Inhibition by Z-Pro-D-Leu of development of tolerance to and physical dependence on morphine in mice.

The peptide-Z-Pro-D-Leu, injected daily in mice receiving morphine chronically, was found to prevent development of physical dependence as measured by changes in body temperature and body weight due either to abrupt or to naloxone-induced withdrawal. On the other hand, administration of Z-Pro-D-Leu only on the last day of morphine treatment did not alter the overt signs of withdrawal. Daily administration of Z-Pro-D-Leu was also effective in blocking the development of tolerance to the analgesic and the hypothermic effects of subsequent challenge doses of morphine. However, the peptide treatment did not alter the acute effects of a challenge dose of morphine on either analgesia or body temperature. No effects on memory were noted, as evaluated in a one-trial passive avoidance task. Clinical implications of the use of Z-Pro-D-Leu are discussed.     

Blockage of narcotic-induced dopamine receptor supersensitivity by cyclo(Leu-Gly).

We have previously reported that the administration of cyclo(Leu-Gly) to mice prior to morphinization blocked the development of tolerance to the analgesic effects of morphine as well as the development of some signs of physical dependence. In the present series of experiments, the effect of the same peptide treatment on changes in dopamine receptor sensitivity induced by chronic morphine treatment were determined. Changes in dopamine receptor sensitivity were determined by measuring (i) the effect of the dopamine agonist apomorphine on locomotor activity and (ii) the hypothermic response to another dopamine agonist, piribedil. Mice that had received the chronic morphine treatment were found to require significantly less apomorphine to produce an increase in locomotor activity, and they exhibited a significantly greater hypothermic response to piribedil than did morphine-naive mice. The injection of 0.2 mumol of cyclo(Leu-Gly) per mouse 2 hr prio to morphine treatment prevented this increased response to both dopamine agonists. Administration of the peptide after the tolerance and dependence had developed did not alter morphine tolerant and dependent states states or the enhanced response to apomorphine or piribedil. It is concluded that dopamine receptor supersensitivity may be involved in the development of narcotic tolerance and physical dependence.     

Essential role for RGS9 in opiate action

Regulators of G protein signaling (RGS) are a family of proteins known to accelerate termination of effector stimulation after G protein receptor activation. RGS9-2, a brain-specific splice variant of the RGS9 gene, is highly enriched in striatum and also expressed at much lower levels in periaqueductal gray and spinal cord, structures known to mediate various actions of morphine and other opiates. Morphine exerts its acute rewarding and analgesic effects by activation of inhibitory guanine nucleotide-binding regulatory protein-coupled opioid receptors, whereas chronic morphine causes addiction, tolerance to its acute analgesic effects, and profound physical dependence by sustained activation of these receptors. We show here that acute morphine administration increases expression of RGS9-2 in NAc and the other CNS regions, whereas chronic exposure decreases RGS9-2 levels. Mice lacking RGS9 show enhanced behavioral responses to acute and chronic morphine, including a dramatic increase in morphine reward, increased morphine analgesia with delayed tolerance, and exacerbated morphine physical dependence and withdrawal. These findings establish RGS9 as a potent negative modulator of opiate action in vivo, and suggest that opiate-induced changes in RGS9 levels contribute to the behavioral and neural plasticity associated with chronic opiate administration.     

The effect of opiates on the intestinal immune response to cholera toxin in mice

We studied the effect of opiates on the intestinal immunoglobulin A response in mice. C57BL mice were orally immunized by two doses of 10 micrograms of cholera toxin, 2 weeks apart. Experimental groups received subcutaneous injections of morphine, either 10 or 20 mg/kg/day, in two divided doses. Morphine was given for 4 days, starting 1 day prior to each cholera toxin dose. Intestinal secretions were collected by lavage 1 week after the last cholera toxin dose, and assayed for specific anticholera toxin antibody and total immunoglobulin A. Results were expressed as units of anticholera toxin per nanogram immunoglobulin A. It was found that morphine, 20 mg/kg/day, reduced the response from 30.9 +/- 3.11 to 9.78 +2- 1.42 units/ng (M +/- SEM; p less than 0.0001). 10 mg/kg/day of morphine slightly reduced the immune response to 21.38 +/- 3.51 units/ng (M +/- SEM), but failed to achieve statistical significance. Naloxone administration prior to morphine injections abolished the inhibitory effects of morphine. Morphine administration had no effect on the response to a booster dose of cholera toxin 3 months after the initial cholera toxin immunization and morphine administration. It is concluded that morphine has a significant inhibitory effect on the intestinal immune response, but does not effect long-term mucosal immunological memory. The effect is probably mediated by a specific opiate receptor, as it is blocked by naloxone. This effect may have clinical implications.     

Twenty-four-hour intragastric pH: tolerance within 5 days of continuous ranitidine administration

OBJECTIVE: To investigate further the phenomenon of pharmacological tolerance to H2-receptor antagonists, we undertook a study of the antisecretory effect of ranitidine with continuous daily administration. METHODS: A total of 28 healthy male volunteers were given ranitidine 150 mg q.i.d. for 5 days. Twenty-four-hour intragastric pH monitoring was performed predosing and on days 1 and 5 of ranitidine administration. Serial blood samples were collected on days 1 and 5 of ranitidine administration for pharmacokinetics. RESULTS: Mean 24-h intragastric pH was 2.62 predosing, 4.22 on day 1 of ranitidine administration and 3.28 on day 5 (p = 0.001, ranitidine day 1 vs. day 5). Intragastric pH was >3, 4, and 5 for 69.9%, 54.3%, and 35.9%, respectively, on day 1 of ranitidine administration and was 45.8%, 30.1%, and 20.8% on day 5 (p<0.005 for each comparison). Subjects' Helicobacter pylori status did not affect the antisecretory effect of ranitidine. There was no alteration in ranitidine pharmacokinetics to account for its reduced antisecretory effect. CONCLUSION: This study has demonstrated a statistically significant reduction in the antisecretory effect of ranitidine within 5 days of continuous administration which is not explained by altered ranitidine pharmacokinetics. This is further evidence for the development of a form of pharmacological tolerance to an H2-receptor antagonist within a few days of continuous daily dosing.     

Prolonged morphine treatment relieves thermal hyperalgesia in rats with sciatic nerve constriction injury

We examined the effects of chronic morphine treatment with regular intermittent administration in a modified chronic constriction injury (CCI) model of the rat sciatic nerve originally introduced by Bennett and Xie. A painful neuropathy was induced over the left hind limb with sciatic nerve ligation, and sham surgery was done on the opposite side in male Sprague-Dawley rats. Paw-with-drawal latency (PWL) was obtained one day before surgery (pre-op baseline) and on the fourth day after surgery (post-op) to assure the development of thermal hyperalgesia. Morphine hydrochloride (5, 10, 15, and 20 mg/kg per day) was subcutaneously administered for 7 days to four experimental groups. The control group received normal saline rather than morphine under the same injection protocol. PWLs were evaluated on days 5, 7, 9, and 11 of the treatment. PWL decreased to 50-60% of the pre-op baseline or sham limb on the fourth day after surgery. Morphine's ability to reverse PWL appeared dose-related, and no tolerance developed during treatment with chronic intermittent administration. This may indicate that prolonged use of intermittently-administered morphine can be a feasible regimen for relief of neuropathic pain.     

Acute thyroid hormone withdrawal rapidly increases the thyrotropin beta and alpha-subunit messenger ribonucleic acids in mouse thyrotropic tumors

We have abruptly discontinued T3 administration to hypothyroid mice bearing thyrotropic tumors and measured increases in tumor steady state TSH subunit mRNA levels with time. Hypothyroid mice bearing the thyrotropic tumor TtT97 were injected daily with T3 (10 micrograms/100 g BW, ip, daily) for 10 days. Groups of mice (n = 3) were killed on the day of the last T3 injection (day 0) and 1, 2, 3, or 5 days after stopping T3 treatment. Plasma T3 concentrations fell to subnormal values between days 1 and 2 after stopping T3 treatment. Plasma TSH and total TSH subunit concentrations were 3% of untreated hypothyroid control concentrations on day 0, and rose 4-fold between days 1 and 2 and 20-fold by day 5 (P less than 0.01). Plasma total alpha-subunit concentrations were 28% of untreated hypothyroid control concentrations on day 0, rose to 158% of baseline values by day 2, and rose 3-fold by day 5 (P less than 0.001). Tumor TSH beta and alpha-subunit mRNA levels were 5% and 52% (P less than 0.01) of hypothyroid control levels on day 0. TSH beta mRNA levels rose nearly 9-fold between days 1 and 2 (P less than 0.01). alpha-Subunit mRNA levels rose to 135% of initial values by day 2 and to 144% of initial values by day 3 (P less than 0.05). Changes in tumor TSH subunit mRNA levels paralleled changes in plasma subunit glycoprotein concentrations. Increases in tumor subunit mRNA levels after abruptly stopping T3 treatment occurred rapidly, predominantly between 24-48 h after stopping T3, and TSH beta mRNA was considerably more responsive than alpha-subunit mRNA.     

Defective tolerance to the toxic and metabolic effects of interleukin 1

The effect on food intake, body weight, and survival of mice given recombinant lipopolysaccharide (LPS), tumor necrosis factor/cachectin (TNF), or interleukin 1 (IL-1) (5 micrograms/mouse, ip, twice daily) was studied. All agents induced a rapid reduction of food intake and body weight after 1 day of treatment. Unlike TNF and LPS, IL-1 given as two daily administrations of 5 micrograms was lethal within 3 days. Mice treated with LPS or TNF rapidly developed tolerance to their anorectic effect, whereas tolerance to IL-1 required a longer time to develop and was not complete. We investigated the possible roles of changes in serum corticosterone and glucose in the effects of LPS, TNF and IL-1. A single injection of LPS, TNF, or IL-1 markedly increased serum corticosterone levels after 2 h. After only 2 days of chronic treatment, mice given LPS or TNF were refractory to induction of serum corticosterone by a subsequent injection of LPS or TNF, but mice given IL-1 for 2 days were still fully responsive to IL-1. IL-1, unlike TNF and LPS, induced a marked hypoglycemic response. Repeated administration of IL-1 sensitized to its hypoglycemic effect. This lack of adaptation to the increase of serum corticosterone and hypoglycemia was also observed when IL-1 was given at lower, nonlethal doses (0.25-1.0 microgram) and for a longer period (up to 8 days). The defective tolerance to the metabolic and toxic effects of IL-1 in this experimental model indicates that there are major differences between the in vivo biological responses to IL-1 and TNF.     

Morphine analgesia in mice of different ages.

The analgesic response to 5 mg per kg morphine sulfate was examined in male C57BL/6J mice aged from 2 months to 28 months by the tail-flick assay. The baseline latency of old mice was slightly lower than that of young mice. There was a smaller increase in latency after morphine in old mice than in young mice. Decreased analgesic response in old mice was accompanied by a slower rate of uptake of labeled morphine into the blood after i.p. injection and a longer half-life of the drug. There was no difference between young and old mice in the number of narcotic receptors in the brain.     

Central serotonergic neurons are differentially required for opioid analgesia but not for morphine tolerance or morphine reward

Opioids remain the most effective analgesics despite their potential adverse effects such as tolerance and addiction. Mechanisms underlying these opiate-mediated processes remain the subject of much debate. Here we describe opioid-induced behaviors of Lmx1b conditional knockout mice (Lmx1bf/f/p), which lack central serotonergic neurons, and we report that opioid analgesia is differentially dependent on the central serotonergic system. Analgesia induced by a kappa opioid receptor agonist administered at the supraspinal level was abolished in Lmx1bf/f/p mice compared with their wild-type littermates. Furthermore, compared with their wild-type littermates Lmx1bf/f/p mice exhibited significantly reduced analgesic effects of mu and delta opioid receptor agonists at both spinal and supraspinal sites. In contrast to the attenuation in opioid analgesia, Lmx1bf/f/p mice developed tolerance to morphine analgesia and displayed normal morphine reward behavior as measured by conditioned place preference. Our results provide genetic evidence supporting the view that the central serotonergic system is a key component of supraspinal pain modulatory circuitry mediating opioid analgesia. Furthermore, our data suggest that the mechanisms of morphine tolerance and morphine reward are independent of the central serotonergic system.     

Long-term sensitization to the activation of cerebral delta-opioid receptors by the deltorphin Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 in rats exposed to morphine.

In experiments to evaluate responses to the activation of cerebral delta-opioid receptors, repeated daily injection of the selective delta-opioid agonist Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 ([D-Ala2]deltorphin II) into rat brain resulted in the development of tolerance, whereas repeated daily injection or continuous infusion of morphine resulted in sensitization to the behavioral activating effects of the delta-opioid agonist. Although the rats did not modify their spontaneous locomotor activity after morphine withdrawal, they became markedly hyperresponsive to the locomotor and stereotypy-producing effects of a challenge dose of the delta-opioid agonist. Sensitization to activation of delta-opioid receptors persisted for at least 60 days after discontinuing morphine treatment. These results show that the development of tolerance and long-term sensitization to opioids involves delta-opioid as well as mu-opioid receptors.     

口服抗痛粉的镇痛作用研究

目的证明口服抗痛粉是否有强镇痛效应。方法口服抗痛粉，用辐射热甩尾法测定大鼠痛阈。结果口服30、60、120mg／kg三种剂量的抗痛粉产生明显的剂量镇痛效应关系。60、120mg／kg抗痛粉的镇痛效应与2mg／kg吗啡的镇痛效应近似。结论口服抗痛粉可以和外周吗啡受体结合产生镇痛作用，60、120mg／kg浓度的抗痛粉所产生的强镇痛效应在90min内几乎与吗啡2mg／kg的相同。     

In vivo activation of a mutant mu-opioid receptor by antagonist: future direction for opiate pain treatment paradigm that lacks undesirable side effects

Based on the in vitro ability of opioid antagonists to activate a mu-opioid receptor mutant, S196A, we reasoned that when expressed in the appropriate sites in vivo, this mutant receptor could be used to elicit the analgesic effects of the opioids without the accompanying side effects, such as tolerance and dependence. To test this hypothesis, we introduced the S196A mutation into the mouse mu-opioid receptor by a knock-in strategy to test the ability of the opioid antagonist to produce analgesic effects. In these homozygous mice, we observed increased intrinsic efficacy of opioid analgesics with two antinociceptive tests: hot water tail-withdrawal and acetic acid-induced writhing tests. Opioid antagonists, such as naloxone and naltrexone, elicited antinociceptive effects similar to that of partial agonists. Most importantly, chronic treatment of the homozygous mice with naltrexone did not produce the expected tolerance response, whereas less physical dependence was observed than with chronic morphine treatment. Such in vivo properties suggest the possibility of using the S196A mutant of the mu-opioid receptor and opioid antagonists to minimize the spectrum of unwarranted side effects in pain management when opiate analgesics are used.     

Tolerance to Ethanol Hypothermia in Inbred Mice: Genotypic Correlations with Behavioral Responses

Hypothermia was studied 5 min before, and 30 and 60 min after intraperitoneal administration of ethanol (3 g/kg) in 20 inbred strains of mice. Ethanol was given daily for 8 days, and temperatures were taken on Days 1, 3, 5, and 8. Tolerance was indexed by the reduction in hypothermia over days. There were large strain differences in baseline temperature, the hypothermic effect of ethanol, and in development of tolerance to hypothermia. Some strains of mice (DBA/1J, DBA/2N, MA/MyJ, and PL/J) did not develop tolerance to the hypothermic effect of ethanol. Initial sensitivity to the hypothermic effect of ethanol was significantly genetically correlated with tolerance development, indicating control of these responses by common genes. Ethanol-induced changes in activity and ataxia, as well as blood ethanol concentrations, were also assessed. Although there were significant strain differences in activity reduction, ataxia, blood-ethanol concentrations, and changes in these parameters during the course of chronic treatment, none of these variables could explain the genetic differences in hypothermic sensitivity and tolerance.     

Multiple opiate receptors: [3H]ethylketocyclazocine receptor binding and ketocyclazocine analgesia.

The receptor binding of the kappa agonist [3H]ethylketocyclazocine to brain homogenates in vitro and ketocyclazocine (kappa) analgesia in vivo has been investigated and compared to morphine, a mu agonist. Saturation analysis of [3H]ethylketocyclazocine binding in both mice and rats yielded biphasic Scatchard plots similar to those of opiate mu agonists, antagonists, enkephalins, and endorphins. Treatment of brain membranes with monovalent and divalent cation, chelating agents, protein-modifying reagents, and enzymes affected [3H]ethylketocyclazocine binding in a manner similar to that of [3H]morphine. Naloxazone, a long-acting antagonist that selectively abolished high-affinity [3H-DAla2,Met5]enkephalinamide binding in vivo, also selectively blocked high-affinity [3H]ethylketocyclazocine binding. Evaluation of analgesia with writhing and tail-flick assays in animals whose high-affinity binding sites were blocked by naloxazone demonstrated a 6- to 7-fold increase in median effective dose (ED50) values of ketocyclazocine. This decrease in analgesic potency was comparable to morphine's decreased potency in similarly treated mice. These biochemical and pharmacological results suggest that the analgesic properties of both kappa and mu agonists may be mediated through the same subpopulation of receptors, the high-affinity binding sites.     

Paradoxical LH and prolactin responses to naloxone after chronic treatment with morphine

A series of experiments was conducted in adult male rats to study the development of tolerance to and dependence on morphine in the neural processes controlling LH and prolactin secretion. The central mechanisms controlling both these hormones became tolerant following chronic application of the opiate agonist; this was seen in the form of diminished responsiveness to the agonist with time. There was an apparently greater degree of tolerance in the mechanisms regulating LH secretion than in those regulating prolactin secretion. In parallel experiments, the opiate antagonist naloxone was used to test for the development of dependence in rats chronically treated with morphine. While behavioural signs of physical dependence (withdrawal) were evident, the LH and prolactin responses proved to be the same as those observed in response to acute administration of opiate agonists (i.e. naloxone respectively decreased and increased serum LH and prolactin concentrations in animals chronically treated with morphine). This paper may represent the first report of such paradoxical responses to naloxone. It also demonstrates that opioid tolerance and dependence may exist as two separate phenomena in vivo.     

Melatonin enhances the rewarding properties of morphine: involvement of the nitric oxidergic pathway

Melatonin has different interactions with opioids including the enhancement of the analgesic effects of morphine and also reversal of tolerance and dependence to morphine. The present study assessed the effect of melatonin on morphine reward in mice using a conditioned place preference (CPP) paradigm. Our data showed that subcutaneous administration of morphine (1-7.5 mg/kg) significantly increased the time spent in the drug-paired compartment in a dose-dependent manner. Intraperitoneal (i.p.) administration of melatonin (1-40 mg/kg) alone did not induce either CPP or conditioned place aversion (CPA), while the combination of melatonin (5-20 mg/kg) and sub-effective dose of morphine (0.5 mg/kg) led to rewarding effect. We further investigated the involvement of the nitric oxidergic pathway in the enhancing effect of melatonin on morphine CPP, by a general nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). L-NAME (1 and 5 mg/kg, i.p.) alone or in combination with morphine (0.5 mg/kg) did not show any significant CPP or CPA. Co-administration of L-NAME (5 mg/kg) with an ineffective combination of melatonin (1 mg/kg) plus morphine (0.5 mg/kg) produced significant CPP that may imply the similarity of action of melatonin and L-NAME and involvement of the nitric oxidergic pathway in this regard. Our results indicate that pretreatment of animals with melatonin enhances the rewarding properties of morphine via a mechanism which may involve the nitric oxidergic pathway.     

beta-Endorphin: cross tolerance to and cross physical dependence on morphine.

The effects of beta-endorphin on the antinociceptive responses and abrupt withdrawal jumping in morphine-dependent mice were studied. Mice were rendered morphine dependent by implantation of a morphine pellet (75 mg base) for 3 days. The analgesic response to beta-endorphin decreased after morphine pellet implantation, as evidenced by an eight-fold increase in the median antinociceptive dose of morphine was found. In small doses (0.09-0.17 mug per mouse), beta-endorphin suppressed abrupt withdrawal jumping. Met-enkephalin, even in high doses (200 mug per mouse), did not suppress abrupt withdrawal jumping.     

Chronic continuous nicotine exposure during periadolescence does not increase ethanol intake during adulthood in rats

BACKGROUND: Nicotine has been considered as the gateway drug, because many teenagers experience cigarette smoking before seeking out other drugs. By using an animal model system, we assessed the effects of chronic continuous nicotine exposure during periadolescence on ethanol intake during young adulthood. METHODS: Periadolescent Sprague-Dawley rats (35 days old) were used at the beginning of this study. These animals received subcutaneous implantation of nicotine pellets (15 or 25 mg in 21-day time-release pellets) or placebo pellets (0 mg of nicotine) on postnatal day 35. Beginning on postnatal day 53, the animals received various concentrations of ethanol solution during their active period (5:00 PM to 9:00 AM) starting with 2% (v/v, 4 days), then 5% (5 days), 8% (6 days), and 10% (6 days). Between 9:00 AM and 5:00 PM of the same day, the ethanol solution was replaced by regular tap water. The amounts of ethanol solution and regular water were measured daily. RESULTS: The analyses showed that ethanol intake (grams per kilogram of body weight) in the nicotine 15 and 25 mg groups did not differ from that in the nicotine 0 mg group, and no sex difference was found in ethanol intake. However, ethanol intake was increased as a function of the treatment days. CONCLUSIONS: The results showed that chronic continuous nicotine exposure during adolescence did not increase ethanol intake in rats during young adulthood.     

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Abstract

Calmodulin levels were determined in brain areas (cerebellum, cortex, diencephalon, medulla + pons and striatum) of immature, mature and old mice one hour after they were injected with morphine (10 mg/kg, s.c.) and 3 days after morphine pellet (75 mg) implantation (during tolerance). Acutely, morphine caused significant (two-fold) increases in calmodulin in striatal regions from immature and old mice, and increased calmodulin after morphine administration to mature mice occurred only in the cerebellum. After tolerance to morphine had developed, calmodulin levels in all brain regions were the same as those from placebo-implanted mice.