Footshock- and psychological-stress prevent the development of tolerance to spinal but not supraspinal morphine.

The site of action involved in the suppression by exposure to footshock (FS)- and psychological (PSY)-stress of the development of antinociceptive tolerance to morphine has been investigated. Daily treatment with 10 mg/kg, s.c.; 3 micrograms, i.t.; and 5 micrograms, i.c.v. of morphine, regardless of the administration route, resulted in the development of tolerance. Daily exposure to FS- or PSY-stress suppressed the development of tolerance to s.c. and i.t. administered morphine but not that to i.c.v. administered morphine. Pretreatment with 2 mg/kg, i.p. of nor-binaltorphimine (nor-BNI) abolished the suppressive effect of PSY-stress on the development of tolerance to morphine given s.c. The suppression by PSY-stress was also antagonized by 2 micrograms, i.t. of nor-BNI and not by 2 micrograms, i.c.v. of nor-BNI. Thus, the development of tolerance in the spinal cord due to interaction of morphine at mu-opioid receptors can be suppressed by exposure to these stresses, probably through the descending signals from the supraspinal area, and activation of kappa-opioid receptors in the spinal cord could also participate in the suppression by PSY-stress.     

Attenuation of the antinociceptive action of the selective kappa-opioid receptor agonist, U-50,488H by ICS-205-930

The antinociceptive action of s.c. administered U-50,488H was antagonized by s.c. administered ICS-205-930, a selective 5-HT3 receptor antagonist. To characterize the site of interaction, U-50,488H and ICS-205-930 were administered either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). When U-50,488H was administered i.c.v., its antinociception action was antagonized by ICS-205-930 given either i.c.v. or i.t., increasing the ED50 values of U-50,488H by approximately twofold from 48 to 98 and 90 nmol/mouse, respectively. However, when U-50,488H was administered i.t., its antinociception action was not antagonized by ICS-205-930 given either i.c.v. or i.t. These findings suggest that i.c.v., but not i.t. administered U-50,488H may release serotonin both supraspinally and spinally to interact with 5-HT3 receptors to produce antinociception.     

Disability of development of tolerance to morphine and U-50,488H, a selective kappa-opioid receptor agonist, in neuropathic pain model mice

We examined the analgesic and anti-allodynic effects of morphine and U-50,488H (trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide methanesulfonate salt), a selective kappa-opioid receptor agonist, and the development of tolerance to their effects in neuropathic pain model mice induced by sciatic nerve ligation (SNL). In the tail-pinch method, morphine at 10 mg/kg, s.c. produced a weak analgesic effect in SNL mice; however, U-50,488H at 5 mg/kg, s.c. produced an analgesic effect equipotent to that in normal mice. In contrast, morphine produced an adequate analgesic effect when given either intracerebroventricularly (i.c.v.) or intrathecally (i.t.), but U-50,488H only produced analgesia when given i.t. Repeated administration of morphine (either i.c.v. or i.t.) or U-50,488H (either s.c. or i.t.), did not induce tolerance to the effect. In the static allodynia test with an application of von Frey filaments, both compounds given s.c. suppressed the allodynic effect, but in the dynamic allodynia test involving lightly stroking the plantar surface with a cotton bud, only U-50,488H produced an anti-allodynic effect. Repeated administrations of both compounds did not develop tolerance to these anti-allodynic effects. Thus, U-50,488H was found to be a highly effective at blocking hyperalgesia and allodynia in nerve injury, and these findings suggest that kappa-opioid receptor agonists are attractive pharmacological targets for the control of patients with neuropathic pain.     

Maintenance of acute morphine tolerance in mice by selective blockage of kappa opioid receptors with norbinaltorphimine.

In this study we investigated the effect of the highly selective kappa opioid antagonist, norbinaltorphimine (norBNI) on the development of tolerance to a single dose of morphine. Mice were pretreated with 100 mg/kg of morphine sulfate (morphine), s.c. and 2 h later, norBNI (20 mg/kg s.c.) was administered and various times after this pretreatment, antinociceptive ED50 value of morphine was determined in the tail-flick assay. Twenty-four and 72 h after morphine injection, ED50 values of morphine were significantly increased by about 2.5-fold from those of their control mice that received saline instead of the tolerance-inducing dose of morphine. In a second set of experiments, animals were pretreated similarly with morphine and norBNI and 72 h after morphine injection, various opioid agonists were applied by the i.c.v. or i.t. route to see whether or not any cross-tolerance had developed to these agonists. The ED50 of i.c.v.-administered morphine was significantly greater than that of the non-pretreated controls. A small degree of cross-tolerance was observed with U-50,488H but not with DPDPE [D-Pen2,D-Pen5]enkephalin (DPDPE) at the supraspinal site. At the spinal site, tolerance to morphine was not observed. These results suggest that antagonism at kappa opioid sites after morphine administration, modulates positively the development of opioid tolerance.     

Effects of nor-binaltorphimine on the development of analgesic tolerance to and physical dependence on morphine.

The effects of a highly selective kappa antagonist, nor-binaltorphimine (nor-BNI), on the development of tolerance to morphine analgesia and physical dependence on morphine were examined. Pretreatment with nor-BNI (5 mg/kg s.c.) 2 h prior to injection of morphine or a selective kappa agonist, U-50,488H, significantly antagonized the analgesic effect of U-50,488H, but not morphine analgesia in mice. The development of tolerance to morphine analgesia was significantly potentiated by pretreatment of mice with nor-BNI 2 h prior to morphine treatment during chronic morphine treatment for 5 days. Additionally, the pretreatment with nor-BNI during chronic treatment with the high dose of morphine for 5 days significantly potentiated the naloxone-induced body weight loss in morphine-dependent mice and rats. These findings suggest that inactivation of the kappa opioid system may potentiate the development of tolerance to morphine analgesia in mice and may aggravate the naloxone-precipitated body weight loss in morphine-dependent mice and rats.     

The role of vasopressin on the effect of U-50,488 to block the development of morphine tolerance and physical dependence

U-50,488, a selective κ-opioid receptor agonist, has been reported to inhibit the development of antinociceptive tolerance to morphine in mice, rats and guinea pigs, but the mechanism involved in this action remains unknown. Since U-50,488 has been reported to supress the plasma vasopressin level, we investigated the role of vasopressin with U-50,488 in the male Sprague Dawley rat in this study. Animals (230–270 g) were chronically treated with morphine (10 mg/kg, i.p.) twice a day for 6 days in order to induce tolerance to antinociceptive effect measured by tail-flick test. Withdrawl symptoms were precipitated by naloxone (10 mg/kg, i.p.) on day 7. U-50,488 (i.p.) or AVP (i.p. or i.c.v.) or U-50,488 and AVP was (were) coadministered with chronic morphine to investigate their effects on morphine tolerance and dependence. We found that coadministration of 8 mg/kg U-50,488 (i.p.) with morphine almost completely block morphine tolerance and partially block withdrawal symptoms. In contrast, coadministration of AVP (0.3 μg/kg, i.p., or 0.01 μg, i.c.v.) with morphine and U-50,488, the effects of U-50,488 to block morphine tolerance and dependence were reversed. In addition, treatment of AVP antagonist (dPTyr(Me)AVP, 0.5 μg/kg, i.p. or 0.5 μg, i.c.v.) has the similar effect as U-50,488 to block morphine tolerance. In summary, the effect of U-50,488 to block morphine tolerance and dependence may relate to its inhibitory effect on AVP release. Received: 20 February 1996 / Accepted: 14 October 1996     

Activation of c-fos expression in the heart after morphine but not U-50,488H withdrawal

1. In the present work we have studied in the heart the expression of Fos, the protein product of the c-fos proto-oncogene and the adaptive changes in noradrenergic neurons after naloxone or nor-binaltorphimine (nor-BNI) administration to morphine or U-50,488H pretreated rats. 2. Male rats were implanted with placebo (na?ve) or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received saline s.c., naloxone (5 mg kg(-1) s.c.) or nor-BNI (5 mg kg(-1) i.p.). Other groups of rats were rendered tolerant/dependent on U-50,488H by injecting the drug twice daily (15 mg kg(-1) i.p.) for 4 days. Control animals received saline. On day 5 the animals were injected with vehicle i.p. or nor-BNI (5 mg kg(-1) i.p.). 3. Using immunohistochemical staining of Fos, present results indicate that morphine withdrawal induced marked Fos immunoreactivity (Fos-IR) within the cardiomyocyte nuclei. Moreover, Western blots analysis revealed a peak expression of c-fos in right and left ventricle after naloxone induced withdrawal in parallel with an increase in noradrenaline (NA) turnover. 4. However, after nor-BNI administration to rats chronically treated with U-50,488H, we found a decrease in the NA turnover. In addition, the administration of nor-BNI to rats chronically treated with U-50,488H or morphine did not induce modifications in the Fos-IR, in the heart. 5. These results demonstrated that morphine withdrawal induces the expression of Fos protein, as well as an enhancement of noradrenergic activity in the heart. In contrast to morphine U-50,488 withdrawal produces no changes in Fos-IR in parallel with a decrease in NA turnover, indicating that the kappa-opioid receptors are not involved in the molecular adaptive mechanisms responsible for the development of opioid dependence in the heart.     

Effects of morphine in rats treated chronically with U-50,488 H, a kappa opioid receptor agonist

The pharmacological effects of morphine, namely analgesic, hyperthermic and cataleptic effects, were assessed in rats rendered tolerant to U-50,488H, a kappa opioid receptor agonist. Male Sprague-Dawley rats were injected intraperitoneally with U-50,488H (25 mg/kg) twice a day for four days. The rats which served as controls were injected similarly with the vehicle. Chronic administration of U-50,488H resulted in the development of tolerance to its analgesic and hypothermic effects, but not to its diuretic effect. The development of tolerance to the pharmacological effects of U-50,488H was associated with decreased binding of [3H]ethylketocyclazocine [( 3H]EKC) to brain and spinal cord membranes. The decreased binding of [3H]EKC in U-50,488H-treated rats was due to changes in the Bmax value; the Kd values remained unaltered. Intraperitoneal administration of morphine (8 mg/kg) to rats produced analgesia (as determined by the tail-flick test) and hyperthermia. A dose of 50 mg/kg of morphine produced cataleptic response. The intensity of analgesic, hyperthermic and cataleptic effects of morphine were unaltered in rats tolerant to U-50,488H. The development of tolerance to analgesic and hypothermic effects of U-50,488H were associated with down-regulation of brain and spinal cord kappa opioid receptors. Finally, U-50,488H does not confer cross-tolerance to morphine, a predominantly mu opioid receptor agonist.     

Functional interaction among opioid receptor types: up-regulation of mu- and delta-opioid receptor functions after repeated stimulation of kappa-opioid receptors

It has been widely accepted that repeated administration of kappa-opioid receptor agonists leads to the development of antinociceptive tolerance. The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride ((-)U-50,488H) on the mu- and delta-opioid receptor agonist-induced antinociception and G-protein activation in mice. The mice were injected either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) pretreated with saline or (-)U-50,488H once a day for seven consecutive days. Two hours after the last injection, the mice were challenged by either mu- or delta-opioid receptor agonist for the antinociceptive assay. Repeated treatment with (-)U-50,488H (s.c. or i.c.v.) significantly enhanced antinociceptive effect of both mu-opioid receptor agonist (morphine) and delta-opioid receptor agonists ([d-Ala2]deltorphin (DELT) and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dime thyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) compared to saline-treated groups. Under these conditions, repeated s.c. injection of (-)U-50,488H significantly enhanced both mu- and delta-opioid receptor agonist-stimulated [35S]GTPgammaS binding in the membrane of the thalamus. On the contrary, either repeated administration of morphine (s.c. or i.c.v.) or SNC-80 failed to affect the kappa-opioid receptor agonist-induced antinociception and G-protein activation. Taken together, these results suggest that repeated stimulation of kappa-opioid receptor markedly increases the functional mu- and delta-opioid receptors, whereas repeated stimulation of either mu- or delta-opioid receptor had no direct effect on kappa-opioidergic function in mice.     

The novel kappa-opioid receptor agonist TRK-820 has no affect on the development of antinociceptive tolerance to morphine in mice

The effects of the novel kappa-opioid receptor agonist 17-cyclopropylmethyl-3,14beta-dihydroxy-4, 5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl)acrylamido+ ++]morphinan hydrochloride (TRK-820) on the development of antinociceptive tolerance to morphine were investigated in mice and compared with those of trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)-cyclohexyl) benzenacetamide methane sulfonate hydrochloride (U-50,488H), a well-defined exogenous kappa-opioid receptor agonist. Morphine (1. 25-20 mg/kg, s.c.) produced a dose-related antinociceptive effect in the 51 degrees C warm-plate test. Daily treatment with morphine (10 mg/kg, s.c.) resulted in the development of antinociceptive tolerance. The development of antinociceptive tolerance to morphine was dose-dependently suppressed by the co-administration of U-50, 488H (1-10 mg/kg, s.c.) with morphine, but not TRK-820 (0.003-0.03 mg/kg, s.c.). These results suggest that TRK-820-sensitive kappa-opioid receptor subtypes may not be involved in modulating the development of antinociceptive tolerance to morphine.     

Attenuation of morphine tolerance after antisense oligonucleotide knock-down of spinal mGluR1

1. Chronic systemic treatment of rats with morphine leads to the development of opioid tolerance. This study was designed to examine the effects of intrathecal (i.t.) infusion of a metabotropic glutamate receptor 1 (mGluR1) antisense oligonucleotide, concomitant with chronic morphine treatment, on the development of tolerance to morphine's antinociceptive effects. 2. All rats received chronic (6 day) s.c. administration of morphine to induce opioid tolerance. Additionally, rats were treated with either mGluR1 antisense (AS), missense (MIS) or artificial cerebrospinal fluid (ACSF) by i.t. infusion via chronically implanted i.t. catheters connected to osmotic mini-pumps. The effects of acute i.t. or s.c. morphine on tail-flick latencies were assessed prior to and following chronic s.c. morphine treatment for all chronic i.t. infusion groups. mGluR1 protein level in the spinal cord was determined by Western blot analysis for all treatments, assessing the efficiency of knock-down with AS treatment. 3. Acute i.t. morphine dose-dependently produced antinociception in the tail-flick test in na?ve rats. Systemic morphine-treated rats administered i.t. ACSF or MIS developed tolerance to i.t. morphine. Chronic i.t. infusion with mGluR1 AS significantly reduced the development of tolerance to i.t. morphine. 4. In contrast to i.t. morphine, tolerance developed to the antinociceptive effects of s.c. morphine, in all i.t. infusion groups, including the mGluR1 AS group. 5. The spinal mGluR1 protein level was dramatically decreased after mGluR1 AS infusion when compared to control animals (na?ve and ACSF-treated animals). 6. These findings suggest that the spinal mGluR1 is involved in the development of tolerance to the antinociceptive effects of morphine. Selective blockade of mGluR1 may be beneficial in preventing the development of opioid analgesic tolerance.     

Spinal opioid analgesic effects are enhanced in a model of unilateral inflammation/hyperalgesia: possible involvement of noradrenergic mechanisms.

We have examined the spinal analgesic activity of opioid agonists and antagonists in a model of short term, unilateral, carrageenan-induced inflammation/hyperalgesia. Rats received a single s.c. injection of carrageenan (2-6 mg in saline) 3-24 h prior to testing hindpaw withdrawal latencies to noxious thermal stimuli. Dose-response curves for intrathecally administered agonists with mu- and/or delta-opioid activity were shifted to the left for inflamed hindpaws when compared to contralateral non-inflamed paws. The selective kappa-receptor agonist U-50,488H had no activity in this analgesic assay on either inflamed or non-inflamed paws when administered intrathecally. However, systemic administration of U-50,488H did produce significant elevations of paw withdrawal latencies in inflamed paws. The alpha 2-adrenoceptor agonist clonidine also produced dose-dependent antinociception in the paw withdrawal assay after systemic or intrathecal administration. Inflamed hindpaws were significantly more sensitive to the antinociceptive effect of morphine on inflamed hindpaws was blocked by the opioid antagonist naloxone or the alpha 2-adrenoceptor antagonist idazoxan. The effect of clonidine was only blocked by idazoxan. Antagonists alone had no significant effect on withdrawal latencies. The data indicate that the analgesic action of opioids during conditions of inflammation may depend on an interaction with spinal noradrenergic pathways.     

A selective kappa-opioid agonist, U-50,488H, blocks the development of tolerance to morphine analgesia in rats

The development of tolerance to morphine analgesia was completely blocked by the coadministration of a selective kappa-opioid agonist, U-50,488H at doses of 3.2 or 10 mg/kg i.p. These doses of U-50,488H exerted no analgesic effect by themselves and did not affect the analgesia induced by 10 mg/kg of morphine. The analgesic effect of morphine was restored when 10 mg/kg of U-50,488H was coinjected in morphine-tolerant rats. These findings suggest that activation of the kappa-opioid system prevents the development of tolerance to morphine analgesia.     

Beta-lactam antibiotic prevents tolerance to the hypothermic effect of a kappa opioid receptor agonist

Beta-lactam antibiotics are the only clinically approved drugs which directly increase glutamate uptake. They activate the glutamate transporter subtype 1 (GLT-1), the protein responsible for 90% of glutamate uptake in the mammalian brain. The capacity of GLT-1 to clear extracellular glutamate suggests that glutamate transporter activators be explored for therapeutic approaches to clinical conditions caused by increased glutamatergic transmission. One of the most common drug effects mediated by increased glutamatergic signaling is opioid tolerance. Therefore, we tested the hypothesis that a beta-lactam antibiotic (ceftriaxone), by increasing glutamate uptake, prevents tolerance to hypothermia induced by a kappa opioid receptor agonist (U-50,488H). A single injection of U-50,488H (20mg/kg, s.c.) caused significant hypothermia in rats. Tolerance to the hypothermic effect of U50,488H was induced by injecting U50,488H (20mg/kg) twice daily for 7days. Pretreatment with ceftriaxone (200mg/kg, i.p.) for 7days did not alter the acute hypothermic response to U50,488H (20mg/kg) but did prevent tolerance to U50,488H-induced hypothermia. Central administration of dl-threo-beta-benzyloxyaspartic acid (TBOA) (0.2mumol, i.c.v.), a glutamate transporter inhibitor, abolished the effect of ceftriaxone. These results identify a functional interaction between ceftriaxone and U50,488H in vivo and provide pharmacological evidence that a beta-lactam antibiotic abolishes tolerance to hypothermia induced by a kappa opioid receptor agonist.     

Further evidence for the implication of a kappa-opioid receptor mechanism in the production of psychological stress-induced analgesia

The analgesic effect induced by exposure to psychological stress, using a communication box (psychological stress-induced analgesia, PSY-SIA), was completely antagonized by 10 min pretreatment with 0.5, 1 and 2 mg/kg of nor-binaltorphimine and with 0.5 and 1 mg/kg of Mr2266, selective kappa-opioid receptor antagonists, in the tail pinch method. Neither footshock (FS)- nor forced swimming (SW)-SIA was affected by these antagonists. The selective delta-opioid receptor antagonist naltrindole, at doses up to 20 mg/kg, had no appreciable effect on PSY-SIA. Daily morphine treatment, 10 mg/kg, s.c., resulted in tolerance to the analgesic effect, and concurrent exposure to PSY-stress suppressed the development of morphine tolerance. The substitution of treatment with U-50,488H for PSY-stress still resulted in analgesia on the initial day; and likewise, the suppression by U-50,488H of the development of morphine tolerance was replicated by PSY-stress. Pretreatment with nor-binaltorphimine antagonized the suppressive effect of PSY-stress on the development of morphine tolerance without affecting the analgesic effect of morphine per se. These results provide further evidence that PSY-SIA involves the mediation by kappa-opioid receptor mechanisms.     

Effects of U-50,488H on isolated atria from rats chronically treated with the κ-opioid agonist,U-50,488H

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Effect of kappa-opioid receptor agonists on morphine analgesia in morphine-naive and morphine-tolerant rats

The effect of i.p. administration of kappa-opioid receptor agonists, bremazocine, tifluadom and U-50,488H on morphine (8 mg/kg i.p.)-induced analgesia in morphine-naive and morphine tolerant male Sprague-Dawley rats was determined using the tail-flick test. The tolerance to morphine in the rats was induced by s.c., implantation of six morphine pellets during a 7-day period. Implantation of morphine pellets resulted in the development of tolerance as evidenced by the decrease in the analgesic response to morphine when compared to placebo pellets implanted rats. Bremazocine (0.3, 1.0 and 3.0 mg/kg) and U-50,488H (16 mg/kg) antagonized morphine-induced analgesia in morphine-naive rats while tifluadom (8 and 16 mg/kg) potentiated the effect. In morphine-tolerant rats, bremazocine (3 mg/kg) and U-50,488H (16 mg/kg) potentiated morphine-induced analgesia. Tifluadom at any of the doses had no effect on morphine-induced analgesia in morphine-tolerant rats. These results provide evidence that different kappa-opioid agonists modify morphine-induced analgesia differentially in morphine-naive and morphine-tolerant rats.     

Analgesia produced by intrathecal administration of the kappa opioid agonist, U-50,488H, on formalin-evoked cutaneous pain in the rat

The antinociceptive activity of the selective k opioid agonist U-50,488H, given intrathecally (i.t.) against chemically induced cutaneous pain in rats, was assessed from cumulative dose-response experiments and the formalin test. Three successive i.t. doses of 5, 10 and 35 nmol of U-50,488H produced a gradual reduction of pain scores which was statistically significant at all observation periods. This effect was antagonized significantly by 3 mg/kg i.p. of the opiate antagonists, naloxone and WIN 44,441-3. The analgesia profile showed a clear dose-response relationship. A dose producing 50% ‘maximum posible analgesia’ of 6.20 nmol (95% confidence interval: 3.05–12.59 nmol) was calculated. The results indicated that cutaneous pain of a chemical/inflammatory nature is highly sensitive to activation of k receptors of the spinal cord dorsal horn.     

Interaction of supraspinally administered interferon-alpha with opioid system in the production of antinociception

We examined the antinociceptive effect of intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administered of interferon-alpha (IFN-α) using the tail-flick analgesic test in mice. IFN-α administered i.c.v. but not i.t. showed an antinociceptive effect in a dose-dependent manner (0.05–5 μg). To examine the possible interaction with brain opioid system, IFN-α was cotreated with either β-endorphin or morphine. Combined i.c.v. treatment of IFN-α (0.5 μg) with β-endorphin (0.125 μg) caused a synergistic antinociceptive effect. And also, the synergistic interaction maintained at least for 60 min after the co-treatment of IFN-α and β-endorphin. However, the combined treatment of i.t. IFN-α with i.c.v. β-endorphin showed neither an additive nor a synergistic antinociceptive profile. The i.c.v. co-treatment of IFN-α with morphine (0.2 μg) showed an additive antinociceptive effect only. The i.t. administered IFN-α did not show any additive antinociceptive effect when morphine was administered i.c.v. simultaneously. Taken together, our results suggest that supraspinally co-administered IFN-α and β-endorphin may produce antinociception synergistically via interaction of IFN-α with supraspinal β-endorphin sensitive opioid receptors.     

Effects of spinal versus supraspinal administration of cyclic nucleotide-dependent protein kinase inhibitors on morphine tolerance in mice

The consequences of becoming tolerant to the analgesic effects of morphine include increased risk of unwanted side effects, such as respiratory depression, because the patient is required to take larger doses of the opioid to get the same relief from pain. Many studies suggest that phosphorylation plays a role in the neuroplasticity associated with opioid tolerance. This study examines the effect of inhibiting cyclic nucleotide-dependent protein kinase activity in the brain or spinal cord of morphine-tolerant mice. KT5720, a cyclic adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, or KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, was centrally administered in morphine-tolerant and placebo-treated mice prior to a systemically administered challenge dose of morphine. KT5720 completely reversed morphine tolerance in the tail-flick assay when the pretreatment was administered intracerebroventricularly (i.c.v.); KT5823 had no effect on morphine via this route. When either of these drugs was administered intrathecally (i.t.), the activity of morphine was greatly diminished in the tolerant animals, with no effect on morphine antinociception in the placebo group. These data suggest that cAMP-dependent protein kinase activity may be upregulated in the brain with morphine tolerance, and that this upregulation is critical to the expression of tolerance to the antinociceptive effects of morphine. In the spinal cord, however, the activity of cyclic nucleotide-dependent protein kinases, and possibly their substrate proteins, may be affected by chronic morphine exposure such that inhibition of these kinases produces hyperalgesia.