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J Barg

Publications and source records attributed to J Barg.

71 records · Page 4Linked to original sources
PubMed1991

The prenatal development profile of expression of opioid peptides and receptors in the mouse brain.

Although the postnatal development of opioid systems of mammalian brain has been well studied, little is known about the ontogeny of and relationship between embryonic (E) opioid peptides and their receptors. Moreover, a simultaneous assessment of levels of the 3 classes of opioid peptides and their putative receptors during embryonal development has not been made. To this end, the ontogeny of opioid peptides and receptors in mouse brain were examined during the period E11.5 to postnatal day 1 (P1). Met-enkephalin, dynorphin and beta-endorphin immunoreactivity were detected before their putative opioid receptors. beta-Endorphin can be discerned as early as E11.5, whereas mu binding was first observed at E12.5. Although dynorphin and Met-enkephalin were measurable at the same time as beta-endorphin, kappa-receptors were not detected until E14.5 and delta sites were not found at all prenatally. Differences in immunoreactivity levels of the 3 peptides occur with dynorphin being lower than Met-enkephalin and beta-endorphin, consistent with a low Bmax for kappa binding. Expression of the 3 opioid peptides as well as mu and kappa opioid receptors rapidly increase in parallel from E14.5 to E18.5. Interestingly, levels of beta-endorphin diminish by P1, the stage at which a sharp rise of mu receptors occurs. In a comparative study of the binding of beta-endorphin 1-31, its truncated form (1-27) and their N-acetyl derivatives to E14.5 brain membranes, beta-endorphin 1-31 exhibited the highest affinity.

Animals↗
PubMed1991

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

Animals↗
PubMed1991

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.

Aging↗
PubMed1991

Transient expression of opioid receptors in defined regions of developing brain: are embryonic receptors selective?

The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1991

Up-regulation of delta opioid receptors in neuroblastoma hybrid cells: evidence for differences in the mechanisms of action of sodium butyrate and naltrexone.

Opioid binding in subcellular fractions from neurohybrid cells was assessed using two models of up-regulation. Homologous up-regulation was achieved by treating NG108-15 cells with the opioid antagonist naltrexone. Na butyrate was added to NCB-20 cell cultures to affect heterologous up-regulation. In both paradigms light and heavy membranes were resolved by concanavalin A (con A) pretreatment of cells followed by density centrifugation. [3H][D-Ala2,D-Leu5]enkephalin (DADLE) and [3H]diprenorphine Bmax values for these fractions increased without changes in affinity. In contrast to 48 h of antagonist treatment, 5 min of exposure to naltrexone down-regulated heavy membrane delta sites. Under both conditions of up-regulation, inhibition of LM [3H]DADLE specific binding by 5'-guanylylimidodiphosphate was enhanced suggesting greater receptor coupling to guanine nucleotide binding regulatory proteins. Although attenuated by addition of cycloheximide, [3H]DADLE binding to total homogenates increased upon naltrexone treatment of NG108-15 cells. Heavy membrane Bmax values were also augmented in the presence of cycloheximide and naltrexone for 48 h. Activities of beta-glucuronidase and beta-hexoseaminidase were diminished in total homogenates and subcellular fractions from naltrexone-treated cells, suggesting an opioid-induced alteration in lysosomal enzyme trafficking. Comparable receptor down- and up-regulation and attenuation of lysosomal enzyme activity were elicited by the delta-selective opioid peptide antagonist (allyl)2 Tyr-Aib-Aib-Phe-Leu-OH. These results suggest that homologous up-regulation entails initial down-regulation and blockade of receptor degradation.

Binding Sites↗
PubMed1990

Depolarization regulates selectively the expression of different opioid receptors: a decreased number of kappa receptors in chronically activated guinea pig brain cell cultures.

Previous studies about the differential effect of chronic membrane depolarization on the expression of mu and delta opioid receptors prompted us to investigate whether the same treatment regulates also the expression of kappa opioid receptors. Embryonic guinea pig brain cells that exhibit in culture a high density of kappa receptors were treated for different time periods with potassium chloride, and the number of these receptors was determined with the universal opioid ligand [3H]diprenorphine. In a second set of experiments the cultures were treated with the sodium channel activator veratridine and opioid receptors were determined with the selective kappa ligand [3H]U69,593. The results indicate that chronic membrane depolarization (for 3 or 6 days) decreases significantly the number of kappa receptors, with no effect on the affinity of the ligands. Taken together with our previous reports, it is suggested that neuronal activation has a selective and possibly opposite regulatory role on the expression of the 3 opioid receptors.

Animals↗
PubMed1990

Differential effect of mu, delta, and kappa ligands on G protein alpha subunits in cultured brain cells.

Rat and guinea pig fetal brain cell cultures and immunoblotting techniques were used to study the effect of receptor selective opioids on the level of the membrane-bound alpha i and alpha o GTP binding protein subunits. Incubation of rat hindbrain cultures with the mu selective peptide DAGO decreased the amount of both alpha proteins. The reduction observed was equivalent to 36% in alpha o and 41% in alpha i. On the other hand, incubation of rat forebrain cultures with this peptide had an opposite effect, increasing the alpha o and alpha i levels by 66% and 68%, respectively. This differential effect of the peptide on the G proteins at the two brain areas may reflect the selective interaction at the receptor level; DAGO induced a fast and effective receptor down-regulation (50% decrease in Bmax) in hindbrain but not in forebrain cultures. Moreover, delta and mu selective ligands differed in their effect, as indicated by the finding that the delta selective peptide DPDPE increased the amount of both alpha proteins in hindbrain cultures by 40%. Similar experiments conducted with guinea pig brain aggregate cultures indicated that the kappa selective agonist U50,488 decreased the amount of the membrane bound alpha i protein subunit by 56%. The results thus indicate that opioid agonists, interacting selectively with the three types of opioid receptors, induce a complex repertoire of changes in the immunoreactive levels of the membrane-bound alpha GTP binding protein subunits in various CNS structures.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1989

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.

Adenylyl Cyclases↗
PubMed1989

Expression of the three opioid receptor subtypes mu, delta and kappa in guinea pig and rat brain cell cultures and in vivo.

Expression of the three opioid receptor subtypes mu, delta and kappa in aggregating cell cultures prepared from embryonic guinea pig or rat brains was compared with the in vivo expression of the receptors in the brain of developing and adult animals of the same species. At the day of culturing, one third of the receptors in the brain of guinea pig embryos were of the kappa type. In culture, however, the aggregating brain cells acquired within 14 days a high percentage (75%) of kappa receptors. As only 28% of the receptors in the adult guinea pig brain are of this subtype, an attempt was made to further analyse the specificity of this developmental process. In guinea pig, the 2-fold increase in kappa receptors in culture was accompanied with a decline in both the percentage and the density (per protein) of mu and delta subtypes. In contrast, a marked increase in delta receptors was observed in rat whole brain, forebrain or hindbrain cultures. Thus, the developmental pattern of the three receptor subtypes in rat brain cultures, but not in guinea pig, was similar to that in vivo. These and additional experiments suggest that at the developmental stage taken to prepare the cultures, neurons expressing opioid receptors were already programmed in the rat but not in guinea pig brain.

Animals↗
PubMed1989

Developmental profile of kappa, mu and delta opioid receptors in the rat and guinea pig cerebellum.

The phenotypic expression of the three opioid receptors kappa, mu and delta was determined in the rat and guinea pig cerebellum during ontogeny. In both species, the increase in cerebellum weight was accompanied with a gradual increase in the total number of opioid receptors. At the same time, however, the receptor concentration (femtomoles per milligram protein) fluctuated, and finally declined by 3.3- and 2.0-fold in the adult rat and guinea pig cerebella, respectively. In the guinea pig, kappa receptors constituted about 80% of the receptors at the earliest developmental stage tested, day 20 of the embryo, but then there was a sharp decrease in this value, resulting from an increase in both mu and delta receptors. This decrease was, however, transient, and the percentage of the kappa receptors increased again to reach 77% of the receptors in the adult cerebella. A similar transient increase in the percentage of mu and delta receptors was observed during the development of the rat cerebellum, although the receptor density in this species was 1/4-1/8 that of the guinea pig. Cultures of aggregating guinea pig cerebellar cells were used to further study the factors regulating the expression of the three opioid receptors in this brain region. The possibility that the development in vivo of cells expressing different opioid receptors is associated with the ontogeny of the endogenous opioid peptides is discussed.

Aging↗
PubMed1984

Up-regulation of opiate receptors by opiate antagonists in neuroblastoma-glioma cell culture: the possibility of interaction with guanosine triphosphate-binding proteins.

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5'-triphosphate (GTP) analogue guanosine 5'-[beta, gamma-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in delta type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.

Animals↗
PubMed1984

Characterization and down-regulation of opiate receptors in aggregating fetal rat brain cells.

Aggregating brain cells prepared from embryonic rats bind radioactive opiates in a stereospecific manner. The drug selectivity, receptor content during culturing and down-regulation of these apparent opiate receptors were studied in aggregates prepared from the embryonic hindbrain or forebrain. The receptor content in hindbrain but not forebrain aggregates was increased up to 3-fold after 21 days in culture. Differences between the receptors of the two types of aggregates were also observed in the affinity of opiate alkaloids and D-Ala2,D-Leu5-enkephalin (DADL). The potent opiate alkaloid etorphine induced down-regulation of opiate receptors in aggregates prepared from either brain region whereas DADL was a potent down-regulator in the forebrain but not in the hindbrain aggregates and morphine had no effect in both tissues. The implications of these results concerning the control of various types of opiate receptors in the whole animal are discussed.

Animals↗
PubMed1983

Down-regulation of opiate receptors in serum-free cultures of aggregating fetal brain cells.

Brain cells from rat embryos that were cultured in serum-free medium form aggregates which possess stereospecific binding of radioactive opiates. The specificity of these opiate receptors and their regulation by opiate alkaloids and enkephalin was studied. D-ala2, D-leu5-enkephalin (DADL) induced down-regulation of opiate receptors in aggregates prepared from the forebrain but had no effect on hindbrain aggregates, whereas the potent alkaloid etorphine induced down-regulation of the receptors in both tissues. Interestingly, morphine did not induce down-regulation in both tissues. The significance of these findings and their relationship to the control of different types of opiate receptors are discussed.

Animals↗
PubMedDate not supplied

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.

Animals↗