Mu Opioid Binding Sites Research Articles

COMT Val108/158Met genotype affects the mu-opioid receptor system in the human brain: Evidence from ligand-binding, G-protein activation and preproenkephalin mRNA expression

Recent data from [11C]carfentanil ligand-PET indicate that in the human brain, the availability of mu-opioid (MOP) receptor binding sites is affected by the Val108/158Met polymorphism of the catechol-O-methyltransferase (COMT) gene. This prompted us to validate the impact of COMT Val108/158Met on MOP receptors in human post-mortem brain. [3H]DAMGO receptor autoradiography was performed in frontal cortex, basal ganglia, thalamus and cerebellum (8 Met/Met, 6 Met/Val, 3 Val/Val). With respect to genotype, numbers of MOP binding sites in COMT Met108/158 homozygous and Val108/158Met heterozygous cases were higher than in Val108/158 homozygous. Differences were significant in the caudate nucleus (Val/Met vs. Val/Val), nucleus accumbens (Val/Met vs. Val/Val) and the mediodorsal nucleus of the thalamus (Met/Met vs. Val/Val). In the thalamus, this was corroborated by DAMGO-stimulated [35S]GTPγS autoradiography. Moreover, stepwise multiple regression taking into account various covariables allowed to confirm the COMT genotype as the most predictive factor in this structure.As a mechanism how COMT might exert its action on MOP receptors, it has been suggested that at least in striatopallidal circuits COMT Val108/158Met impacts on enkephalin, which is capable of reciprocally regulating MOP receptor expression. Thus, we assessed preproenkephalin mRNA by in situ hybridization. In the striatum, mRNA levels were significantly higher in COMT Met108/158 homozygous cases indicating that MOP binding sites and enkephalin are regulated in parallel. Moreover, the transcript was not detectable in the thalamus. Thus, mechanisms other than an enkephalin-dependent receptor turnover must be responsible for COMT-related differences in MOP binding site availability in the human brain.

Autoradiographic mapping of mu-opioid receptors during opiate tolerance and supersensitivity in the rat central nervous system.

In this autoradiographic study we have analysed the regional changes in the density of mu-opioid receptors produced by the chronic administration of sufentanil alone and after concurrent administration with nimodipine. mu-Opioid receptors in the central nervous system (CNS) of rats were labelled using 5 nM [3H]DAMGO. Sufentanil, a high-efficacy agonist, was administered for 7 days by chronic infusion (2 microg/h). Another group of animals received a simultaneous infusion of sufentanil (2 microg/h) and nimodipine (1 microg/h) for 7 days. These two drug regimes have been previously shown to induce tolerance and supersensitivity to the analgesic effect of the opioid, respectively. Our results clearly demonstrate that opioid tolerance is associated with a generalised down-regulation of mu-opioid binding sites throughout the brain and the spinal cord. Compared with the findings in tolerant animals, the CNS of animals supersensitive to sufentanil showed less down-regulation of mu-opioid receptors, to the extent that, particularly in brain areas related to nociception, such as the somatosensory cortex, central grey, raphe magnus nucleus and dorsal horn of the spinal cord, no down-regulation occurred. These neurochemical findings may contribute to the functional interaction between nimodipine and sufentanil that we have previously observed in analgesic studies.

Pharmacokinetic-pharmacodynamic analysis of the EEG effect of alfentanil in rats following beta-funaltrexamine-induced mu-opioid receptor "knockdown" in vivo.

The objective of this investigation was to determine the influence of pre-treatment with the irreversible mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) on the pharmacokinetic-pharmacodynamic (PK/PD) relationship of alfentanil in rats. The PK/PD correlation of alfentanil (2 mg x kg(-1) intravenously in 20 min) was determined in chronically instrumented rats using amplitudes in the 0.5-4.5 Hz frequency band of the EEG as pharmacodynamic endpoint. Beta-FNA was administered intravenously (10 mg x kg(-1)) either 35 min or 24 h prior to the PK/PD experiments. Pre-treatment with beta-FNA had no influence on the pharmacokinetics of alfentanil. The in vivo concentration-EEG effect relationships, however, were steeper and shifted towards higher concentrations with no difference between the 35-min and the 24-h pre-treatment groups. Analysis of the data on basis of the operational model agonism revealed that the observed changes could be explained by a 70-80% reduction in alfentanil efficacy in beta-FNA pre-treated rats. This is consistent with results from an in vitro receptor bioassay showing a 40-60% reduction in the number of specific mu-opioid binding sites in the brain. This investigation confirms the validity of a previously postulated mechanism-based PK/PD model for the effect of synthetic opiates in rats.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu 2 specificity. However, some of these ligands are able to block partially (40–60%) the high affinity (putative mu 1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu 2) sites.

Quantitative Autoradiography of Mu-Opioid Receptors in the CNS of Alcohol-Naive Alcohol-Preferring P and -Nonpreferring NP Rats

M cBRIDE, W. J., E. CHERNET, D. L. M cKINZIE, L. LUMENG AND T.-K. LI. Quantitative autoradiography of mu-opioid receptors in the CNS of alcohol-naive alcohol-preferring P and -nonpreferring NP rats. ALCOHOL 16(4) 317–323, 1998.–The densities of mu-opioid binding sites in the CNS of alcohol-naive adult male P and NP rats ( N = 9 each line) were examined using quantitative autoradiography. Coronal sections (20 μm) were prepared from frozen brains and incubated in 5 nM [ 3H]DAMGO to label mu-opioid receptor sites. Nonspecific binding was determined in the presence of 1 μM DAMGO. The amount of [ 3H]DAMGO binding was (a) 20–25% higher in the olfactory tubercle, nucleus accumbens shell and core, and basolateral and lateral amygdaloid nuclei; (b) 15% higher in the lateral septal intermediate nucleus and caudate-putamen patches; and (c) 10–30% lower in the pyramidal and radiatum layers in the CA1 region of the anterior dorsal hippocampus, ventral dentate gyrus and CA1 pyramidal layer of the posterior hippocampus, and posterior medial cortical amygdaloid nucleus of the P compared to the NP rat. No line differences were found in any of the other regions examined (e.g., the cerebral cortical subregions and layers, thalamic nuclei, ventral tegmental area, ventral pallidum, lateral hypothalamus, other regions of the hippocampus, and several subcortical structures). The innate differences in the amount of binding to mu-opioid recognition sites in certain limbic structures, such as the nucleus accumbens, amygdala, and olfactory tubercle, of the P and NP lines may be factors contributing to their disparate alcohol drinking characteristics.

Changes of mu opioid receptor binding sites in rat brain following electroacupuncture.

With [3H]-ohmefentanyl as a ligand, autoradiographic technique was used to observe the effect of electroacupuncture (EA) on mu opioid receptor binding sites in the brain areas related to pain modulation. The results were as follows: (1) The distribution of the mu receptor in the rat central nervous system was consistent in general with the results reported previously. (2) After EA of Tsu-San-Li, the mu receptor binding sites were increased significantly in the following examined structures: the caudate nucleus, septal nucleus, medial preoptic area, amygdalaoid nucleus, periaqueducal gray, interpeduncular nucleus, nucleus raphe magnus, and cervical and lumbar enlargements. The results indicate that EA is able to increase mu binding sites in the brain areas related to analgesia, suggesting the enhancement of mu receptor function by EA.

Electron microscopic distribution of mu opioid receptors on noradrenergic neurons of the locus coeruleus.

The distribution of mu opioid receptors was examined by light and electron microscopic autoradiography in the locus coeruleus of the rat following in vitro labelling with the iodinated agonist [125I]FK-33824. At the light microscopic level, specific mu opioid binding sites were concentrated over the perikarya and dendrites of neurons that were tyrosine hydroxylase-immunopositive in adjacent sections. Accordingly, both the number of tyrosine hydroxylase-immunoreactive neurons and the density of labelled mu receptors decreased markedly throughout the rostrocaudal extent of the nucleus following treatment with the catecholaminergic neurotoxin 6-hydroxydopamine. By electron microscopy, specifically labelled receptors were detected both inside and on the surface of locus coeruleus neurons. Intracellular sites were found by resolution circle analysis to be highly concentrated within the endoplasmic reticulum and Golgi apparatus, suggesting that the ligand recognizes both glycosylated and preglycosylated forms of receptor. The remainder were found mainly over the cytoplasmic matrix or intracytoplasmic vesicles, and were attributed to newly synthesized or recycled receptors in transit. Cell surface receptors were present over both dendritic and perikaryal membranes of noradrenergic cells. These were most highly concentrated opposite abutting axon terminals, suggesting the existence of receptor 'hot spots' at sites of putative endogenous ligand release. However, only a small proportion of these sites was associated with synaptic specializations. Furthermore, an important contingent was detected opposite non-axonal elements, such as dendrites and glial cells, suggesting that mu opioid ligands act mainly parasynaptically on locus coeruleus neurons. Finally, approximately 5% of labelled receptors were associated with axoglial interfaces, indicating that a minor action of mu opioids in the locus may be presynaptic and/or glial.

Morphine-6-O-beta-D-glucuronide but not morphine-3-O-beta-D-glucuronide binds to mu-, delta- and kappa- specific opioid binding sites in cerebral membranes.

We investigated the nature of interaction of morphine-3-O-beta-D-glucuronide (M3G) and morphine-6-O-beta-D-glucuronide (M6G) with opioid binding sites at the mu-, delta- and kappa-opioid receptors (mu-OR, delta-OR and kappa-OR) in cerebral membranes. Saturation binding experiments revealed a competitive interaction of M6G with all three opioid receptors. Inhibition binding experiments at the mu-OR employing combinations of morphine and M6G resulted in a rightward shift of the IC50 for morphine proportional to the M6G concentration, thus strengthening the finding of competitive interaction of M6G at the mu-opioid binding site. Data in absence and presence of M6G were included in a three-dimensional model. Compared to a model with one binding site a model with two binding sites significantly improved the fits. This might indicate that different mu-OR subtypes are involved. Hydrolysis of M6G to morphine was investigated and did not occur. Therefore the effects of M6G on binding to the mu-OR were due to M6G and not due to morphine. In contrast, M3G at the three opioid receptors was found to inhibit binding being about 300 times weaker than morphine. This effect was well explained by the amount of contaminating morphine (about 0.3%) identified by HPLC. We conclude that M6G binds to mu-, delta- and kappa-OR in a competitive manner. Some of our results on the mu-OR suggest two binding sites for agonists at the mu-OR and that M6G binds to both sites. Our results suggest that the high potency of M6G as an analgesic is mediated through opioid receptors. In contrast, M3G does not interact with the mu-, delta- or kappa-OR. We therefore doubt that any effect of M3G is mediated via opioid receptors.

Downregulation of mu-opioid binding sites following chronic administration of neuropeptide FF (NPFF) and morphine.

The effect of continuous ICV infusion of NPFF (10 micrograms/microliter) and morphine (40 micrograms/microliter) on mu-opioid binding sites was examined in rats using the in vitro radiolabeled techniques of whole-brain homogenate receptor binding and quantitative autoradiography. Mu receptors were labeled with [3H][D-Ala2-MePhe4,Glyol5] enkephalin in the homogenate binding experiments and with [125I][D-Ala2-MePhe4,Gly-ol5]enkephalin in autoradiographic studies. In homogenate binding studies, chronic administration of NPFF or morphine significantly downregulated mu receptors by 20% and 44%, respectively. Quantitative autoradiographic experiments demonstrated downregulation of mu opioid receptors in specific brain nuclei for both NPFF- and morphine-treated animals. Within the striatum and several nuclei of the thalamus, the mu receptors of the NPFF- and morphine-treated animals were decreased by 20-30% and 38-73%, respectively. These results suggest that NPFF may modulate opioid-mediated responses in part by altering the density of mu-opioid receptors.

Distribution of neuropeptide FF (FLFQPQRFamide) receptors in the adult rat spinal cord: effects of dorsal rhizotomy and neonatal capsaicin

By using quantitative autoradiography and highly selective iodinated ligands, we quantified modifications in neuropeptide FF binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8 segments) and lumbar (L3-L5 segments) enlargements in two models: (i) rats neonatally treated with capsaicin; (ii) rat submitted 15 days before to unilateral dorsal rhizotomies. We comparatively analysed the distribution of mu-opioid binding sites in the same animals. We have shown that the [125I]YLFQPQRFamide (neuropeptide FF sites) labelling is not significantly modified following selective damage of fine afferent fibres by neonatal capsaicin treatment. In the cervical and lumbar enlargements, capsaicin-treated/control binding ratios for [125I]YLFQPQRFamide were 0.90 and 0.86, respectively. While unilateral dorsal rhizotomy induced a drastic decrease in [125I]FK-33-824 labelling in the side ipsilateral to the lesion as compared to the intact side of (yielding ratios of 0.29 and 0.31 for cervical and lumbar levels, respectively), [125I]YLFQPQRFamide labelling was not significantly modified, yielding ratios of 0.98 and 0.91 for cervical and lumbar levels, respectively. These data suggest that, in contrast with a majority of mu-opioid receptors, neuropeptide FF receptors are not located on fine primary afferent fibers carrying nociceptive information from the fore- or hindlimb in the rat. This preferential postsynaptic localization, together with the reported "morphine modulating" action of this peptide, support the proposal of a role for neuropeptide FF in intraspinal modulation of nociceptive input.

Neuropeptide receptors in developing and adult rat spinal cord: An in vitro quantitative autoradiography study of calcitonin gene‐related peptide, neurokinins, μ‐opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors

A number of neuroactive peptides including calcitonin gene-related peptide (CGRP), substance P, neurokinin B, opioids, somatostatin (SRIF), galanin, neurotensin and vasoactive intestinal polypeptide (VIP) have been localized in adult rat spinal cord and are considered to participate either directly and/or indirectly in the processing of sensory, motor and autonomic functions. Most of these peptides appear early during development, leading to the suggestion that peptides, in addition to their neurotransmitter/neuromodulator roles, may possibly be involved in the normal growth and maturation of the spinal cord. To provide an anatomical substrate for a better understanding of the possible roles of peptides in the ontogenic development of the cord, we investigated the topographical profile as well as variation in densities of [125I]hCGRP alpha, [125I]substance P/neurokinin-1 (NK-1), [125I]eledoisin/neurokinin-3 (NK-3), [125I]FK 33-824 ([D-Ala2, Me-Phe4, Met(O)ol5]enkephalin)/mu-opioid, [125I]galanin, [125I]T0D8-SRIF14 (an analog of somatostatin); [125I]neurotensin and [125I]VIP binding sites in postnatal and adult rat spinal cord using in vitro quantitative receptor autoradiography. Receptor binding sites recognized by each radioligand are found to be distributed widely during early stages of postnatal development and then to undergo selective modification to attain their adult profile of distribution during the third week of postnatal development. The apparent density of various receptor sites, however, are differently regulated depending on the lamina and the stage of development studied. For example, the density of mu-opioid binding sites, following a peak at postnatal day 4 (P4), declines gradually in almost all regions of the spinal cord with the increasing age of the animal. [125I]substance P/NK-1 binding sites, on the other hand, show very little variation until P14 and then subsequently decrease as the development proceeds. In the adult rat, most of these peptide receptor binding sites are localized in relatively high amounts in the superficial laminae of the dorsal horn. To varying extents, moderate to low density of various peptide receptor binding sites are also found to be present in the ventral horn, intermediolateral cell column and around the central canal. Taken together, these results suggest that each receptor-ligand system is regulated differently during development and may each uniquely be involved in cellular growth, differentiation and in maturation of the normal neural circuits of the spinal cord. Furthermore, the selective localization of various receptor binding sites in adult rat spinal cord over a wide variety of functionally distinct regions reinforces the neurotransmitter/modulator roles of these peptides in sensory, motor and autonomic functions associated with the spinal cord.

Hypothalamic changes in norepinephrine release in rats with estradiol valerate-induced polycystic ovaries.

Chronic anovulation and polycystic ovaries (PCO) can be induced by a single i.m. injection of estradiol valerate (EV, 2 mg in oil) in the rat. Constant exposure to high plasma levels of estradiol provokes a neurotoxic effect on the hypothalamic neurons, including those from the arcuate nucleus. Because of the important participation of hypothalamic norepinephrine (NE) in the regulation of GnRH release and the possible noxious effect of prolonged exposure of these neurons to estradiol, our interest was to study the activity of the noradrenergic neurons innervating the hypothalamus. We analyzed the biosynthesis, content, and release of NE from the noradrenergic nerve terminals of the hypothalamus during the PCO condition. We found a decrease in tyrosine hydroxylase (TH) activity and in the content of dopamine (DA) in the anterior hypothalamus after 2 mo of EV injection, whereas dopamine-beta-hydroxylase (D beta H) was increased without changes in NE content. No variations in TH activity or in DA and NE contents in the medial hypothalamus were observed, but a decrease in D beta H activity was evident. After 2 mo of EV administration, an increase in the electrically induced release of NE from anterior hypothalamic blocks incubated in vitro was detected; this effect was not evidenced in the medial hypothalamus. After 5 mo of EV administration, release of NE increased in anterior hypothalamic blocks but decreased in medial hypothalamic tissue. The inhibitory effect of morphine on NE release found in control animals was increased in the hypothalamus from PCO rats, suggesting an increased number of mu-opioid binding sites in noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and distribution of a cloned rat mu-opioid receptor.

We have cloned and expressed a rat brain cDNA, TS11, that encodes a mu-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 +/- 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 +/- 0.2 nM) approximately beta-endorphin (Ki = 0.7 +/- 0.05 nM) approximately morphine (Ki = 0.8 +/- 0.5 nM) approximately [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 +/- 0.5 nM) uch much greater than U50,488 (Ki = 910 +/- 0.78 nM) > [D-Pen2,5]- enkephalin (Ki = 3,170 +/- 98 nM) > dextrorphan (Ki = 4,100 +/- 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a mu-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) in COS-7 cells 50 microM 5'-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 +/- 0.5 nM) to a lower-affinity state (IC50 = 89.0 +/- 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned mu-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the mu-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of mu-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.

Chronic I.C.V. infusion with monoclonal anti-NPFF IgG up-regulates mu opioid binding sites in rat brain: An autoradiographic study

0-0.47, p<O.05) with this behavioral measure across the combined patient groups. These results extend on our earlier postmortem brain findings and demonstrate that increased levels of these G proteins also occur in peripheral cells from BAD but not MDD subjects. Furthermore, the present findings offer additional support to the notion that disturbances in G-protein function play an important role in the pathophysiology of BAD.

Retinoic acid-induced differentiation of human neuroblastoma SH-SY5Y cells is associated with changes in the abundance of G proteins.

Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH-SY5Y cells: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased. This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-imido)triphosphate [Gpp(NH)p; 10 nmol/L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prostaglandin E1 (PGE1) receptors and Gs alpha, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1-stimulated adenylate cyclase activity, but significantly reduced amounts of Gs alpha were found. This down-regulation is paralleled by a decrease in the stimulatory activity of Gs alpha as assessed in S49 cyc- reconstitution assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetalins: opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries.

A synthetic peptide combinatorial library made up of 52,128,400 hexapeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual peptides with mu receptor affinity were found using an iterative process for successively determining the most active peptide mixtures. Upon completion of this iterative process, the three peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).

Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2 (NPFF), downregulates mu opioid binding sites in rat brain

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 μg/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the B max of mu binding sites (labeled by [ 3H][ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin) from 262 ± 12 to 192 ± 12 fmolmg protein, and increased the K d from 1.1 to 2.3 nM . Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [ 3H][ d-Ala 2-MePhe 4,Gly-ol 5]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 μg/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance. Viewed collectively with previous findings that chronic ICV infusion of anti-NPFF IgG upregulates mu receptors, these data provide additional evidence that the density of CNS mu receptors is tonically regulated by NPFF in the extracellular fluid. The action of NPFF to decrease mu receptors is consistent with an antiopioid role for this peptide; however, the fact that NPFF (administered into the lateral ventricle) did not appreciably alter expression of morphine tolerance and dependence contrasts with previous findings and reinforces the view that this effect is most reliably seen after third ventricle administration.

Long-term changes in striatal opioid systems after 6-hydroxydopamine lesion of rat substantia nigra

The effects of unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway on striatal opioid peptides and receptors were determined at different time-intervals, from three days up to 24 weeks, post-lesion. Mu, delta and kappa opioid binding site densities in the ipsilateral caudate-putamen were decreased by 25–50% in rats which exhibited a greater than 90% loss of dopamine uptake sites. Differentiation of radioligand binding to kappa 1 and kappa 2 subtypes demonstrated a selective loss of kappa 2 sites post-lesion. The onset of significant 6-hydroxydopamine lesion-induced changes in striatal opioid binding sites was delayed with respect to the loss of dopamine uptake sites. Furthermore, maximal loss of dopamine uptake sites was apparent within seven days post-lesion, but not until two to four weeks for mu, delta and kappa sites. In animals which exhibited an incomplete loss of dopamine uptake sites (less than 80%) there was no significant change in opioid binding site density. Striatal proenkephalin and prodynorphin messenger RNA levels were increased and decreased, respectively, after complete 6-hydroxydopamine lesion. Modulation of peptide messenger RNA levels was apparent within seven days and was maintained up to 24 weeks post-lesion. In contrast, proenkephalin and prodynorphin messenger RNA levels were unchanged in animals which exhibited an incomplete loss of striatal dopamine uptake sites. Taken together, these observations suggest that the majority of mu, delta and kappa 2 opioid binding sites are localized on non-dopaminergic elements in the caudate-putamen, but that substantia nigra innervation plays a role in the control of striatal opioid receptor expression. The 6-hydroxydopamine lesion-induced decreases in striatal opioid binding site density may, in part, be a function of agonist-induced receptor downregulation. Alternatively, both opioid receptor and peptide expression in the caudate-putamen may be directly, but independently, regulated by ventral mesencephalic neurons.

Effects of ovarian hormones on brain opioid binding sites in castrated female rats.

These experiments were performed to analyze whether treatments of ovariectomized female rats with ovarian steroid regimens able to induce either an increase (positive feedback effect) or a decrease (negative feedback effect) of serum levels of luteinizing hormone (LH) have some impact on the characteristics of mu-opioid binding sites in circumscribed areas of the brain. The increase of serum levels of LH elicited by a treatment with estradiol benzoate (EB) plus progesterone (P; positive feedback effect) was accompanied by a significant decrease in the number of mu-binding sites in the hypothalamus and in the corpus striatum. The decrease in serum levels of LH induced by a treatment with EB alone (negative feedback effect) brought about a significant increase of the number of mu-binding sites in the thalamus and in the hippocampus. These results seem to suggest that the release of LH induced by EB plus P may involve a decrease of hypothalamic mu-binding sites. Apparently, the inhibitory effect on LH release exerted by EB alone does not involve any change of the density of these binding sites in the hypothalamus.

Differential coupling of opioid binding sites to guanosine triphosphate binding regulatory proteins in subcellular fractions of rat brain.

In this study, we present evidence for the occurrence of mu, delta, and kappa opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5'-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and Mn++ was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with alpha-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low Km GTPase activity in SPM was greater than in microsomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more mu opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.