Immunosuppressive effects of chronic morphine treatment in mice

In this report we describe the immunomodulatory effects of subcutaneous morphine pellets in mice, a model commonly used in the study of opiate tolerance and dependence. Mice given a single 75 mg morphine pellet displayed marked atrophy and reduced cellularity of the spleen and thymus, and an attenuated lymphocyte proliferative response to T- and B-cell mitogens (concanavalin A and bacterial lipopolysaccharide, respectively). These immunosuppressive effects were observed 72 hr following implantation of the pellet, a time point by which the mice also had developed tolerance to the antinociceptive effect of the pellet. Splenic and thymic atrophy with reduced mitogen-induced lymphocyte proliferative responses and opiate tolerance were also apparent in mice subjected to a multiple pellet implantation schedule. However, implantation of a pellet containing 37.5 mg morphine did not suppress mitogen-stimulated lymphocyte proliferation, which was slightly elevated in this group. These findings concur with other observations suggesting immunosuppression with morphine tolerance. Furthermore, we suggest that chronic morphine treatment acts as a pharmacologic stressor that mimics behavioral stress.     

Effects of hypophysectomy and ACTH on opiate tolerance and physical dependence.

In opiate-naive hypophysectomized rats, the enhanced potency of parenterally injected morphine was almost completely normalized by chronic ACTH in doses that maintained adrenal weight. Following morphine pellet implantation, the amount of morphine required for antinociception, catalepsy, and colonic temperature effects in tolerant rats was identical among hypophysectomized and sham-operated rats whether or not chronic ACTH was administered. However, brain levels of morphine were greater in tolerant, hypophysectomized rats, an effect blocked by chronic ACTH. Hypophysectomy thus appears to enhance the magnitude of tolerance development by a mechanism that is reversed by doses of ACTH that maintain adrenal weight. On the other hand, in sham-control rats this dose and schedule of ACTH were without effect on morphine potency in opiate-naive or tolerant rats or on brain levels of morphine. In general, hypophysectomy and/or ACTH did not modify the intensity of abstinence signs following naloxone-precipitated withdrawal. Therefore, little evidence was obtained to suggest a direct, commanding role of the pituitary in chronic opiate effects. Instead, a secondary adrenal involvement may be important.     

Reduced tolerance to morphine thermoregulatory effects in senescent rats

Age-related differences in the thermoregulatory response to morphine have been shown in rats. To determine if these age-related differences would be reflected in the acquisition of tolerance, we studied morphine tolerance induced by either a single morphine dose or implantation of a morphine pellet. precipitated withdrawal was also analyzed by inducing withdrawal with naloxone in morphine-pelleted rats. Senescent (26 or 27 month old), mature (10 or 11 month old) and young (3 or 4 month old) male Fischer 344 rats were restrained and changes in rectal temperature were monitored for six hours after morphine administration. Only mature and young rats exhibited increased hyperthermic responses to a second low dose of morphine (5 mg/kg s.c.). Only young rats became tolerant after a single higher morphine dose (25 mg/kg s.c.). All age groups showed tolerance three days after morphine pellet implantation. Hypothermia was equivalent in all age groups when withdrawal was induced by naloxone in morphine-pelleted rats. These results indicate that older rats were more resistant to the acquisition of tolerance to the thermic effects of morphine; however; with continued morphine treatment, rats became tolerant regardless of age.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Effects of Electroconvulsive Shock on Tetrahydrobiopterin and GTP-Cyclohydrolase Activity in the Brain and Adrenal Gland of the Rat

The effects of a single and repeated electroconvulsive shock (ECS) (300 mA, 0.2 s) on tetrahydrobiopterin (BH4) levels and GTP-cyclohydrolase activity in the brain and adrenal glands of rats were examined. Twenty-four hours after the last ECS treatment (one/day for 7 days), biopterin levels were significantly elevated in the locus coeruleus, hippocampus, frontal cortex, hypothalamus, ventral tegmental area, and adrenal gland. There were no changes in biopterin levels after a single application of ECS. GTP-cyclohydrolase activity was significantly increased in the locus coeruleus, frontal cortex, hippocampus, hypothalamus, and adrenal gland 24 h after repeated ECS and remained elevated in certain tissues up to 8 days after the last treatment. Kinetic analysis of adrenal and locus coeruleus GTP-cyclohydrolase 1 day after 7 days of ECS showed significant changes in both Km and Vmax values. These data suggest that the long-term increases in BH4 levels and GTP-cyclohydrolase activity after repeated ECS may play a part in the mediation of the antidepressant effects of ECS.     

Comparison of the effects of specific and nonspecific inhibition of nitric oxide synthase on morphine analgesia,tolerance and dependence in mice

The effects of L-Canavanine, a selective inducible nitric oxide synthase (NOS) inhibitor and N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, on pain threshold and morphine induced analgesia, tolerance and dependence in mice were investigated and compared. Morphine was administered by subcutaneous implantation of a pellet containing 40 mg free base and withdrawal was precipitated by intraperitoneal (i.p.) injection of naloxone (2 mg/kg). L-Canavanine (200 mg/kg, i.p.) did not affect the pain threshold, morphine-induced analgesia and the induction and expression phases of morphine tolerance and dependence. L-NAME (20 mg/kg, i.p.) significantly (p < 0.05) enhanced the pain threshold, potentiated morphine-induced analgesia and attenuated the expression phase of morphine dependence which has been characterized by withdrawal signs and body weight loss, but did not modify the induction phase of morphine tolerance and dependence. It is concluded that constitutive NOS isoforms which were inhibited by L-NAME may be involved specifically in the mechanisms of morphine induced analgesia, tolerance and dependence.     

Reinstatement of precipitated narcotic withdrawal hypothermia in the rat

Withdrawal hypothermia can be induced in rats by injection of naltrexone 72 h after subcutaneous implantation of a morphine pellet. At 45 days after implantation the same dose of naltrexone is without effect on body temperature and the animals are normally sensitive to the hypothermic effect of acute morphine administration. This acute administration of morphine re-sensitizes the animals to naltrexone so that administration of the antagonist again causes withdrawal hypothermia. These results are consistent with the view that narcotic dependence can be reinstated in previously dependent, but not naive, animals by acute administration of the narcotic.     

Ethanol suppression of naloxone-induced withdrawal in morphine-dependent rats

The technique of morphine pellet implantation was used to produce physical dependence on morphine in male rats. The number of “wet dog” shakes occurring within a period of 30 minutes during naloxone-precipitated (1.0 mg/kg, s.c.) withdrawal in four-day morphine implanted rats was determined after either acute or chronic treatment with ethanol. An acute dose of ethanol administered prior to withdrawal had no significant effect on the withdrawal response whereas chronic administration of ethanol during the development of dependence on morphine significantly suppressed the naloxone-precipitated withdrawal response to 44–57 percent of the control response. Analysis of brain and plasma for morphine concentration four days following dependence development showed no significant differences between morphine controls and those animals treated with both morphine and ethanol. Pentobarbital, another central nervous system depressant, demonstrated no effect on the withdrawal response, whether administered acutely or chronically during the development of dependence.     

Dissociation of tolerance to the analgesic and hypothermic effects of morphine by using thyrotropin releasing hormone

The effects of thyrotropin releasing hormone (TRH) on tolerance to the analgesic and hypothermic effects of morphine were determined in male Swiss Webster mice. The tolerance to morphine was induced by SC implantation of a morphine pellet containing 75 mg morphine free base for 3 days. Subcutaneous injections of TRH (4 mg/kg) twice a day inhibited tolerance to the analgesic effect of morphine, as evidenced by a greater degree of analgesia in TRH treated mice as compared with similarly treated vehicle injected controls. The same treatment, however, failed to modify tolerance to the hypothermic effect of morphine. These effects were produced with alterations in brain or plasma levels of morphine. It is concluded that tolerance to the two pharmacological effects of morphine may involve separate mechanism.     

Inability of cyclo (Leu-Gly) to facilitate the development of tolerance to and physical dependence on morphine in the rat

The effect of cyclo (Leu-Gly), an analog of melanotropin release inhibition factor on the development of tolerance to and physical dependence on morphine in the rat was investigated. Administration of cyclo (Leu-Gly) (1 μg/rat/day) prior to and during morphine pellet implantation failed to facilitate the development of tolerance to the analgesic and hypothermic effects of morphine. Similarly the development of dependence on morphine was not facilitated by cyclo (Leu-Gly) as evidenced by changes in body weight and body temperature observed during abrupt withdrawal of morphine. These studies do not lend support to the previous observations that cyclo (Leu-Gly) and other related peptides facilitate the development of tolerance to and physical dependence on morphine.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

Serum IL-10 involved in morphine tolerance development during adjuvant-induced arthritis

Opioid receptors play an important role in modulation of hyperalgesia in inflamed tissues, but chronic morphine application induces such side effects as tolerance. There is near communications between cytokines and mu opioid receptor expression. This study was aimed to assess the role of serum IL-10 in morphine tolerance development during adjuvant-induced arthritis. Adjuvant arthritis (AA) was induced on day 0 by single injection of Complete Freund’s Adjuvant (CFA) into the rats’ hindpaw. Hyperalgesia, edema, and spinal mu opioid receptor (mOR) variations were assessed on 0, 7, 14, and 21 days of the study. For assessment of the morphine tolerance development, morphine effective dose (4 mg/kg) was administered from the 14th day after CFA injection and continued until the morphine post-dose paw withdrawal latency (PWL); it did not significantly differ from the baseline. For assessment of the effects of IL-10 on tolerance induction, a neutralizing dose (ND50) of anti-IL-10 was administered daily during different stages of the study. AA induction in the right hindpaw of rats resulted in unilateral inflammation and hyperalgesia within 21 days of the study. Anti-IL-10 antibody administration in the AA rats induced marked elevation of hyperalgesia compared to the AA control group. Our data also indicated that morphine effective anti-hyperalgesic dose significantly decreased in the AA rats compared to the control group, which this symptom was aligned with spinal mu opioid receptor (mOR) expression increase during AA. Moreover, there was a significant difference in morphine tolerance induction between the AA and control rats, and our results also demonstrated that IL-10 played an important role in tolerance-induction process. It can be concluded that morphine tolerance slowly progressed when administered morphine effective dose was reduced during AA chronic inflammation. On the other hand, it seems that increased level of serum IL-10 may affect morphine tolerance development during inflammation.     

Attenuation of morphine analgesia by serum from morphine-treated rabbits.

We have previously shown that repeated s.c. implantation of rabbits with pellets containing morphine or certain other narcotics evokes production of a serum morphine-binding component that has the characteristics of an immunoglobulin. We have, in the study reported here, given rabbit anti-morphine pellet serum (anti-MP) to rats i.v. at a dose equal to 20% of their blood volume. Effects on morphine analgesia as measured by the hot plate were studied following a 5 mg/kg dose of morphine. The anti-MP serum and an antiserum to a morphine-protein conjugate both significantly attenuated the analgesic response to morphine on the first day after serum treatment when compared to results in rats given control rabbit serum. Subsequent testing of the same rats (on Days 2, 9, and 48) with no additional serum treatment showed no differences in response.     

Ethanol-induced hypothermia. Cross tolerance with morphine

The development of tolerance to ethanol-induced hypothermia and hypnosis, and cross-tolerance with morphine was studied in mice and rats. Ethanol significantly decreased the body temperature in rats (3.0 and 3.2 g/kg) and in mice (3.5 and 4.0 g/kg). Chronic administration of ethanol resulted in the tolerance not only to ethanol hypothermia but also to hypothermic effects of morphine in examined animals. Implantation of morphine pellets caused the development of cross tolerance to ethanol-induced hypothermia in rats but not in mice. The hypnotic effect of ethanol was significantly shorter in chronic alcoholized rats but not in morphine-implanted rats. Neither chronic ethanol administration nor implantation of morphine pellets changed the duration of ethanol-induced hypnosis in mice. These results seem to support the hypothesis on the opiate-like mechanism of ethanol action.     

Cross tolerance to etorphine in rats tolerant to morphine-induced antidiuresis

Previously in the analgesic tail flick assay, mice and rats implanted with morphine pellets were shown to be highly tolerant to subcutaneously administered morphine but not to etorphine. The present purpose was to see whether the same differential response would be found to the antidiuretic response of morphine and etorphine in water-loaded rats because the presence of such a differential response would be of value in studying mechanisms of tolerance. Etorphine injected subcutaneously was about 1000x more potent than morphine in producing an antidiuretic response. Following chronic administration of morphine by pellet implantation, where the pellets remained in place during the drug challenge, profound tolerance developed to the antidiuretic effect of both morphine and etorphine. The dose-response curves for both were shifted to the right in non-parallel fashion with decreased slopes and antidiuretic efficacies. The large degree of tolerance developed to the antidiuretic effect of etorphine in morphine pellet implanted rats in contrast to the lack of development of tolerance to etorphine in the tail flick assay indicated that different mechanisms of development of tolerance exist for the two responses.     

Chronic morphine treatment increases the number, but decreases the affinity of [3H]-U69,593 binding sites in the rat heart.

In the present study, the effects of chronic morphine treatment on the binding properties of tritiated U69,593, a specific kappa-ligand, in the rat heart were determined. Adult rats were given morphine through osmotic pumps at a rate of 80 micrograms/hour supplemented by daily injection of morphine at increasing doses for 9 days. The increase in colonic temperature to morphine, used as an indicator of development of tolerance in the rat, was measured daily. It was shown that, on day 7 following morphine treatment, the rats developed tolerance to morphine as indicated by an attenuated hyperthermic response to morphine. The [3H]-U69,593 specific binding properties were determined by direct receptor binding assay. The binding sites increased gradually and reached a significantly higher level at day 10. Scatchard analysis showed that both Bmax and Kd increased at day 10 following morphine treatment, indicating an increase in number of sites and a reduction in affinity to the kappa-ligand. Acute morphine injection at a dose of 10 mg/kg did not cause any significant alteration of [3H]-U69,593 binding sites. Two days after withdrawal of morphine, the [3H]-U69,593 binding sites returned to the original level. The finding of the present study indicates that there is an up-regulation of number, but a reduction in affinity of kappa-binding sites following chronic morphine treatment.     

Morphine withdrawal modifies antinociceptive effects of acute morphine in rats

Repeated opioid use is known to cause tolerance of antinociceptive effects. Whether opioid abstinence modifies antinociceptive effects is unknown. Here we reported that morphine withdrawal for 18 h and 4 days after repeated morphine treatment largely reduced tail-flick latencies compared with control, while the rats showed severe withdrawal syndromes. However, the latencies and withdrawal syndromes were restored to control level at 20 days withdrawal. Similarly, antinociceptive effects of acute morphine were decreased at 18 h and further decreased at 4 days but restored to control level at 20 days withdrawal. Behavioral stress that was given to the rats at 18 h withdrawal further reduced tail-flick latencies and antinociceptive effects. Conversely, the glucocorticoid receptor antagonist RU38486 increased tail-flick latencies and antinociceptive effects at 4 days withdrawal. These results suggest that morphine withdrawal could evoke behavioral stress to modify antinociceptive effects, implicating a significant influence of opioid abstinence on chronic pain treatment.     

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats

Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4-7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.     

Differential analgesic cross-tolerance to morphine between lipophilic and hydrophilic narcotic agonists

Mice were rendered tolerant to morphine by the subcutaneous implantation of one 75 mg morphine pellet. Seventy-two hours post-pellet implantation, the animals were evaluated in the tail-flick assay for analgestic tolerance and cross-tolerance to subcutaneously administered morphine, normorphine, methadone, etorphine and intracerebroventricularly administered morphine. With the pellet remaining in situ during testing, there was the expected analgestic tolerance to peripherally administered morphine and analgesic cross-tolerance to normorphine. However, with the pellet in situ during testing, there was a surprising lack of analgesic tolerance to intracerebroventricularly administered etorphine or methadone. In contrast, removal of the morphine pellet 3 hours prior to the analgesic evaluation apparently unmasked the expression of tolerance and cross-tolerance as evidenced by a three fold, parallel shift to the right of the analgesic dose-response curve for subcutaneously administered etorphine and methadone and a seven fold shift for intracerebroventricularly administered morphine. These data emphasize that a more rigorous evaluation of tolerance development methodologies need be explored and support the suggestion that removal of the morphine-inducing pellet prior to analgesic determinations results in a distinct state of “tolerance” quite different from that observed with the pellet remaining in situ during testing.     

Intragastric administration of cyclo(Leu-Gly) inhibits the development of tolerance to the analgesic effect of morphine in the rat

The effect of intragastric administration of cyclo(Leu-Gly), a cyclic dipeptide derived from melanotropin release inhibiting factor (Pro-Leu-Gly-NH2), on the development of tolerance to the analgesic effect of morphine in the rat was determined. The tolerance to morphine in the rat was induced by subcutaneous implantation of four morphine pellets during a 3-day period. The rats which served as controls were implanted with placebo pellets. The analgesic response to a challenge dose of morphine was determined by the tail-flick test. The tail-flick latencies were determined before and then every 30 min for 180 min. The analgesic response was computed by determining the area under the time-response curve. Implantation of morphine pellets resulted in the development of tolerance as evidenced by decreased analgesic response to morphine in morphine pellet implanted rats as compared to placebo pellet implanted rats. Chronic intragastric administration of cyclo(Leu-Gly) (4 to 16 mg/kg) inhibited the development of tolerance to morphine. A dose of 8 mg/kg of cyclo(Leu-Gly) completely blocked the tolerance to morphine. The study provides for the first time evidence that intragastric administration of a cyclic peptide can inhibit the development of tolerance to morphine, and that effective neuropeptides and their analogs can be developed as potential drugs to inhibit opiate-induced tolerance.