Naloxone Binding Research Articles

D-alanine 2]-methionine enkephalinamide binding to rat brain slices: Differences from opiate alkaloid binding and reduction by chronic morphine treatment

Binding of a metabolically stable enkephalin analog, [d-alanine 2]-methionine-enkephalinamide (DALA), was characterized in rat brain slices. Unlike morphine or naloxone binding, which showed positive cooperativity in these preparations, DALA binding did not exhibit homotropic activation. The amount of DALA bound was less in artificial cerebrospinal fluid (150 mM sodium) than in Tris buffer. Reduced binding was observed in both media with slices obtained from animals rendered tolerant to morphine by pellet implantation. The results indicate that DALA and opiate drugs occupy the same binding sites, but that the characteristics of the binding of the enkephalins and the alkaloids are different.

MORPHINE ANALGESIA AND CEREBRAL OPIATE RECEPTORS: A DEVELOPMENTAL STUDY

1 Development of the analgesic response to morphine and ontogenesis of central opiate receptors were analyzed in rats 5 to 120 days old. 2 The analgesic effect of morphine increased until day 15, after which it decreased to reach a plateau at about day 30. With phenoperidine, on the other hand, the analgesic effect increased until day 15, remained constant between day 15 and day 30 after which it decreased slowly. 3 The ratio of the amounts of morphine in blood over those in brain increased about 3 fold between day 15 and day 30. 4 Opiate receptors were detected in the brain of newborn rats: stereospecific binding of [3H]-naloxone at 10 and 50 nM indicated the presence of low and high affinity binding sites. 5 The number of [3H]-naloxone binding sites increased rapidly during the second and third week after birth. Their affinity for several opiates remained constant throughout development. 6 These results indicate that the analgesic activity of opiates varies with age: until day 15, the analgesic effect of opiates increases in parallel with the number of opiate brain receptors. Then, the formation of the blood brain barrier introduces an additional step in the regulation of opiate activity.

Opiate receptors on mesolimbic dopaminergic neurons

Following electrocoagulation of dopaminergic cell bodies of the mesolimbic system, [ 3H]naloxone binding decreases significantly in septum and nucleus accumbens. The latter effect is also observed after local injection of 6-hydroxydopamine. These findings indicate that opiate receptors are localized presynaptically on dopaminergic neurons from limbic areas. They could mediate presynaptic inhibitions of dopamine release which would account for a large number of behavioral effects of morphine in single or repeated administrations.

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Abstract—Using [3H]naloxone at a concentration of 4.5 nm, the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P2 fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo‐labile than type II. [3H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (Kd) of 2 and 16 nm, respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons‐medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.

Opiate receptor: cooperativity of binding observed in brain slices.

Kinetic constants for binding of 3H-labeled morphine and naloxone were determined from Hill plot analyses and from experiments in which the concentration of tritiated drug was constant and that of nonlabeled drug varied. With brain slices, the binding of either drug exhibited strong positive cooperativity (Hill slope greater than 3); this was not observed in brain homogenates. Thus, in slices the relationship between opiate binding site and ligand may be more relevant physiologically and pharmacologically.

In vitro pharmacology of the opioid peptides, enkephalins and endorphins

In the guinea-pig ileum methionine-enkephalin, normophine and morphine are equipotent in depressing electrically evoked contractions; leucine-enkephalin has about 25% of the activity. The mouse vas deferens is more sensitive to the enkephalins which are 30 to 60 times more potent than morphine. Fragments of β-lipotropin 61–91 (β-endorphin) having sequences up to LPH 76 are more potent in the mouse vas deferens than in the guinea-pig ileum but β-endorphin is about equipotent in the two preparations. None of the peptides has antagonist activity. Methionine-enkephalin and normophine are equipotent in inhibiting [ 3H]-naloxone binding by homogenate guinea-pig brain in the absence of Na + while leucine-enkephalin has only 25% of this activity. In the giunea-pig ileum, naloxone antagonises normorphine and the enkephalins equally well whereas in the mouse vas deferens about ten times more naloxone is required for the enkephalins than for normorphine. Methionine-enkephalin depresses output of acetylcholine in the guinea-pig ileum and of noradrenaline in the mouse vas deferens.

Sympathetic ganglion cell x neuroblastoma hybrids with opiate receptors

A number of cultured cells of nerve or smooth muscle origin have been surveyed for stereospecific binding of either [ 3H] naloxone or [ 3H]dihydromorphine. Of the cell lines tested, three somatic cell hybrids, TCX17, NX1 and Z5, exhibited substantial specific binding of both naloxone and dihydromorphine. In addition, specific binding of [ 3H]naloxone by the hybrid clone 108cc15 was confirmed. The neuroblastoma, smooth muscle, glial, and other hybrid cell lines demonstrated little or no specific binding. Specific binding of [ 3H] dihydromorphine is dose dependent. Dissociation constants for the three hybrid clones range from 15 to 90 nM and maximal binding values range from 75 to 225 fmoles/mg of protein. The order of effectiveness of other opiates in displacing bound ( 3H]dihydromorphine in TCX17 cells agrees well with that found in brain. Sodium ions selectively inhibit agonist binding (dihydromorphine) but not antagonist binding (naloxone), while potassium and magnesium salts have little effect on either.

Ontogenetic development of [ 3H]naloxone binding in rat brain

To study the ontogenesis of an apparent opiate receptor, the high affinity, stereospecific binding of [ 3H]naloxone was measured in whole brains and several brain regions of the developing rat. Stereospecific binding of [ 3H]naloxone was first detected in whole rat brain at 15 days of gestation and by birth attained 40% of adult specific concentration of binding sites. Scatchard analysis indicated that developmental changes in stereospecific binding reflect increases in the number of sites with the equilibrium dissociation constant for [ 3H]naloxone remaining constant. At birth, caudal regions of the brain attained relatively higher levels of the binding site than the cortical regions. Treatment of pregnant rats with morphine or naloxone during the last week of pregnancy did not significantly affect the stereospecific binding of [ 3H]naloxone in the brains of their offspring.

Opiate receptor binding—enhancement by opiate administration in vivo

Administration of opiate agonists and antagonists to mice produces a dose-dependent 50–100 per cent enhancement of stereospecific [ 3H]dihydromorphine or [ 3H]naloxone binding to brain homogenates within 5 min. Three opiate antagonists are 10–1000 times more potent in eliciting this increase in binding than their structurally analogous agonists. Naloxone, the antagonist with the least agonist activity, is the most potent drug in producing receptor enhancement. Implantation of morphine pellets in mice increases receptor binding 30–100 per cent for 2 108 hr with no time-dependent trend. Drug-induced receptor binding enhancement appears to involve an increase in number of binding sites rather than a change in receptor affinity. Sodium, which increases binding of opiate antagonists in normal mouse brain homogenate, fails to increase binding of [ 3H]naloxone in homogenates derived from naloxone-injected mice.

Opiate receptors in mice: Genetic differences

The concentration of opiate receptors in the brains of mice was determined by means of a naloxone-binding assay. The strains of mice used in these experiments were C57BL/6By, BALB/cBy, their reciprocal F 1 hybrids, and 7 recombinant-inbred strains derived by inbreeding from the F 2 generation. These strains could be divided into 3 groups on the basis of the number of opiate receptors: high (CXBH); low (CXBK); and intermediate (all the other strains). The difference in stereospecific binding of naloxone reflects a difference in the total number of receptor sites rather than in the affinity for the drug. The recombinantinbred strains also differ in their analgesic response to morphine, as previously determined by the tail-flick assay. The differences in the number of opiate receptors are not enough to account for the genetic difference in analgesic responsiveness. Both these parameters appear to be under different genetic control, and at least 2 genetic determinants may be involved in regulating the level of opiate receptors.

Differential effects of sodium on two types of opiate binding sites

Effects of Na + on saturable naloxone binding were studied in vitro using particulate fractions obtained from rat brain homogenates. Na + stimulated the saturable naloxone binding in thalamus-hypothalamus regions, but inhibited it in cerebellum. These findings strongly support the hypothesis that two types of naloxone binding sites exist in brain tissues.

Discrimination by temperature of opiate agonist and antagonist receptor binding

Variations in incubation temperature can markedly differentiate opiate receptor binding of agonists and antagonists. In the presence of sodium increasing incubation temperatures from 0° to 30° reduces receptor binding of 3H-naloxone by 50% while tripling the binding of the agonist 3H-dihydromorphine. Lowering incubation temperature from 25° to 0° reduces the potency of morphine in inhibiting 3H-naloxone binding by 9-fold while not affecting the potency of the antagonist nalorphine. At temperatures of 25° and higher the number of binding sites for opiate antagonists is increased by sodium and the number of sites for agonists is decreased by sodium with no changes in affinity. By contrast, in the presence of sodium lowering of incubation temperature to 0° increases opiate receptor binding of the antagonist naloxone by enhancing its affinity for binding sites even though the total number of binding sites are not changed.

Genetically determined differences in the effects of morphine on mice

We have compared a variety of well known behavioral actions of morphine across four strains of laboratory mice. These measures included locomotor activation, analgesia, tolerance, dependence and drug-seeking. We found that analgesia and drug-seeking did not vary greatly among strains, although some statistically significant differences were found. The interstrain rank ordering for locomotor activity, tolerance and dependence was different for each of these measures. This suggests that each of these effects of morphine may depend upon separate mechanisms, perhaps with each under independent genetic control. Other experiments were carried out to determine whether or not there were differences in the in vivo brain uptake of an opiate or the in vitro binding of naloxone to ‘stereospecific receptors’. No significant interstrain differences could be found.

Isolation

A method is described for extraction from bovine pituitary glands of a substance that shows several properties characteristic of opiates. The material inhibits the twitch tension of the electrically stimulated guinea pig longitudinal muscle-myenteric plexus preparation and of the electrically stimulated mouse vas deferens. This inhibition is reversed and blocked by the opiate antagonist naloxone. The extract also inhibits binding of the opiate agonist etorphine and the opiate antagonist naloxone to stereospecific binding sites in synaptic membranes of guinea pig brain. The inhibition of naloxone binding is decreased by Na + in the manner characteristic of opiate agonists. The physiologic role of the pituitary opioid remains to be investigated.

Stereospecific binding of narcotics to brain cerebrosides

Cerebrosides were shown to bind etorphine and naloxone stereo-specifically with high affinity. The relative potency of several narcotic analgesics in preventing the binding of etorphine and naloxone to cerebrosides correlated well with their reported intraventricular analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Explanations for the apparent proteo-like behavior of the opiate receptor are provided.

Properties of opiate-receptor binding in rat brain.

[(3)H]Naloxone, a potent opiate antagonist, binds stereospecifically to opiate-receptor sites in rat-brain tissue. The binding is time, temperature, and pH dependent and saturable with respect to [(3)H]naloxone and tissue concentration. The [(3)H]naloxone-receptor complex formation is bimolecular with a dissociation constant of 20 nM. 15 Opiate agonists and antagonists compete for the same receptors, whose density is 30 pmol/g. Potencies of opiates and their antagonists in displacing [(3)H]naloxone binding parallel their pharmacological potencies.