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A class of opioid receptor active derivatives of oxymorphone has been synthesized using a common enaminone intermediate. The derivatives have heterocyclic groups fused to the 6,7-positions of the morphinan system and all were synthesized in high yield. A pyrazolo derivative is an agonist for the mu and delta receptors and an antagonist for the kappa receptor.

1. Chronic administration of L -arginine (200 mg/kg, IP) twice a day for 4 days decreased the antinociceptive response to subcutaneously, but not to intracerebroventricularly, administered morphine in male Swiss-Webster mice, as measured by the tail-flick test. 2. The decreased antinociceptive response to morphine was reversed by concurrent administration of N G -nitro- L -arginine ( L -NNA) (5 mg/kg, IP), an inhibitor of nitric oxide synthase. 3. The concentrations of morphine in mice treated chronically with L -arginine and then given morphine (10 mg/kg, SC) were determined in the peripheral tissues. L -Arginine treatment significantly increased the concentration of morphine in spleen and lungs, did not modify it in liver, kidneys and urine. L -NNA by itself had no effect on the distribution of morphine in peripheral tissues but reversed the changes induced by chronic treatment with L -arginine. 4. Acute administration of L -arginine (200 mg/kg, IP) did not modify either the morphine antinociception or the morphine distribution in peripheral tissues. 5. Previous studies from this laboratory indicated that chronic treatment with L -arginine decreases the concentration of morphine in several brain regions and spinal cord of mice. 6. The facts that chronic treatment with L -arginine does not alter antinociception induced by ICV administered morphine and it increases the concentration of morphine in peripheral tissues while decreasing it in brain regions after peripheral administration of morphine suggest that the decreased antinociception induced by subcutaneously administered morphine may be related to its decreased entry into the brain.

The effects of acute and chronic administration of cocaine on the antinociception and tolerance to the antinociceptive actions of mu-(morphine), kappa-(U-50,488H), and delta-([D-Pen2,D-Pen5]enkephalin; DPDPE), opioid receptor agonists were determined in male Swiss-Webster mice. Intraperitoneal injection of 40 mg/kg of cocaine by itself produced weak antinociceptive response as measured by the tail-fick test but the lower doses were ineffective. Administration of morphine (10 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (10 microg/mouse, ICV) produced antinociception in mice. Cocaine (20 mg/kg) potentiated the antinociceptive action of morphine and DPDPE but had no effect on U-50,488H-induced antinociception. Administration of morphine (20 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (20 microg/mouse, ICV) twice a day for 4 days resulted in the development of tolerance to their antinociceptive actions. Tolerance to the antinociceptive actions of morphine and U-50,488H was inhibited by concurrent treatment with 20 or 40 mg/kg doses of cocaine; however, tolerance to the antinociceptive action of DPDPE was not modified by cocaine. It is concluded that cocaine selectively potentiates the antinociceptive action of mu- and delta- but not of the kappa-opioid receptor agonist. On the other hand, cocaine inhibits the development of tolerance to the antinociceptive actions of mu- and kappa- but not of delta-opioid receptor agonists in mice.

The effects of noribogaine, a metabolite of ibogaine, on the development of tolerance to the antinociception action of morphine was determined in male Swiss-Webster mice. Ibogaine is an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga. Morphine tolerance in mice was developed by two different methods. Mice were rendered tolerant to morphine either by subcutaneous implantation of a pellet containing 25 mg morphine free base for 4 days or by injecting morphine (20 mg/kg, s.c.) twice a day for 4 days. Placebo pellet implanted mice or vehicle injected mice served as controls. To determine the effect of intraperitoneally administered noribogaine on tolerance development, the drug was injected in the appropriate dose twice a day. In pellet implanted mice, a dose of 20 mg/kg of noribogaine attenuated the tolerance to morphine whereas lower doses had no effect. Similarly, in mice given multiple injections of morphine, noribogaine attenuated tolerance development at 20 and 40 mg/kg doses. Previous studies from this laboratory had shown that ibogaine at 40 and 80 mg/kg doses inhibited tolerance to morphine. Because noribogaine could attenuate morphine tolerance at lower doses than ibogaine, it is concluded that the attenuating effect of ibogaine on morphine tolerance may be mediated by its conversion to noribogaine, a more active metabolite.

Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ d -Pen2, d -Pen5]enkephalin (20 μg/mouse) twice a day for 2 days. This procedure resulted in down-regulation of binding sites for [ 3 H ][ d -Pen2, d -Pen5]enkephalin as evidenced by a 52% decrease in the Bmax value. Twice daily injections of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine (MK-801) (0.1 mg/kg, i.p.) or [(−)3-SR,4a-RS,8a-SR-6-(phosphonomethyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] (LY 235959) (2 mg/kg, i.p.), the noncompetitive and competitive antagonist of the N-methyl- d -aspartate (NMDA) receptor, respectively, for 2 days did not alter the Bmax or Kd value of [ 3 H ][ d -Pen2, d -Pen5]enkephalin binding to the mouse brain. Concurrent treatment of MK-801, but not of LY 235959 with [ d -Pen2, d -Pen5]enkephalin, reversed the decreases in Bmax value of [ 3 H ][ d -Pen2, d -Pen5]enkephalin. Twice daily injections of [ d -Ala2,Glu4] deltorphin II (20 μg/mouse) for 2 days caused an increase in the Kd value, but not the Bmax value of [ 3 H ][ d -Pen2, d -Pen5]enkephalin to bind to brain membranes. Concurrent treatment of [ d -Ala2,Glu4]deltorphin II with LY 235959 reversed the increase in Kd value of [ 3 H ][ d -Pen2, d -Pen5]enkephalin binding induced by multiple injections of [ d -Ala2,Glu4]deltorphin II, but MK-801 had no effect. The results suggest that multiple injections of δ1- and δ2-opioid receptor agonists down-regulate δ1-opioid receptors of the brain by modifying Bmax and Kd values of [ 3 H ][ d -Pen2, d -Pen5]enkephalin binding, respectively. MK-801 and LY 235959 reverse δ1- and δ2-opioid receptor agonists-induced down-regulation of brain δ1-opioid receptor, respectively, apparently by different mechanisms. It is concluded that short term treatment of mice with δ1-opioid receptor agonist down-regulates brain δ1-opioid receptors by decreasing Bmax of the ligand which is partially reversed by concurrent treatment with MK-801 but not by LY 235959. On the other hand, short term treatment of mice with δ2-opioid receptor agonist down-regulates brain δ1-opioid receptors by increasing Kd of the ligand which is partially reversed by concurrent treatment with LY 235959 but not by MK-801.

1. 1. The time course of the effect of chronic administration of morphine on the activity of nitric oxide synthase (NOS) in the brain regions and spinal cord of the mouse was determined. The effect of naltrexone by itself on the NOS activity and that induced by morphine also were determined. 2. 2. Male Swiss Webster mice were implanted subcutaneously with a pellet containing 25 mg of morphine free base for 4 days. Placebo pellet implanted mice served as controls. 3. 3. Twenty-four hours after treatment with morphine, NOS activity decreased in the cerebellum, midbrain, cortex and remainder of the brain as well as in the spinal cord. Forty-eight and 72 hr after the treatment with morphine, NOS activity increased in the cerebellum and cortex, but no change was observed in the other brain regions and spinal cord. Twenty-four hours after morphine pellet removal (withdrawal), NOS activity in all brain regions and the spinal cord had returned to normal. 4. 4. Implantation of a pellet containing 10 mg of naltrexone did not alter NOS activity in any brain region or spinal cord for 24, 48 and 72 hr or 24 hr after removal of the pellet. 5. 5. Implantation of a naltrexone pellet in conjunction with a morphine pellet blocked the changes in NOS activity in the brain region and spinal cord induced by morphine. 6. 6. It is concluded that the initial decrease in NOS activity by morphine may be related to enhanced motor activity, whereas the increase in NOS activity in certain brain regions may be associated with tolerance-physical dependence development. Additionally, the changes in central NOS activity by morphine appear to be mediated by opioid receptors because they were blocked by concurrent treatment with naltrexone.

Fusion of an azole moiety at C-6 and C-7 of naltrexone (1) is illustrated by the synthesis of the title compound 8. Bromination of 3-O-methylnaltrexone led to the 1,7α-dibromo derivative which reacted with thiourea to attach the 2-aminothiazole ring to C-6 and C-7 of naltrexone. After converting the amino and alcohol groups to trimethylsilyl derivatives, the aromatic bromo group was removed by halo-lithium interchange with butyllithium, followed by hydrolysis with water. In the final step of the synthesis, the methyl ether was cleaved by boron tribromide to generate 8. An alternate synthesis of 8 commenced with 3-O-acetylnaltrexone (9). Bromination of 9 in acetic acid in the presence of hydrobromic acid produced a mixture of 3-O-acetyl-7α-bromonaltrexone (10) and 7α-bromonaltrexone (11), both, as hydrobromides. Reaction of this mixture with thiourea furnished 8 (62% from 1). While 1H and 13C chemical shifts of all compounds are reported, those of 11 hydrobromide and 8 dihydrochloride were established unequivocally.

Male Sprague-Dawley rats were rendered tolerant to U-50,488H by twice-daily injections of the drug (25 mg/kg, i.p.) for 4 days. In tolerant rats, the binding of [3H]MK-801 was increased in pons and medulla and corpus striatum but decreased in midbrain and hippocampus and was due to changes in Bmax values. In U-50,488H-abstinent rats, the binding of [3H]MK-801 was increased in pons and medulla and hippocampus, and decreased in midbrain and amygdala. In hippocampus, the Bmax of [3H]MK-801 was increased but the Kd was decreased whereas in amygdala and pons and medulla, the changes were due to alterations in the Bmax values. Previous studies have shown that NMDA receptor antagonists block the tolerance to the analgesic action of U-50,488H in rodents. The present studies demonstrate differential changes in the NMDA receptors of brain regions of U-50,488H-tolerant and -abstinent rats.

The effects of the competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, LY235959, were determined on the analgesic and hypothermic effects as well as on the development of tolerance to these effects of U-50,488H, a kappa-opioid receptor agonist in mice and rats. In the mouse, a single injection of LY235959 given 10 min prior to U-50,488H did not modify the analgesic action of the latter. Similarly, chronic administration of LY235959 twice a day for 4 days did not modify U-50,488H-induced analgesia in mice. Repeated pretreatment of mice with LY235959 dose-dependently attenuated the development of tolerance to the analgesic actions of U-50,488H. In the rat, LY235959 by itself produced a significant analgesia and prior treatment of rats with LY235959 enhanced the analgesic action of U-50,488H. Similar effects were seen with the hypothermic action. Pretreatment of rats with LY235959 attenuated the development of tolerance to the analgesic but not to the hypothermic action of U-50,488H. These results provide evidence that LY235959 produces differential actions on nociception and thermic responses by itself and when given acutely with U-50,488H in mice and rats. However, when the animals are pretreated with LY235959, similar inhibitory effects are observed on the development of tolerance to the analgesic action of U-50,488H in both the species. These studies demonstrate an involvement of the NMDA receptor in the development of kappa-opioid tolerance and suggest that the biochemical consequences of an opioid's interaction with the opioid receptor are not the only factors that contribute to the acute and chronic actions of opioid analgesic drugs.

Bhargava, H. N., G.-M. Zhao, J.-T. Bian, Y. Nan, S. P. Upadhyaya, X. Wei, W. J. Dunn, III and L. Bauer. Effects of some 7-arylidene and 7-heteroarylidene morphinan-6-ones on the antinociceptive activity of [ d -Pen 2 , d -Pen 5 ] enkephalin and [ d -Ala 2 , Glu 4 ]deltorphin II and on multiple opioid receptors. peptides 18(5) 695–701, 1997.—The in vivo and functional effects of several 7-arylidene and 7-heteroarylidene morphinan-6-ones were determined at the μ -, δ -, and κ -opioid receptors using the guinea pig brain membranes, guinea pig ileum (GPI), and mouse vas deferens (MVD). In vivo effects included the antagonism by these compounds given subcutaneously on the antinociceptive actions of intracerebroventricularly injected [ d -Pen 2 , d -Pen 5 ]enkephalin (DPDPE) and [ d -Ala 2 , Glu 4 ]deltorphin II (deltorphin II), the highly selective putative δ 1 - and δ 2 - opioid receptor agonists. Finally, the partition coefficients of these compounds were estimated (CLOGP) and determined experimentally at pH 7.4 in the 1-octanol/water system. Compared with E-7-benzylidenenaltrexone (BNTX), most compounds except for E-7-(4-chlorobenzylidene)naltrexone, were more potent at δ -opioid receptors than at the μ -opioid receptor, whereas, in comparison to the κ -opioid receptor, the activities of the E-7-arylidene or E-7-heteroarylidene naltrexone derivatives at the δ -receptor were in the following order, where the 7-substituents were: 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- > 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene > 4-chlorobenzylidene. In the MVD preparation, the potencies at the δ -opioid receptor, in comparison to BNTX, were in the following order, where the 7-substituents were: benzylidene = 1-methyl-2-imidazolylmethylene- > 4-fluorobenzylidene- = 3-pyridylmethylene- = 4-pyridylmethylene-. All compounds antagonized δ 1 and δ 2 -opioid receptor agonist-induced analgesia. The antagonist potencies at the δ 1 -opioid receptor were in the following order, where the 7-substituents were: benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolylmethylene- ≈ 4-pyridylmethylene-, whereas at the δ 2 -opioid receptor, the order was benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolylmethylene- > 4-pyridylmethylene. In general, all compounds exhibited greater potency at the δ 2 - than δ 1 -opioid receptor. The computed partition coefficients were, as expected, greater than the apparent log P values, which were determined experimentally. Generally, the lipophilicity values in decreasing order were: 4-chlorobenzylidene- > 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- = 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene-. In general, the benzylidene and 4-pyridylmethylene derivatives, which have medium lipophilicities, were equally effective at the δ 1 - and δ 2 - receptors; the 3-pyridylmethylene and 1-methyl-2-imidazolylmethylene derivatives had lower lipophilicities and were more selective for the δ 2 - than δ 1 - receptor; the 4-chlorobenzylidene and 4-fluorobenzylidene derivatives were more lipophilic and had intermediate activity. The plot of pED 50 values for the in vivo tests for the δ 1 - and δ 2 - receptors showed that the two receptors are not independent with respect to this series of compounds.

Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin, [3H][D-Pen2, D-Pen5]enkephalin, and [3H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [3H]labeled ligands of μ-([D-Ala2, MePhe4, Gly-ol5]enkephalin; DAMGO), δ ([D-Pen2, D-Pen5]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [3H]DAMGO, [3H]DPDPE and [3H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was δ- ≥ κ- ≥ μ-, with lung parenchyma exhibiting the greatest binding capacity for δ- and μ- receptors compared to the other regions examined. The Kd values showed small differences between ligands and regions tested. The μ- and δ-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, κ-opioid receptor density was augmented in sensitized lung parenchyma but an increase in Kd values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.

The effects of chronic administration and subsequent withdrawal of mu-, kappa- and delta-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at mu-, kappa- and delta-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla (76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of mu- and kappa-opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to mu- and kappa- but not to delta-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by mu-, kappa- and delta-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to mu-, kappa- and delta-opioid receptor agonists.

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N -methyl- D -aspartate receptors following multiple intracerebroventricular injections of [ D -Pen 2 , D -Pen 5 ]enkephalin and [ D -Ala 2 , Glu 4 ]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ D -Pen 2 , D -Pen 5 ]enkephalin and [ D -Ala 2 , Glu 4 ]deltorphin II, the selective agonists of the δ 1 - and δ 2 -opioid receptors, on the binding of [ 3 H]MK-801, a noncompetitive antagonist of the N -methyl- D -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ D -Pen 2 , D -Pen 5 ]enkephalin or [ D -Ala 2 , Glu 4 ]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [ 3 H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [ 3 H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ D -Pen 2 , D -Pen 5 ]enkephalin treated mice. In [ D -Ala 2 , Glu 4 ]deltorphin II treated mice, the binding of [ 3 H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B max value of [ 3 H]MK-801. It is concluded that tolerance to δ 1 - and δ 2 -opioid receptor agonists is associated with up-regulation of brain N -methyl- D -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N -methyl- D -aspartate receptors antagonists block tolerance to the analgesic action of δ 1 - and δ 2 -opioid receptor agonists.

The effects of 7-nitroindazole (7-NI), an inhibitor of the neuronal nitric oxide synthase (nNOS) which does not increase blood pressure, on tolerance to the antinociceptive activity of mu-(morphine), kappa-(U-50,488H) and delta-([D-Pen2, D-Pen5]enkephalin, DPDPE) opioid receptor agonists were determined in mice. Male Swiss-Webster mice were made tolerant by twice daily injections of morphine (20 mg/kg, s.c.), U-50,488H (25 mg/kg, i.p.) or DPDPE (20 micrograms/mouse, i.c.v.) for 4 days. When tested on day 5, tolerance to their antinociceptive activity was evidenced by decreased response in chronic drug treated mice in comparison to vehicle-injected mice. Concurrent administration of 7-NI (20, 40 or 80 mg/kg, i.p.) with DPDPE did not modify the development of tolerance to the antinociceptive action of DPDPE. However, 7-NI (40 or 80 mg/kg, i.p.) inhibited the development of tolerance to the antinociceptive activity of morphine and U-50,488H but the lower dose of 7-NI (20 mg/kg, i.p.) was not effective. Chronic administration of 7-NI by itself did not modify the acute response to morphine, U-50,488H or DPDPE. It is concluded that a specific inhibitor of nNOS can inhibit tolerance to the antinociceptive activity of mu- and kappa- but not of delta-opioid receptor agonists in mice.

A number of (E)-7-arylidenenaltrexones were synthesized by azeotropic distillation of water from a benzene solution of naltrexone and an aromatic aldehyde (benzaldehyde, 4-chloro- and 4-fluorobenzaldehyde, 3-and 4-pyridinecarboxaldehyde and 1-methyl-2-imidazolecarboxaldehyde) using piperidine as a catalyst. In addition, (E)-7-benzylidenenaloxone was prepared by the previously published Claisen-Schmidt condensation using sodium hydroxide in methanol. The stereochemistry of these arylidene derivatives 3–9 was determined to be (E) by means of nuclear Overhauser enhancement experiments. The 13C nmr spectra of (E)-3–9 are recorded in deuteriochloroform and those of the hydrochlorides in deuteriodimethyl sulfoxide.

The specific binding of [3H]GBR 12935 to crude synaptosomal membranes of brain regions and spinal cord of morphine tolerant and abstinent rats was investigated. Male Sprague-Dawley rats were implanted with 6 morphine pellets each containing 75 mg of morphine base during a 7-day period. Placebo pellet implanted rats served as controls. Rats sacrificed without removal of the pellet were considered tolerant whereas those from which pellets were removed 16 hr prior to sacrificing were labeled abstinent. The binding of [3H]GBR 12935 was initially determined at a 1 nM concentration in all brain regions and spinal cord, which was followed by the determination of Bmax and Kd values in the corpus striatum, a highly enriched region for the dopamine transporter. In morphine tolerant rats, the binding of [3H]GBR 12935 was increased in the hypothalamus (182%) but was decreased in the corpus striatum (34%) and spinal cord (30%). The decrease in binding in the corpus striatum was due to an increase in the Kd value of [3H]GBR 12935. However, during morphine withdrawal, the binding of [3H]GBR 12935 was still higher in the hypothalamus (255%) but was decreased in the hippocampus (53%). Thus, chronic administration of morphine results in changes in the dopamine transporter function in selected brain regions and the spinal cord, and these changes are dependent upon whether or not the animals are undergoing the abstinence syndrome.

1. 1. The effects of morphine tolerance and abstinence on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptors were determined in brain regions and spinal cord of the mouse. 2. 2. Male Swiss-Webster mice were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of a pellet containing 75 mg of morphine base for 3 days. Placebo pellet-implanted mice served as controls. In tolerant (nonabstinent) mice, the pellets were left intact at the time of sacrificing whereas, in the abstinent mice, the pellets were removed 6 hr prior to sacrificing. 3. 3. The binding of [3H]MK-801 to membranes prepared from spinal cord and brain regions (cortex, pons-medulla, hypothalamus, hippocampus, amygdala, striatum, and midbrain) was determined by using a 5 nM concentration of the ligand in the presence of 30 μM glycine and 50 μM of glutamate. 4. 4. In nonabstinent morphine-tolerant mice, the binding of [3H]MK-801 was decreased in pons-medulla and hypothalamus, but was increased in the spinal cord in comparison to that in placebo controls. The reduction in binding in pons-medulla was due to a decrease in the Bmax value; the Kd value remained unchanged. The binding of [3H]MK-801 was increased in the hippocampus of morphine-abstinent mice. 5. 5. These studies demonstrate that NMDA receptors of brain regions and spinal cord are differentially affected in morphine-tolerant and abstinent mice.

The effects of NG-nitro- l -arginine ( l -NNA) and NG-monomethyl- l -arginine ( l -NMMA), two potent inhibitors of nitric oxide synthase (NOS) on the development of tolerance to the analgesic action of [ d -Ala2,Glu4]deltorphin II (deltorphin II), a δ2-opioid receptor agonist, and morphine, a μ-opioid receptor agonist, were determined in mice. Male Swiss-Webster mice were rendered tolerant to deltorphin II by twice daily ICV injections of the drug for 4 days. Tolerance to morphine was induced by twice daily SC injections of the drug for 4 days. Multiple injections of deltorphin II (20 μg/mouse) or morphine (15 mg/kg) resulted in the development of tolerance to their analgesic action as evidenced by decreases in the response in comparison to mice injected with vehicle. Concurrent administration of l -NNA or l -NMMA (2, 4, or 8 mg/kg, IP) had no effect on the development of tolerance to the analgesic action of deltorphin II. However, the same doses of l -NNA or l -NMMA inhibited the development of tolerance to the analgesic action of morphine. Acute treatment with l -NNA or l -NMMA did not modify deltorphin II- or morphine-induced analgesia in mice. It is concluded that NOS inhibition attenuates tolerance to the analgesic action of morphine but not to that of deltorphin II, a δ2-opioid receptor agonist, in the mouse.

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a mu-opioid receptor agonist, of U-50,488H, a kappa-opioid receptor agonist of [D-Pen2, D-Pen3]enkephalin (DPDPE), a delta 1-opioid receptor agonist, and of [D-Ala2,Glu4]deltorphin II (deltorphin II), a delta 2-opioid receptor agonist in mice. Morphine administered ICV (2.5 micrograms/ mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 micrograms/mouse; ICV), and deltorphin II (15 micrograms/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of mu-, kappa-, delta 1-, or delta 2-opioid receptor agonists.

The effect of chronic administration of naltrexone, an opioid receptor antagonist, on the activity of the dopamine transporter in brain regions and spinal cord was determined. Male Sprague-Dawley rats

Two potent inhibitors of nitric oxide synthase (NOS), namely, NG-nitro-L-arginine (NNA) and NG-monomethyl-L-arginine (NMMA) were administered intracerebroventricularly (i.c.v.) in morphine-dependent mice to investigate their effects on abrupt withdrawal and naltrexone-precipitated abstinence signs. Male Swiss-Webster mice were rendered dependent on morphine by subcutaneous implantation of a morphine pellet containing 75 mg of morphine base. Mice implanted with placebo pellets served as controls. NMMA or NNA administered i.c.v. had minimal effects on body weight loss and hypothermia that occur during abrupt withdrawal of morphine. When administered i.c.v., both NNA or NMMA (0.1, 1 and 10 micrograms/mouse) dose-dependently inhibited naltrexone-induced stereotyped jumping behavior in mice. I.c.v. administration of NMMA also attenuated withdrawal induced fecal pellet formation. This effect, however, was not dose-dependent. In conclusion, these results suggest that brain NO plays an important role in the expression of behavioral signs of morphine withdrawal syndrome. In addition, these results support the idea that NOS inhibitors may be potentially useful in the treatment of opioid withdrawal syndrome.

Because low pH treatment is known to alter the coupling of G-proteins to brain receptors, and little is known about such an effect in the spinal cord, the present study was undertaken to examine whether preincubation of rat spinal cord membranes at low pH (pH 4.5) alters opioid receptor binding characteristics and sodium fluoride (NaF)-stimulated adenylyl cyclase (AC) activity (as a function of G s mediated). [ 3 H][ d -Ala 2 ,MePhe 4 ,Gly-ol]enkephalin (DAMGO) and [ 3 H]ethylketocyclazosine (EKC) were used to label μ- and κ-opioid receptors, respectively. AC activity was determined using ATP as substrate and cAMP formed was quantified. Low pH treatment of membranes did not affect the μ- and κ-opioid binding characteristics in rat spinal cord. However, the low pH treatment significantly reduced the NaF-stimulated AC activity in rat spinal cord. It is concluded that low pH treatment causes selective changes in the functional coupling of G s -proteins to AC without affecting the opioid receptor binding characteristics in the spinal cord.

1. 1. The effect of NG-monomethyl- l -arginine (NMMA), an inhibitor of nitric oxide synthase (NOS), on the development of tolerance to and physical dependence on morphine was determined in the rat. 2. 2. Male Sprague-Dawley rats were rendered tolerant to and dependent on morphine by the subcutaneous implantation of four morphine pellets (each containing 75 mg morphine base) during a 3 day period. Placebo pellet implanted rats served as controls. 3. 3. Chronic administration of morphine resulted in the development of tolerance to the analgesic action of morphine. Twice daily injections of NMMA (4 or 8 mg/kg) attenuated the tolerance to morphine as evidenced by higher analgesic response in NMMA treated than in vehicle treated morphine tolerant rats. 4. 4. Chronic administration of morphine also resulted in the development of physical dependence as evidenced by the appearance of a variety of symptoms including stereotyped jumping response following naltrexone injection. Concurrent treatment with NMMA inhibited naltrexone-induced jumping response but other responses like fecal boli formation, wet dog shakes, teeth chattering, rearing and ejaculations were not modified. 5. 5. It is concluded that inhibition of NOS can attenuate the development of tolerance to, and physical dependence on, morphine in the rat. However, it appears that higher doses of NOS inhibitors are required in the rat than in the mouse for blockade of both tolerance and physical dependence processes.

Previous studies in this laboratory and elsewhere have provided evidence that compounds acting as delta opioid receptor agonists exhibit marked immunostimulatory potential. Conversely, the delta opioid receptor antagonists have previously been shown to demonstrate immunosuppressive effects as assessed by proliferation of T-cells following allogeneic or xenogeneic stimulation. The present study was performed to further characterize this immunosuppressive activity using the compounds benzylidene naltrexone (BNTX), naltrindole (NTI), and naltriben (NTB). In vitro exposure to BNTX resulted in an apparent dose-related suppression of B-cell proliferation, cytokine production by T-helper cells, and natural killer (NK) cell activity, with statistically significant suppression observed at concentrations between 1 and 10 microM. NTI was also immunosuppressive for all immune function parameters examined, although this compound was less active than BNTX. In vitro exposure to the structurally related compound NTB had no significant effect on any immune function examined in this study. In all cases, immunosuppression occurred in the absence of any detectable alteration in cellular viability, suggesting a specific immunosuppressive effect rather than overt toxicity.

The effect of morphine tolerance/dependence and abstinence on the preproenkephalin (PPE) gene expression was determined in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were rendered tolerant and physically dependent on morphine by SC implantation of six pellets, each containing 75 mg of morphine base, during a 7-day period. Placebo pellet-implanted rats served as controls. In tolerant rats, the pellets were left in place at the time of sacrifice whereas in abstinent rats, the pellets were removed 16 h prior to sacrificing. The levels of PPE mRNA were determined in brain regions (striatum, cortex, pons-medulla, hypothalamus, amygdala, and midbrain) and spinal cord. The levels of PPE mRNA increased significantly in the cortex (62%) and the spinal cord (352%) of morphine-tolerant rats when compared to placebo pellet-implanted control rats. In other brain regions, the levels of PPE mRNA in placebo and morphine-tolerant rats did not differ. On the other hand, in morphine-abstinent rats, the levels of PPE mRNA increased in the striatum (62%) and hypothalamus (34%) but were decreased in pons-medulla (68%), midbrain (51%), and spinal cord (36%) in comparison to the placebo controls. The results clearly demonstrate differential changes in enkephalin gene expression in brain regions and spinal cord of the abstinent and nonabstinent morphine-tolerant/dependent rats.

Nitric oxide synthase (NOS) activity was determined by the rate of conversion of [3H]arginine to [3H]citrulline in brain regions (midbrain, hypothalamus, cerebellum, hippocampus, corpus striatum, cortex, pons-medulla and amygdala) and spinal cord of male Swiss Webster mice and male Sprague-Dawley rats. In mice, high activity of NOS was found in cerebellum, hypothalamus and midbrain; intermediate activity in pons-medulla, hippocampus, amygdala, corpus striatum and cortex; and low activity in the spinal cord. In rat, highest activity of NOS was observed in cerebellum followed in decreasing order by midbrain, hypothalamus, cortex, striatum, pons-medulla, hippocampus and spinal cord. In all tissues, NOS activity was higher in rat than in mouse. Analysis of the Eadie-Hofstee plot indicated that the Vmax and Km values of the enzyme in cortex and cerebellum of rats were higher than in mouse tissues. These studies show differential distribution of NOS activity in mouse and rat brain regions and spinal cord and higher activity of NOS in rat brain regions and spinal cord compared to mouse tissues.

The immunomodulatory potential of the indole alkaloids ibogaine and harmaline was examined in a panel of in vitro immune function assays. These assays were chosen to assess T-cell regulatory and effector function, B-cell function, macrophage function, and natural killer-cell function. The in vitro exposure to either ibogaine or harmaline resulted in a dose-related suppression of all immune functions examined except macrophage function. This suppression was noted at various concentrations in different assays, but was generally only associated with high concentrations (10-100 mumol/l).

1. The effects of morphine tolerance and abstinence on the characteristics of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H]MK-801, were determined in the brain regions and spinal cord of the rat. 2. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of six morphine pellets during a 7-day period. In tolerant (non-abstinent) rats, the pellets were left intact at the time of sacrificing, whereas in the abstinent rats the pellets were removed 16 hr prior to sacrificing. 3. The binding of [3H]MK-801, an NMDA receptor antagonist, to membranes prepared from spinal cord and brain regions (cortex, striatum, amygdala, hippocampus, hypothalamus, midbrain and pons-medulla) was determined using 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In non-abstinent morphine tolerant rats, the binding of [3H]MK-801 was decreased by 40 and 33% in the midbrain and spinal cord, respectively, in comparison with their placebo controls. In morphine abstinent rats, the binding of [3H]MK-801 was decreased by 42, 29 and 50% in hypothalamus, midbrain and spinal cord, respectively, in comparison with their placebo controls. The binding of [3H]MK-801 to other brain regions and spinal cord of morphine tolerant and abstinent rats did not differ from their respective placebo controls. 5. Thus, these studies demonstrate, for the first time, that in the presence of glutamate and glycine, NMDA receptors of selected brain regions and spinal cord are down-regulated in rats treated chronically with morphine.

The present studies examined the hypothesis that the N-methyl- d-aspartate (NMDA) receptor-nitric oxide (NO) pathway might be involved in the acute and chronic actions of Δ9-tetrahydrocannabinol (THC). The ability of dizocilpine (MK-801), a competitive NMDA receptor antagonist and NG-monomethyl- l-arginine (l-NMMA), an inhibitor of NO synthase enzyme to modify the analgesic and hypothermic responses following the acute and chronic treatment of animals with THC was determined in male Swiss-Webster mice. Intraperitoneal administration of THC (5, 10 and 20 mg/kg) produced dose-dependent analgesic and hypothermic effects. MK-801 at 0.1 gg/kg i.p. attenuated the analgesic but not the hypothermic responses to THC (10 and 20 mg/kg, i.p.). The effects of various doses of MK-801 (0.03, 0.1 and 0.3 mg/kg, i.p.) on the analgesic and hypothermic responses to a 10 mg/kg, i.p. dose of THC was also determined. All the doses of MK-801 antogonized the analgesic but not the hypothermic effects of THC. The chronic treatment of animals with THC (10 mg/kg, i.p.) twice daily for 4 days produced tolerance to its analgesic and hypothermic effects. Pretreatment of animals with MK-801 (0.03–0.30 mg/kg, i.p.) did not affect the development of tolerance to the analgesic or the hypothermic action of THC. The pretreatment of animals with l-NMMA (2–8 mg/kg, i.p.), did not alter the analgesic or hypothermic effects of THC. Also, it did not modify the tolerance to its pharmacological actions. It is concluded that non-competitive antagonism of NMDA receptor by MK-801 selectively antagonized the analgesic action of THC and that the mechanisms in the analgesic response and tolerance to THC may be different. Finally, NO does not appear to be involved in the acute or chronic actions of THC.

Male Swiss-Webster mice were rendered tolerant to morphine by subcutaneous implantation of a morphine pellet, each containing 75 mg morphine base, for 3 days. Mice implanted with placebo pellets served as controls. A high degree of tolerance to the analgesic effect of morphine developed as evidenced by decreased analgesic response to various doses of morphine. Δ 9 -Tetrahydrocannabinol (5, 10 and 20 mg/kg i.p.) produced dose-dependent analgesic and hypothermic effects in mice implanted with placebo pellets. A significant decrease in the analgesic effects of tetrahydrocannabinol was observed in morphine-tolerant mice as compared to placebo controls. Mice were rendered tolerant to Δ 9 -tetrahydrocannabinol by injecting the drug (5, 10, or 20 mg/kg i.p.) twice daily for 4 days. Vehicle-injected mice served as controls. Tolerance to the analgesic and hypothermic effects of Δ 9 -tetrahydrocannabinol in mice injected chronically with the drug was evidenced by the decreases in the intensity of these responses when compared to those observed in vehicle-injected controls. Morphine produced dose-dependent analgesic and hypothermic effects in mice injected chronically with vehicle but the intensity of these effects was significantly lower in mice injected chronically with Δ 9 -tetrahydrocannabinol. These results indicate that a possible interaction exists between Δ 9 -tetrahydrocannabinol and the μ-opioid receptors and that a substantial tolerance to analgesic and hypothermic effects of morphine develops in Δ 9 -tetrahydrocannabinol-tolerant mice. To investigate whether the chronic administration of Δ 9 -tetrahydrocannabinol alters the μ-opioid receptors in the central nervous system (CNS), the binding of a selective μ-opioid receptor agonist, [ 3 H][ D -Ala 2 , MePhe 4 , Gly-ol 5 ]enkephalin ([ 3 H]DAMGO), to whole brain and spinal cord μ-opioid receptors was determined in Δ 9 -tetrahydrocannabinol-tolerant animals. The B max and K d values of [ 3 H]DAMGO in brain or the spinal cord of Δ 9 -tetrahydrocannabinol-tolerant mice were not altered. Thus, evidence is presented for the existence of bidirectional cross- tolerance between morphine and Δ 9 -tetrahydrocannabinol in the mouse without changes in μ-opioid receptors of the central nervous system.

The effects of the methyl esters of L-tyrosine (L-Tyr-OMe) and L-tryptophan (L-Trp-OMe) on the analgesic action of trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidin)cyclohexyl]-benzene acetamide methane sulfonate (U-50,488H), a kappa-opioid receptor agonist, were determined in male Swiss-Webster mice using the tail-flick test. Intraperitoneal injections of U-50,488H produced a dose-dependent analgesic response. The analgesic response to all doses of U-50,488H was potentiated by L-Tyr-OMe at 200 mg/kg injected intraperitoneally 30 min prior to the injection of U-50,488H. The effect of various doses of L-Tyr-OMe (50, 100 and 200 mg/kg) on the analgesia produced by 20 mg/kg of U-50,488H was also determined. The lowest dose (50 mg/kg) of L-Tyr-OMe did not modify U-50,488H-induced analgesia but the two higher doses enhanced it significantly. L-Tyr-OMe by itself at all the doses tested had no effect on the tail-flick latency. L-Trp-OMe (200 mg/kg) enhanced the analgesic action of 10 and 20 mg/kg doses of U-50,488H but not that induced by a 5 mg/kg dose. The analgesia induced by 20 mg/kg of U-50,488H was potentiated by L-Trp-OMe at 100 and 200 mg/kg but not by a 50 mg/kg dose. L-Trp-OMe by itself also did not alter the tail-flick latency. Previously, the studies in this laboratory have shown that L-Try-OMe potentiates morphine, a mu-opioid receptor agonist-induced analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)

The analgesic and hypothermic responses to U-50,488H (25 mg/kg IP), a kappa opiate receptor agonist, were determined in male Sprague-Dawley rats aged 4, 8, and 24 weeks. In addition, the characteristics of the binding of [3H]ethylketocylazocine (EKC) to kappa opiate receptors in whole brain and spinal cord of rats of three age groups were also determined. Administration of U-50,488H produced an age-related increase in the analgesic response in the rat, i.e., the older rats exhibited a higher intensity of analgesic response than the younger rats. U-50,488H also produced a hypothermic response. The response in 4- and 24-week-old rats was similar, but that in 8-week-old rats was smaller than the rats in the other two age groups. [3H]EKC bound to whole brain and spinal cord membranes of rats at a single high affinity site. The Bmax value of [3H]EKC in the brain and spinal cord of 24-week-old rats was significantly lower than in 4- and 8-week-old rats; however, the Kd values did not differ. It is concluded that kappa opiate receptor agonist produces age-related increase in its analgesic response and that such effects are not related to the characteristics of kappa receptors in the brain and spinal cord.

The effect of chronic administration of morphine to rats on the N-methyl-D-aspartate (NMDA) receptors labeled with [3H]MK-801, a non-competitive antagonist, was determined in brain regions and spinal cord. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of 6 morphine pellets during a 7-day period. Each pellet contained 75 mg of morphine free base. Animals serving as controls were similarly implanted with placebo pellets. This procedure resulted in the development of a high degree of tolerance and physical dependence on morphine. Two sets of rats were used. In one, the pellets were left intact at the time of sacrifice (tolerant) and in the other the pellets were removed 16 h prior to sacrificing (abstinent). The binding constants, Bmax and Kd values of [3H]MK-801 were determined in cortex, hippocampus, hypothalamus, corpus striatum, midbrain and spinal cord. In the absence of glycine and glutamate, [3H]MK-801 bound to tissue membranes at a single high affinity site. The Bmax and Kd values of [3H]MK-801 were not altered in any of the tissues of the morphine abstinent rats. The Bmax value of [3H]MK-801 was significantly decreased in cerebral cortex of morphine tolerant rats as compared to their placebo controls but the Kd values did not change. In other brain regions and spinal cord of morphine tolerant rats and their placebo controls, the Bmax and Kd values of [3H]MK 801 did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of lysergic acid diethylamide (LSD) to alter immune function after direct in vitro exposure was examined. It was demonstrated that LSD is able to suppress the proliferation of B-lymphocytes; the production of the cytokines IL-2, IL-4, and IL-6; and the induction of cytotoxic T-lymphocytes at a concentration of 100 microM. In vitro exposure to LSD had differential effects on natural killer (NK) cell activity, with significant enhancement of both basal and IL-2-augmented NK cell function at concentrations between 0.0001 and 0.1 microM, and suppression of NK response at 100 microM. These results demonstrate that LSD may have a direct effect on components of the immune system at concentrations that may be reached upon human exposure.

The potential of synthetic and natural amphetamines to modulate cellular immune effector and regulatory mechanisms was evaluated in an in vitro exposure system. Murine splenic lymphocytes and elicited peritoneal macrophages were cultured with 0.0001-100 microM of amphetamine sulfate, methamphetamine hydrochloride, or the (S) or (R) isomers of cathione hydrochloride. T-lymphocyte regulatory function was assessed by quantitating the production of cytokines, and T-lymphocyte effector function was assessed by the induction of cytotoxic T-lymphocytes (CTL). B-lymphocyte function was measured by proliferation, and natural immunity was assessed by quantitating basal and IL-2 augmented natural killer (NK) cell activity. None of the compounds tested had any direct effect on cellular viability. Exposure to amphetamine resulted in a significant suppression of IL-2, but not IL-4, production by T-lymphocytes, as well as a suppression of B-lymphocyte proliferation only at the highest amphetamine concentration examined. NK cell function was slightly suppressed by amphetamine exposure, but was enhanced by methamphetamine exposure. Conversely, exposure to either (S) or (R) isomers of cathinone resulted in stimulation of IL-2 production, B-lymphocyte proliferation, and CTL induction. No significant effect of cathione was noted on NK cell function. These data suggest that natural and synthetic amphetamines exhibit differential immunomodulatory activity following in vitro exposure.

1. 1. The effects of naltrexone pellet implantation and removal on the analgesic and hypothermic effects of morphine and the binding of 3 H- d -Ala 2 , MePhe 4 , Gly-ol 5 -enkephalin (DAMGO) to μ-opiate receptors in rat brain regions and spinal cord were determined. 2. 2. Male Sprague-Dawley rats were implanted subcutaneously with a pellet containing 10 mg of naltrexone for 7 days. Placebo pellet implanted rats served as controls. The pellets were removed on day 8, and the analgesic and hyperthermic effects were determined in the rat 24 hr later. Morphine produced a dose-dependent analgesic and hyperthermic responses in rats implanted with placebo pellets. Enhanced analgesic and hyperthermic responses to morphine were produced in rats implanted with naltrexone pellets. 3. 3. The binding constants ( B max and K d values) of [ 3 H]DAMGO in regions of the brain (amygdala, hypothalamus, striatum, midbrain, hippocampus, pons + medulla and cortex), and spinal cord of rats with naltrexone pellet left intact or removed were determined. The B max values of [ 3 H]DAMGO were increased in all brain regions and spinal cord of rats in which the naltrexone pellets were left in place or removed prior to sacrificing. However, the K d values of [ 3 H]DAMGO were unaffected by naltrexone treatment. 4. 4. It is concluded that enhanced analgesic and hyperthermic response to morphine is produced in rats implanted with naltrexone pellets and such alterations in the pharmacological responses are due to up-regulation of μ-opiate receptors in all the brain regions and spinal cord. Additionally whether the pellets were left intact (receptors blocked) or removed (receptors not blocked), the μ-opiate receptors were up-regulated in spinal cord and all the regions of the brain.

The effect of thyrotropin releasing hormone (TRH) administered either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) on the analgesic and hypothermic actions of U-50,488H, a highly selective kappa-opiate agonist, was determined in male Swiss Webster mice. Intraperitoneal administration of U-50,488H (8-32 mg/kg) produced a dose-dependent analgesia as assessed by the tail-flick test. Similarly, U-50,488H also produced a dose-dependent hypothermia in mice. TRH was administered s.c. 15 min or i.c.v. 5 min prior to U-50,488H injection. TRH (1,3 and 10 mg/kg, s.c.) dose-dependently attenuated the analgesic effect of U-50,488H (32 mg/kg), whereas TRH at these doses displayed almost complete blockade of the hypothermic effect of U-50,488H. Similarly, TRH (0.03, 0.3 and 1 microgram/mouse; i.c.v.) dose-dependently attenuated the analgesic and hypothermic actions of U-50,488H, indicating the central component in the action of TRH. TRH alone in doses used showed no change in either basal tail-flick latency or body temperature, demonstrating the lack of effect of this drug alone on pain and temperature responsiveness. Studies have shown that TRH does not modify morphine or beta-endorphin-induced analgesia in animals nor does it affect the binding of mu-opiate agonist or antagonist to brain membranes. Previous studies from this laboratory have indicated that kappa-opiates but not mu-opiates inhibit the binding of [3H][3-MeHis2]-TRH to brain membranes. The present studies clearly show that TRH modulates the pharmacological actions mediated by kappa-opiate agonists in mice. Thus, these studies provide further in vivo evidence for an acute interaction between TRH and kappa-opiates.

The effects of naltrexone injected intravenously (i.v.) on the pharmacological actions and distribution of i.v. injected morphine in brain regions and spinal cord of male Sprague-Dawley rats were determined. Naltrexone (0.625- and 2.5-mg/kg doses) antagonized the analgesic and hyperthermic effects of morphine (10-mg/kg dose). For distribution studies, naltrexone (0.625- and 2.5-mg/kg doses) was co-administered with morphine via indwelling catheters. Rats were sacrificed at various times after drug injection and the concentration of morphine in brain regions (hypothalamus, hippocampus, cortex, pons and medulla, amygdala, midbrain and corpus striatum), spinal cord and serum was determined by radioimmunoassay. The concentration of morphine in various brain regions was found to be time dependent. Initially, at 5 min, the highest concentration of morphine was found in the hypothalamus and the lowest in the striatum. In cortex and spinal cord, the concentration of morphine was significantly higher in comparison to the other brain regions at 30- and 60-min time points. Co-administration of lower dose of naltrexone (0.625 mg/kg) did not significantly alter the distribution of morphine in brain regions and spinal cord with some exceptions. The higher dose of naltrexone (2.5 mg/kg) increased the concentration of morphine in several brain regions and spinal cord. The ratio of the concentration of morphine in brain region or spinal cord to serum was decreased by naltrexone. It is concluded that naltrexone also alters the distribution of morphine in the central nervous system.

The effect of morphine tolerance-dependence and abstinence on the characteristics of dopamine D 2 receptors in brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with 6 morphine pellets for a 7-day period, each containing 75 mg of morphine free base. Rats implanted with placebo served as controls. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to a challenge dose of morphine. Similarly, the development of physical dependence was evidenced by a decreased in body weight and colonic temperature after morphine pellet removal (withdrawal). The binding characteristics ( B max and K d values) of [ 3 H]spiroperidol to dopamine D 2 receptors were determined in the tissues of morphine-tolerant and morphine-abstinent rats. In the tolerant rats, the pellets were left intact at the time of sacrificing, whereas, in the abstinent rats the pellets were removed 18 h prior to sacrificing. The binding of [ 3 H]spiroperidol was determined in membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups. [ 3 H]Spiroperidol bound to brain regions and spinal cord at a single high affinity site. The B max or the K d values in brain regions and spinal cord of morphine-tolerant and -abstinent rats did not differ from their respective placebo controls. The behavioral responses to a selective dopamine D 2 receptor agonist, 2-bromo-α-ergocryptine were also determined in the morphine-abstinent rats. In morphine-abstinent rats, increased behavioral activity, such as total distance travelled, number of movements, and the number of stereotypic movements was seen as compared to placebo controls. The dose of 2-bromo-α-ergocryptine which by itself had no effect on any type of behavioral activity in placebo-treated rats, increased the total distance travelled, horizontal activity, number of movements, and movement time in morphine-abstinent rats. Although in morphine-tolerant or morphine-abstinent rats, the characteristics of [ 3 H]spiroperidol binding to dopamine D 2 receptors in brain regions and spinal cord were unchanged, the supersensitivity was observed to behavioral responses of 2-bromo-α-ergocryptine, a selective dopamine D 2 receptor agonist. These results provide an evidence for behavioral responses of 2-bromo-α-ergocryptine, a selective up-regulation in morphine abstinent rats. Previously, we have show that dopamine D 1 receptors are unaffected in morphine tolerant rats but are modified in morphine-abstinent rats. Thus, in the morphine abstinent process a significant difference was noted in the biochemical characteristics of dopamine D 1 and D 2 receptors.

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a kappa-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1-13), whereas the heart had the lowest level. The levels of dynorphin (1-13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1-13) were elevated only in the midbrain. The levels of dynorphin (1-13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1-13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1-13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the mu- and kappa-opiate induced tolerance-dependence and abstinence processes.

Previously it was demonstrated that intravenously administered morphine produced greater analgesic but lower hyperthermic responses to morphine in 24-week-old rats in comparison to 8-week-old rats. The differential pharmacological responses to morphine could not solely be attributed to the pharmacokinetic parameters, namely area under the serum morphine concentration-time curve, serum levels of morphine extrapolated to zero time, half-life, mean residence time, apparent volume of distribution at the steady state, terminal rate constant and total body clearance of morphine in serum. In order to determine whether the differences in pharmacological responses to morphine in rats from two age groups are related to differential distribution of morphine in the central nervous system, in the present study, the time course of the distribution of morphine in brain regions (hypothalamus, hippocampus, cortex, pons and medulla, amygdala, midbrain and corpus striatum), spinal cord and serum following intravenous injection of 10 mg/kg dose to 8- and 24-week-old male Sprague-Dawley rats was determined. Morphine injected intravenously produced a greater analgesic but less intense hyperthermic effect in 24-week-old rats in comparison to 8-week-old rats. In most of the brain regions and spinal cord, with few exceptions, the concentration of morphine was found to be greater in 24-week-old rats than in 8-week-old rats. Similarly, the ratio of the concentration of morphine in brain region or spinal cord to serum was significantly higher in rats from the older age group. The studies demonstrate that the altered pharmacological responses to intravenously administered morphine to rats of differing ages may be related to the higher concentration of morphine in the central nervous system of older rats, which in turn may be related to the differences in the blood-brain barrier to morphine in the two age groups.

Phencyclidine hydrochloride (PCP) was tested for its ability to alter a variety of immune effector and regulatory functions in vitro. B 6 C 3 F 1 murine splenic lymphocytes or elicited peritoneal macrophages were cultured in vitro with medium only or medium containing 10 −10 –10 −4 M PCP. Macrophages cultured with or without PCP were stimulated with lipopolysaccharide, and production of interleukin 6 (IL-6) and tumor necrosis factor (TNF) was assessed by bioassay. Cytotoxic T-cell effector function was determined following 5-day lymphocyte co-culture with tumor stimulator cells in the presence of PCP. In addition, the ability of T-lymphocytes to produce specific immunoregulatory cytokines IL-2 and IL-4 in the presence of PCP was quantitated by bioassay. B-lymphocyte function was determined by quantitating lymphocyte proliferation following stimulation with anti-IgM antibody and murine IL-4. Natural immunity was assessed by culturing lymphocytes with or without PCP for 24 h, then quantitating basal and IL-2 augmented natural killer (NK) cell activity. In the absence of effects on cell viability, significant suppression of IL-2 production by T-cells was noted at pharmacologically relevant PCP concentrations (1 μM). In vitro concentrations of 10 μM suppressed the generation of specifically sensitized cytotoxic T-cells. In addition, PCP significantly suppressed both IL-2-augmented NK function as well as B-lymphocyte proliferation. By comparison, macrophage IL-6 production was not affected by any concentration of PCP examined in this study.