Failure of MIF-1 or naloxone to reverse ischemic-induced neurologic deficits in gerbils

MIF-1 and naloxone exert similar actions in several situations. Since naloxone, at a dose of 1 mg/kg IP, has been reported to reverse the neurologic deficits of gerbils whose right common carotid artery had been occluded, MIF-1 was tested under the same conditions and the effects compared with naloxone. Doses of 0.1, 1.0, and 10.0 mg/kg IP of MIF-1 and naloxone did not significantly alter the signs of either moderate or severe neurologic deficits. Thus, the results of this study with gerbils do not add evidence for the use of these opiate antagonists in strokes.     

Naloxone-like actions of MIF-1 do not require the presence of the pituitary

The actions of peripherally administered MIF-1 (Pro-Leu-Gly-NH₂) and naloxone in blocking the effects of morphine in the tail-flick test were measured across a wide range of five doses in hypophysectomized and intact mice. The presence of the pituitary gland failed to influence the response to MIF-1 or naloxone. Both hypophysectomized and intact mice were significantly affected by these two compounds at doses of 0.01, 0.1, 1.0, and 10.0 mg/kg IP. The greatest effect of MIF-1 occurred at 100 mg/kg, but naloxone was lethal at this dose. Preliminary experiments with other tests showed that at 10 mg/kg, naloxone, but not MIF-1, was effective in preventing the Straub-tail reflex and in precipitating withdrawal-jumping in mice implanted with morphine pellets. Only minimal activity was shown by MIF-1 in preventing blockade of electrically induced contractions of the guinea pig ileum by morphine. Neither compound was active in the frog-righting test. In summary, the results emphasize the differential actions of MIF-1 as an opiate antagonist and demonstrate that the pituitary is not required for their mediation.     

Similar antagonism of morphine analgesia by MIF-1 and naloxone in Carassius auratus

Prolyl-leucyl-glycinamide (MIF-1), the C-terminal tripeptide of oxytocin, and naloxone were administered intracranially (IC) to goldfish (Carassius auratus) in doses of 0.001, 0.01, 0.1, 1.0 and 10.0 mg/kg and compared to a diluent control group for their ability to reduce the effects of morphine (30 mg/kg IC) in an assay measuring analgesia to electric shock. Threshold levels of pain were determined by the voltage necessary to produce an agitated swimming response (ASR). Both MIF-1 and naloxone were found to significantly reduce the analgesic effects of morphine when compared to the diluent control group. Similar dose-response curves in an apparent sine-wave pattern were noted with both MIF-1 and naloxone when comparisons were made both at 20 minutes after administration of morphine and over the entire 150 minutes of the experiment. The results support the evidence that MIF-1 can act as an opiate antagonist.     

Tyr-MIF-1 augments benzodiazepine receptor binding in vivo

Behavioral and limited neurochemical evidence indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems. A previous study using in vitro techniques indicated that MIF-1 (Pro-Leu-Gly-NH2) and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), peptides with anti-opiate activity, enhanced GABA-stimulated benzodiazepine binding. To assess the activity of these peptides under in vivo conditions, we evaluated their effects on benzodiazepine receptor binding as determined by specific uptake of the benzodiazepine antagonist [3H]-Ro15-1788. Tyr-MIF-1, at a dose of 1 mg/kg IP, significantly augmented benzodiazepine binding in cortex and hippocampus but not in cerebellum, hypothalamus, or pons-medulla. Increases in binding were due in large part to increased apparent affinity at the receptor. At none of the doses of MIF-1 (0.1, 1 and 10 mg/kg) or at the highest (10 mg/kg) and lowest (0.1 mg/kg) doses of Tyr-MIF-1 was there any significant alteration in benzodiazepine binding in any region evaluated. These results indicate that peptide-benzodiazepine receptor interactions may also occur in vivo.     

Acute administration of MIF-1 or Tyr-MIF-1 inhibits haloperidol-induced catalepsy in rats

The effects of MIF-1 (Pro-Leu-Gly-NH2) and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) on haloperidol-induced catalepsy were studied in order to examine the influences of both peptides on central dopaminergic function. In the first experiment, several variables were tested. It was found that the optimal effect was achieved with a dose of 1.0 mg/kg haloperidol injected SC an hour before testing for catalepsy and 1.0 mg/kg MIF-1 injected SC 30 min before testing. In the second experiment, Tyr-MIF-1 as well as MIF-1 were injected as single injections at four doses. Catalepsy was inhibited in an inverted U-shape dose-response relationship with the maximal effect of each peptide occurring at 1.0 mg/kg. The results indicate that when careful attention is given to dose, both MIF-1 and Tyr-MIF-1 can activate dopaminergic neuronal activity after acute administration.     

Radioimmunoassay of MIF-1/Tyr-MIF-1-like material in rat pineal

The hypothalamic peptide MIF-1 (Pro-Leu-Gly-NH₂) was coupled to thyroglobulin and injected into rabbits. The resulting antiserum reacted with the tetrapeptide Tyr-MIF-1 to a greater extent than with the tripeptide MIF-1, presumably because of a better conformation for antibody binding. By radioimmunoassay (RIA), immunoreactive MIF-1/Tyr-MIF-1-like material was found in the pineal gland of each of the 100 rats examined. The tendencies for slightly higher levels in pineals obtained from rats kept in constant darkness for two weeks, from rats in a normal light cycle decapitated at noon, or from rats which had been hypophysectomized were not statistically significant. Gel filtration of pineal extracts on a column of Sephadex G-10 revealed that by RIA one immunoreactive peak eluted near MIF-1 and oxytocin, and another peak near Tyr-MIF-1. The results suggest the presence in pineal tissue of an MIF-1-like material as well as a novel peptide containing Tyr-Pro-Leu-Gly-NH₂ or a closely related structure for which oxytocin is unlikely to be the precursor.     

Assessment of SNC 80 and naltrindole within a conditioned taste aversion design

Although compounds with relative selectivity for the mu and kappa opiate receptors subtypes have been reported to condition taste aversions, it is not known whether systemically administered delta compounds have the ability to produce aversions. To that end, female Long-Evans rats were adapted to water deprivation and were given pairings of a novel saccharin solution and various doses of the selective delta agonist SNC 80 (0.32-10.0 mg/kg; Experiment 1) or the selective delta antagonist naltrindole (1.0-18.0 mg/kg; Experiment 2). For comparison, the relatively selective mu agonist morphine (Experiment 1) and mu antagonist naloxone (Experiment 2) were assessed under identical conditions. Both SNC 80 (Experiment 1) and naltrindole (Experiment 2) were effective as unconditioned stimuli within this design, inducing dose-dependent taste aversions with repeated conditioning trials. Although at no dose did animals injected with SNC 80 differ from those injected with morphine, aversions induced by SNC 80 were acquired at a faster rate than those induced by morphine. Subjects injected with naloxone drank significantly less than those injected with naltrindole at the 10 mg/kg dose, and aversions induced by naloxone at 5.6 and 10 mg/kg were acquired at a faster rate than those induced by naltrindole. Although the basis for opioid agonist- and antagonist-induced taste aversions is not known, the differences between aversions induced by SNC 80 and naltrindole and those induced by morphine and naloxone, respectively, may be a function of their relative selectivity for specific opiate receptor subtypes.     

Characterization of binding sites for N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in rat brain

Binding sites for N-Tyr-Pro-Leu-Gly-NH₂ (Tyr-MIF-1), a novel peptide structurally related but immunoreactively different from Pro-Leu-Gly-NH₂ (MIF-1), were investigated. The presence of Tyr-MIF-1-like material in brain tissue has previously been demonstrated by RIA and its levels were shown to vary with the diurnal cycle and after pinealectomy. We now demonstrate a high affinity binding site in rat brain that is saturable and specific for Tyr-MIF-1. Crude P2 synaptosomal fractions from rat brains were incubated with ¹²⁵I-Tyr-MIF-1 in the presence or absence of μM unlabelled Tyr-MIF-1 (nonspecific binding). Binding reached equilibrium at 30–40 min at 23°C and at about 4 hr on ice, after which it was relatively stable for at least 18 hr. None of the other peptides (including MIF-1) or amino acid residues tested were found to effectively compete for ¹²⁵I-Tyr-MIF-1 binding. Binding was linear with protein from 280 μg to at least 1.1 mg protein per tube. Scatchard analysis of the striatum-thalamus revealed the presence of binding sites with an apparent K_D of 91 nM and maximum number of sites in the range of 45 fmol/mg tissue. Analysis of several brain areas revealed a differential distribution of the binding sites with relatively high concentrations in cortex, striatum, and amygdala and low concentrations in pons-medulla. Together with the previously published RIA results, the demonstration of a receptor for Tyr-Pro-Leu-Gly-NH₂ supports the concept of the presence of this novel peptide and its receptor in the brain.     

Biphasic effects of MIF-1 and Tyr-MIF-1 on apomorphine-induced stereotypy in rats

The effects of several doses of MIF-1 and Tyr-MIF-1 (0.2, 0.5, 1.0 and 5.0 mg/kg, SC) on the stereotypic behavior induced by various doses (0.08, 0.2, 0.5, 1.0 and 2.0 mg/kg, SC) of apomorphine (APO) were tested in rats. MIF-1 increased the stereotypic behavior induced by 0.5 and 1.0 mg/kg of APO, but decreased the stereotypic behavior induced by 2.0 mg/kg of APO. Tyr-MIF-1 showed a biphasic effect similar to that exerted by MIF-1. The results suggest that the type of response to MIF-1 and Tyr-MIF-1 after APO is influenced by the activity of central dopaminergic neurons.     

Opiate antagonists enhance the working memory of rats in the radial maze

Two experiments tested the influence of the opiate antagonists naloxone and naltrexone on the spatial working memory of rats in a 12-arm radial maze. In Experiment 1, ten rats were serially forced to visit six randomly selected arms, then were removed from the maze for delays of either 30, 60, or 240 minutes, and then returned to the maze for a free-choice memory test with all 12 arms available. Five minutes into the delay, rats were injected intraperitoneally (IP) with either physiological saline or naloxone (1 mg/kg). When injected with naloxone the rats revisited forced-choice arms less often than when injected with saline during a subsequent free-choice test. In Experiment 2, twelve rats showed a similar facilitation of working memory when injected with the opiate antagonists naltrexone (0.3 mg/kg) and naloxone (1 mg/kg) in comparison to a saline control condition. These findings demonstrate the beneficial effects that opiate antagonists exert on working memory-based performance in the radial maze. They may also resolve conflicting reports about the influence of opiate antagonists on radial maze performance, by suggesting that the choice of measurement and testing conditions are crucial for detecting these effects in working memory procedures.     

Effects of MIF-1 and three related peptides on reserpine-induced hypothermia in mice

MIF-1, Tyr-MIF-1, pGlu-Leu-Gly-NH₂, and cyclo-Leu-Gly were tested for 3 hr (60, 120, and 180 min) at 3 doses (0.1, 1.0, and 10.0 mg/kg IP) in mice pretreated 18 hr earlier with reserpine. Reversal of hypothermia was significant only with MIF-1 (p < 0.05) at 120 and 180 min but not with the 2 analogs (pGlu-Leu-Gly-NH₂ and cyclo-Leu-Gly) reported to more active in other tests than MIF-1 or with Tyr-MIF-1, a recently described brain peptide. Thus, the results demonstrate that the relative potency of related peptides can differ depending upon the experimental situation.     

Tyr-MIF-1 and MIF-1 are active in the water wheel test for antidepressant drugs

MIF-1 [Pro-Leu-Gly-NH2] and Tyr-MIF-1 [Tyr-Pro-Leu-Gly-NH2] were tested in a system in which antidepressant drugs are known to result in increased wheel turning as mice attempt to escape from a small tank of water. One hr after injection, both peptides were found to cause a significant increase of the number of rotations of the wheel at doses as low as 0.01 mg/kg IP, the dose-response pattern for MIF-1 resembling an inverted-U. DSIP and morphine, by contrast, decreased the number of rotations. Under the conditions tested, neither MIF-1 nor Tyr-MIF-1 reversed the effect of morphine. The results demonstrate that MIF-1 and Tyr-MIF-1 are active in another test for antidepressants.     

MIF-1 is active in a chronic stress animal model of depression

MIF-1 was tested in an animal model of depression that used unpredictable chronic stress. In this paradigm, rats received either no stressors or a daily protocol of a variety of stressors for 20 days, during which time daily, intraperitoneal injections of various compounds were given. The tricyclic antidepressant imipramine (5 mg/kg) and low doses (0.1 and 1.0 mg/kg) of MIF-1 significantly increased activity and decreased defecation in an open field on day 21. No dose of naloxone (0.01-10.0 mg/kg) acted as an antidepressant. A high dose (10.0 mg/kg) of MIF-1 significantly increased the effects of chronic stress and produced hyperalgesia. Chronically-stressed rats were significantly more analgesic than controls. The results indicate that MIF-1 can act as an antidepressant in this model.     

Naloxone as a stimulus in drug discrimination learning: generalization to other opiate antagonists.

Nonopiate dependent animals were trained to discriminate the opiate antagonist naloxone (1 mg/kg) from distilled water within the conditioned taste aversion baseline of drug discrimination learning. Specifically, rats injected with naloxone prior to a saccharin-LiCl pairing, and with its vehicle prior to saccharin alone, rapidly acquired the drug discrimination, avoiding saccharin following the administration of naloxone and consuming saccharin following its vehicle after only three conditioning trials. Once the discrimination was acquired, generalization tests revealed that the opiate antagonists diprenorphine and naltrexone and the mixed opiate agonist/antagonist nalorphine completely generalized to the naloxone cue at doses of 1.8, 5.6 and 18 mg/kg, respectively. That discriminative control was established with a low dose of naloxone (i.e., 1 mg/kg) and other compounds with opiate antagonist activity generalized to the naloxone cue suggest that the stimulus effects of naloxone were likely mediated through the opiate receptor. Because each of these compounds are reported to bind to the mu receptor (with varying affinities and varying degrees of selectivity), the stimulus properties of naloxone are likely mediated at this specific receptor subtype.     

Effects of naloxone on morphine induced sedation and hyperactivity in the hamster

Three experiments investigated the effects of naloxone on morphine elicited changes in hamster locomotor activity. In Experiment 1, a prior subcutaneous injection of naloxone (0.4 mg/kg) converted morphine (15 mg/kg) elicited hypoactivity into hyperactivity: Compared with saline controls, naloxone pretreated animals were hyperactive following a subcutaneous injection of morphine. Experiment 2 investigated the effects of four doses of naloxone (0, 0.04, 0.1, 0.4 mg/kg) on morphine elicited hyperactivity. Results indicated that naloxone reversal of morphine elicited hyperactivity is directly related to dose of naloxone. In Experiment 3, naloxone (0.4 mg/kg) was administered one and two hours after a morphine injection. Compared with saline controls, morphine treated animals were hypoactive for approximately 40 minutes after each of the naloxone injections. Results are discussed in terms of a modified dual-action hypothesis.     

Blockade of brain dopamine receptors antagonizes the anti-immobility effect of MIF-1 and Tyr-MIF-1 in rats

Previous studies have shown effects of MIF-1 (prolyl-leucyl-glycinamide) and Tyr-MIF-1 (tyrosyl-prolyl-leucyl-glycinamide) in animal models of depression and also effects on dopaminergic function. These observations prompted us to examine whether the effects of the two peptides in the behavioral 'despair' test were modulated by dopamine antagonists. MIF-1 and Tyr-MIF-1, at the small dose of 0.01 mg/kg i.p. (24, 5 and 1 h before the test), produced a significant anti-immobility effect. This effect was antagonized by a single injection of either haloperidol or sulpiride, two dopamine receptor blockers. The same low dose of the tricyclic antidepressant desipramine was without significant effect in this test. The results indicate that Tyr-MIF-1, like MIF-1, is active in the behavioral despair test for antidepressants and that at least some of the CNS actions of these peptides are mediated by dopamine receptors.     

Patterns of drinking in the rat following the administration of opiate antagonists

Durations of drinking were recorded for water-deprivated rats as they drank to satiety, following SC injections of naloxone (0.1–10.0 mg/kg), naltrexone (0.1–10.0 mg/kg) or saline vehicle. The results provided evidence for the effects of opiate antagonists on the temporal pattern of drinking exhibited by water-deprived animals. A separate, time-sampling procedure was used to supplement the drinking duration data, and showed that the opiate antagonists may suppress water consumption during a period 2.5–7.5 min after the start of the initial drinking bout. A second experiment confirmed that the pattern of drinking displayed during schedule-induced polydipsia in the rat is resistant to any suppressant effect of a moderate dose of an opiate antagonist. The similiarity between opiate receptor blockade and water preloading in their effect on drinking in response to water deprivation, and lack of effect on schedule-induced polydipsia is discussed. Opiate antagonists may affect drinking principally by imposing a thirst satiety signal.     

Naloxone attenuates incubated sucrose craving in rats

Rationale Cue-induced craving precedes drug relapse and contributes to eating disorders. Opiate antagonists have been demonstrated to be effective at reducing cravings for drugs and food. Craving, as defined as responding for a stimulus previously associated with a reward, increases, or incubates, over forced abstinence in an animal model of relapse. Objectives This paper aims to determine anticraving effects of the opiate antagonist, naloxone, on the incubation of sucrose craving. Methods 106 male Long-Evans rats lever pressed for 10% sucrose solution 2 h/day for 10 days. On either day 1 or 30 of forced abstinence, rats responded in extinction for 6 h and then were injected (ip) with either saline or naloxone (0.001, 0.01, 0.1, 1, or 10 mg/kg). The rats then responded for 1 h for presentation of a tone + light cue previously presented with every sucrose delivery during self-administration training. Results The rats responded more in extinction and following saline on day 30 vs day 1 (an incubation of craving). Except for a trend for a decrease in responding following 10 mg/kg on day 1, naloxone was primarily effective on day 30. On day 30, naloxone significantly reduced responding at all doses except for 0.1 mg/kg. Conclusions The time-dependent increase in sensitivity to an opiate antagonist is consistent with time-dependent changes in the opiate system following forced abstinence from sucrose. These changes may partly underlie the incubation of sucrose craving. In addition, these findings could be used to support the use of naloxone as an anticraving medication in protracted abstinence.     

Effects of naloxone and its quaternary form on fluid consumption in rats

Three studies were performed on albino rats to determine the effects of naloxone and its quaternary derivative, naloxone methylbromide, on fluid consumption. The doses of the quaternary naloxone were equated with naloxone by molarity and effectiveness in order to facilitate direct comparisons. All rats were deprived of food and water for 12 hr and exposed to a 20% sucrose solution for a 2 hr period. In Experiment 1, a low (0.01 mg/kg) dose of naloxone or an equated dose of quaternary naloxone was given ICV and immediate access allowed to the fluid on four consecutive days. Animals receiving naloxone were not significantly different from controls, and rats receiving quaternary naloxone exhibited seizures, resulting in decreased consumption. In Experiment 2, the low dose of naloxone or the equated dose of quaternary naloxone was given IP for four consecutive days and neither was significantly different from controls. In Experiment 3, animals were given an IP dose of either 1 mg/kg naloxone, a 1 mg/kg or 50 mg/kg dose of quaternary naloxone, or saline and tested for a single 2 hr period. The doses of 1 mg/kg naloxone and 50 mg/kg quaternary naloxone produced significantly less drinking than controls. In all studies, the initial 30 min period produced the most drinking. Suppression of drinking by a dose of 50 mg/kg quaternary naloxone suggested, in contrast to other studies, that it may cross the blood-brain barrier at high doses.     

MIF-1 does not act like naloxone in antagonizing the cardiovascular activity of leucine-enkephalin in the conscious dog

MIF-1 (Pro-Leu-Gly-NH2), a hypothalamic tripeptide, has been demonstrated to stimulate naloxone in antagonizing the effects of opioid peptides in a number of experimental systems including enkephalin-induced analgesia in the tail-flick assay, beta-endorphin induced hypothermia and hypomotility, deprivation-induced drinking, and analgesia in goldfish. MIF-1, however, has no effect upon the activity of enkephalins in the mouse vas deferens or enkephalin binding in the rat striatum. We have studied the interactions of MIF-1 with Leu5-enkephalin (Leu5-ENK) in the conscious, chronically instrumented dog. Although naloxone inhibits both the elevations of heart rate and blood pressure produced by IV Leu5-ENK in the conscious state and the depressions in these variables produced by Leu5-ENK after pentobarbital anesthesia, MIF-1 has no effect upon the Leu5-ENK response in either state. However, both naloxone and MIF-1 seem to raise mean arterial pressure in the conscious dog. These results indicate that MIF-1 does not act like naloxone in antagonizing the peripheral effects of Leu5-ENK and lend further support to the existence of mechanistic differences among opiate-mediated behavior, analgesia, and cardiovascular activity.