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.     

Purification Of Mouse and Human Interferons: Detection Of Subunit Structure

During the purification of mouse and human interferons, multiple active components have been detected. Mouse interferon was purified over 500-fold by differential precipitation, centrifugation, gel chromatography, and isoelectric focusing. On electrofocusing, two molecular forms (A and B) were noted. Form B (pI 7.35) had a molecular weight of about 38,000 and Form A (pI 7.15), which was equally active, a molecular weight of 19,000. Purified Form B was dissociable into Form A, but the reverse reaction occurred to a much lesser extent. Human interferon, purified about 1500-fold, is also composed of multiple molecular forms. Form B (pi 5.60) had a molecular weight of about 24,000 and Form A (pi 5.35), which may contain up to 85% of the total activity, a molecular weight of 12,000. Both forms appear to have equal specific activities. The dissociation of both human and mouse interferons into subunits appeared to take place during dialysis versus low salt (0.01 M tris pH 7.A). The data are consistent with the idea that the native molecule exists as a dimer of similar or identical subunits. Dimer formation, which probably occurs within the cells, does not seem to lead to a measurable cooperative effect between the subunits.     

Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.     

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH₄)₂SO₄ precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.     

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.     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

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.     

一种新阿片肽的分离纯化

报道了一种新阿片肽OP1的分离纯化。将重组毕赤酵母经适宜的生长和表达培养后,所得的发酵液经离心得无细胞上清液,上清液经超滤后过Sephadex G-10柱。将经Sephadex G-10柱所得具有阿片活性的粗组分用HPLC-MS分析,根据阿片肽N-端均有一个酪氨酸残基,且在肽链的第三或第四位上有一个芳香族氨基酸残基这一性质,依据分子量确定活性组分中可能存在的所有阿片肽,然后根据这些阿片肽的等电点,利用AKTA Purifier 100快速纯化系统的DEAE-阴离子交换纤维素柱将其进一步分离,活性组分再用Sephasil peptide C18反相高压液相柱分离得到活性组分OP1肽,鉴定纯度后测定其氨基酸组成。最后确定该肽的一级序列为YPFPGPIRYG,该阿片肽序列目前尚未见报道。     

Binding Interactions with the Complementary Subunit of Nicotinic Receptors

The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)₂(β4)₃. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.     

Evidence for Disulfide Bonds in Membrane-Bound and Solubilized Opioid Receptors

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane-bound and digitonin-solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D-Ala2-D-Leu5-enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin-solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2. [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.     

Delta Opioid Receptors: The Link between Exercise and Cardioprotection

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

大鼠Gs alpha亚基的原核表达和纯化

用 PCR的方法 ,在大鼠 Gs alpha亚基的 C端引入了 6个外源组氨酸 (即 6×His- Tag)并以此为纯化标记 .构成的表达载体 p QE60 /rat Gsα( L)在大肠杆菌 BL2 1 ( DE3)中获得了稳定的表达 .经 DEAE- Sephacel离子交换柱和 Ni- NTA Agarose亲和层析获得纯化的具有较高 [35S]- GTPγS结合活力的重组大鼠 Gs alpha亚基