Mu Sites Research Articles

Activation-induced cytidine deaminase targets SUV4-20-mediated histone H4K20 trimethylation to class-switch recombination sites

Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin (Ig) genes. Association of AID with RNA Pol II and occurrence of epigenetic changes during Ig gene diversification suggest participation of AID in epigenetic regulation. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. Here, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that AID binding to the IgH locus promotes an increase in H4K20me3. In 293F cells, we demonstrate interaction between co-transfected AID and the three SUV4-20 histone H4K20 methyltransferases, and that SUV4-20H1.2, bound to the IgH switch (S) mu site, is replaced by SUV4-20H2 upon AID binding. Analysis of HIGM2 mutants shows that the AID truncated form W68X is impaired to interact with SUV4-20H1.2 and SUV4-20H2 and is unable to bind and target H4K20me3 to the Smu site. We finally show in mouse primary B cells undergoing class-switch recombination (CSR) that AID deficiency associates with decreased H4K20me3 levels at the Smu site. Our results provide a novel link between SUV4-20 enzymes and CSR and offer a new aspect of the interplay between AID and histone modifications in setting the epigenetic status of CSR sites.

P5.033 Bringing HIV Testing to the People- Benefits of Mobile Unit Testing in Lima, Peru, 2007–2009

BackgroundMobile unit (MU) HIV testing is an alternative method of providing healthcare access. We compared demographic and behavioural characteristics, HIV testing history, and HIV prevalence between people seeking testing at...

Muoniated radical states in the group 16 elements: Computational studies

Recent experimental studies on positive muon implantation in silicon, selenium, and tellurium have been interpreted on the basis that the primary paramagnetic species observed is XMu ( X = S , Se, or Te), the muonium-substituted analog of the appropriate diatomic chalcogen monohydride radical. However, temperature-dependent signal visibility, broadening, and hyperfine shift effects remain puzzling. The interplay of degeneracy, spin–orbit coupling, and vibrational averaging in these species makes them computationally challenging despite their small size. In this work computational studies are carried out on all hydrogen isotopomers of the series OH, SH, SeH, and TeH. Several different methodological approaches are compared, and the effects of wavefunction symmetry, spin–orbit coupling, and zero-point vibrational corrections on the isotropic and anisotropic components of the hyperfine interaction are examined. Additionally, some models of the Mu site in rhombic sulfur are briefly considered.

Separation of Binding Affinity and Intrinsic Activity of the Potent μ-Opioid 14-Methoxymetopon

Receptor binding studies of 5,14-O-dimethyloxymorphone (14-methoxymetopon) in brain membranes have established its high affinity for mu-binding sites, but its analgesic potency far exceeds the modest increase in binding affinity relative to other opioids. The current study has established the selectivity of [(3)H]14-methoxymetopon for mu sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine mu-opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [(3)H]14-methoxymetopon for the variants expressed in Chinese hamster ovary cells was quite high, with K(D) values around 0.2 nM for all of the variants with the exception of mMOR-1F (K(D) of 1.2 nM). The affinity for most of the expressed variants was greater than that seen in the brain membranes (K(D) of 0.99 nM). Functionally, in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays with the MOR-1 variants, 14-methoxymetopon and the mu-opioid peptide [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) showed similar efficacies, as determined by maximal stimulation, but 14-methoxymetopon was up to 65-fold more potent than DAMGO. The greatest difference was seen with mMOR-1E and the least with mMOR-1C, which displayed only a 10-fold difference. These potency differences in the stimulation of [(35)S]GTPgammaS binding far exceeded the differences in binding affinity. The differences between 14-methoxymetopon and DAMGO remained after normalizing the potency shifts based upon receptor binding affinities and varied from 1.2-fold with mMOR-1C to 21-fold for mMOR-1 and 42-fold with mMOR-1F. Thus, 14-methoxymetopon is a potent agonist against all of the mMOR-1 splice variants, but its potency ranged widely despite similar binding affinities for most of the variants and may give insight into its unusual pharmacological profile.

Developmental and regional alteration of kappa-opioid receptors in seizure-susceptible EL mouse brain.

The binding of [3H]ethylketocyclazocine ([3H]EKC) under the suppression of mu and delta sites in the brain of EL mice (seizure-susceptible) was examined to determine the relationship between seizures and the dynorphinergic system. The density of kappa-opioid receptors in the cerebrum of adult EL mice during interictal periods significantly increased, without changes in apparent affinities, compared with that of adult ddY mice (seizure-nonsusceptible; the mother strain of EL). Subsequently, the binding of 0.8 nM [3H]EKC in 8 brain regions was examined in the 2 strains. The [3H]EKC binding in 25-day-old EL mice that had no seizures significantly increased in the hippocampus and amygdala. At the age of 50 days, EL mice displayed abortive seizures, and the number of kappa sites in EL mice was significantly greater in the hippocampus, amygdala and cerebral cortex. It was further shown that the binding of [3H]EKC in 150-day-old adult EL mice during interictal periods was markedly increased in the hippocampus, amygdala, cerebral cortex and striatum, compared with the corresponding regions in ddY mice. The up-regulation of kappa receptors in the EL mouse brain may result from deficits in endogenous dynorphins, which could be involved in the pathogenesis of seizure diathesis and seizure manifestations in the EL mouse.

Opiate receptor binding, guinea pig ileum activity, and prolonged analgesia induced by the brain peptide Tyr-W-MIF-1 and two potent analogs

Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH 2) is a peptide isolated from human and bovine brain with opiate activity. Two synthetic analogs of Tyr-W-MIF-1 were compared with the parent compound, DAMGO and Tyr-MIF-1 for (a) binding to opiate receptors (b) activity in the guinea pig ileum assay, and (c) analgesic activity after ICV administration to the rat. All compounds tested showed preferential binding to mu receptors. Tyr-MIF-1 showed relatively low affinity, but high selectivity (∼400-fold selectivity for mu over delta and more than 500-fold selective for mu over kappa sites). Tyr-W-MIF-1, which induces prolonged analgesia after ICV administration, showed affinity for mu sites that was 10-fold higher than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa sites. Two cyclized analogs of Tyr-W-MIF-1 approached the potency of DAMGO in several assays. The analogs showed 50- and 20-fold increases in affinity (IC 50 of 6 and 18 nM) for the mu site relative to Tyr-W-MIF-1, with 50-fold selectivity for mu over delta sites, and lack of binding to non-opiate Tyr-MIF-1 sites. The analogs were active in the guinea pig ileum assay with IC 50s of 10 and 30 nM. Dose-dependent analgesia after ICV administration of one of the analogs persisted for about an hr after 1 μg, for 3 hr after 25 μg, and for more than 6 hr after 100 μg. For both analogs, analgesia after 1 μg was similar to that seen with DAMGO. Thus, an endogenous brain peptide (Tyr-W-MIF-1) that does not contain the sequence Tyr-Gly-Gly, together with its structural analogs, can provide a range of selectivity patterns for binding to opiate receptors and potent opiate agonist activity.

Novel distribution of sodium-dependent [ 3H]naloxone binding sites in rat brain

Sodium is known to increase opioid antagonist binding (1). Although the alkylating agent N-ethylmaleimide (NEM) inhibits normal [ 3H]naloxone binding, it has no effect on sodium-dependent [ 3H]naloxone binding (2). These studies were performed to determine whether NEM would also selectively reveal sodium-dependent [ 3H]naloxone binding sites in tissue sections and whether sodium-dependent sites have the same anatomical distribution as normal [ 3H]naloxone binding sites. Displacement binding assays showed that sodium-dependent [ 3H]naloxone binding sites were low affinity mu sites uncoupled from G proteins. Autoradiographic studies showed that the anatomical distribution of sodium-dependent [ 3H]naloxone binding was distinct from that of normal [ 3H]naloxone binding, with the highest levels in the bed nucleus of the stria terminalis, lateral hypothalamus, lateral amygdala, median raphe nucleus and parabrachial nucleus.

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to opioid receptors in frog brain membrane preparations.

A tritiated heptapeptide, [3H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3H]Met-enkephalin-Arg6-Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent KD value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The KD determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (nH = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met-enkephalin-Arg6-Phe7 binding reflects a kappa 2- and/or delta-subtype specificity of the heptapeptide. Binding to a kappa 1 and/or mu site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg6-Phe7 in frogs cannot be ruled out. The [3H]-Met-enkephalin-Arg6-Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.

Mu, delta, and kappa opiate receptor binding of Tyr-MIF-1 and of Tyr-W-MIF-1, its active fragments, and two potent analogs

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH 2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 μM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (K i = 71nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH 2, show high affinity as well as potent analgesic activity.

Multiple opiate receptor subtypes are involved in the stimulation of growth hormone release by beta-endorphin in female rats.

The growth hormone (GH) secretory response to beta-endorphin and the involvement of opiate receptor subtypes in this response were determined in diestrous female rats. The involvement of the mu (mu), delta (delta) and/or kappa (kappa) site was determined by administering specific antagonists for each of these sites prior to beta-endorphin. beta-Funaltrexamine (1 or 5 micrograms) was administered to block mu sites, ICI 154,129 (5 or 25 micrograms) blocked delta sites and nor-binaltorphimine (8 micrograms) blocked kappa sites. The ability of these antagonists to block GH secretion following intravenous morphine administration was also determined. The opiate antagonists and beta-endorphin were administered into the lateral ventricle. A dose-response study for beta-endorphin indicated that 0.5 micrograms beta-endorphin was the minimum stimulatory dose for GH release, producing an approximately 4-fold increase in circulating levels of GH; lower doses of beta-endorphin did not stimulate secretion. All three antagonists were capable of blocking the stimulatory effects of beta-endorphin. These results provide evidence that all three opiate receptor subtypes are involved in the stimulatory effect of beta-endorphin on GH release.

Opioid-inhibited adenylyl cyclase in rat brain membranes: lack of correlation with high-affinity opioid receptor binding sites.

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.

Effect of brain lesions on [3H]ohmefentanyl binding site densities in the rat striatum and substantia nigra.

We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent changes in the subcellular distribution of rat brain mu-opioid receptors and GTP binding regulatory proteins.

The relative subcellular distributions of mu-opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1-day-old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of mu-opioid sites were assessed using [3H] [D-Ala2,Me-Phe4,Gly-ol5]enkephalin. P1 LMs possessed 43% of the total mu-opioid binding detected compared to 16% in the adult. Although NaCl inhibited mu binding in LMs to a greater extent than in HMs, age-dependent differences were not observed. P1 LM mu binding possessed greater sensitivity to 5'-guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Go alpha protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Go alpha after one- or two-dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular mu sites than adult LMs.

Where are the mu receptors that mediate opioid analgesia? An autoradiographic study in the HAR and LAR selection lines.

One line (strain) of mouse has been selectively bred in our laboratory for 15 generations to exhibit a very high sensitivity to levorphanol-induced analgesia on the hot plate assay (HAR or high antinociceptive response line). Concurrently, a second line (LAR or low antinociceptive response line) has been bred in the opposite direction, i.e., to exhibit a very low sensitivity under the same conditions. This has resulted in a 7-fold difference in sensitivity between HAR and LAR mice as a result of changes in gene frequency. Receptor autoradiographic studies with 3H-DAGO were carried out in the central gray to find receptor populations differing greatly in density between HAR and LAR mice to parallel their in vivo sensitivity differences: such receptors would then be implicated in mediating in vivo analgesia. The caudal portions of the dorsal raphe nucleus (DRN) showed 1.5- to 2-fold differences in density of mu sites, while the periaqueductal gray (PAG) showed relatively small differences. These results strongly suggest that mu receptors in a portion of the DRN are involved in mediating analgesia due to systemically administered opioids in this population of mice.

Modulation of the binding characteristics of hypothalamic mu opioid receptors in rats by gonadal steroids

Recent evidence suggests that the effects of the opioids on gonadotropin release may depend on the endocrine status existing in the experimental animal. In the brain, the effects of the opioids are exerted through the interaction with different classes of opioid receptors (mu, delta, kappa, etc.). Among these, the mu receptors appear to be particularly relevant to the control of gonadotropin secretion. Different groups of experiments have been performed in the rat in order to analyze whether changes of circulalting levels of sex steroids may have an impact on the binding characteristics of hypothalamic mu opioid receptors, as evaluated by a receptor binding assay performed on plasma membrane preparations, using [ 3H]dihydromorphine as a mu ligand. In a first series of experiments, it has been observed that the ontogenesis of hypothalamic mu opioid receptors is different in male and in female rats: the concentration of mu sites, similar in animals of the two sexes at 16 days of age, increases in females, but not in males, between day 16 and day 26 of life. This sexual difference persists in 60-day old animals, when the brain is fully mature. It has also been observed that the pattern of maturation of hypothalamic mu receptors can be reversed by neonatal castration of males and by neonatal testosterone treatment of females. In a second series of experiments, it has been shown that in the hypothalamus of regularly cycling female rats the concentration of mu receptors varies during the different phases of the estrous cycle. In particular, a rather high density of mu sites during diestrus day 2 and the morning of the day of proestrus was found; this is followed by a progressive decline during the afternoon of the day of proestrus and the day of estrus, with a minimum value of the concentration of mu receptors being recorded in the first day of diestrus. These fluctuations seem to be linked to the physiological changes of serum levels of ovarian steroids: in fact, in a third series of experiments, it has been found that the positive feedback effect on LH release, exerted by the treatment of ovariectomized female rats with estrogens plus progesterone, is accompanied by a significant decrease of the concentration of hypothalamic mu opiod receptors; treatments with estrogens alone, able to induce a negative feedback effect on LH secretion, are not associated with modifications of hypothalamic mu receptors. These data seem to indicate that hypothalamic mu receptors may be involved in the positive but not in the negative feedback control of LH secretion. Finally, it has been observed that during pregnancy there is a significant increase of the concentration of hypothalamic mu opioid receptors followed by a return towards control levels during the postpartum period. On the other hand, the simulation of the levels of sex steroids present during the first half of pregnancy, obtained through a long-term treatment of ovariectomized animals with estrogens plus progesterone, was not accompanied by any modification of the concentration of hypothalamic mu receptors. On the basis of these last results, the authors would then be inclined to suggest that, during pregnancy, the concentration of hypothalamic mu opioid receptors is not linked to endocrine phenomena, but may be related to the stressful conditions represented by pregnancy itself and by the oncoming delivery. The present findings strongly suggest that, in particular conditions, a strict link may exist between the endocrine environment and the binding characteristics of mu opioid receptors at hypothalamic level.

High resolution radioautographic localization of [ 125I]FK-33-824-labelled mu opioid receptors in the spinal cord of normal and deafferented rats

Recent data have shown that [ 125I] d-Ala 2, MePhe 4, Met(o)ol 5-enkephalin (FK-33-824) is a highly selective and specific mu opioid receptor ligand [Moyse et al. (1986) Peptides 7, 351–355]. This probe was used here to investigate the detailed radioautographic distribution of mu sites at various levels of the spinal cord. [ 125I]FK-33-824 binding sites were localized by both tritium-sensitive film and liquid emulsion radioautography in the spinal cord of naive and deafferented rats. In naive animals, high densities of mu sites were apparent within laminae I–II at all levels of the dorsal horn, with higher levels of labelling seen in layer IIi as compared to IIo in the lumbar segment. Laminae III–IV contained about half the quantities of binding observed in superficial layers. Relatively high densities of sites were also seen over lamina VI in the upper cervical cord and throughout Clarke's column. Within the latter, [ 125I]FK-33-824 binding clearly spared the large perikarya of the spinocerebellar neurons. In the ventral horn, [ 125I]FK-33-824 binding was mainly concentrated in layer IX, at the level of cervical and lumbar enlargements. Labelled sites were confined to the neuropil, mostly sparing the soma of motoneurons. Significant decreases in [ 125I]FK-33-824 binding in laminae I–II (55%) and III–IV (28%) were detected four days following cervical (C3–C7) or lumbar (L1–L6) rhizotomies. These decrements were most evident at seven days post-lesion at C3–C7 levels (93 and 76% in laminae I–II and III–IV, respectively) and recovered slightly thereafter up to 28 days post-lesion. In contrast, dorsal rhizotomies did not influence mu labelling in either the ventral horn or Clarke's column. These results confirm the association of mu opioid binding sites with dorsal primary afferent fibres and demonstrate the presence of mu sites in Clarke's column and lamina IX of the ventral horn. These findings suggest that endogenous opioids in the spinal cord play a role in sensory motor integration as well as in the modulation of primary nociceptive inputs.

Bacteriophage Mu sites and functions involved in the inhibition of λ:: Mini-Mu growth

To better understand the nature of the mini-Mu-directed process which results in inhibition of λ:: mini-Mu growth we characterized spontaneous deletion mutants of the λ:: mini-Mu phage. On the basis of analysis of the deletion endpoints, mini-Mu replication functions, and integration and inhibition properties, the λ:: mini-Mu deletion mutants were divided into five classes which define the Mu sites and functions involved in λ::mini-Mu growth inhibition. Class 1 mutants, which still exhibit λ::mini-Mu growth inhibition, collectively delete all the Mu late functions encoded by the mini-Mu. Class 2 and 5 mutants, which show cis-dominant defects in inhibition and integration, delete the right and left mini-Mu attachment sites, respectively. Phages of Classes 3 and 4, which delete the Mu B or A and B genes, respectively, show recessive defects in growth inhibition. The properties of these mutants define the Mu replication functions, A and B, and the Mu attachment sites as essential for the inhibition of λ:: mini-Mu growth. The observation that the sites and functions essential for Mu replication also have requisite roles in the inhibition of λ:: mini-Mu growth suggests that inhibition results from mini-Mu-promoted replicative interference of λ::mini-Mu development.

Apparent down-regulation of rat brain mu- and kappa-opioid binding sites labelled with [3H]cycloFOXY following chronic administration of the potent 5-hydroxytryptamine reuptake blocker, clomipramine.

This study examined the effect of chronic clomipramine administration on opioid mu- and kappa-binding sites. Clomipramine (5 mg kg-1 day-1) or saline was administered to rats via osmotic minipumps for 3 days or 28 days. Lysed-P2 brain membranes were prepared and preincubated for 60 min without (control membranes) or with 1 microM of the mu-selective acylating agent, 2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid azole-HC1 (BIT), to deplete membranes of mu-binding sites. [3H]6-Desoxy-6 beta-fluoronaltrexone ( [3H]cyclo FOXY) was used to label mu and kappa-binding sites. Weighted nonlinear least squares analysis of cycloFOXY binding surfaces permitted determination of the Kd and Bmax values of mu- and kappa-binding sites in control and treated rats. Subacute (3 days) administration of rats with clomipramine had no significant effect on [3H]cycloFOXY binding. Chronic (28 days) administration of clomipramine produced a small (approximately 10%) but statistically significant decrease in the Bmax. These findings are discussed in reference to other studies that have examined the effect of chronic antidepressant administration on opioid receptors, and speculate that the endogenous opioid systems may play a role in obsessive-compulsive disorder.

14.beta.-(Bromoacetamido)morphine irreversibly labels .mu. opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond

Paradoxical and subtype‐specific effects of opiate antagonists on the expression of opioid receptors in rat brain cultures

Cultures of aggregating fetal rat brain cells express mu, delta, and kappa opioid receptors. The potent and long-lasting opioid antagonist naltrexone was used to investigate whether different regulatory mechanisms are involved in the expression of the three receptor subtypes. In cultures treated for seven days, naltrexone increased dose-dependently the binding of 3H-diprenorphine to the three receptor subtypes, with the mu sites being affected at a lower concentration than the other two; A Scatchard analysis indicated that this receptor up-regulation was obtained by an increase in the BMax, with no significant change in the affinity of the ligand to the receptors. In contrast to these effects in cultures treated for 7 days, it was surprising to find that a 48 hr treatment with naltrexone had an apparent converse and subtype-specific influence; the antagonist decreased significantly the binding of 3H-diprenorphine to both mu and delta receptors but had no effect on kappa sites. Two other opioid antagonists, naloxone and levallorphan, had a similar effect. Further analysis of naltrexone's mode of action was obtained by studying its effect on the adenylate cyclase activity. Of several inducers of this enzyme, the beta-adrenergic inducer isoproterenol gave the highest increase in cyclic AMP. Naltrexone had no significant effect on the basal adenylate cyclase activity but it altered the pattern of cyclic AMP formation in isoproterenol-stimulated cultures. Overall, the results indicate that in addition to its classic antagonistic activity, naltrexone exhibits in fetal brain aggregates some properties associated with opiate agonists.