Morphine-tolerant Rats Research Articles

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine

The addition of narcotic analgesics in vitro to nerve ending preparations from rat caudate nucleus in an assay of adenylate cyclase activity (AC) resulted in an inhibition of basal AC only at drug concentrations of 10− 4M or higher, and no inhibition of dopamine-stimulated (DA) AC at these drug concentrations. The acute administration of morphine at a moderately high dose (60 mg/kg) produced an increase in striatal cAMP levels, and increases in basal and DA-AC in caudate nerve-endings. In morphine-tolerant rats, striatal cAMP levels and basal AC were similar to control values, while DA-AC was elevated. These results suggest: (1) that opiates do not act directly on DA-AC, the ‘dopamine receptor’, and (2) that the observed behavioural DA sensitivity in tolerant animals may be produced by the DA-AC supersensitivity.

Action of reserpine in morphine-tolerant rats: absence of an antagonism of catecholamine depletion.

The mechanism of resistance to the catecholamine-depleting effects of reserpine in morphine-tolerant rats was examined. In vitro, morphine did not alter reserpine inhibition of 3 H-norepinephrine uptake by synaptic vesicles. Similarly, uptake by vesicles isolated from morphine-tolerant animals was as susceptible to reserpine inhibition as that of corresponding preparations from saline-treated controls. Consistent with earlier reports, intraperitoneally administered reserpine only partially reduced brain catecholamine levels in most rats treated chronically with morphine. However, reserpine injected intravenously depleted norepinephrine and dopamine in experimental animals to the same extent as controls treated by either route of administration. The amount of reserpine which actually reached the brain was estimated by intraperitoneal injection of 3 H- reserpine. Animals chronically treated with morphine and with only partially reduced norepinephrine concentrations exhibited tritium levels less than one-half those of controls. It is concluded that an increased resistance to reserpine cannot be correlated with central nervous system tolerance to morphine. Rather, systemic inactivation of reserpine may be enhanced as a result of chronic morphine treatment.

Effects of intraventricular brain injections of neurotransmitters on colonic temperature in morphine-tolerant rats

The possible sensitivity changes caused by morphine tolerance in the postsynaptic receptors of putative neurotransmitters involved in the central thermoregulatory mechanisms were studied by intraventricular brain injections of 5-HT, NA, DA, ACh, physostigmine, pilocarpine and carbachol into 20-day old morphine-tolerant and control rats. Temperature acclimation effects were ruled out by keeping the animals in a thermoneutral environment (32°C) during the periods in which morphine would ordinarily have caused hypothermia at lower environmental temperatures. At 22–24°C, the hypothermia produced by ACh, physostigmine and both in combination was significantly greater in morphine-tolerant animals, while there were no significant differences between tolerant and control animals in the colonic temperature responses to the other substances tested. It is suggested that cholinergic receptors of pronounced ACh specificity are induced in the central thermoregulatory neurones of immature rats by repeated administration of morphine for 6 days.

The effect of morphine on rat brain catecholamines: Turnover in vivo and uptake in isolated synaptosomes

The Acute administration of morphine and other narcotic analgesic drugs to animals produces transient changes in the levels of biogenic amines in the central nervous system (WAY and Shen, 1971). Among the most striking acute responses are the depletion of dopamine from the striatum (GuNNE et al., 1969) and norepinephrine from the hypothalamus (VOGT, 1954; Reis et al., 1969). As measured by the accumulation of 14C-dopamine and -norepinephrine from 14C-tyrosine in vivo, the biosynthesis of dopamine is transiently increased after a single injection of morphine, especially in the hypothalamus and striatum (Clouet and Ratner, 1970). A more pronounced increase in the rates of dopamine biosynthesis was found in the same brain regions of morphine-tolerant rats. The present report is the results of the exploration of the relationship between brain biogenic amines and opiates in three aspects: (1) biosynthesis of catecholamines; (2) tyrosine hydroxylase levels; and (3) synaptosomal uptake of catecholamines.

Acute morphine tolerance and physical dependence in rats. 4th Report: Electron microscopic observations of the adrenal cortex in acute morphine tolerant rats

Acute morphine tolerance and physical dependence in rats. 4th Report: Electron microscopic observations of the adrenal cortex in acute morphine tolerant rats

Modification of serotonin metabolism in rat brain after acute or chronic administration of morphine

The steady state concentration of 5-hydroxyindoleacetic acid (5-HIAA) was elevated in rat brain for at least 4 hr after administration of a single dose of morphine sulfate (30 mg/kg, s.c.), and for more than 40 hr after subcutaneous implantation of pellets of morphine alkaloid. The concentration of 5-HIAA returned to normal 3 days after pellet implantation at a rate that paralleled the development of tolerance to the analgesic and other overt actions of morphine. Morphine did not modify the steady state concentration of serotonin under any of the treatment conditions. The turnover of brain serotonin was increased significantly during the 90-min period following a single injection of morphine sulfate (30 mg/kg, s.c.), as indicated by an increased rate of accumulation of 5-HIAA after blockade of efflux of 5-HIAA by probenecid in morphinetreated animals. As judged either by the rate of accumulation of 5-HIAA after administration of probenecid, or by the rate of accumulation of serotonin after treatment with pargyline, an increase in turnover of serotonin was evident in brains of tolerant rats 72 hr after pellet implantation. The rate of efflux of 5-HIAA from the brain was the same in control and morphine-tolerant rats. These results indicate that changes in brain serotonin metabolism are associated with both the acute effects of morphine and with morphine tolerance.

Temperature effects of intraventricular serotonin, norepinephrine and pilocarpine in the morphine-tolerant rat

Studies were undertaken to examine the possibility that changes occur in the responsiveness of thermoregulatory neurons in the anterior hypothalamus to endogenously released neurotransmitters upon the development of tolerance to morphine. In experiments conducted at an environmental temperature of 20–21°C, tolerance produced by the subcutaneous implantation of morphine alkaloid pellets failed to alter the temperature response of rats to intraventricular injections of 5-HT (10 μg), NE (20 μg) or pilocarpine (100 μg). It is concluded that tolerance development to morphine-induced hypothermia is not a result of changes in the postsynaptic sensitivity to the putative neurotransmitters in the thermoregulatory center.

The effect of cholinergic antagonists on increases of spontaneous locomotor activity and body weight induced by the administration of morphine to tolerant rats.

Increases both in spontaneous locomotor activity and in body weight were observed after the administration of morphine-HCl (100 mg/kg, i. p.) to tolerant rats. These signs were inhibited by pretreatment of the morphine-tolerant rats with cholinergic antagonists; the inhibition by scopolamine was greater than that by methscopolamine or atropine. These results suggest that both central and peripheral cholinergic mechanisms participate in the increases both in spontaneous locomotor activity and in body weight of tolerant rats after morphine administration.

Disturbed patterns of behaviour in morphine tolerant and abstinent rats.

1. Eating, drinking and spontaneous motor activity were studied in rats receiving large daily doses of morphine. These forms of behaviour were largely suppressed when the rats were made abstinent and were restored when morphine was given again.2. Compensation for depressions of behaviour during abstinence did not seem sufficient to account for all the stimulant effects of morphine in tolerant rats. Morphine also had slight stimulant actions in non-tolerant rats.3. In tolerant rats, the repeated pairing of the effects of morphine with the re-emergence of behaviour such as eating and drinking may intensify the rewarding value of the drug.

Change of copper concentration in the central nervous system after morphine treatment and effect of copper on morphine analgesia in rats

Morphine increased the concentration of copper in the spinal cord. This was antagonized by simultaneous injection of levallorphan, which injected alone did not influence the copper content of nervous tissues. In morphine tolerant rats, the copper content of the cerebral cortex was lower than that of control animals 4 hr after the last injection of morphine. Intraperitoneal injection of copper sulphate enhanced the analgesic action of morphine as evidenced by a rise in the threshold tail clip pressure. The relations of morphine analgesia, morphine tolerance and copper metabolism are discussed.

N-demethylation of N-14C-methyl-codeine in morphine tolerant and nontolerant rats and mice.

SummaryN-Demethylation of codeine by morphine tolerant male rats decreased significantly as compared to nontolerant male rats to the level of nontolerant female rats; whereas in the nontolerant female rats, female mice, and tolerant male mice, it was not changed.

Uptake of dihydromorphine- 3H by synaptosomes

Tritium-labeled dihydromorphine (DHM- 3H) was accumulated in vitro by the synaptosomal fraction (nerve endings) of rat cerebral cortex. Particles derived from nerve endings by osmotic shock exhibited a higher concentration of DHM- 3H than did other subcellular fractions of brain when the isolated fractions were incubated with the drug in vitro . Nalorphine in large concentrations reduced the uptake of DHM- 3H by all fractions to the same degree. No difference was apparent in the uptake of DHM- 3H by subcellular fractions from non-tolerant and morphine-tolerant rats. The uptake of DHM- 3H by nerve endings was attributed to a binding phenomenon, although an active transport process may provide the drug an access to the binding sites in the intact tissue.

Effects of acute and chronic morphine tolerance of the serum copper, ceruloplasmin and hepatic copper contents.

Former papers from this laboratory reported that on treatment with sodium diethyldithiocarbamate (DDC), a dopamine-β-hydroxylase inhibitor and a chelating agent for copper, the analgesic action of morphine was potentiated in rats (1, 2). Although the possibility that effect of DDC on morphine analgesia might be related to catecholamine metabolism cannot be completely excluded, we clarified that the changes in copper metabolism of the blood and tissues in rats could be responsible for the potentiation of morphine analgesia by DDC and that fluctuation of the copper metabolism in rats by the treatment of morphine may be involved in the manifestation of analgesia and development of tolerance to the analgesia (2, 3). Some authors have postulated that the development of acute and chronic tolerance to the analgesic action of morphine in rats appears to involve RNA and/or protein synthesis (4-6), because tolerance is prevented by drugs which inhibit RNA or protein synthesis. The increase in the ceruloplasmin content in the serum, due to its synthesis in the liver after injection of copper, was also inhibited by actinomycin D and cycloheximide (7). Since the plasma copper content increases in animals which are tolerant to morphine analgesia (2), it seemed interesting to examine the effects of repeated intravenous injections or chronic treatment with morphine on the content of the serum copper protein, ceruloplasmin, during development of tolerance to the analgesic action of morphine. The present paper reports changes in the levels of serum copper, ceruloplasmin, hepatic copper and biliary excretion of copper in rat tolerated to the analgesic action of morphine caused by repeated intravenous or by chronic subcutaneous injections of morphine (acute or chronic morphine tolerant rat, respectively).

Plasma copper levels and their significance in morphine analgesia and tolerance

Morphine lowered plasma copper content. However after of sodium diethyldithiocarbamate, which decreased the copper level markedly and potentiated morphine analgesia, it caused no further decrease. This action of morphine was antagonized by levallorphan which elevated plasma copper concentration. In morphine tolerant rats, there was a marked increase in plasma copper level which was partially reversed by morphine withdrawal. When sodium diethldithiocarbamate was administered to the morphine tolerant animals, morphine analgesia reappeared, and the elevated copper level was markedly decreased. These results indicate that the changes in copper level in plasma, or the mechanisms mediating these alterations and/or the results caused by these fluctuations may be related to the manifestation of morphine analgesia and tolerance.

Reduction of the antinociceptive effect of 5-hydroxytryptophan in morphine tolerant rats.

The effect of 5-hydroxytryptophan on pain threshold was studied in rats both tolerant and nontolerant to the analgesic action of morphine as assessed using a procedure of electrical stimulation. The compound elevated pain threshold and exhibited an additive effect with morphine analgesia in nontolerant rats. A marked reduction of the antinociceptive action of the serotonin precursor as well as absence of the additive effect with morphine was observed in rats tolerant to the analgesic. These results are discussed in terms of the possible mechanism of action of serotonin on morphine effects.

AN INCREASED SENSITIVITY TO ALCOHOL IN RABBITS TREATED WITH A CERTAIN FACTOR CONTAINED IN A MICROSOMAL SUBFRACTION OF THE BRAIN

Cochin and Kornetsky (1) showed that tolerance to the effects of a single injection of morphine in the rat as measured by the hot plate reaction could be demonstrated for periods up to one year following that single injection. Thus, they postulated that tolerance to narcotic drugs might well be an immune phenomenon or a mechanism resembling the antigen-antibody reaction. Later, Kornetsky and Kiplinger (2, 3) attempted a passive transfer of a hypothetical factor of tolerance in serum from morphine-tolerant animals (rat, dog, monkey and man) to nontolerant animals (mouse). Although they were unsuccessful in transferring the tolerance, they succeeded in demonstrating the presence of certain transferable factors in the serum of morphine-tolerant animals, since under condition of those studies, potentiation of the depressant and an analgesic action of morphine was observed. The role of the potentiating substance in the serum of the morphine-tolerant animal and the mechanism by which it potentiates morphine are as yet unknown. On the other hand, Kornetsky and Cochin (4) reported that serum from morphinetolerant rabbits attenuated morphine analgesia in mice and they assumed that the immune mechanisms involved may partially account for some of the phenomena associated with tolerance to morphine. Ungar and Cohen (5) also showed that administration of extracts of the brain taken from morphine-tolerant rats and dogs conferred tolerance on mice. The presence of the factors in the blood of morphine-tolerant animals that have attenuating or enhancing effects on morphine in nontolerant animals is an interesting finding, although the immune reaction hypothesis is no more than speculation. During a study of the effect of the treatment with a homogenate of the brain from alcohol-tolerant rabbits, on the duration of alcohol anesthesia following the intravenous injection of a test dose of alcohol, it was noted that an increased sensitivity and/or a decreased or a biphasic change in the sensitivity to alcohol was observed depending on the individual rabbit. Based on these conflicting evidences the present investigation was undertaken to define the change of sensitivity to alcohol using the normal rabbit as the recipient and the donor species.

Lack of transfer of morphine tolerance by administration of rat cerebral homogenates.

SummaryTransfer of tolerance to the analgesic effects of morphine to mice by injection of cerebral homogenates from morphine-tolerant rats was studied. After such pretreatment the mice were injected with a test dose of morphine and then tested for analgesia using the hot plate method. Neither the dose-response curve nor the mean increase in reaction time was significantly altered by pretreatment with the cerebral homogenate from morphine-tolerant rats. The possible reasons for the lack of confirmation of results obtained by other workers were discussed.

A comparison of 6.14.endoetheno-7-(1-hydroxy-1-cyclohexylethyl)-tetrahydro-oripavine (A potent analgesic derived from thebaine) with morphine

The biological activities of M125 (6.14.endoetheno-7-(1-hydroxy-1-cyclohexylethyl)-tetrahydro-oripavine) and morphine have been compared by studying the changes produced by their repeated administration to rats in (1) the in vitro N- demethylating activity of the liver microsomal enzymes and (b) the pharmacological response to the drugs. M125 is N- demethylated by liver microsomal preparations under the same conditions required for the N- demethylation of morphine. After the repeated administration of M125 rats became tolerant to M125 itself and also to morphine. Similarly, morphine tolerant rats were tolerant to M125. The in vitro N- demethylation of both compounds was depressed in liver microsomal preparations from both M125 and morphine tolerant rats. After withdrawal of M125 from tolerant rats the pharmacological response of the animals to the drug and the liver microsomal N- demethylating activity returned to normal within twenty-five days. N- allyl normorphine inhibited the N- demethylation of morphine more strongly than that of M125 but the nature of the inhibition was the same in each case. It is suggested that M125 produces its biochemical and pharmacological effects by similar mechanisms to morphine.

Studies on the intracellular distribution and tissue binding of dihydromorphine-7,8-H3 in the rat.

SummaryDihydromorphine-7,8-H3 of high specific activity was prepared by the catalytic tritriation of the double bond of morphine and purified. Studies of the intracellular distribution of radioactivity in the brains and livers of rats injected with the labeled drug showed that most of it was in the soluble fraction. However, the brain nuclear fraction consistently contained 10-20% of the radioactivity of the homogenate. The administration of nalorphine had no effect on tissue levels or intracellular distribution. Binding of dihydromorphine to brain and liver homogenates and to serum proteins was demonstrated by equilibrium dialysis. The degree of binding was similar in the different tissues and not affected by the addition of nalorphine. Tissue homogenates from morphine-tolerant rats exhibited the same amount of binding as those from control rats.

Induction of morphine tolerance by material extracted from brain of tolerant animals

Administration of extracts of brain taken from morphine-tolerant rats and dogs conferred tolerance on mice. This was shown by the significantly lowered analgesic response to morphine in mice treated with tolerant rat brain as compared with untreated controls and animals which received extracts on normal brain. Induced tolerance was also demonstrated by a significant shift in the dose-response curve of morphine. The degree of tolerance depended upon the amount of brain received by the mice. The effect of a single injection of brain extract lasted at least ten days. The factor responsible for the induction of morphine tolerance is water soluble, insoluble in ethanol and acetone, and is dialyzable. On partition with phenol and under the influence of enzymes, it behaves like a small protein or peptide.