Differentiation of beta-adrenoceptors in right atrium, diaphragm and adipose tissue of the rat, using stereoisomers of propranolol, alprenolol, nifenalol and practolol.

2-Diazomorphine-bovine serum albumin (2-DAM-BSA) was prepared by diazotizing p-aminobenzoyl-BSA to morphine. Rabbits immunized with 2-DAM-BSA produced antibodies directed to morphine. A 50 percent reduction in ³H-morphine binding required 4.4 pmol of morphine, and 60, 225, and 350 pmol of normorphine, morphine-3-glucuronide, and codeine, respectively. A radioimmunoassay for brain morphine is described, validated, and used to determine if naloxone alters brain morphine in morphine pelleted mice. The apparent biological half-life of morphine in brain was approximately 52 hours between 24 and 72 hours after pellet implantation, and decreased to 1.25 hours after pellet removal. Naloxone (10 mg/kg) administered 24, 48, or 72 hours after implantation and in doses of 1.0–100 mg/kg administered at 48 hours resulted in either no significant change, or, in a few experiments, increased the brain concentration of morphine. The present experiments could not detect a fraction of total brain morphine that is reduced by naloxone.     

Inhibition of abstinence syndrome in opiate defendent mice by cyclo (His-Pro)

Intracerebral administration of cyclo (His-Pro), the postulated metabolite of thyroliberin (TRH, pGlu-His-Pro-NH₂) inhibited the naloxone induced withdrawal responses in morphine dependent mice. Mice were rendered dependent on morphine by the subcutaneous implantation of a pellet (containing 75 mg of morphine free base) for three days. Six hours after pellet removal, the naloxone ED₅₀ for the jumping response was found to be higher in mice injected with cyclo (His-Pro) compared with that of vehicle controls. Similarly, the hypothermic response observed following 50 μg/kg of naloxone given given 6 h after pellet removal or that seen with 100 μg/kg of naloxone given 24 h after pellet removal from morphine-dependent mice was inhibited by cyclo (His-Pro). Previously, we have shown similar results with TRH on the morphine abstinence syndrome. It appears, therefore, that cyclo (His-Pro) may be the active metabolite of TRH and analogs of cyclo (His-Pro) may be useful in blocking the symptoms of the opiate abstinence syndrome.     

Short-term tolerance to morphine: Effects of indomethacin

Short-term tolerance to opiates has been demonstrated in as little as three hours after priming with a single dose of morphine in naive animals. Tail-flick latency in mice and changes in plasma corticosterone in rats were the indicators tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hrs. subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hrs. prior to estimation of tail-flick latency and ED 50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. The effects of indomethacin, 5 or 10 mg/kg, were examined on both systems. Rats and mice were pretreated with indomethacin at 2.25 or 3 hrs., respectively, before morphine-priming. In all cases, indomethacin did not produce alterations in responses previously observed in correspondently treated controls.     

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.     

Dissociation of morphine withdrawal diarrhea and jumping in mice by the peripherally selective opioid antagonist SR 58002 C

Mice were rendered physically dependent by repeated administration of morphine, 25 mg/kg s.c., 5 times daily for 4 days, and on the 5th day, 2 h after the last morphine dose, they were challenged with a s.c. injection of either naloxone, 25 mg/kg, or the peripherally selective opioid antagonist SR 58002 C (N-methyl levallorphan mesilate), 75 mg/kg. Naloxone provoked jumping and diarrhea in all the animals; mice challenged with SR 58002 C presented no significant jumping but a high frequency of withdrawal diarrhea. When naloxone, 12 mg/kg, or SR 58002 C, 50 mg/kg, were given s.c. in combination with repeated morphine as above, mice which had received naloxone with morphine presented virtually no diarrhea or jumping upon naloxone challenge; those repeatedly treated with morphine plus SR 58002 C were substantially protected from naloxone-precipitated diarrhea, but not jumping. These results further support the remarkable selectivity for peripheral opioid receptors of SR 58002 C, even after repeated high-dose treatment, and are strongly consistent with the primary role of a local intestinal mechanism in the development and expression of opioid withdrawal diarrhea in mice. The in vivo dissociation of central and peripheral components of dependence on morphine is illustrated, apparently for the first time.     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

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.     

Naloxone reversal of morphine elicited hyperactivity

When naloxone is administered during morphine elicited hyperactivity, hyperactivity is reversed and hypoactivity occurs in its place. The present experiment tested the hypothesis that this effect is the result of morphine induced supersensitivity to naloxone. Two groups of hamsters received equivalent pretreatment with 15 mg/kg morphine (Groups M/M and M/S) for three days while a third group received saline (Group S/S). During subsequent testing one group received a morphine injection (Group M/M) while the others received saline (Groups M/S and S/S) before being placed in running wheels for a three hour session. Two hours later half the animals in each group received an injection of 0.4 mg/kg naloxone and half received saline. Naloxone produced hypoactivity in animals running under the influence of morphine (Group M/M), but neither in those with an equivalent history of morphine pre-treatment (Group M/S), nor in saline controls (Group S/S). These results are inconsistent with the hypothesis under test, but congruent with a modified dual-action hypothesis.     

Short-term tolerance to morphine: effects of diazepam, phenobarbital, and amphetamine

Short-term tolerance to morphine, which can be demonstrated in as little as 3 hours after a single administration of the opiate, was examined in animals chronically pretreated with diazepam, phenobarbital, or amphetamine. Tail-flick latency in mice and changes in plasma corticosterone in rats were the parameters tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hours subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hours prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. Chronic treatment with diazepam or amphetamine in either species did not significantly alter short-term tolerance development by either parameter. However, with phenobarbital pretreatment, the plasma corticosterone response was attenuated and short-term tolerance to morphine's analgesic effects did not occur. Further studies in morphine-pelleted mice showed that analgesic tolerance occurred similarly in all groups. This suggests that barbiturates may delay the process.     

Dopamine receptor sensitivity after repeated morphine administrations to rats.

It was studied in rats, if chronic morphine treatment induces a supersensitivity of dopamine receptors in brain. The rats were treated twice daily for 8–11 days with single doses of morphine, increasing from 10 to 20 mg/kg i.p. The experiments were carried out 16–20 hours after the last injection of morphine. After chronic morphine treatment, the potency of apomorphine in lowering the striatal dopamine turnover was increased. On the other hand, apomorphine was not more potent in inducing stereotypies (sniffing, licking, gnawing) after chronic morphine administration than in saline controls. Finally, dopamine activated the adenylate cyclase in striatal homogenates of rats after chronic morphine treatment to a similar extent as in homogenates of control rats. The results suggest that a supersensitivity of dopamine receptors in brain is not necessarily involved in symptoms of an increased dopaminergic activity after chronic morphine application.     

Effects of naloxone-precipitated withdrawal after a single dose of morphine on catecholamine concentrations in guinea-pig brain

The effect of naloxone-precipitated withdrawal after acute morphine was studied on the concentrations of noradrenaline (NA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and on the metabolite/parent amine ratios MHPG/NA, DOPAC/DA and HVA/DA, in eight regions of the guineapig brain. Guinea-pigs were treated with a single dose of morphine sulphate (15 mg/kg s.c.) or saline (control) and 2h later with naloxone hydrochloride (15 mg/kg s.c.) to precipitate withdrawal. The animals were decapitated at 0.5 h or 1 h after naloxone injections and their brains analysed for monoamine concentrations by HPLC-ECD. At 0.5 h after naloxone-precipitated withdrawal NA and MHPG levels, and the MHPG/NA ratio, were increased in the hypothalamus, and the NA levels were increased in the hypothalamus, medulla/pons and cortex 1 h after naloxone. Naloxoneprecipitated withdrawal also produced increased DA metabolism in the cortex, midbrain and medulla 0.5 h later, and in the cortex, hypothalamus and striatum 1 h later. Hence naloxone-precipitated withdrawal from acute morphine treatment produced a complex pattern of increased synthesis and metabolism of NA and DA which varied over time and with the brain region examined.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

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.     

Evidence for sedative effects of low doses of morphine in mice involving receptors insensitive to naloxone

Low doses of morphine (0.30–2.5 mg/kg) decrease in a dose-dependent manner spontaneous climbing behaviour in mice. This effect is not modified by administration of naloxone at doses up to 1.25 mg/kg. These morphine doses do not modify the locomotor activity but, when they are associated with naloxone (0.5 mg/kg), an obvious inhibition occurs. In rats, a hyperactivity follows the akinesia produced by a morphine administration (10 mg/kg). This hyperactivity is changed into a significant hypokinesia when the animals are treated with naloxone (0.05 mg/kg). These results might reveal a dual effect of low doses of morphine, the excitatory effect of morphine being antagonized by naloxone whereas no action on the sedative effect is observed.     

Evidence for an underlying opponent process during morphine elicited hyperactivity in the hamster

Two experiments investigated the effects of naloxone on morphine elicited hyperactivity in the hamster. In Experiment 1, naloxone (0.4 mg/kg) administered two hours after morphine (15 mg/kg) produced sedation in animals running at high rates under the influence of morphine. Saline control animals running at comparable rates were unaffected by naloxone. In Experiment 2, naloxone administered two hours after morphine converted morphine elicited hyperactivity into sedation. These results are discussed in terms of a modified dual-action hypothesis which holds that morphine elicited hyperactivity masks an underlying opponent process.     

Effects of suckling and of a long interval after ovariectomy on hypothalamo-hypophyseal responsiveness to naloxone, morphine and GnRH in beef cows

The response of serum luteinizing hormone (LH) to morphine, naloxone and gonadotropin-releasing hormone (GnRH) in ovariectomized, suckled (n=4) and nonsuckled (n=3) cows was investigated. Six months after ovariectomy and calf removal, the cows were challenged with 1mg, i.v. naloxone/kg body weight and 1 mg i.v. morphine/kg body weight in a crossover design; blood was collected at 15-minute intervals for 7 hours over a 3-day period. To evaluate LH secretion and pituitary responsiveness, 5 mug of GnRH were administered at Hour 6 on Day 1. On Days 2 and 3, naloxone or morphine was administered at Hour 3, followed by GnRH (5 mug/animal) at Hour 6. Mean preinjection LH concentrations (3.6 +/- 0.2 and 4.7 +/- 0.2 ng/ml), LH pulse frequency (0.6 +/- 0.1 and 0.8 +/- 0.1 pulses/hour) and LH pulse amplitude (2.9 +/- 0.5 and 2.9 +/- 0.6 ng/ml) were similar for suckled and nonsuckled cows, respectively. Morphine decreased (P < 0.01) mean serum LH concentrations (pretreatment 4.2 +/- 0.2 vs post-treatment 2.2 +/- 0.2 ng/ml) in both suckled and nonsuckled cows; however, mean serum LH concentrations remained unchanged after naloxone. Nonsuckled cows had a greater (P < 0.001) LH response to GnRH than did suckled cows (area of response curve: 1004 +/- 92 vs 434 +/- 75 arbitrary units). We suggest that opioid receptors are functionally linked to the GnRH secretory system in suckled and nonsuckled cows that had been ovariectomized for a long period of time. However, gonadotropin secretion appears not to be regulated by opioid mechanisms, and suckling inhibits pituitary responsiveness to GnRH in this model.     

Chimeric peptide of Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception.

A synthetic chimeric peptide of Met-enkephalin and FMRFamide (YGGFMKKKFMRFa), based on MERF was synthesized. This peptide was tested for possible antinociceptive effects using the tail flick test in mice. The effect of the chimeric peptide on morphine antinociception and development of tolerance to the antinociceptive action of morphine was also investigated. The chimeric peptide produced significant, dose-dependent antinociception (40, 60 and 90 mg/kg) in the tail flick test. Pretreatment with naloxone (5 mg/kg, IP) significantly attenuated the antinociceptive effect induced by the chimeric peptide (90 mg/kg, IP), indicating involvement of an opioidergic mechanism. In combination experiments with morphine, the antinociceptive dose of the chimeric peptide (60 mg/kg, IP) potentiated morphine (7 mg/kg, IP) antinociception. A low dose of the chimeric peptide (10 mg/kg, IP), that did not produce significant antinociception on its own, also potentiated morphine antinociception. In the tolerance studies, male albino mice received twice daily injections of morphine (20 mg/kg, IP) followed by either saline (0.1 ml) or chimeric peptide (80 mg/kg, IP) for a period of 4 days. A control group received twice daily injections of saline (0.1 ml) for the same period. When tested on Day 5, tolerance to antinociceptive action of morphine (15 mg/kg, IP) was evidenced by decreased response in chronic morphine plus saline treated mice compared to control group. Concurrent administration of chimeric peptide (80 mg/kg, IP) with morphine significantly attenuated the development of tolerance to the antinociceptive action of morphine. The preliminary results of this study demonstrate that peripherally administered chimeric peptide can produce dose dependent, naloxone reversible, antinociception; potentiate morphine antinociception and attenuate morphine tolerance, indicating a possible role of these type of amphiactive sequences in antinociception and its modulation. These chimeric peptides may also prove to be useful tools for further ascertaining the role of FMRFa family of peptides in mechanisms leading to opiate tolerance and dependence.     

Piracetam modifies morphine and related drug-induced elevation of serum corticosterone concentration in rats

Piracetam (2-oxo-l-pyrrolidine acetamide, UCB 6215) or physiological saline solution was injected intravenously to female rats; after 60 min the animals were decapitated and blood was collected. Piracetam in doses of 100, 300 and 600 mg/kg resulted in a progressive suppression of serum corticosterone concentration (Cpd B) as compared to the controls. Morphine (5 and 10 mg/kg), nalorphine (5 and 10 mg/kg) and naloxone (0.5 mg/kg) induced a significant rise of Cpd B 30 min after subcutaneous injection, however, this could be prevented by 300 mg/kg piracetam given intraperitoneally 60 min prior to decapitation. Piracetam was ineffective in reducing the effects of high doses of morphine (20 mg/kg) and nalorphine (20 mg/kg). The drug had no effect on either ether stress or electric footshocks induced activation of the pituitary-adrenocortical system. In vitro the drug had no effect on pituitary ACTH release following exposure to crude hypothalamic extract. It is concluded that the effect of piracetam on the pituitary-adrenocortical axis is mediated through hypothalamic or extrahypothalamic brain structures and influences one of the effects of morphine and related drugs.     

Discriminative effects of morphine administered intracerabrally in the rat

Rats were trained in a two-choice discrete trial avoidance paradigm to discriminate between saline and 3.0 mg/kg of morphine administered S.C. The microinjection of 0.3–3.0 μg of morphine into the lateral ventricle produced discriminative effects equivalent to those of the systemic training dose as measured by responding on the morphine-appropriate choice lever. Discriminative effects equivalent to those of the morphine training dose were not consistently produced by administration of morphine into the periaqueductal gray, lateral septum or dorsomedial thalamus in doses as high as 10 μg. However, the discriminative effects of systematically administered morphine were blocked by 10–30 μg of naloxone administered intracerebrally at all of the brain sites tested. Thus, the primary site at which morphine acts to produce discriminative effects in the rat is central, although the specific brain areas mediating these effects remain unidentified. The actions of naloxone could be the result of diffusion of the drug into the ventricular system or into the systemic circulation.     

Blockade of morphine supersensitivity by an antisense oligodeoxynucleotide targeting the delta opioid receptor (DOR-1)

An antisense oligodeoxynucleotide (ODN) targeting 20 bases of the coding sequence of the cloned delta opioid receptor (DOR-1), a mismatched ODN (different from the antisense ODN at 4 bases) or saline was administered to 3 groups of CD-1 mice implanted with naltrexone pellets (7.5 mg) for 7 days. Morphine supersensitivity (i.e., increased potency as defined by decreased morphine ED₅₀ values) was observed 24 h after pellet removal (day 8) in mice treated with saline or mismatch ODN, but not in antisense ODN treated mice. Antisense ODN alone had no effect on basal nociceptive thresholds or morphine analgesia but reduced the analgesic potency of the delta₂ opioid agonist [D-Ala²]deltorphin II. These data suggest that the delta₂ opioid receptor system participates in the adaptive changes contributing to increased morphine potency following chronic naltrexone treatment.