Naloxone Binding Sites Research Articles

On the mechanism of opioid-oestradiol interactions

Characteristics of opioid binding and possible relationships between oestradiol and opioid binding sites were studied in rat oestrogen sensitive tissues (uterus, preoptic area-anterior hypothalamus, median eminence-basal hypothalamus). Naloxon (Nal) and oestradiol (Oe) bindings were assessed by in vitro saturation analyses. In 800 g supernatants of both uterine and hypothalamic tissues homogenates high affinity ( K d : 2−4 × 10 −9M) and low capacity [ 3H]Nal binding sites were found. These binding sites were sedimented from 800 g supernatant by further centrifugation at 10 5 g for l h. In competition studies [ 3H]Nal binding was completely prevented by morphine, while met-enkephalin and leu-enkephalin caused only a partial inhibition. [ 3H]Nal binding was increased by ovariectomy and decreased by Oe treatment (10μg/100g b.wt) in both tissues. The cytoplasmic [ 3H]Oe binding in the studied tissues seems to be affected by the naloxone binding system. After in vitro saturation of naloxone binding sites by naloxone the [ 3H]Oe binding to low affinity sites (type II) in hypothalamus as well as in uterus has been increased by 8- and 2-fold, respectively. These results indicate the presence of specific [ 3H]Nal binding in rat uterus with similar properties to those found in the hypothalamus. Furthermore an interaction between opioid and oestradiol receptor systems could be also suggested.

Conformation and electrostatic potential surfaces of opiates: Relationship to µ- and λ-site binding.

Molecular mechanics energy minimization and electrostatic potential surface calculations have been used to examine a series of opioid compounds that interact with the µ opoid receptor and a recently discovered high-affinity naloxone binding site (the λ site). The compounds studied include members of the morphinan, 4,5-epoxymorphinan, and benzomorphan families. All compounds bind with a high affinity at the µ opiate receptor site, but only the 4,5-epoxymorphinans bind tightly at the λ site. The results suggest that conformational differences in the various families do not satisfactorily explain the observed trends in binding affinity at the λ site. However, electrostatic potential surfaces for a representative subset of these opioids exhibit patterns that allow us to classify members as high-affinity or low-affinity λ-site ligands in good agreement with the experimental results. The procedures used in this work may be useful in defining characteristics that impart selectivity for opiate receptor subtypes such as the µ, δ, and κ receptors.

Direct effects of the adrenergic neurotoxin DSP4 on central opiate receptors: implications for neuroendocrine studies.

We have shown that the noradrenergic neurotoxin DSP4 can acutely abolish naloxone-induced secretion of LH in the rat. A part of this influence is clearly related to an unexpected interaction with hypothalamic opiate ( [3H]-naloxone) binding sites. Injection of DSP4 (50 mg/kg) can severely reduce opiate binding assayed in vitro. In addition, the drug is very potent in blocking opiate receptors in vitro, as determined in slice and homogenate assays. Consideration must now be given to the possibility that many previous studies on acute effects of DSP4 undoubtedly involved the opiatergic system in addition to noradrenergic terminals. Thus, DSP4 is not the drug of choice for experiments designed to probe catecholamine-opiate interactions in the control of LH release.

Specific binding of3H-naloxone with isolated rat enterocytes

Specific 3H-naloxone binding with isolated rat enterocytes has been demonstrated. This binding was selectively inhibited by different opiates and proved to be irreversible, temperature-dependent and sodium azide-sensitive. The presence of naloxone binding sites on enterocytes indicates a possible involvement of opiates in the regulation of enterocyte functions.

Characterization of a labile naloxone binding site (lambda site) in rat brain.

A high-affinity binding site selective for naloxone and other 4,5-epoxymorphinans (lambda site) has been previously described in rat brain. Following homogenization of freshly dissected brain, the lambda sites convert from a high-affinity to a low-affinity state. When measured with [3H]naloxone, the decay is very rapid at 20 degrees C (t 1/2 less than 2 min), whereas it is progressively slowed at lower temperatures. Proteinase inhibitors, antoxidants, and sulfhydryl group-protecting agents failed to prevent this conversion. Kinetic measurements of mu and lambda binding at varying temperatures demonstrated that the decrease in lambda binding does not coincide with the concurrent increase in mu binding and that the loss of high-affinity lambda binding at 20 degrees C can be partially restored when the temperature is lowered to 0 degrees C. The low-affinity state of the lambda site is rather stable in the Tris buffer homogenates and is susceptible to digestion by a protease. The (-)-isomer of WIN 44,441, a benzomorphan drug, binds to lambda sites with moderate affinity (dissociation constant, KD = 63 nM), whereas the (+)-isomer does not (KD greater than 10,000 nM), thus establishing stereoselectivity of the binding process. Neither the high-affinity nor the low-affinity state of lambda binding is significantly affected by the presence of 100 mM sodium chloride or 50 microM Gpp(NH)p, (a GTP analog), which is in contrast to the dramatic effect of these agents on the established opioid receptor system. Naltrexone, naloxone, nalorphine, and morphine (in this order of decreasing potency) bind to the lambda site in vivo in intact rat brain over dosage ranges that are commonly employed in pharmacological studies.

Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain

Using adjacent section autoradiography-immunocytochemistry, the distribution of [ 3H]naloxone binding sites was studied in relation to neuronal systems containing [Leu]enkephalin, dynorphin A, or β-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of [ 3H]naloxone, the pharmacological (μ-like) properties of which appear unaltered. In contrast, specific binding of the δ ligand [ 3H]D-Ala 2,D-Leu 5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, we have noted associations between [ 3H]naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex; for example, in caudate-putamen, patches of opioid receptors did not correspond to the distribution of enkephalin immunoreactivity, but there was a correspondence between subcallosal streaks of binding sites and enkephalin. The complexity of the association between [ 3H]naloxone binding sites and the multiple opioid systems, and previous reports of colocalization of μ and κ receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post-translational processing in determining the physiological consequences of opioid neurotransmission.

Increase in rat cortical [ 3H]naloxone binding site density after chronic administration of antidepressant agents

Given chronically, the first and second generation antidepressants imipramine and citalopram, and antidepressant neuroleptics levomepromazine and chlorprothixene, elevated moderately but significantly (by 15–30%) the density of [ 3H]naloxone binding sites in rat cerebral cortical membranes. There were only small non-significant changes in K d values.

Monoaminergic antagonists which block naloxone-induced release of luteinizing hormone bind selectively to hypothalamic opiate receptors

The possibility that adrenergic receptor antagonists which prevent naloxone-induced release of luteinizing hormone (LH) in vivo exert their action by direct competition with naloxone for hypothalamic opiate receptors was investigated in vitro in immature female rats. First, 26-day-old rats were injected with prazosin, an α 1-adrenergic blocker, or yohimbine, an α 2-adrenergic blocker, before receiving naloxone (2.5 mg/kg body wt.). Both adrenergic antagonists prevented naloxone-provoked LH secretion in a dose-dependent manner with yohimbine exhibiting a slightly greater potency. In a separate experiment hypothalami from 26-day-old rats were removed, membrane pellets prepared and incubated with [ 3H]naloxone in the presence of increasing concentrations of naloxone or various monoamine-active substances. Phentolamine, prazosin and yohimbine were the most effective competitors for naloxone binding sites while pronethalol, methysergide and metergoline were far less effective. These findings parallel the relative inhibitory potencies of these compounds in vivo for preventing naloxone-induced LH release as shown here and in a previous report 5. Clonidine and l-phenylephrine, both α-adrenergic agonists, also showed activity in the binding assay. Surprisingly, α-methyl-p-tyrosine and 5-hydroxytryptophan, substances which substitute for monoamine precursors early in the biosynthetic pathway, also displaced [ 3H]naloxone from hypothalamic receptors. These results offer a mechanism for the modulating effects of monoamine-active drugs on opiate antagonist-induced LH release and may have significance for inhibition of LH secretion by endogenous opiates.

Opiate receptors on lymphocytes and platelets in man

Receptors for opiates, opiate-like substances, and their antagonists, such as naloxone (a close chemical and conformational congener of morphine), on brain cell homogenates and neuroblastoma × glioma hybrid cells in tissue culture have been reported. The present study on the binding of [ 3H]naloxone to lymphocytes and platelets freshly isolated from the peripheral blood of 39 healthy adult human volunteers showed that (1) [ 3H]naloxone bound to lymphocytes and platelets at 4°C, reaching equilibrium in 30 min, and was not removed by washing (three times) with the suspending medium; (2) the binding of [ 3H]naloxone to cells decreased in the presence of increasing amounts of unlabeled naloxone, approaching a plateau; (3) significant amounts of the radioligand remained bound in the presence of micromolar quantities of the unlabeled ligand; and (4) in the absence of Na + ions, 1 to 10 nmol of morphine hydrochloride for 10 6 lymphocytes, and 1 to 25 nmol of morphine hydrochloride for 10 8 platelets, decreased the binding of [ 3H]naloxone by 43 to 57%. It is concluded that at least some of the [ 3H]naloxone binding sites on human lymphocytes and platelets are specific opioid receptor sites of the μ type (Enkephalins define the δ sites.) The observations on the binding of naloxone to cells do not appear to be artifacts. Opioid receptor sites on lymphocyte and platelet membranes may have properties similar to those on nerve cell membranes.

Restoration of mean arterial pressure in endotoxic shock by meptazinol: Contributions from lysosomal membrane stabilisation, opiate antagonism and noradrenaline release

Meptazinol elevated mean arterial pressure in rats which had been treated with endotoxin. The drug also reduced the titer of circulating lysosomal enzymes. This effect was secondary to the restoration of mean arterial pressure (MAP). In vitro, meptazinol stabilised lysosomal membranes, increased noradrenaline release and interacted with the opiate receptor (naloxone-binding site) as an antagonist. The relevant contributions of these phenomena to the restoration of MAP are discussed.

Levorphanol-sensitive [ 3H]naloxone binding in developing brainstem following prenatal morphine exposure

Levorphanol-sensitive [ 3H]naloxone binding was measured in brainstems of fetuses and postnatal off-spring of rats injected with morphine or saline or pairfed with morphine-injected animals during the last half of gestation. Brainstem receptor concentrations in saline-injected animals decreased by 25% from gestation day 21 to postnatal day 60, while [ 3H]naloxone binding affinity doubled during the same interval. Chronic morphine exposure decreased receptor concentration at the 21st day of gestation by 28–48%, without changing binding affinity. However, the number and affinity of [ 3H]naloxone binding sites in morphine-injected animals were not different from controls at 60 days postnatally.

Opiate binding to brain slices and ontogenesis of hypothalamic [3H]naloxone binding sites

We have developed a radioligand binding assay based on the use of hypothalamic slices and have examined the ontogenesis of [3H]naloxone binding sites in male and female rats. [3H]NAL binding is reversible, saturable, stereospecific, of high affinity, readily displaceable by morphine and is sensitive to phenoxybenzamine. These characteristics suggest that [3H]NAL readily binds to opiate receptors in brain slices. With this assay we have demonstrated that: (a) there is an age-related increase in opiate binding sites in rat hypothalamus, (b) there are sex differences in the binding affinity of the sites and (c) the values of Bmax are approximately 2–5-fold higher than the levels previously reported from assays with brain homogenates.

Synthesis and pharmacological studies of 4,4-disubstituted piperidines: a new class of compounds with potent analgesic properties.

A series of 4,4-disubstituted piperidines has been synthesized and evaluated for analgesic activity. Several of these analogues show analgesic potency comparable to morphine in the mouse writhing and tail-flick tests. A number of compounds exhibit high affinity for [3H]naloxone binding sites in rat brain membranes. Among the most potent derivatives are compounds 15 and 48. Although opiate-like, attempts to modify this activity with various substituents have failed to produce antagonistic properties. A few of these analogues also show marked long-lasting serotonin antagonism in the guinea pig serotonin toxicity test and the DL-5-hydroxytryptophan induced head-twitch model in the mouse.

Ontogenesis of opiate binding sites and radioimmunoassayable beta-endorphin and enkephalin in regions of rat brain.

The postnatal changes in the levels of radioimmunoassayable enkephalin and beta-endorphin, as well as the densities of [3H]methionine-enkephalin and [3H]naloxone binding sites in rat cerebellum, brainstem and whole forebrain were determined. The opiate peptides and the opiate binding sites reached their highest levels at the first week postpartum in the cerebellum, at the second week in the brainstem and at the third week in the whole forebrain. This finding is in line with the developmental profiles of other well-established neuronal pathways which also showed a caudal-to-rostral sequence of development. Moreover, there was a close relationship between the elevation and decline in the amounts of opiate binding sites and in the levels of opiate peptides in each brain region. These observations are consistent with other evidence which suggests that enkephalin and beta-endorphin are functioning as neurotransmitters or neuromodulators in the central nervous system.

U-50488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse

U-50,488H is a chemically novel analgesic that is a potent opioid-like agent on the mouse tail flick and electrically stimulated guinea pig ileum tests. U-50,488H is a very weak competitor for naloxone binding sites in brain and ileum. However, the drug has high affinity for kappa receptor binding sites revealed by competition for EKC sites in the presence of dihydromorphine. Morphine has both supraspinal and spinal sites of action since it was a potent analgesic after both intracranial and intraspinal injections. However, U-50,488H works predominantly at the spinal level. Dynorphin may be an endogenous ligand at this site. Studies on cat dorsal horn neurons suggest that U-50,488H analgesia may be due to an increase in threshold for neuron excitation.

Development of methionine-enkephalin and naloxone binding sites in regions of rat brain

The development of opiate receptors in whole forebrain, brain stem and cerebellum of developing rats was measured by specific [ 3H]methionine-enkephalin ([ 3H]Met-Enk) and [ 3H]naloxone binding. The level of opiate receptor as determined by the binding of these two opiates varied with brain region as well as with age; however, the amount of [ 3H]naloxone bound in the same brain region obtained from animals of the same age was greater than that of [ 3H]Met-Enk. Kinetic analyses of these two opiate binding sites in newborns of the same age revealed that they differed in their apparent affinities and receptor numbers. Moreover, both morphine and naloxone were equally active in competing for [ 3H]Met-Enk binding in all 3 brain regions during the first month after birth, whereas methionine-enkephalin was only about 30–50% as active as morphine in competing for [ 3H]naloxone binding in that period. These results suggest that in the rat brain there are two types of opiate receptors and that the heterogeneity of opiate receptors is already apparent during early postnatal life.

Opiate receptor development in midbrain and forebrain of posthatch chicks

Specific binding of [ 3H]naloxone to midbrain and forebrain was examined from chicks which were either 1, 7, 14, 21 or 28 posthatch days of age. While there was a significant age-related increase in the total number of [ 3H]naloxone binding sites in both brain regions, the increase was greater in magnitude in forebrain than in midbrain. In both brain regions, there was a concomitant age-related decrease in the density of receptor sites without any alteration in receptor affinity for naloxone. The decrease in receptor density was more rapid in onset and was greater in magnitude in midbrain than in forebrain.

Seasonal variation in the apparent number of binding sites for 3H-opioid agonists and antagonists

Throughout the year, a radioreceptor assay was used to investigate the number of binding sites for several opioid ligands in C57BL/6J mouse brain homogenates. The mean number of sites observed for 3H-morphine and 3H-dihydromorphine was about one third less than the mean for 3H-D-ala 2-met 5-enkephalinamide, 3H-naloxone or 3H-naltrexone. Some seasonal variation was observed in all the opioids studied; the seasonal fluctuation observed in the apparent number of naloxone binding sites was the greatest. These seasonal alterations may play a role in the annual pattern of several physiological processes in which endorphin systems are directly or indirectly involved.

Effect of lithium and sodium ions on opiate- and dopamine-receptor binding.

The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-3H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [3H]naloxone and [3H]DHM. Binding of [3H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [3H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.

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.