Reversal of the effects of centrally-administered morphine on colonic motility in dogs by the benzodiazepine receptor antagonist RO 15-1788

The effects of intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of morphine on jejunal and colonic motility were investigated in conscious dogs chronically prepared with strain gage transducers and compared to those of i.c.v. DAGO, a highly selective opiate mu agonist. Morphine i.v. (100 micrograms/kg) and i.c.v. (10 micrograms/kg) administered 3 hrs after a meal stimulated colonic motility for 3-5 hrs and induced a phase 3 on the jejunum, which appeared after a 15-60 min delay following i.c.v. administration. These effects were reproduced by DAGO administration at doses of 2 micrograms/kg i.v. and 0.2 micrograms/kg i.c.v. The effects of i.v., but not those of i.c.v., morphine and DAGO on jejunal and colonic motility were blocked by a previous administration of naloxone (100 micrograms/kg i.v.). The colonic stimulation but not the jejunal phase 3 induced by i.c.v. morphine and DAGO were blocked by RO 15-1788 (1 mg/kg i.v.), a selective benzodiazepine antagonist. The colonic stimulation induced by i.v. morphine or DAGO was not modify by i.v. RO 15-1788. It is concluded that i.c.v. administration of mu agonist involved benzodiazepine but not opiate receptors to stimulate colonic motility in dogs.     

Prostaglandin hyperalgesia, V: a peripheral analgesic receptor for opiates

Prostaglandin E2 injected in the rat paw causes hyperalgesia which is antagonized by local injections of opiate and opiate antagonists. In the present investigation in rats it is shown that naloxone has an analgesic effect at doses as low as 2 micrograms/site, injected into the rat hind paw. At a dose that has no analgesic effect (1 microgram/site) naloxone antagonized the analgesia produced by either local or systemic administration of morphine. Local administration of levorphanol (50 micrograms/site) caused a 50% reduction in the intensity of the hyperalgesia induced by prostaglandin E2. A dose four times greater of its isomer, dextrorphan, had little analgesic effect. The present results support the suggestion that this peripheral analgesia is the result of an action of opiates in receptors located at the nociceptors.     

Interaction of D-Ala2-[Tyr-3,5-3H]enkephalin(5-D-Leu) with opiate receptors of rat brain

Some kinetic features of D-Ala2-[Tyr-3.5-3H]enkephalin (5-D-Leu) binding to opiate receptors of rat brain were studied. It was shown that the Leu-enkephalin D analog interacts with the high and low affinity binding sites of opiate receptors, the equilibrium constants being equal to 0.71 and 8.4 nM, respectively. The rate constant for the label association with the high affinity binding sites in 2 . 10(8) M-1 min-1; those for the label dissociation from the opiate receptor binding sites with high and low affinities are 7.2 . 10(-3) and 0.16 min-1, respectively. Hence, the half-life time of these complexes is 95.7 and 4.3 min, respectively. Na+, K+ and Li+ markedly decrease the specific finding of the label, while Mg2+, Mn2+ and Ca2+ at the concentrations studied markedly increase its specific binding. It is concluded that the Leu-enkephalin D-analog under study acts as a morphine agonist and reveals a much higher affinity for rat brain opiate receptors than does Leu- or Met-enkephalin. This makes it a useful tool for study of the enkephalin reception under normal and pathological conditions.     

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Hyperthermia induced by morphine administration to the VTA of the rat brain: an effect dissociable from morphine-induced reward and hyperactivity

Morphine action at opiate receptors in the ventral tegmental area (VTA) of the rat brain has been implicated in the production of increased locomotor activity and in morphine's rewarding properties. In the present experiments, bilateral administration of morphine (18 micrograms tapped into the tips of 28 gauge cannulae) into the VTA resulted in an increase in body temperature in rats. This effect was both reversed and blocked by a systemic injection of the opiate receptor blocker, naloxone, suggesting that it was due to morphine action at opiate receptors. The neuroleptic, pimozide, injected systemically four hours prior to morphine administration completely blocked the increased locomotor activity but had no effect on the hyperthermia. These data demonstrate that the hyperthermia was not brought about by the increased physical activity. Furthermore, these results suggest that while morphine-induced reward and increased locomotor activity may be mediated by an interaction of morphine and the ascending mesolimbic dopamine system, the hyperthermia is not. In an additional experiment, the effect of systemic injections of the central neurotransmitter receptor antagonists, scopolamine, phenoxybenzamine, and methergoline, on the hyperthermia induced by morphine in the VTA was investigated. Only the serotonin antagonist, methergoline, attenuated the hyperthermia.     

Stability of opiate receptors of wet and lyophilized preparations of rat brain membranes

The effects of incubation of rat brain membranes at 0 degrees C on the specific binding of mu-ligands (naloxone, morphine) and the delta-ligand (D-Ala2, D-Leu5-enkephalin) to opiate receptors were studied. The effects of lyophilization of rat brain membranes on the properties of the opiate receptors were determined. The lyophilized brain membrane preparations revealed an extraordinarily high stability as compared to "wet" membranes. The experimental results suggest that morphine and D-Ala2, D-Leu5-enkephalin binding both to the high affinity and low affinity sites has different nature and point to the utility of stable and standard preparations of lyophilized membranes for the use in the receptor analysis of opiate and opioid peptides.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

家兔中枢内源性阿片样物质在紧张性高血糖反应中的作用

本工作观察家兔内源性阿片样物质在紧张性高血糖反应中的作用。通过向家兔侧脑室内注射阿片受体阻断剂纳洛酮或羧基肽酶 A 的抑制剂 D-苯丙氨酸以分别减弱或加强脑内内源性阿片样物质的作用。结果表明,纳洛酮能使由乙醚或2-脱氧葡萄糖所引起的高血糖反应减弱,而使由胰岛素所引起的低血糖反应加强并延搁其回复过程。D-苯丙氨酸表现为相反的效应。在已对吗啡形成耐受的家兔,2-脱氧葡萄糖所引起的高血糖反应也呈减弱。这些结果提示,脑内内源性阿片样物质与紧张性高血糖反应有关。     

Binding sites of opiates and endogenous opioids in the oocytes of the toad Bufo viridis

The binding of labelled naloxone, morphine and (D-Ala2,D-Leu5)enkephalin (DADL) to oocyte membranes of the toad Bufo viridis was investigated. The opiate antagonist naloxone binds to the membranes much more effectively than morphine or DADL. The binding of [3H]naloxone is reversible and saturating. The bound [3H]naloxone is readily replaced by unlabelled naloxone or bremazocine (kappa-agonist), far less effectively by morphine (mu-agonist) and SKF 10.047 (sigma-agonist) and is not practically replaced by DADL (delta-agonist), beta-endorphin (epsilon-agonist) and other neuropeptides. Analysis of experimental results in Scatchard plots revealed two types of binding sites with a high (Kd = 15 nM) and low (Kd = 10(3) nM) affinity for naloxone. The number of sites responsible for the binding of naloxone possessing a high affinity is 16 pmol-/mg of oocyte homogenate protein, i.e., 20-50 times as great as in the toad or rat brain. Trypsin and p-chloromercurybenzoate decrease the binding of [3H]naloxone. The oocyte extract is capable of replacing the membrane-bound [3H]naloxone, on the one hand, and of inhibiting the smooth muscle contracture of the rabbit vas deferens, on the other. This inhibition is reversed by naloxone and can also be induced by bremazocine, but not by morphine, DADL and SKF 10.047. In all probability oocytes contain compounds that are similar to opiate kappa-agonists. It may also be possible that these compounds mediate their effects via specific receptors and are involved in the control over maturation of oocytes and early development of toad eggs.     

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.     

Restraint stress enhances morphine-induced analgesia in the rat without changing apparent affinity of receptor

Antagonism of morphine analgesia (tail-flick assay) by naloxone was assessed quantitatively by in vivo "apparent" pA2 determination in unstressed rats and in rats subjected to restraint stress. Restrained rats had a higher baseline tail-flick latency than did unstressed (unrestrained) animals, and were more sensitive to the analgesic effect of morphine, as reflected in lower morphine ED50s. There was no significant difference between apparent pA2 values of unstressed and restrained rats using pA2 regression line analysis. This suggests that while stress enhances the analgesic effect of morphine, it does not appreciably alter opiate receptor affinity for naloxone under the conditions of this study.     

The effect of di- and trivalent metal ions on the binding of [3H-Tyr1, D-Ala2, D-Leu5] enkephalin to opiate receptors from the rat brain

The influence of Ca2+, Mg2+, Mn2+, Sr2+, La3+, Nd3+, Sm3+, Eu3+, and Gd3+ ions on the binding of labeled, stable enkephalin analogue, [3H-Tyr1, D-Ala2, D-Leu5]enkephalin, to opiate receptors of the rat brain membrane preparations has been investigated. The formation of the complex can be described by a scheme involving at least two independent binding sites. The high affinity site does not discriminate the divalent and trivalent metal ions: all examined cations enhanced the enkephalin affinity for this site. The ligand binding to the low affinity site is potentiated only by Mn2+, Mg2+, and lathanoides. The maximal concentration of the binding sites of the above two types is not affected by the cations. The increase in the ionic strength of the solution entails a decrease in the affinity of the ligand for the high affinity binding site. It is shown that the effect of both di- and trivalent metal cations on the [3H-Tyr1, D-Ala2, D-Leu3] enkephalin binding is mediated through one cation attachment site on the respective enkephalin receptor.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Morphine-6-glucuronide, a potent mu agonist

The 3- and the 6-glucuronides of morphine have been examined in binding studies and in vivo. The 3-glucuronide had poor affinity in all binding studies whereas the 6-glucuronide potently labeled mu, but not delta or kappa receptors with affinities similar to morphine. Microinjections of the 3-glucuronide directly into the periaqueductal gray were without effect. The 6-glucuronide, on the other hand, was up to 20-fold more potent than morphine following microinjections in the same region. High doses of the 6-glucuronide produced profound seizure activity. All 6-glucuronide actions were sensitive to the opiate antagonist naloxone.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Mu-opiate binding and morphine antagonism by octapeptide analogs of somatostatin

A series of cyclic conformationally restrained octapeptide analogs of somatostatin were examined for their ability to inhibit the binding of tritiated mu, kappa, and delta opiate receptor ligands. Several of these substances were found to have high affinity for mu opiate receptors while having very low affinity for both kappa and delta receptors. Previous suggestions that somatostatin analogs exhibit opiate antagonist activity led to a study of the ability of the two most potent compounds to inhibit morphine analgesia in rats after intracerebroventricular injection. One of the compounds significantly antagonized morphine analgesia although the other displayed severe toxicity. These two compounds differed in that the very toxic compound had previously been found to possess significant somatostatin activity. It thus appears that the structural requirements for toxicity and somatostatin activity can be differentiated from those for opiate activity.     

Opiate binding sites and endogenous opioids in Bufo viridis oocytes

Binding sites with high affinity for [3H]naloxone, but not for [3H]morphine and [3H] (D-Ala2, D-Leu5) enkephalin, have been found in membranes of Bufo viridis oocytes. The binding is reversible and saturable. Bound [3H]naloxone is easily displaced both by unlabeled naloxone and bremazocine, much worse by morphine and SKF 10,047; (D-Ala2, D-Leu5) enkephalin and beta-endorphin practically fail to displace [3H]naloxone. Scatchard analysis is consistent with the existence of two classes of binding sites with Kd 15 nM and 10(3) nM. The number of binding sites with high affinity for naloxone is 16 pmol/mg protein of homogenized oocytes which is 20-50-fold higher than in, toad or rat brain. Oocyte extract displaces [3H]naloxone bound with oocytes' membranes and inhibits electrically evoked contractions of the rabbit vas deferens. This inhibition is reversed by naloxone. It is suggested that compounds similar to opiate kappa-agonists exist in oocytes. It cannot be ruled out that they participate via specific receptors in the regulation of oocyte maturation and egg development.     

Alteration of brain stem auditory evoked potentials after intracerebroventricular administration of met-enkephalin in rabbits.

Brainstem auditory evoked potentials (BAEPs) were elicited by binaural click stimulation and recorded from the rabbits with chronically implanted electrodes and a cannula for intracerebroventricular injection (i.c.v.). 400 BAEPs were averaged off line. The registration was carried out before and after i.c.v. injection of met-enkephalin (2.5 or 25 nmol), naloxone (20 micrograms), or i.v. injection of morphine (1.0, 2.0, 5.0 mg/kg b.w.). Enkephalin caused shortening of interpeak latency time, naloxone caused its lengthening, while the effect of morphine was not unidirectional. Enkephalin caused increase in the surface area below the negative peaks located in the range of 4.5-7.5 ms from the first positive peak, naloxone caused its decrease while the effect of morphine was also in this respect not unidirectional. It is concluded that opiate receptors are involved in the modulation of the auditory brainstem responses.     

Neuropharmacological actions of enkephalin after systemic administration.

A pentapeptide, methionine-enkephalin, which interacts with opiate receptors in the brain, was found to markedly potentiate the behavioral effects of DOPA when administered intraperitoneally into mice. These effects, which were even more striking with D-alanine-2-methionine-5-enkephalin but less with morphine, persisted at least two hours after systemic injection of the peptide. Only a weak effect of enkephalin was seen in a serotonin potentiation test. Systemic injections of enkephalin resulted in a significant reduction of footshock-induced fighting and slight reduction in audiogenic seizures in mice. The results suggest that the CNS effects observed after systemic administration of enkephalin may involve the dopaminergic receptor mechanism.     

The biochemical and behavioral effects of phospholipase A2 and morphine microinjections in the periaqueductal gray of the rat

In order to characterize the in vivo action of phospholipase A2 (PLA2) on opiate receptors and opiate-induced behaviors, the effects of injections of PLA2 into the periaqueductal gray region (PAG) of the rat were assessed on free fatty acid (FFA) release, opiate-binding levels, and morphine-induced behaviors. Rats received bilateral PAG injections of 2 micrograms of PLA2 while anesthetized. One hour later, regions around the cannulae tracts in PLA2-treated rats contained over 2.5 times more FFA than saline-injected controls, and 3H-dihydromorphine binding was reduced on average more than 70%. In another series of experiments, conscious rats were given 2 micrograms of PLA2 prior to 10 micrograms of morphine through cannulae chronically implanted into the PAG. PLA2 did not significantly attenuate morphine-induced analgesia as measured by the tail-flick test to radiant heat, but did prevent the explosive motor behavior observed following morphine injections alone. PLA2 by itself did not induce analgesia, but did cause explosive motor behavior 2 hr after the injections. Neither lysophosphatidylcholine nor trypsin resulted in motor seizures following PAG injections. It was concluded that the behavioral effects of PLA2 result from the unique properties of the enzyme, rather than generalized membrane damage, and that the opioid sites and mechanisms that mediate analgesia are different from those associated with explosive motor behavior.