Effect of Comenic Acid on the Activation of G Proteins by Agonists of Opioid Receptors in Plasma Membranes from the Rat Brain

We studied the effect of comenic acid on transmembrane opioid signaling (in particular, on that mediated by -opioid receptors, with the use of an agonist of these receptors, [D-Ala²]-leucine enkephalin (DALE), and mediated by -opiate receptors, with the use of morphine). It was demonstrated that comenic acid modulates agonist-dependent binding of [³⁵S]GTPS in plasma membranes from the rat brain. The effect of comenic acid on the activation of G proteins via -opioid receptors possessing high affinity for DALE depended on the ion composition of the medium: in the presence of K⁺ the activation decreased, while in the presence of Na⁺ it remained invariable. Under the influence of comenic acid, the agonist-dependent activation of G proteins mediated by receptors with low affinity for DALE was intensified in the presence of both Na⁺ and K⁺. Using morphine, we observed opposite effects: in the presence of Na⁺ or K⁺ comenic acid decreased or increased, respectively, the agonist-dependent activation of G proteins. We suggest that due to the comenic acid-induced modulation the relative intensity of activation of G proteins, which control signal pathways activated by opioid receptors of different types, can be significantly changed.     

Hydroxyethylation of Comenic Acid

A small jar fermentor was developed in order to investigate the effect of oxygen supply on hydrocarbon fermentation. Several indices to oxygen transfer were examined with this small jar fermentor. Conditions for suitable oxygen supply were examined in l-glutamic acid fermentation from hydrocarbon by use of shaking flasks and these small jar fermentors. The data indicated that the rate of oxygen transfer ought to be more than 14.3 × 10^?7 mole/ml·min in order to obtain satisfactory results. The coefficient of oxygen transfer rate (K_La/H) decreased as the fermentation went on, so the supply of oxygen enriched gas mixture was effective to increase the production of l-glutamic acid.     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....     

Radioligands for Probing Opioid Receptors

Abstract

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains β-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu⁵] enkephalin, three [Met⁵] enkephalins, one [Met⁵] enkephalyl-Arg-Arg-Val-NH₂ (metorphamide or adrenorphin), one [Met⁵] enkephalyl-Arg-Gly-Leu and one [Met⁵] enkephalyl-Arg-Phe. [Leu⁵] enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to α- and β-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are μ, δ and ?. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.     

Mechanism of Action of Nalidixic Acid on Conjugating Bacteria

When nalidixic acid, a specific and effective inhibitor of cellular deoxyribonucleic acid synthesis, is added to conjugating bacteria at any time during mating, it stops genetic transfer provided the donor bacterium is sensitive to the drug. When this inhibition is released by the removal of the nalidixic acid, transfer does not resume at the point on the chromosome where it was stopped, but begins again at the transfer origin. Curves relating the effects of various low doses of nalidixic acid to the frequency of recombination reveal that several "hits" are necessary to inhibit recombination for early markers. The number of required "hits" decreases as the distance of the marker from the transfer origin increases. Transfer between drug-resistant cells may also be inhibited by nalidixic acid. The effect of high drug doses on matings between resistant cells is similar to that of low drug doses on matings with a sensitive male.     

Stress in Morphine-Addicted Rats: Antistressor Properties of Comenic Acid

In chronic experiments carried out on 128 rats we demonstrated that the withdrawal syndrome revealed in morphine-addicted animals is combined with the development of stress. This was reflected in an increase in the total duration of grooming behavior and in changes in its temporal pattern. In morphine-addicted animals, only the initial and final phases of the seven successive grooming phases remained clearly pronounced, whereas intermediate stages were lost. A recycling of the initial grooming stage, which could result in self-inflicted damage to the skin, was observed. Under conditions of complete discontinuation of morphine, disturbances of grooming were even intensified, and during 18 days after discontinuation of the drug such disturbances gradually disappeared. Injections of comenic acid against the background of discontinuation of morphine significantly accelerated the process of normalization of both the duration and pattern of grooming (9 days after discontinuation of the drug). In intact rats, comenic acid did not induce noticeable disturbances of grooming. We conclude that comenic acid possesses antistressor properties under conditions of the withdrawal syndrome.     

Arrest of Morphine Withdrawal Syndrome Using Comenic Acid: an Experimental Study on Rats

Chronic experiments on rats with experimental morphine addiction demonstrated that injections of comenic acid are capable of significantly arresting the withdrawal syndrome after morphine discontinuation. Neither habituation nor addiction to comenic acid were developed. Optimum doses of comenic acid have been selected (34 to 50 mg/kg per day).     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Subunit Structure and Purification of Opioid Receptors

Abstract

Considerable evidence indicates the existence of multiple types of opioid receptors. The three major types have been named mu, delta and kappa. The earlier evidence was based on pharmacological as well as membrane binding experiments. This paper will emphasize more recent studies using solubilized opioid binding sites.

Several laboratories, including our own, have succeeded in separating kappa receptors from other types. A similar separation of mu from delta receptors has not yet been achieved. By crosslinking experiments with ¹²⁵I- human beta-endorphi we have been able to provide strong evidence for differences in molecular size between the major binding components of mu (65K) and delta (53K) receptors… It is not yet established whether the difference resides in the in the protein or carbohydrate portion of these glycoproteins. These results suggest that the three major types of opioid receptors represent distinct molecular entities.

An active opioid binding protein solubilized from bovine striatal membranes has been purified to apparent homogeneity. The major purification steps involve affinity chromatography and lectin chromatography on immobilized wheat germ agglutinin. The purified material gave a single band of molecular weight 65K Da on SDS-PAGE. Its specific activity for opioid binding was ca. 13000 pmol/mg protein and its properties are those of a component of the mu receptor.     

Affinity Labeling Mu Opioid Receptors With Novel Radioligands

1. A series of novel opiate ligands based upon 6α-naloxamine have been examined in opioid receptor binding assays.2. Coupling an ethylamine spacer alone to 6-α-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa₁ opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives.3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [¹²⁵I]NalAmiBen and the azido derivative [¹²⁵I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [¹²⁵I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [¹²⁵I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [¹²⁵I]NalAziBen binding showed a band at approximately 70–80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [¹²⁵I]NalAmiBen.     

螺环哌啶类似物作为DOR激动剂的特征结构研究

阿片受体激动剂与特定阿片受体亚型结合,常用来治疗与外伤、癌症或心脏病相关的严重疼痛,是十分有吸引力的药用物质．阿片受体有3种经典亚型(δ, κ, μ),均有与其对应的激动剂．δ阿片受体(DOR)激动剂因其还有明显的抗焦虑、抗抑郁和器官保护作用,是非常有前景的药物．本文研究了一批共102个N-取代螺环哌啶类似物作为δ阿片受体激动剂的分子,采用比较分子力场(CoMFA)和比较分子相似性指数(CoMSIA)两种分析方法对所有分子进行了三维定量构效关系(3D-QSAR)研究,其中基于疏水场和氢键供体场参数建立的CoMSIA模型最佳,其模型结果为：Q²=0.501,R²_ncv=0.787,R²_pre=0.780,证明模型自我吻合良好,同时有较强的内部及外部预测能力．而模型的等势线图分析表明,在R₁处引入疏水性的取代基及在R₂处引入亲水性的取代基或氢键供体基团对提高激动剂活性有利．这些结论有助于更好地理解N-取代螺环哌啶类似物作为DOR激动剂的机理,为新型的δ阿片受体激动剂的设计和优化提供一定的指导．     

β-N-Oxalylamino-L-Alanine Action on Glutamate Receptors

beta-N-Oxalylamino-L-alanine (L-BOAA) is a non-protein excitatory amino acid present in the seed of Lathyrus sativus L. This excitotoxin has been characterized as the causative agent of human neurolathyrism, an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue-culture, and in vitro receptor binding investigations revealed that L-BOAA might mediate acute neurotoxicity through quisqualate (QA)-preferring glutamate receptors. The present study demonstrates the stereospecific action of L-BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L-BOAA was most active in displacing thiocyanate (KSCN)-sensitive specific tritiated (RS)-alpha-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding (i.e., QA receptor) (Ki = 0.76 microM) with a rank-order potency of QA greater than kainate greater than N-methyl-D-aspartate (NMDA). By contrast, the nonneurotoxic D-BOAA isomer (100 microM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L-BOAA. Scatchard analysis of L-BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L-BOAA, 265 nM) without a significant change in QA-receptor density, and Hill plots yielded coefficients approaching unity. Differential L-BOAA concentration-dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. (ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit

Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 microM cocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential kappa-agonists ethylketocyclazocine, dynorphin A1-13, dynorphin A, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] -benzeneacetamide (U-50,488), and (-)-5,9-dimethyl-2'-OH-2-tetrahydrofurfuryl-6,7-benzomorphan [(-)-MR 2034], whereas (+)-MR 2035 [the (+)-enantiomer of (-)-MR 2034] was ineffective. In contrast, the preferential delta-agonists Leu-enkephalin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalin (DADLE) as well as the mu-agonists morphine, normorphine, D-Ala2-Gly-ol5-enkephalin (DAGO), and beta-casomorphin 1-4 amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 microM) was much more potent than ethylketocyclazocine (1 microM) or DADLE (1 microM). (-)-N-(3-furylmethyl)-alpha-noretazocine [(-)-MR 2266], 1 microM, a preferential kappa-antagonist, antagonized the effect of ethylketocyclazocine more potently than (-)-naloxone or (+)-MR 2267 [the (+)-enantiomer of (-)-MR 2266]. Given alone, (-)-MR 2266 slightly and (+)-MR 2267 (1 microM each) greatly enhanced NA release, apparently due to alpha 2-adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Action of Nalidixic Acid on Escherichia coli V. Possible Mutagenic Effect

Cook, Thomas M., (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), William A. Goss, and William H. Deitz. Mechanism of action of nalidixic acid on Escherichia coli. V. Possible mutagenic effect. J. Bacteriol. 91:780-783. 1966.-With a streptomycin-dependent organism, Escherichia coli ATCC 11143, it has been shown that exposure to nalidixic acid, under conditions permitting some bactericidal action, results in an increased number of streptomycin-independent bacteria among the survivors. This effect is evident only with proliferating cultures, and is related to the concentration of nalidixic acid and the duration of exposure.     

Effects on Inflammation of Newly-Synthesized Hexapeptide with Affinity to Opioid and Nociceptin Receptors

Following the discovery of the N/OFQ/NOP system and its modulatory role in physiological and pathophysiological processes, intensive study has started to find selective NOP ligands with hypothetic therapeutic potential. Among the agonists, a hexapeptide Ac-RYYRWK-NH₂ has been identified. It expresses high NOP receptor affinity and selectivity. Its molecule was used as a template, in which Tyr⁵ was substituted by original β²-tryptophan analogue (S)-2-(1-methyl-1H-indol-3-yl)propionic residue (compound HP3) The new compound activates both NOP and opioid receptors. Having in mind that classical opioids, as well as nociceptin, are involved in modulating pain and inflammation, we examined the anti-inflammatory effect of newly-synthesized peptide HP3 on carrageenan-induced peripheral inflammation, and compared it with that of indomethacin (3 mg/kg). It was found that HP3 in dose 40 μg/kg exerts weaker anti-inflammatory action in the first 180 min of the experiment, but is equally effective with indomethacin 3 mg/kg at the end of the observation. The HP3 effect is due mainly of the activation of opioid receptors.     

Role of Opioid Receptors on Food Choice and Macronutrient Selection in Meat-Type Chick

The present study was designed to examine the role of opioid receptors on food choice and macronutrient selection in neonatal chicks. In this study, 13 experiments designed, experiments 1–3 for effect of specific opioid receptors on appetite and experiments 4–13 on effect of opioid receptors on food choice and macronutrient selection in meat-type chick. In experiment 1, chicken intracerebroventricular (ICV) injected with 125, 250 and 500 pmol of DAMGO (µ-opioid receptor agonist). Experiment 2 was conducted to investigate the effect of DPDPE (δ-opioid receptor agonist) at doses of 20, 40 and 80 nmol. In experiment 3 ICV injection of the U-50488H (κ-opioid receptor agonist, of 10, 20 and 40 nmol) was done. In experiment 4, birds injected with saline and different diets: standard diet without fat, diet containing nutrient energy 20 % higher than standard, diet containing nutrient energy 20 % lower than standard and standard diet containing fat were offered to them to investigate desire of chicken to diets. Experiments 5–7 were similar to experiment 4, except, birds ICV injected with 125, 250 and 500 pmol of DAMGO. In experiments 8–10 chicken received ICV injection of DPDPE (20, 40 and 80 nmol). The experiments 11–13 was similar to previous experiments which birds injected with different doses of U-50488H (10, 20 and 40 nmol), respectively. Then the cumulative food intake measured until 180-min post injection. According to the results, ICV injection of DAMGO diminished food intake while DPDPE and U-50488H increased appetite (P < 0.05). Despite anorexigenic effect, ICV injection of DAMGO increased birds desire to eat fat containing standard diet compared to the standard diet without fat (P < 0.05). These findings suggest endogenous opioids governing preferences for fat rich foods.