ODC-polyamine system is involved in morphine analgesia

alpha-Difluoromethylornithine (DFMO) directly infused into a brain-lateral ventricle (12.5, 25 and 50 micrograms/rat) dose- and time-dependently inhibited brain ODC activity. While having no influence per se on pain threshold, DFMO significantly inhibited the analgesic activity of morphine (15 mg/kg i.p.), this effect being obtained when brain ODC activity was reduced by at least 80%. On the other hand, DFMO had no influence on number and affinity of brain opiate binding sites. Morphine per se neither modified whole brain ODC activity nor significantly affected the ODC inhibitory effect of DFMO. In more discrete brain areas (midbrain, brainstem) morphine actually increased ODC activity. The present results indicate that brain ODC/polyamines system may play a role in the analgesic activity of opioids, probably at a post-receptorial level or through a non-opiate receptor-linked mechanism.     

"Paradoxical" analgesia and aggravated morphine dependence induced by opioid antagonists

Chronic treatment with naloxone (Nx) or naltrexone (Ntx) induces paradoxical analgesia. In the present study, the effects of chronic treatment with opioid receptor antagonists, such as nor-binaltorphimine (nor-BNI) for kappa and naltrindole (NTI) for delta receptors, on analgesic response using the hot plate test and on morphine physical dependence in rats were examined. The hot plate latency was significantly increased by pretreatment with Nx (5 mg/kg, s.c.), nor-BNI (20 mg/kg, i.p.) or NTI (20 mg/kg, i.p.) for 5 days. After chronic pretreatment with these antagonists, the rats were treated with morphine-admixed food (0.5 mg/g of food) for 3 days. Chronic pretreatment with Nx and NTI significantly increased Nx precipitated body weight loss in morphine dependent rats, while chronic pretreatment with nor-BNI produced small increase. These results indicate that chronic treatment with nor-BNI or NTI as well as with Nx induces obviously paradoxical analgesia, and that chronic blockade of mu or delta may enhance the development of physical dependence on morphine.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Possible participation of endogenous opioid peptides on the mechanism involved in analgesia induced by vouacapan.

The involvement of opioid peptides in the mechanism of action of vouacapan, a new experimental compound extracted from seeds of Pterodon poligalaeflorus Benth, was investigated both in mice utilizing acetic acid writhing response and in rats utilizing inflammatory hyperalgesia induced by carrageenan and modified Randall-Selitto method. Vouacapan, in both models, caused a dose-dependent analgesia when injected p.o., s.c. and i.p. The analgesic effect was partially blocked by naloxone, nalorphine and n-methyl-nalorphine. Significant tolerance to analgesic effect was observed following repeated administration of vouacapan or morphine. On the last day of treatment, cross administration revealed symmetrical and asymmetrical cross-tolerance between vouacapan and morphine, in rats and mice, respectively. We conclude that a release of endorphins could be involved in the analgesic mechanism of vouacapan in both models tudied.     

GABA in morphine analgesia and tolerance

Drugs affecting various steps of GABA transmission exhibit analgesia in a variety of experimental models in animals; this analgesic response generally requires high doses of the drugs and does not appear to be opiate-like since the GABAergic analgesia is naloxone-insensitive and lacks dependence liability. The outcome of the analgesia response is variable when opiate and GABAergic drugs are administered together; however, directly acting GABA receptor stimulants and GABA-transaminase inhibitors generally enhance the analgesic effect of opiates. The development of newer GABAergic drugs with greater potency and specificity may offer an alternative to opiate analgesics. The results obtained over the years, on the possible involvement of the GABA system in morphine tolerance and dependence are equivocal. Studies on region-specific changes in opiate-GABA interaction as well as opiate-GABA-benzodiazepine interaction are needed to further elucidate the role of GABA on opiate system.     

Endothelin receptor antagonists restore morphine analgesia in morphine tolerant rats

Several neurotransmitter mechanisms have been proposed to play a role in the development of morphine tolerance. The present study provides evidence for the first time that endothelin (ET) antagonists can restore morphine analgesia in morphine tolerant rats. Tolerance to morphine was induced by subcutaneous implantation of six morphine pellets during a 7-day period. The degree of tolerance to morphine was measured by determining analgesic response (tail-flick latency) and hyperthermic response to morphine sulfate (8 mg/kg, subcutaneously (s.c.)) in placebo and morphine pellet implanted rats. The maximal tail-flick latency in morphine pellet-vehicle treated rats (7.54 s) was significantly lower (P<0.05) when compared to placebo pellet-vehicle treated rats (10s), indicating that tolerance developed to the analgesic effect of morphine. In separate sets of experiments, ET antagonists, BQ123 (10 microg, intracerebroventricularly (i.c.v.)) and BMS182874 (50 microg, i.c.v.) were administered in placebo and morphine tolerant rats. BQ123 was injected twice daily for 7 days and once on day 8. BMS182874 was administered only on day 8. Morphine (8 mg/kg, s.c.) was administered 30min after BQ123 or BMS182874 administration. It was found that both BQ123 and BMS182874 potentiated morphine analgesia in placebo and morphine tolerant rats. BQ123 potentiated tail-flick latency by 30.0% in placebo tolerant rats and 94.5% in morphine tolerant rats compared to respective controls. BMS182874 potentiated tail-flick latency by 30.2% in placebo tolerant rats and 66.7% in morphine tolerant rats. Morphine-induced hyperthermic effect was also potentiated by BQ123 and BMS182874. The duration of analgesic action was also prolonged by BQ123 and BMS182874. The effect of BMS182874 was less as compared to BQ123. BQ123 and BMS182874 are selective ET(A) receptor antagonists. Therefore, it is concluded that ET(A) receptor antagonists restore morphine analgesia in morphine tolerant rats.     

MIF-1 and Tyr-MIF-1 antagonize morphine and opioid but not non-opioid stress-induced analgesia in the snail, Cepaea nemoralis

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and naloxone on morphine and warm and cold stress-induced increases in the latency of the thermal (40 degrees C hot plate) avoidance behaviors of the terrestrial snail, Cepaea nemoralis, were examined. All three substances blocked the morphine- and warm stress-induced opioid analgesia, while having no effects on non-opioid cold stress-induced analgesia. Tyr-MIF-1 had a significantly greater inhibitory effect than MIF-1. These results indicate that MIF-1 and Tyr-MIF-1 antagonize the antinociceptive effects of exogenous opiates and opioid-mediated analgesia in snails in a manner analogous to that described for mammals. This raises the possibility of an evolutionary conservation of functional opioid antagonists.     

Role of the hypothalamus in inhibiting the hypophysis-adrenal cortex system through the feedback mechanism

Chronic experiments were made on intact rabbits and rabbits with destroyed paraventricular and ventromedial nuclei of the hypothalamus to explore the hydrocortisone-induced inhibition of the stressor response of the pituitary-adrenocortical system. Intravenous injection of hydrocortisone in a dose of 100 micrograms/kg 5 minutes before immobilization stress led to inhibition of corticosteroid elevation induced by immobilization of the animals. Inhibition of the stressor reaction was maximal in intact animals, less in rabbits with destroyed ventromedial nuclei, and further less in animals with destroyed paraventricular nuclei. The paraventricular and ventromedial nuclei of the hypothalamus are necessary for inhibition of the pituitary-adrenocortical system by the feedback mechanisms.     

Dynorphin-A-(1–13) antagonizes morphine analgesia in the brain and potentiates morphine analgesia in the spinal cord

Intrathecal injection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1–13) (1.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick latency in the rat. The analgesic effect of the composite dynorphin/morphine was dose-dependent in serial dilutions so that a composition of 1/8 of the analgesic dose of dynorphin and 1/3 that of morphine produced an analgesic effect equipotent to full dose of either drug applied separately. The analgesic effect induced by dynorphin/morphine mixture was not accompanied by motor dysfunction and was easily reversed by a small dose (0.5 mg/kg) of naloxone. Contrary to the augmentatory effect of dynorphin on morphine analgesia in the spinal cord, intracerevroventricular (ICV) injection of 20 nmol of dynorphin-A-(1–13) exhibited a marked antagonistic effect on the analgesia produced by morphine (120 nmol, ICV). The theoretical considerations and practical implications of the differential interactions between dynorphin-A-(1–13) and morphine in the brain versus spinal cord are discussed.     

Bovine endozepine potentiates morphine analgesia in mice

This paper describes the influence of bovine endozepine (BEP) on the analgesic effect of morphine. The intraventricular administration of BEP between doses of 2-4 nmole in mice resulted in the potentiation of the morphine-analgesic effect. The basic pain threshold did not change when BEP was given alone, thus indicating that BEP is unable to elicit analgesic effect alone. Intravenous injection of BEP in the dose range of 5-7.5 mg/kg also potentiated the analgesic effect of morphine. These observations suggest that endozepine can act as a regulator of pain.     

Phorbol ester suppression of opioid analgesia in rats

Protein kinase C (PKC) has been shown to be an important substrate in intracellular signal transduction. Very little is known concerning its possible role in mediating opiate-induced analgesia. In the present study, 12-O-tetradecanoylphorbol 13-acetate (TPA), a selective activator of PKC, was injected intrathecally (ith) to assess its influence on the analgesia induced by intrathecal injection of the mu opioid agonist PL017, the delta agonist DPDPE and the kappa agonist 66A-078. Radiant heat-induced tail flick latency (TFL) was taken as an index of nociception. TPA in the dose of 25-50 ng, which did not affect the baseline TFL, produced a marked suppression of opioid antinociception, with a higher potency in blocking mu and delta than the kappa effect. In addition, mu and delta agonists induced remarkable decreases in spinal cyclic AMP (cAMP) content whereas the kappa effect was weak. The results suggest a cross-talk between the PKC system and the signal transduction pathway subserving opioid analgesia.     

Ageing,opioid analgesia and the pineal gland

The effects of ageing on day-night rhythms of analgesia was examined with young (1–2 months), mature (8–12 months) and old (20–30 months) mice. Significant age-related declines were observed both in the absolute levels and diel rhythms of morphine analgesia, with the most pronounced changes occuring at night. Administration of the pineal hormone, melatonin, augmented day-time levels of analgesia in all age classes and reversed the age-related decline in nocturnal morphine analgesia in old mice. Inhibition of pineal function in young mice by either exposure to light pulses or treatment with benserazide mimicked the effects of ageing on nocturnal morphine analgesia. These findings suggest that the pineal gland and melatonin are involved in modulating diel rhythms of analgesia and have an influential role on age-related changes in opioid responses.     

Effect of opioid agonist-antagonist interaction on morphine dependence in rats

Morphine dependence was induced by treatment with morphine-admixed food (0.25mg/g of food) for 7 days. Withdrawal was precipitated by injecting naloxone (0.5mg/kg, s.c.). Rats treated with morphine exhibited body weight loss upon the naloxone injection. When morphine-dependent rats were injected subcutaneously with morphine, codeine, meperidine and pentazocine 30 min before the naloxone injection, these drugs significantly suppressed the naloxone-precipitated loss of body weight in a dose-dependent manner. However, body weight loss induced through coadministration of naloxone and Mr-2266 BS were not suppressed by morphine pretreatment. These results suggest that opioids protect against naloxone-precipitated loss of body weight, and that mu and kappa opiate receptors play an important role in the protection against naloxone-precipitated withdrawal.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

The fos proto-oncogene protein:regulation by morphine in the rat hypothalamus

Mechanisms by which opiates alter neuronal functions, including neuroendocrine functions, are not well defined. We have previously demonstrated that morphine rapidly and transiently increases expression of the proto-oncogene c-fos in the rat caudate-putamen. This regulation of the c-fos gene by morphine may represent a portion of the intracellular cascade coupling activation of opiate receptors on the cell surface to subsequent alterations in neuropeptide gene expression. In the present study, we have focussed on effects of morphine on c-fos expression in the ventromedial hypothalamus, which contains estrogen-concentrating neurons and a large number of neurons expressing the opioid proenkephalin and Proopiomelanocortin. The hypothalamus has been identified as a "final common pathway" between the remainder of the central nervous system and the pituitary gland. As a marker for c-fos expression, we have detected pp50 c-fos (FOS) protein immunocytochemically, using a polyclonal antibody to the M peptide of FOS, and revealed an intense nuclear stain in many neurons. Labeled nuclei were drawn by camera lucida from 12 matched sections (one side only) covering the rostral and middle levels of the ventromedial nucleus of six rats given morphine and six given phosphate buffered saline. Morphine treatment significantly increased the number and density of immuno-labeled nuclei in the ventromedial nucleus, but not in the arcuate nucleus. These results suggest effects of morphine (directly or indirectly) on neurons in the ventromedial hypothalamic nucleus, despite the relative absence of morphine receptors in this nucleus. These results may also provide an anatomical basis for neuroendocrine alterations following morphine treatment.