Effect of β-FNA on Opiate δ Receptor Binding

Abstract: (β-FNA, the β -fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of δ-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the S receptor. In this paper we examine the effect of (β -FNA on the binding of the prototypic δ agonists, Leuenkephalin and d -Ala²- d -Leu⁵-enkephalin, its metabolically stable analogue, and show that treatment of membranes with β -FNA does lead to alterations in the in vitro properties of δ receptors.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

管花马兜铃化学成分的研究

从管花马兜铃中分得马兜铃酸-A,7-甲氧基马兜铃酸-A,马兜铃内酰胺-β-D-葡萄糖甙,尿囊素和Eupomatenoid-7。     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Redistribution of cell surface transferrin receptors prior to their concentration in coated pits as revealed by immunoferritin labels

Summary Immunocytochemistry has been used to study distribution of cell surface transferrin receptors in erythroid, leukemic (K562) cells. The cells were fixed and labelled with monoclonal (OKT-9) anti-transferrin receptor antibodies; the antibody-labelled receptors were then detected by either immunofluoresceinor immunoferritin-antimouse-antibody conjugates. Typically, the immunoferritin labels were distributed diffusely at the non-coated regions of the cell surface as well as concentrated in the clathrincoated pits. To examine further this pattern of distribution, cells were labelled at 0° C and then warmed to 37° C for zero to 30 min prior to fixation. The majority of the immunoferritin labels were initially dispersed in small groups at the non-coated regions of the cell surface (mean = 6 immunoferritin labels/cluster), but larger groups were common subsequent to incubation at 37° C (mean = 13 immunoferritin labels/cluster). However, the size of immunoferritin labels in the coated pits was unchanged (mean = 12 immunoferritin labels/pit). Immunoferritin labels were typical in coated and uncoated vesicles l min after warming to 37° C, but common in endosomes, multivesicular bodies and lysosomes by 30 min. It appears that single cell-surface receptors form large aggregates prior to their concentration in coated pits. Coated vesicles, uncoated vesicles, and endosomal vacuoles may together form the non-lysosomal compartment where the internalized receptors might be dissociated from the ligands (antibodies).     

Evidence for the appearance of an uncoupled form of the beta-adrenergic receptor distinct from the internalized receptor

Agonist treatment of C6-glioma cells induces two altered states in beta-adrenergic receptors, a low affinity for the hydrophilic antagonist CGP-12177 and a low affinity for agonists like isoproterenol. We present evidence that, in cells not treated to inhibit receptor internalization, the two properties occur with a different time course, the low affinity for isoproterenol preceding that for CGP-12177. In that the low affinity for CGP-12177 is due to the internalization of the receptor, the results indicate that uncoupling of the receptor, indicated by the low affinity for isoproterenol, occurs while the receptor is still located on the cell surface. Removal of the agonist leads to reappearance of the receptor to the plasma membrane followed by loss of the uncoupled state.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.