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C J Coscia

Publications and source records attributed to C J Coscia.

At least 73 records · Page 4Linked to original sources
PubMed1985

Differential up-regulation of microsomal and synaptic membrane mu opioid receptors.

Naltrexone was administered to rats for 7 days by osmotic minipump (5 mg/kg/day) and thereupon, forebrain mu opioid receptor levels in subcellular fractions were monitored by homologous displacement of [3H]D-ala2-mePhe4-gly-ol5 enkephalin binding. Microsomes displayed increases in mu receptor concentrations that were twofold greater than those associated with synaptic plasma membrane fractions (92 vs. 51%). Levels in crude membranes rose 77%. Binding affinities were unchanged.

Animals↗
PubMed1985

Substantial reduction of noradrenaline in kitten visual cortex by intraventricular injections of 6-hydroxydopamine does not always prevent ocular dominance shifts after monocular deprivation.

Ten kittens had cannulas inserted into their lateral ventricles for daily injections of 6-hydroxydopamine (6-OHDA). At 5-6 weeks of age one eye was sutured shut, and one week later recordings were made from the visual cortex to assay the ocular dominance of a sample of cells. In six kittens the injections of 6-OHDA were continued until the day before recording, while in four kittens the injections were stopped around the time of eye suture, on the assumption that continued injections of 6-OHDA over several days has effects that are not specific to the noradrenaline (NA) system and that the two procedures might show different results. In all animals the concentration of NA in the visual cortex near the site of recording was reduced by approximately 90%. In all animals the ocular dominance histograms recorded from the visual cortex were shifted so that the majority of cells (83 +/- 13%) were dominated by the open eye. There were no substantial differences between the two groups of experimental animals or between the experimental animals and two control animals that had cannulas implanted and ascorbate alone injected without 6-OHDA. We conclude that the concentration of NA in the visual cortex can be reduced substantially by injections of 6-OHDA into the lateral ventricle without preventing the shift in ocular dominance that usually occurs after suturing shut the eyelids of one eye.

Animals↗
PubMed1985

Stereospecific opiate-binding sites occur in coated vesicles.

We prepared clathrin-coated vesicles from bovine forebrain utilizing sucrose or deuterium oxide-Ficoll density gradient centrifugation followed by permeation chromatography. Homogeneity was monitored by electron microscopy (EM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). EM revealed that the predominant (up to 98% of the total) organelles were coated vesicles and empty hexagonal baskets. Diameters of the coated vesicles ranged from 37 to 120 nm with a mean of 65.2 +/- 2.2. Upon SDS-PAGE of the coated vesicle fraction, the most prominent band appeared at 180,000 daltons. There were also three additional bands at 100,000, 50,000 and 35,000 daltons, giving the overall pattern characteristic of coated vesicles. Both 0.5 nM tritiated naltrexone and etorphine displayed specific binding to coated vesicles. Naltrexone binding in coated vesicles from gradient fractions was increased 2.5-fold over the original 100,000 X g pellet. An additional 4-fold enrichment in specific binding was observed after permeation chromatography which was concomitant with an increase in the volume density of coated vesicles in electron micrographs. Naltrexone binding was stereospecific and etorphine binding was inhibited by 100 mM NaCl (40%). Both naltrexone and etorphine binding were inhibited by 50 microM guanyl-5'-yl imidodiphosphate (40 to 50%). In summary, purified bovine brain-coated vesicles contained high affinity stereospecific opiate alkaloid-binding sites with characteristic opioid binding properties.

Animals↗
PubMed1985

Differential ontogeny of multiple opioid receptors (mu, delta, and kappa).

We investigated the postnatal ontogeny of opioid receptors in rat brain under assay conditions which, when combined with computerized analysis, effectively reflect the developmental profile of high affinity binding to mu, delta, and kappa subpopulations. Concentrations of mu sites were assessed with the selective ligand 3H-[D-ala2,mePhe4,gly-ol5]enkephalin (DAGO). The other two sites were analyzed in binding assays with less selective radioligands but in the presence of specific unlabeled ligands which suppress cross-reactivity. We utilized 3H-[D-ala2,D-leu5]enkephalin (DADL) in the presence of 10 nM DAGO to label delta sites and 3H-ethylketocyclazocine (EKC) in the presence of 100 nM DADL + 100 nM [D-ala2,mePhe4,Met(0)ol5]enkephalin to detect kappa receptors. After birth, the density (femtomoles per milligram of wet weight) of mu sites declined for several days and then rose sharply over the next 2 weeks, increasing 2-fold by adulthood. Delta (delta) sites appeared in the second week postnatal and increased more than 8-fold in the next 2 weeks. Levels of kappa receptors were relatively low at birth and increased slowly (2-fold, overall). Computerized analyses of binding data revealed that DAGO and DADL were binding to single populations of sites throughout the postnatal period. DAGO and EKC affinities did not fluctuate in this period, whereas DADL affinities were low for the first week and then rose to adult levels. In summary, mu, kappa, and delta receptors exhibit differential postnatal developmental profiles. The former two are present at birth, whereas the latter appears in the second week. The postnatal increase for all three sites appear to be preceded by the previously demonstrated emergence of opioid peptides.

Animals↗
PubMed1984

Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors.

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.

Animals↗
PubMed1984

Microsomal opiate receptors: characterization of smooth microsomal and synaptic membrane opiate receptors.

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.

Animals↗
PubMed1984

Substantial reduction of cortical noradrenaline by lesions of adrenergic pathway does not prevent effects of monocular deprivation.

We tested the theory that depletion of noradrenaline reduces the plasticity of the visual cortex in kittens by using another method of depletion. Lesions were made in the lateral hypothalamus to interrupt fibers in the dorsal noradrenergic bundle going from the locus ceruleus to the telencephalon. The lesions were induced at approximately 3 1/2 weeks of age in kittens; approximately 2 weeks later one eye was sutured shut, and about 10 days after that cells were recorded in the visual cortex. The location of the lesions was verified histologically, and the effect of the lesions was verified by noradrenaline analyses (high pressure liquid chromatography-electrochemistry) of samples from the visual cortex. The noradrenaline content of the visual cortex was reduced by 70 to 90%. However, the majority of cells recorded in the visual cortex could not be driven through the eye that had been sutured closed. The ocular dominance histograms for cells in the visual cortex were indistinguishable from those of kittens that were monocularly deprived for a similar period in the "critical period" and that had normal amounts of noradrenaline in their visual cortex. Therefore, we conclude that reduction of the noradrenaline content of the visual cortex by 70 to 90% is insufficient, by itself, to prevent the physiological changes that occur in the visual cortex after monocular deprivation.

Animals↗
PubMed1983

Localization of enzymes and alkaloidal metabolites in Papaver latex.

In continuing studies on the metabolic activity of Papaver somniferum, latex has been examined for its enzyme and alkaloidal metabolite content. After an initial centrifugation of latex at 1000g, the pellet which contained a heterogeneous population of dense organelles was further resolved on sucrose gradients. Of the enzymes monitored, acid phosphatase and L-3,4-dihydroxyphenylalanine decarboxylase were found to be in the latex 1000g supernatant, whereas catecholase (polyphenolase) was localized in two distinct organelles within the 1000g sediment. The lighter organelles, sedimenting at 30% sucrose, contained a soluble enzyme which was readily released on organelle plasmolysis, whereas the catecholase found within the heavier organelles, sedimenting at 55-60% sucrose, was membrane bound and showed significant activity only in the presence of Triton X-100. These latter organelles also contained the alkaloids, including morphine and thebaine, and were observed to readily accumulate [14CH3]morphine. The alkaloid precursor, dopamine, was localized in the same dense vesicle fraction as the alkaloids. The rate of uptake of [7-14C]dopamine into these fractions at room temperature, however, was markedly lower than that of morphine. Electron microscopic examination of the organelles of various densities revealed that they possessed different morphology. The results are consistent with the concept that both the 1000g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a subpopulation of dense organelles found in the 1000g sediment have at least a function as a storage compartment for both alkaloids and their catecholamine precursor.

Alkaloids↗
PubMed1983

Differential inhibition of activated tyrosine hydroxylase.

Under conditions of cyclic AMP-dependent protein phosphorylation, tyrosine hydroxylase (EC 1.14.16.2; TH) is activated. Kinetic analysis reveals that, upon activation the affinity of the enzyme cofactor tetrahydrobiopterin, Vmax, as well as the Ki of its putative feedback inhibitor dopamine, are increased. Catecholic inhibitors of rat striatal TH have been assessed for the structural requirements that impart differential sensitivity to activated and control enzyme. By varying cofactor and inhibitor concentrations, Ki's were generated from Dixon plots. Structural analogs of dopamine in which the amino group was fixed in a cis conformation, i.e., 6,7-dihydroxytetrahydroisoquinolines, exhibit the same Ki for activated and nonactivated TH. However, 2-amino-6,7-dihydroxytetralin (ADTN), in which the nitrogen is extended in a fixed trans conformation of the beta-rotamer, exhibited a fourfold increase in Ki upon assaying tyrosine hydroxylase under phosphorylation conditions. By systematically increasing the hydrophobicity of the substituent at C-1 of 1-carboxy-6,7-dihydroxytetrahydroisoquinolines the inhibitory potency was enhanced, suggesting the presence of a hydrophobic region near the catecholic binding site. If the hydrophobic group was rigid as in the catechol estrogens, 2-hydroxy-estradiol and 2-hydroxyestrone, the Ki was relatively low (2 X 10(-5) M) despite the absence of an amino group. Upon activation the Ki increased fourfold. These studies provide insight into the topography of the catecholic binding site on TH and to attendant changes occurring upon activation. The results suggest that the catechol binding site includes both amino group-interacting and hydrophobic regions which are influenced by enzyme activation.

Animals↗
PubMed1983

Ontogeny of benzomorphan-selective (kappa) sites: a computerized analysis.

In an investigation of the postnatal development of kappa opiate receptors, the affinity and capacity of 0.5 nM [3H]-ethylketocyclazocine (EKC) binding in crude rat brain homogenates was measured by displacement with unlabeled EKC, morphine, or D-ala2-D-leu5-enkephalin (DADL). Displacement curves were analyzed using a weighted, non-linear regression, curve fitting computer program. At all stages of development, [3H]-EKC binding fit a two site model significantly better than a one site model. Affinities of EKC, morphine, or DADL for the high affinity [3H]-EKC binding site did not change during the postnatal period. The density of the high affinity [3H]-EKC binding site increased linearly with age, whereas the levels of the low affinity site rose more rapidly during the second week.

Aging↗
PubMed1982

Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity.

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.

Animals↗
PubMed1982

Tetrahydroisoquinolinecarboxylic acids and catecholamine metabolism in adrenal medulla explants.

In a study of the relationship of the tetrahydroisoquinolinecarboxylic acids (TIQCAs) to catecholamine metabolism, we have investigated their effects on cultured rat adrenal medulla explants. Medullae were incubated in medium containing norlaudanosolinecarboxylic acid (NLCA) or 3',4'-deoxynorlaudanosolinecarboxylic acid (DNLCA) (0.5 mM) in the presence and absence of [3H]tyrosine. By paired-ion reverse-phase high pressure liquid chromatography, tissue epinephrine (EPI), norepinephrine (NE), dopamine (DA) and TIQCA were resolved. Endogenous concentrations were measured with electrochemical detection, and radioactivity was assayed by collecting appropriate effluents. Tissue levels of the TIQCAs reached saturating levels of 0.36 mM by about 20 hr. DNLCA elicited a significant decrease (60%) in endogenous DA, NE and EPI at 40 hr, whereas only DA was depressed at 30 hr. NLCA had little effect after 30 or 40 hr. When tissues were maintained in the presence of alpha-methyltyrosine (0.5 mM) for 40 hr, catecholamine levels were depressed to an extent similar to that observed with DNLCA. Incubation with [3H]tyrosine in the presence of TIQCAs revealed inhibition of tyrosine uptake and suggested a reduction in the rate of catecholamine synthesis. These results are consistent with previous data on the inhibition of tyrosine 3-monooxygenase by DNLCA in vitro.

Adrenal Medulla↗
PubMed1982

Tetrahydroisoquinolinecarboxylic acids and regulation of phenylethanolamine N-methyltransferase in cultured adrenal medulla.

Explants of rat adrenal medullae, cultured for up to 48 hr in the presence of 3',4'-deoxynorlaudanosolinecarboxylic acid (DNLCA) or alpha-methyltyrosine, exhibited a 69% increase in phenylethanolamine N-methyltransferase (PNMT) activity as measured in dialyzed homogenates. A related tetrahydroisoquinoline, norlaudanosolinecarboxylic acid (NLCA), when added to the medium did not elevate PNMT activity. No increase in the amount of PNMT was detected by immunochemical titration of homogenates from DNLCA-treated cultured medulla, nor were there changes in rates of synthesis or degradation of the enzyme. Although DNLCA is an inhibitor of tyrosine 3-monooxygenase, it had no effect on PNMT activity when added directly to an incubation mixture in vitro. Kinetic analyses of dialyzed homogenates from explants cultured in the presence of DNLCA revealed that the Vmax of PNMT was higher than that of control tissue. There was no decrease in Km after DNLCA treatment. The increase in PNMT activity appears to be a compensatory response to depletion of medullary catecholamines by DNLCA or alpha-methyltyrosine.

Adrenal Medulla↗
PubMed1982

Demonstration of a slowly dissociating form of bovine hippocampal synaptic membrane opiate receptors.

We studied the binding characteristics of opiate receptors in synaptic plasma membranes isolated from bovine hippocampus. On the basis of kinetic binding studies the interaction of [3H][D-Ala2,D-Leu5]enkephalin (DADL) with its receptor was an extremely slow process. Rates of association and dissociation were determined and a pseudo-first order rate constant for association calculated to be 5.68 x 10(5) l/mol . s at 25 degrees C. The rate of dissociation (t 1/2 = 70 min) was accelerated by GTP and changed from linear to biphasic. The kinetically derived equilibrium dissociation constant (0.29 nM) was considerably lower than the KD obtained from Scatchard and Hill plots (1.24 nM). Measurement of DADL association as a function of temperature yielded a linear Arrhenius plot. Finally, competition binding assays revealed that delta-specific agonists exhibited relatively high potency in displacing [3H]DADL from synaptic plasma membranes receptors whereas mu-specific agonists and antagonists were less effective. These results with bovine hippocampus may be explained by the formation of a slow-dissociating, high affinity agonist conformation of the delta-opiate receptor which has been predicted for the system by the cyclic-allosteric and ternary complex models.

Animals↗
PubMed1982

Differential postnatal development of mu and delta opiate receptors.

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).

Aging↗
PubMed1982

Quinone reductases of higher plants.

NAD(P)H: quinone oxidoreductase (DT-diaphorase) was detected in 100000 x g supernatant fractions of extracts of a wide variety of higher plants. Smaller amounts were also found in microsomes and chloroplast fractions. The enzyme was partially purified from soluble extracts of several plants and the quinone reductase from Catharanthus roseus was enriched 25-fold. Plant quinone reductases have molecular weights in the range of 38000-53000 as determined by gel filtration. The plant enzyme is far less sensitive to dicoumarol than its mammalian counterpart and it is inhibited by superoxide dismutase. Quinone reductase is capable of reducing simple p-benzoquinone and naphthoquinone including vitamins K3 and K1. These results indicate that, although the plant enzyme exhibits a similar substrate specificity, it is distinguishable from mammalian DT-diaphorase particularly with respect to its mechanism of reduction.

Chloroplasts↗