (becomes additive), whereas no change occurs in the ED50 for the i.c.v. or i.t. site itself. The purpose of the present study was to extend these findings in one mouse strain to other strains in order to determine how generally applicable such findings are to the phenomenon of tolerance to morphine. Eight different strains of outbred mice were used in the antinociceptive tail-flick test to quantify the multiplicative interaction of simultaneous administration (i.c.v. and i.t.) in relation to the amount of tolerance developed to morphine administered systemically (s.c.). Even though the control ED50 values for s.c. morphine were similar among strains, the amounts of tolerance developed by 3 days of implantation of a morphine pellet were very different (2-18-fold). A relationship was found between the degree of tolerance developed to s.c. morphine and changes in the multiplicative interaction (as reflected by changes in the i.c.v. plus i.t. morphine ED50 values). In control animals, a multiplicative i.c.v.-i.t. interaction was produced in six of the eight strains, whereas an additive interaction was shown in the remaining two strains. With 3 days of morphine pellet implantation, in three strains which developed a large amount of tolerance to s.c. morphine (7-18-fold), the multiplicative interaction decreased to an additive interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption of compounds from the biliary tree of rats was studied by a retrograde intrabiliary injection (RII) technique. After RII of a number of compounds in volumes that exceeded the maximum distended capacity of the biliary tree, only that volume of RII solution which corresponded to biliary tree capacity remained therein. Furthermore, this latter portion of RII solution was absorbed by a first-order process when the duration of bile duct cannula occlusion was extended. Thus, the present study demonstrates that absorption of retrogradely injected compounds continues during occlusion. Since excretion of i.v. administered compounds into the biliary system was also shown to continue during occlusion, we propose in the present study a concept of bidirectional exchange of solute within the biliary tree during occlusion. As an additional finding, after RII and occlusion, a temporary increase in bile flow was obtained. If the occlusion duration was 3 minutes or less, the bile flow response was of sufficient magnitude to repay alomst exactly the volume of bile that would have been expected to be produced. If the occlusion was 6 and 9 minutes the increased magnitude of the bile flow response was not great enough to repary the larger bile volumes expected and underpayments of -48 and -96 mul were incurred.

The mode of transfer of solutes across the biliary tree epithelium in the reabsorptive direction was studied by the segmented retrograde intrabiliary injection technique in isolated perfused liver of rats. The main purpose was to study the pattern of transit of the radioactive marker compounds through the liver into the venous perfusate. The perfusate was collected as a single pass. All of the marker compounds were quantitatively accounted for in the bile and the perfusate. The apparent volumes of distribution of dextran and sucrose were smaller than that for erythritol. Mannitol was similar to erythritol. The mean transit times were correspondingly: dextran congruent to sucrose < mannitol congruent to erythritol. Dehydrocholate (100 mumol/kg) had no effect on these parameters, but taurocholate (100 mumol/kg) increased the mean transit time for sucrose. This effect was probably related to an increase in biliary tree permeability produced by taurocholate. The present approach appeared to be useful in assessing hepatobiliary function and its alteration by allowing the transit patterns of compounds to be determined in passing from bile through the liver to the blood.

Even though dehydrocholate (DHC) produced an intense choleresis, we found in the rat that the biliary excretion of morphine was inhibited by DHC. The site of action for this inhibitory effect of DHC was investigated in isolated perfused livers in conjunction with intraportal and segmented retrograde intrabiliary injecton of [14C]morphine and [14C]morphine glucuronide (MG). After segmented retrograde intrabiliary injection of morphine, DHC treatment was shown to decrease the amount of MG recovered in bile but increase the amount of MG which appeared in the blood perfusate. That this effect was due to DHC affecting the transport of MG rather than inhibition of the metabolic conjugation of morphine to MG was directly demonstrated. DHC reduced the recovery of MG in bile after administration of MG by SRII. Also in vivo, DHC decreased the recovery of bile of MG give, i.v. DHC produced no consistent effect on the apparent volume of distribution and transit time for morphine and MG and no effect on these transfer parameters for morphine-3-ethereal sulfate. Thus, the effect of DHC can with some degree of specificity be ascribed to an effect on the transport of MG from liver to bile.

The effect of SKF-525-A on the disposition of morphine given by segmented retrograde intrability injection (SRII) was studied in the isolated in situ perfused livers of Sprague-Dawley male rats. SRII of morphine repeated after treatment SKF 525-A (20 mg/kg b. wt. intraportally) showed decreased recovery of morphine glucuronide (MG) in the bile and in the venous outflow. On the other hand, morphine in the venous outflow was increased. Thus, SKF 525-A decreased the total recovery (bile plus venous outflow) of MG. The results after intraportal administration of morphine were consistent with these observations by SRII. These results might indicate inhibition of conjugation by SKF 525-A. However, a similar effect of SKF 525-A was obtained when MG was given by either route. These latter results were interpreted to mean tht SKF 252-A affected a translocation process for MG. Analysis of the liver after the SRII of MG indicated that SKF 525-A caused a 10-fold increase in retention of MG in the liver. Based on the cumulative venous outflow curves, the difference in apparent volumes of distribution, Q, indicated that morphine was taken up by hepatocytes beyond the mannitol, total liver water (intra- and extracellular) space and SKF 525-A decreased this apparent volume of distribution of morphine. Also, SKF 525-A increased the apparent volume of distribution of MG. The mean transit time of morphine was not altered by SKF 525-A whereas that of MG became longer. The evidence indicated that SKF 525-A decreased the egress of MG from liver, a newly recognized mode of action of SKF 525-A.

The effect of the inducing agent trans-stilbene oxide (TSO) on the metabolism and biliary excretion of [14C]morphine was studied in the isolated in situ perfused rat liver. After administration of morphine by intraportal injection or by the segmented retrograde intrabiliary injection technique, the TSO-treated group showed a marked decrease in the biliary recovery of morphine as its glucuronide conjugate [morphine-3-glucuronide (MG)]. However, recovery of MG in the venous outflow of the single pass perfusate was greatly increased. These findings suggested that TSO treatment enhanced the formation of MG from morphine and changed the primary route of hepatic elimination of MG. TSO treatment also decreased the excretion of morphine (as MG) in the bile of anesthetized renal-ligated rats. This decreased biliary function required several days to develop and appeared closely associated with the inductive effect of TSO. After i.v. administration of [14C]MG itself, biliary recovery was also markedly decreased in TSO-treated rats. It is postulated that the effect of the TSO treatment led to either a decrease in canalicular transport of MG into bile or an increase in the efficiency of transfer of MG to the blood at the sinusoidal side of the hepatocyte. Regardless of the mechanism, the results indicate the need to study compartmentalization of drug transport and metabolism functions.

The present study compares the potencies of D-Ala2-D-Leu5-enkephalin (DADL) and beta-endorphin (beta-EP) injection intrathecally and i.c.v. on the inhibition of tail-flick and ice water-induced shaking responses in pentobarbital-anesthetized rats. Peptides were injected stereotaxically into the third ventricle or into the spinal subarachnoid space. The tail-flick response was measured 10 or 20 min after the injection of DADL or beta-EP, respectively, after which the rats were immersed for 5 min in ice water and the number of shakes was counted. Intrathecal DADL and beta-EP were equipotent in inhibiting both the tail-flick and shaking responses. However, treatment with naloxone (2 mg/kg i.p.) shifted the dose-response curve for both responses to the right more for the beta-EP than for DADL. Unlike the equipotency intrathecally, DADL by i.c.v. injection was less potent than beta-EP in inhibiting both the tail-flick and shaking responses. Previous results in the unanesthetized rat had also shown that i.c.v. DADL was less potent than beta-EP in inhibiting the tail-flick response. Thus, the spinal cord is sensitive to both DADL and beta-EP, whereas the supraspinal area is more sensitive to beta-EP. Also, because the potency of i.c.v. beta-EP for inhibiting tail-flick but not shaking was substantially reduced in anesthetized compared to unanesthetized rats, the neuronal substrates involved in inhibition of shaking are different from those of inhibition of the tail-flick. The differential sensitivities to i.c.v. vs. intrathecal administration and the unequal responses to the antagonistic action of naloxone indicate that DADL and beta-EP produce their action through different opioid receptors.

Analgesic ED50 values were determined for s.c. morphine, etorphine, heroin and methadone in mice implanted for 3 days with etorphine pellets. Tolerance to morphine (9-fold) was greater than to the other agents (2- to 4-fold). These results agreed with the previously reported unidirectional non-cross-tolerance effect seen after morphine pellet implantation where tolerance to the hydrophilic agent (morphine) was greater than for the more lipophilic agents (etorphine and heroin). As greater tolerance was found to s.c. morphine than for i.c.v. morphine, this manifestation of tolerance was described as a dispositional tolerance. Experimentally, we were able to observe two additional manifestations of tolerance. After pellet removal, the ED50 values for s.c. morphine, heroin and methadone initially increased before returning to control values. Similarly, both morphine and etorphine ED50 values, after i.c.v. administration, initially increased after pellet removal. Because this second manifestation of tolerance occurred during the rapid disappearance of the tolerance-induction drug, it was called withdrawal tolerance. The third manifestation of tolerance, seen after 3 days of pellet implantation, was characterized by the animal's return to a normal nociceptive sensitivity (control tail-flick latency) in the continued presence of the narcotic pellet. Because this adjustment involved an adaptation by the mice to high brain concentrations of narcotic, this third manifestation of tolerance was designated as a physiologic or homeostatic tolerance. The lability of this new homeostatic state (physiologic tolerance) may be associated with an altered sensitivity to naloxone as evidenced by naloxone-induced jumping (precipitated withdrawal).

The purposes were to show that pentobarbital administered intracerebroventricularly (i.c.v.) antagonized the antinociceptive action of morphine given intrathecally in the tail-flick test in mice, determine whether this antagonistic action involved spinal release of dynorphin A (Dyn A) and demonstrate whether the mechanism involved gamma-aminobutyric acid (GABA) receptors. Previously, it was shown that midazolam given i.c.v. antagonizes morphine-induced antinociception by release of Dyn A in the spinal cord. This antagonism by midazolam is reversed by a variety of benzodiazepine/GABAA receptor complex antagonists given i.c.v. The results indicated that the antagonistic action of pentobarbital given i.c.v. was not due to release of Dyn A based on presumptive tests for Dyn A release. 1) In the tail-flick test, the opioid receptor antagonists, naloxone and nor-binaltorphimine intrathecally, did not reverse the antagonism. 2) Pretreatment with dynorphin antiserum and morphine had no effect. The effect of pentobarbital was inhibited by a benzodiazepine receptor antagonist, flumazenil, given i.c.v. but not by a GABA antagonist, bicuculline s.c. or a chloride ion channel antagonist, picrotoxin, i.c.v. In contrast, the duration of loss of the righting reflex from systematically administered pentobarbital was shortened by bicuculline and picrotoxin but not by flumazenil. These results taken together with the previous results on midazolam suggested that the antagonistic action of pentobarbital on morphine-induced antinociception appeared to involve different GABA receptors from those involved in release of Dyn A by midazolam and those involved in the pentobarbital-induced loss of the righting reflex.

In recent publications we have proposed that dynorphin (Dyn) A(1-17) functions as an antianalgesic modulator to oppose opioid-induced antinociception in mice. In the present experiments using the tail-flick response in mice, other Dyns [Dyn A(1-8), Dyn A(1-13), Dyn A(2-17), Dyn B and alpha- and beta-neoendorphin] when administered intrathecally (i.t.) were shown not to have antianalgesic activity even at high doses (0.5-1 pmol). These Dyns, i.t., did not antagonize the antinociception produced by physostigmine administered i.c.v. or morphine given i.t. These Dyns lacked the intrinsic antianalgesic activity of Dyn A(1-17). However, they had affinity for Dyn A(1-17) receptors as shown in several ways. 1) The antagonism of physostigmine antinociception produced by Dyn A(1-17) given i.t. was reversed by these Dyns given together with Dyn A(1-17). 2) The effect of endogenously released Dyn A(1-17) was reversed. Administered i.c.v., clonidine simultaneously activates antinociceptive and antianalgesic systems [latter mediated spinally by Dyn A(1-17) release]. Thus, these Dyns given i.t. inhibited the action of endogenously released Dyn A(1-17) and allowed the full manifestation of the antinociceptive action of clonidine.(ABSTRACT TRUNCATED AT 250 WORDS)

The antianalgesic effect of dynorphin A (1-17) (dyn A) was demonstrated by i.c.v. administration of agonists (morphine, Tyr-D-Ala2-Gly-N-MePhe4-Gly-ol5, D-Pen2-D-Pen5-enkephalin, beta-endorphin, U50,488H and physostigmine) at 10 min and intrathecal administration of dyn A at 5 min before the tail-flick test in mice. This antianalgesic effect of dyn A was eliminated by 3 or 5.5 hr pretreatment s.c. with 10 or 100 mg/kg of morphine, respectively. This desensitization lasted for about 18 hr. Three-hour pretreatment intrathecally with dyn A also desensitized the mice to dyn A. Previously we had shown that i.c.v. administration of morphine simultaneously activates analgesic and antianalgesic systems and the latter is mediated spinally by dyn A, an endogenous antianalgesic opioid. Present results are consistent with that concept and systemic pretreatment with morphine may release dyn A in the spinal cord to produce the desensitization to the subsequently elicited antianalgesic action of dyn A.

Mapping of the brainstem regions sensitive to beta-endorphin and morphine for antinociception and Met-enkephalin release was performed in rats. Antinociception was assessed by the tail-flick test in pentobarbital-anesthetized rats and the release of immunoreactive Met-enkephalin from the spinal cord was measured by lumbar-cisternal perfusion in urethane-anesthetized rats. The sites in the brainstem most sensitive to beta-endorphin (2 micrograms) for inhibition of the tail-flick response and Met-enkephalin release were located in areas in the caudal medial medulla such as raphe obsacurus nucleus and raphe pallidus nucleus and the adjacent midline reticular formation. Sites in the rostral medial medulla were less sensitive to beta-endorphin for both antinociception and release of Met-enkephalin. The regions 1 mm and more lateral to the midline were not sensitive to beta-endorphin. The sites sensitive to morphine sulfate (4 micrograms) for antinociception were located in areas in the rostral ventromedial medulla such as raphe magnus nucleus, gigantocellular reticular nucleus and gigantocellular reticular nucleus alpha. Raphe obscurus nucleus, which was sensitive to beta-endorphin, was not sensitive to morphine for antinociception. Locus coeruleus was sensitive to morphine and beta-endorphin for antinociception. No sites sensitive to morphine were found for Met-enkephalin release. The correlation between the brainstem sites sensitive to beta-endorphin for the production of antinociception and the release of Met-enkephalin suggests that the antinociception induced by beta-endorphin is mediated by the release of Met-enkephalin. The findings of different brainstem sites sensitive to beta-endorphin and morphine support the hypothesis of different modes of pharmacological actions for beta-endorphin and morphine.

Previous studies in mice demonstrate that, when dynorphin A (1-17) (Dyn A) is administered intrathecally (i.t.) or released spinally (by administration of clonidine or midazolam i.c.v.), i.t. morphine-induced analgesia was reduced. The present aim was to determine whether this antianalgesic action of Dyn A was the result of a spinal or supraspinal site of action by performing studies in spinally transsected mice. The approach was to use anesthetized, acute spinally transsected mice rather than chronic spinally transsected animals to avoid the need for long-term special animal care. The first part of the study evaluated four nonvolatile general anesthetic agents in an attempt to obtain one that did not affect the antianalgesic action of Dyn A, the release of Dyn A, the analgesic action of i.t. morphine (inhibition of the tail-flick response) or the tail-flick latency by itself. alpha-Chloralose (120 mg/kg), urethane (1 g/kg) and pentobarbital (20 or 40 mg/kg) given i.p. did not meet one or more of these requirements. Barbital 400 mg/kg i.p. met the requirements. In the second part of the study, barbital combined with halothane anesthesia was used to perform acute spinal transsection experiments. As in sham controls, the analgesic action of i.t. morphine was undiminished in spinally transsected animals, which indicated that the inhibition of the tail-flick response produced by i.t. morphine was on a spinal reflex response. On the other hand, spinal transsection produced a loss of the action of i.t. Dyn A to antagonize i.t. morphine-induced analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)