Studies on the physical dependence liability of analgesics. 5th report: Electron microscopic studies on the ultrastructural transformation of mitochondria in the cells of zona fasciculata of the adrenal cortex in morphine pellet implanted mice (author's transl)

Influence of morphine on the fine structure of mitochondria in the cells of zona fasciculata of the adrenal cortex in mice in which a morphine pellet was subcutaneously implanted was studied using electron microscope. (1) Transformation of the mitochondrial structure, i.e. swelling, formation of lamellar or concentric circular cristae and decrease of electron density of the mitochondrial matrix, was observed 12 hr after implantation, and changes reached a maximum state at 48 hr. These transformations, however, disappeared wihtin 4 days. (2) On the 4th day after implantation, removal the pellet again produced transformation of the mitochondria with withdrawal symptoms, and a normal state was reverted to 72 hr later. Levallorphan challenge produced similar changes. (3) Injection of morphine into mice after removal of the pellet prevented the appearance of mitochondrial transformation. From the results obtained it may be concluded that there are correlations between the transformation of the mitochondria and physical dependence on morphine in mice.     

Comparative effects of synthetic enkephalinamides and morphine on abstinence responses in morphine-dependent mice

The effects of intracerebroventricular (ICV) administration of two synthetic enkephalinamides, D-Ala²-Met⁵-enkephalinamide and D-Met²-Pro⁵-enkephalinamide, were compared with those of morphine on abstinence responses in morphine dependent mice. Mice were rendered dependent by morphine pellet implantation for three days. When administered six hours after pellet removal, both enkephalinamides and morphine reversed the naloxone-induced abstinence jumping response. On a molar basis, D-Met²-Pro⁵-enkephalinamide was the most potent compound and morphine was the least potent. Thus, substitution of D-Methionine in the two-position and prolineamide in five-position of the naturally occurring enkephalins, methionine- and leucine-enkephalin resulted in potent compound. The enkephalinamides and morphine also prevented hypothermia when injected immediately after or six hours after morphine withdrawal (pellet removal).     

Effects of acute and continuous morphine administration on serum glutamate oxalacetate transaminase and glutamate pyruvate transaminase activities in the mouse.

Mice were exposed continuously to morphine by implanting morphine pellets for 12 hr to 3 days. In mice receiving prolonged morphine pellet implantation, second and third morphine pellets were implanted at days 3 and 6, for a total of 9 days. Results showed morphine pellet implantation for 24 hr caused a 2-fold increase in glutamate oxalacetate transaminase (SGOT) and glutamate pyruvate transaminase (SGPT) activities, as compared with those of the placebo control group. SGOT and SGPT activities were returned to control levels after 3–9 days of morphine pellet implantation. In mice implanted with a morphine pellet for 24 hr, both SGOT and SGPT activities returned to normal levels at 3 and 6 days after the removal of the morphine pellet. In mice receiving morphine pellet implantation for 24 hr with three concurrent administrations of naloxone. 40 mg/kg, s.c., the morphine pellet-induced elevations of SGOT and SGPT activities were attenuated significantly. Acute administration of morphine sulfate by both subcutaneous and intraventricular administration elevated SGOT and SGPT activities. The elevations of SGOT and SGPT activities caused by morphine pellet implantation were prevented completely by hypophysectomy and prevented partially by adrenalectomy. These results substantiate our previous morphological and biochemical findings that initial administration with morphine by either injection or pellet implantation may alter hepatic function and the action may be mediated through the central nervous system.     

The effect of aspartic acid on the intensity of physical dependence in morphine dependent mice

The mice (Balb/C strain) given 2% aspartic acid in 5% saccharose solution or only saccharose solution (p. os) starting 12 hr before the sc morphine pellet implantation until the removal of pellet were rendered physically morphine dependent. During the development of morphine dependence and after the removal of pellets (on 3-d day) spontaneous motor activity and analgesic threshold were measured as reliable abrupt withdrawal signs. Aspartic acid prevented to some extent the appearance of symptoms of physical morphine dependence.     

An improved implantation pellet for rapid induction of morphine dependence in mice.

A new type of morphine implantation pellet for the rapid induction of physical dependence in mice can be prepared by absorbing 7 mg morphine sulphate onto molecular sieves Type 4A (BDH). The small cylindrical pellets can be implanted subcutaneously without trauma and the need for anaesthesia, and are easily removed at any time from the animals. The peak of physical dependence is reached 24 h after implantation, and mortality is negligible. Withdrawal symptoms can be precipitated by intraperitoneal injection of naloxone, without removal of the pellet, and up to 70% of a group of mice show the characteristic urge to jump off a raised platform. This type of pellet has definite advantages over some other sustained-release preparations used in studies on morphine addiction in small animals.     

Tolerance, dependence and lethality in morphine-dependent mice after repeated oral administration of methadone

Mice were rendered tolerant to and dependent on morphine via a morphine pellet implantation. Three days later methadone hydrochloride was administered at a dose of 100mg/kg per os 3 hours after pellet removal and then daily for a total of 5--6 days. This dose of methadone was shown to exhibit a high efficacy for the blockade of morphine abrupt withdrawal jumping and only minimal toxicity. Under these conditions, the level of analgetic tolerance with respect to morphine and methadone and the level of dependence as measured by the naloxone ED50 were initially elevated by the morphine treatment. However, upon substitution with oral methadone these levels declined with time at a rate which did not differ from that of a group of mice receiving only water after morphine pellet removal. Despite these findings, the methadone treatment was associated with an increasing tolerance to methadone lethality during the administration of this narcotic which was nearly double that of a similarly treated water control group by the sixth day. This observation could not be explained by an elevation in the level of cellular tolerance rendered by the methadone treatment since the morphine LD50 was not elevated following identical treatment with morphine and then methadone. The significance of these results is discussed with respect to the role of methadone administration and its metabolism in the modification of tolerance and dependence.     

Aggression during morphine withdrawal: Effects of method of withdrawal,fighting experience,and social role

Offensive and defensive components of aggressive behavior were determined in resident and intruder mice. Withdrawal aggression was measured after the removal of a subcutaneous morphine pellet or after precipitation by naloxone in naive mice and after removal of a morphine pellet in mice with prior fighting experience. In naive mice, removal of a morphine pellet led to increases in attack bites and threats but naloxone-precipitated withdrawal led to decreases in these behaviors and to increases in defensive posturing, escape attempts and vocalizations. Prior fighting experience abolished the enhanced attack behaviors of resident mice following morphine pellet removal, but led to heightened defensive behavior in intruder mice. The behavior of intruder mice appeared more sensitive to naloxone administration than the behavior of resident mice; naloxone influenced not only intruder defensive behavior, but also other non-aggressive behaviors. The social role of the drug recipient and his prior history of aggressive behavior are important determinants of morphine and naloxone effects on aggression.     

Histamine turnover in the brain of morphine-dependent mice

Summary The turnover of brain histamine was examined in mice implanted subcutaneously with a morphine pellet (50 mg free base). The numbers of naloxone-precipitated jumpings and body shakes were maximum 2 and 3 days after implantation, respectively. The brain tele-methylhistamine level significantly increased (50% to 115%) during 12 h3 days after implantation of a morphine pellet, whereas the histamine level remained unchanged. The accumulation of tele-methylhistamine by pargyline treatment was significantly enhanced when pargyline was administered 12 h after implantation, suggesting an enhancement of histamine turnover. However, a similar degree of the tele-methylhistamine accumulation was induced by pargyline during 1–5 days after implantation, as compared with the accumulation in the control mice implanted with a placebo pellet. In mice undergoing morphine withdrawal by either the removal of morphine pellet or the treatment with naloxone 3 days after implantation, the degree of the pargyline-induced telemethylhistamine accumulation or the (S)--fluoromethylhistidine (-FMH)-induced histamine decrease was similar to that observed in the placebo pellet-control mice. The numbers of naloxone-precipitated jumpings and body shakes occurring in mice 3 days after implantation were not significantly affected by any of l-histidine, -FMH or metoprine. These results suggest that turnover of histamine in the brain is enhanced by acute morphine treatment and returns to the normal rate in the stage of chronic treatment and remains unchanged during the state of withdrawal. Send offprint requests to K. Saeki     

Effect of morphine and naloxone on intestinal transit in mice

Morphine caused a dose-dependent slowing of the rate of intestinal transit in mice. This inhibitory effect of morphine was antagonised by naloxone administration. Pretreatment with a single dose of morphine did not induce any detectable tolerance to the inhibitory effect of a second dose of morphine given 5 h later. However, naloxone was more effective in antagonising this inhibitory effect of morphine-pretreated mice than in saline-pretreated animals. Molecular sieve morphine pellet implantation for 24 h induced detectable tolerance to the inhibitory effect of morphine administered 3 h after removal of the pellet. In addition, the antagonistic effect of naloxone was also augmented when compared with blank pellet-implanted control animals. The present study has shown that the enhanced naloxone potency against the inhibitory effect of morphine on intestinal transit was observable before the development of overt tolerance, and that tolerance to the effect of morphine on the small intestine could be induced by implantation of a molecular sieve morphine pellet for 24 h.     

Allyl alcohol partially protects murine hepatic mitochondria against carbon tetrachloride

Male CD-1 mice were treated with carbon tetrachloride (CCl4, 1 ml/kg, i.p.), or allyl alcohol (AA, 0.05 ml/kg, i.p.) or both 24 h prior to sacrifice. The livers were removed, homogenized and mitochondrial preparations were fractionated. Electron micrographs of the mitochondrial pellet from the livers of mice treated with CCl4 showed lysed and fragmented mitochondria as well as a few condensed mitochondria. No normal intact mitochondria were observed. The mitochondrial pellet from AA-treated livers was characterized by a preponderance of condensed mitochondria whether CCl4 was given also or not. The mitochondrial fraction from CCl4-treated mice failed to utilize oxygen whereas that fraction from mice given AA did not differ from control in either its basal oxygen consumption (state 4) or oxygen utilization after the addition of adenosine diphosphate (state 3). State 4 respiration differed slightly but significantly from control in mitochondria from mice given both AA and CCl4 whereas state 3 respiration was greatly and significantly decreased from that of control values. Electron micrographs of liver slices from mice given AA or the combination of AA and CCl4 showed normal mitochondrial morphology. Thus, AA appears to exert an influence, perhaps at the level of the mitochondrial membrane, which is manifested only after their isolation. This effect which results in the condensation of isolated mitochondria serves to partially protect them against CCl4, but it did not prevent the characteristic CCl4-induced centrilobular necrosis.     

The effects of naltrexone on the development of physical dependence on morphine

A single i.p. injection of naltrexone (20 mg/kg) partially inhibited the development of physical dependence upon morphine in mice rendered dependent on morphine by implantation of a pellet containing 75 mg of morphine free base for three days. This was evidenced by an increase in the dose of naloxone (ED50) required to precipitate withdrawal jumping response. The increase in naloxone ED50 was much more pronounced when naltrexone was given prior to and during the course of pellet implantation. Inhibition was also observed when naltrexone was administered one day after the morphine pellet implantation, i.e., after some dependence had already developed. Naltrexone administration prior to and during the development of dependence also inhibited, but only partially, the loss of body weight and hypothermic response observed during abrupt withdrawal of morphine in morphine-dependent mice. The inhibitory effect of naltrexone on morphine dependence development was not associated with changes in brain morphine concentration.     

Effect of propranolol on antinociceptive and withdrawal characteristics of morphine

Propranolol at a dose (10 mg/kg) which did not alter tail-flick latency by itself, did not alter the ED50 of morphine when given 10 min prior to the narcotic. Propranolol at doses of 10 and 25 mg/kg given 10 min prior to naloxone challenge did not significantly alter the frequency of naloxone induced jumping 72 hr after morphine pellet implantation. The ED50 of naloxone in morphine pelleted mice was not altered by treatment with propranolol at 0, 24, and 48 hr after pellet implantation. Naloxone caused hyperactivity in mice when administered 72 hr after morphine pellet implantation. An injection of 25 mg/kg propranolol 10 min prior to naloxone did not block this hyperactivity. In addition, administration of 10 mg/kg of propranolol every 8 hr to rats during withdrawal from morphine failed to alleviate the withdrawal syndrome as evidenced by changes in either body weight or water intake. These data suggest that the beta-adrenergic blocking agent, propranolol, does not alter the antinociceptive activity or lessen the withdrawal syndrome of morphine in rodents.     

Heightened aggressive behavior during morphine withdrawal: effects ofd-amphetamine

The morphine withdrawal syndrome is composed of profound short- and long-term changes in autonomic, somato-motor and affective functions. In mice, morphine withdrawal produces heightened aggressive behavior and alterations in motor behavior; however, it is unclear whether these changes in behavior occur in unison and are dependent on a common mechanism or occur independently. In order to characterize the morphine withdrawal syndrome in mice, male Swiss-Webster mice were housed with female partners for 3–4 weeks before being implanted subcutaneously with morphine or placebo pellets. The pellets were removed 72 h after implantation and behavioral measurements were conducted 5, 48 and 96 h after pellet removal. During these tests, mice receivedd-amphetamine (0.3–10 mg/kg) or saline after which they were assessed for changes in motor behavior and for changes in aggressive behavior while confronting a group-housed male intruder. In morphine-withdrawn mice, frequency of attack behavior was increased by approximately 30% and this effect persisted for at least 4 days. In contrast, explosive jumping was increased and walking and rearing were greatly decreased at the onset of the withdrawal period but declined within the first 24 h and returned to control levels within 48 h of pellet removal.d-Amphetamine maintained the elevated level of aggressive behavior and sharply increased locomotion in morphine-withdrawn mice; in placebo-pelleted mice,d-amphetamine dose-dependently decreased aggressive behaviors while increasing locomotion. The differential time course and the differential modification byd-amphetamine suggest that heightened aggressive behavior is a long-lasting consequence of morphine-withdrawal based on separate mechanisms than the short-lived alterations in motor activity.     

New in vivo evidence for narcotic agonistic property of leucine-enkephalin

Administration of leucine-enkephalin or morphine to mice rendered dependent on morphine by pellet implantation inhibited the naloxone-precipitated abstinence syndrome. The withdrawal jumping response was inhibited by morphine or leucine-enkephalin; however, both failed to inhibit withdrawal defecation and rearing behavior. On a molar basis, leucine-enkephalin was half as potent as morphine in inhibiting the abstinence syndrome. New in vivo pharmacological evidence for narcotic agonist-like activity of leucine-enkephalin is provided.     

PRELIMINARY STUDIES ON INCREASED NALOXONE POTENCY AND ITS RELATIONSHIP TO MORPHINE TOLERANCE AND DEPENDENCE IN MICE

1. Morphine pretreatment (8 0 mg/kg s.c.) induced no overt tolerance to its antinociceptive effect in mice 4 h later, but enhanced the antagonistic potency of naloxone. 2. A molecular sieve morphine pellet implanted for 24 h induced measurable tolerance, but the relative potency of naloxone was not significantly different from that observed after single-dose morphine pretreatment. The development of tolerance and increased naloxone potency do not, therefore, run parallel. 3. Naloxone-precipitated withdrawal symptoms were observed after single-dose morphine and after pellet implantation. However, molecular sieve morphine pellet implantation induced a higher degree of dependence as compared with single dose morphine pretreatment. 4. These results indicate that the rate of development of increased naloxone potency and of morphine tolerance and dependence do not run parallel. This implies that caution must be exercised in regarding increased naloxone potency as a sensitive indicator of the initiation and development of tolerance and dependence to morphine.     

Unidirectional non-cross tolerance to etorphine in morphine-tolerant mice and role of the blood-brain barrier

A unidirectional non-cross tolerance phenomenon to etorphine was observed under conditions of maximal tolerance development to morphine. Using a morphine pellet (75 mg morphine) implantation technique, a 30-fold increase in the analgesic ED50 for subcutaneously (sc) administered morphine was observed. When 30-fold morphine-tolerant animals were examined for cross tolerance to sc etorphine, no change in etorphine's analgesic ED50 was observed. The removal of the tolerance-inducing morphine pellet prior to analgesia testing resulted in the expression of cross tolerance to etorphine. Further characterization of the phenomenon was provided by the reverse experiment in which a large amount of cross tolerance to sc morphine but little tolerance to sc etorphine was observed after etorphine pellet implantation. No change occurred in the analgesic ED50 for intracerebroventricular (icv) administration of etorphine or morphine after the animals were implanted with morphine pellets. These observations implicated the blood-brain barrier as a probable site for the differential expression of narcotic tolerance.     

PKC and PKA inhibitors reinstate morphine-induced behaviors in morphine tolerant mice

Male Swiss Webster mice exhibited antinociception, hypothermia and Straub tail 3h following a 75mg morphine pellet implantation. These signs disappeared by 72h, and the morphine-pelleted mice were indistinguishable from placebo-pelleted ones, although brain morphine concentrations ranged from 200 to 400ng/gm. We previously demonstrated that chemical inhibitors of protein kinase C (PKC) and A (PKA) are able to reverse morphine tolerance in acutely morphine-challenged mice. However, it was not known whether the reversal of tolerance was due to the interaction of kinase inhibitors with the morphine released from the pellet, the acutely injected morphine to challenge tolerant mice, or both. The present study aimed at determining the interaction between the PKC and PKA inhibitors and the morphine released "solely" from the pellet to reinstate the morphine-induced behavioral and physiological effects, 72h after implantation of morphine pellets. Placebo or 75mg morphine pellets were surgically implanted, and testing was conducted 72h later. Our results showed that the intracerebroventricular (i.c.v.) administration of the PKC inhibitors, bisindolylmaleimide I and G?-6976 as well as the PKA inhibitors, 4-cyano-3-methylisoquinoline and KT-5720, restored the morphine-induced behaviors of antinociception, Straub tail and hypothermia in morphine-pelleted mice to the same extent observed 3h following the pellet implantation. The tail withdrawal and the hot plate reaction time expressed as percent maximum possible effect (%MPE) was increased to 80-100 and 41-90%, respectively, in PKC and PKA inhibitor-treated morphine tolerant mice compared to 2-10% in non-treated mice. Similarly, a significant hypothermia (1.3-4.0 degrees C decrease in body temperature) was detected in PKC and PKA inhibitor-treated morphine tolerant mice compared to an euthermic state in non-treated morphine tolerant mice. Finally, the Straub tail score was increased to 1.1-1.6 in PKC and PKA inhibitor-treated tolerant mice, whereas it was totally absent in non-treated animals. It is noticeably that the kinase inhibitors used in the study had no effect in placebo-pelleted mice. Our results provide the first evidence on the ability of PKC and PKA inhibitors to reinstate the behavioral and physiological effects of morphine in non-challenged morphine-tolerant animals.     

Morphine analgesia, tolerance and physical dependence in the adrenalectomized rat

1. Adrenalectomy reduced the median antinociceptive dose (AD50) of morphine in male Sprague-Dawley rats. The antinociceptive effect was assessed by the tail-flick method of D'Amour & Smith (1941).2. Tolerance to the antinociceptive effect of morphine developed in adrenalectomized and sham-operated rats after chronic exposure to morphine. Development of tolerance did not significantly alter the increased sensitivity of adrenalectomized rats to the antinociceptive effect of morphine.3. Adrenal weights were not increased in rats rendered physically dependent on morphine by subcutaneous implantation of a morphine pellet. Withdrawal, induced by intraperitoneal injection of naloxone hydrochloride, 4 mg/kg, or by removal of the implanted pellet, resulted in a rapid increase in adrenal weight.4. In morphine-dependent animals, the incidence of abstinence signs and body weight loss during precipitated withdrawal did not appear to be significantly influenced by adrenalectomy or by corticosterone-pretreatment.     

Immunotoxicological Profile of Morphine Sulfate in B6C3F1 Female Mice

This study was undertaken to investigate a number of immuneparameters which may be compromised with exposure to morphinesulfate. Mice were implanted subcutaneously with 8-, 25-, or75-mg morphine sulfate pellets. Placebo pellets of identicalmakeup to the 75-mg morphine pellet (without morphine of course)were used as a control. Twenty-four hours after implantationof a 75-mg morphine pellet, blood levels reached a peak of 1610ng/ml. Corticosterone increased in parallel with morphine andreached a peak level of 966 ng/ml 24 hr after implantation.The dose response of morphine to increase corticosterone, however,was fiat. The weight of the lymphoid organs, spleen and thymus,and the liver were significantly reduced in the morphine-treatedgroups. Morphine treatment was associated with an increase inserum albumin, SGPT, BUN, and alkaline phosphatase indicativeof hepatic damage. In contrast to increased serum proteins,the C3 component of complement was reduced in a dose-dependentmanner. Leukocyte number in the peripheral blood was significantlyreduced, while erythro-cyte number and hematocrit were bothincreased. The number of B cells and T cells was decreased inmorphine-treated animals. However, the percentage of T cellsrelative to B cells was increased. The primary IgM antibodyresponse to the T-depen-dent antigen, sheep red blood cells,was decreased. Natural killer cell activity was reduced in responseto morphine, as was the phagocytic capacity of Kupffer cells.Host-resistance models of Listeria monocytogenes or Streptococcuspneumoniae showed an increased resistance following administrationof morphine. This increased host resistance, however, was notdue to an increase in antimicrobial action of sera obtainedfrom mice treated with morphine. The majority of morphine'seffects on the immune system exhibited a flat dose response,suggesting that these effects may be mediated secondarily throughcorticosterone.     

Increase in plasma cyclic AMP levels elicited by naloxone in morphine-dependent male mice

Naloxone increased the levels of plasma cyclic AMP in morphine-dependent male mice in which a morphine pellet had been implanted for 72 hr, but not in the nondependent mice. The effect of naloxone on the cyclic AMP levels disappeared 24 hr after the removal of the morphine pellet. Pretreatment with propranolol, hexamethonium or adrenalectomy, but not with atropine or phentolamine inhibited the effect of naloxone. These results suggest that naloxone increases the level of plasma cyclic AMP by releasing catecholamines from the adrenal medulla, thereby activating the adenylate cyclase in the tissues through the stimulation of β-adrenergic receptors. A moderate increase in plasma cyclic AMP was also seen in morphine-dependent mice 1 hr after the removal of the morphine pellet. It is proposed that the increase in the levels of plasma cyclic AMP elicited by naloxone-precipitated or abrupt withdrawal is one of the withdrawal symptoms and probably can be used as a screening method for assessing the degree of physical dependence in laboratory animals.