Localization of the kappa opioid receptor in lipid rafts

Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined whether kappa opioid receptors (KOR) in human placenta and FLAG (DYKDDDDK)-tagged human KOR (FLAG-hKOR) expressed in Chinese hamster ovary (CHO) cells are localized in lipid rafts and whether changes in cholesterol contents affect hKOR properties and signaling. Lipid rafts were prepared from placenta membranes and CHO cells expressing FLAG-hKOR using the Na2CO3 method and fractionation through a sucrose density gradient. The majority of the KOR in the placenta and FLAG-hKOR in CHO cells, determined by [3H]diprenorphine binding and/or immunoblotting with an anti-FLAG antibody, was present in low-density fractions, coinciding with high levels of caveolin-1 and cholesterol, markers of lipid rafts, which indicated that the KOR is localized in lipid rafts. Pretreatment with 2% methyl beta-cyclodextrin (MCD) reduced cholesterol content by approximately 48% and changed the cells from spindle-shaped to spherical. MCD treatment disrupted lipid rafts, shifted caveolin-1 and FLAG-hKOR to higher density fractions, increased the affinity of (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488H) for the hKOR, and greatly increased U50,488H-induced [35S]guanosine 5'-O-(3-thio)triphosphate binding and p42/44 mitogen-activated protein kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with Galphai proteins and MCD treatment reduced caveolin-1 associated with Galphai proteins, which may contribute to the enhanced agonist-induced G protein activation. Caveolin-1 also immunoprecipitated with FLAG-hKOR, but MCD treatment had no effect on the association. Thus, the KOR is located in lipid rafts and its localization in the microdomains greatly affects coupling to G proteins.     

生物玻璃和生物胶原膜联合应用于即刻义齿种植

目的：建立即刻种植动物模型,观察生物玻璃和生物胶原膜在即刻种植中引导骨组织再生、促进骨愈合效果. 方法：实验方法：取成年家犬12只,随机编号后分为4个组,在全麻下分别拔除双侧下颌第一双尖牙,制备近中拔牙窝并即刻植入种植体.前3个组分别应用生物玻璃填塞种植体与拔牙窝骨壁之间的间隙,或在种植床上方覆盖生物胶原膜,并尝试两种方法的联合应用,空白组不采用特殊处理.②观察指标：术后观察记录种植床愈合情况.术后4,8,12周分批处死动物并取其下颌骨标本,采用大体观察、X射线、普通光镜组织学方法和扫描电镜方法观察其在种植床中引起的骨组织再生变化过程和骨结合率,统计比较各种方法的成功率. 结果：①即刻种植成功率：总体成功率为95.8%,其中应用生物玻璃填塞种植体周围间隙可以诱导骨组织再生,其成功率均为100%,在种植床上方覆盖生物胶原膜者成功率为91.7%.②诱导骨组织再生作用：单用生物玻璃或两种方法联合应用诱导骨再生效果均较快,8周时表现骨质沉积量明显较多,其成骨呈多中心性,12周时可以诱导再生成熟的骨组织,并使种植体达到骨结合;应用生物胶原膜成骨呈向心性,成骨速度稍慢,12周时可以引导再生成熟的骨组织,并使种植体达到骨结合;空白组12周时在种植体的上部仍有较大部分软组织存在. 结论：在即刻种植中应用生物玻璃和生物胶原膜均可以诱导骨组织再生,促进骨愈合,获得较高的种植成功率;两者联合应用的成骨效果较单独应用生物胶原膜好.     

Intracisternal thyrotropin-releasing hormone-induced vagally mediated gastric protection against ethanol lesions: Central and peripheral mechanisms

Abstract The vagus is involved in mediating gastric cytoprotection and adaptive cytoprotection. However, the central and peripheral mechanisms through which the vagus expresses its action are still poorly known. Medullary thyrotropin-releasing hormone (TRH) plays an important role in the vagal regulation of gastric function. The stable TRH analogue, RX 77368, micro-injected into the cisterna magna or the dorsal motor nucleus (DMN) of the vagus at a dose that did not influence gastric acid secretion prevented gastric injury induced by intragastric administration of 60% ethanol in conscious or urethane-anaesthetized rats. The cytoprotective action of TRH is mediated through vagal cholinergic release of prostaglandin E₂ (PGE₂). Prostaglandin E₂ action is unrelated to changes in gastric mucosal blood flow (GMBF). In addition, other peripheral mechanisms involve calcitonin gene-related peptide (CGRP) contained in capsaicin sensitive afferent fibres and nitric oxide, both of which mediate the associated increase in GMBF induced by intracisternal injection of RX 77368. These data indicate that medullary TRH induces vagally mediated gastric protection against ethanol lesions. Its action is expressed through the muscarinic dependent release of PGE₂ and nitric oxide, and efferent function of capsaicin-sensitive afferent fibres releasing CGRP.     

(-)U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] induces internalization and down-regulation of the human,but not the rat,kappa-opioid receptor: structural basis for the differential regulation

We showed previously that prolonged activation by (-)U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] led to internalization and down-regulation of the human kappa opioid receptor (hkor), but not the rat kappa opioid receptor (rkor). Herein, we investigated structural determinants in the receptors underlying these differences using chimeric and mutant receptor constructs epitope tagged with FLAG and stably expressed in Chinese hamster ovary cells (CHO). The FLAG-hkor, but not the FLAG-rkor, underwent internalization and down-regulation after exposure to (-)U50,488H. Monensin did not have any effect on the intracellular receptor pool of the FLAG-rkor or rkor with or without (-)U50,488H treatment, indicating that the lack of (-)U50,488H-induced internalization is not due to rapid resurfacing of the rkor. Two chimeric receptors, FLAG-h/rkor and FLAG-r/hkor, were generated, in which the C-terminal domains of the hkor and the rkor were switched. The FLAG-r/hkor displayed significant (-)U50,488H-induced internalization and down-regulation, whereas the FLAG-h/rkor did not, indicating that the C-terminal domain contributes to the differences between the rkor and the hkor. To further characterize, we generated two mutants, FLAG-hkorS358N and FLAG-rkorN358S in which the locus 358 was exchanged. The FLAG-hkorS358N mutant displayed greatly reduced (-)U50,488H-induced internalization and no down-regulation compared with the FLAG-hkor, indicating that Ser358 in the hkor is critical for these processes. However, the FLAG-rkorN358S mutant was internalized, but not down-regulated, demonstrating that N358 prevents the rkor from being internalized, but it may not have a role in the lack of down-regulation of the rkor. In addition, the trafficking of the FLAG-rkorN358S mutant seems to be more complex than the rkor and the hkor.     

Dual ultrastructural localization of mu-opiate receptors and substance p in the dorsal horn

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics.     

Immunolabeling of mu opioid receptors in the rat nucleus of the solitary tract: extrasynaptic plasmalemmal localization and association with Leu5-enkephalin

Activation of the mu opioid receptor (MOR) by morphine within the caudal nucleus of the solitary tract (NTS) is known to mediate both cardiorespiratory and gastrointestinal responses. Leu⁵-enkephalin (LE), a potential endogenous ligand for MOR, is also present within neurons in this region. To determine the cellular sites for the visceral effects of MOR ligands, including LE, we used immunogold-silver and immunoperoxidase methods for light and electron microscopic localization of antisera against MOR (carboxyl terminal domain) and LE in the caudal NTS of rat brain. Light microscopy of coronal sections through the NTS at the level of the area postrema showed MOR-like immunoreactivity (MOR-LI) and LE labeling in punctate processes located within the subpostremal, dorsomedial and medial subnuclei. Electron microscopy of sections through the medial NTS at this level showed gold-silver particles identifying MOR-LI prominently distributed to the cytoplasmic side of the plasma membranes of axons and terminals. MOR labeled terminals formed mostly symmetric (inhibitory-type) synapses but sometimes showed multiple asymmetric junctions, characteristic of excitatory visceral afferents. MOR-LI was also present along extrasynaptic plasma membranes of dendrites receiving afferent input from unlabeled and LE-labeled terminals. We conclude that MOR ligands, possibly including LE, can act at extrasynaptic MORs on the plasma membranes of axons and dendrites in the caudal NTS to modulate the presynaptic release and postsynaptic responses of neurons. These are likely to include local inhibitory neurons and both gastric and cardiorespiratory afferents known to terminate in the subnuclei with the most intense MOR-LI. © 1996 Wiley-Liss, Inc.     

Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries

The ultrastructure of substance P-containing fibers in feline cerebral arteries was examined by combining substance P immunohistochemistry and electron microscopy. At the light and electron microscopic level, positive fibers were observed in the adventitia and at the border between the adventitia and media, but not within the media or the endothelium. The substance P-containing fibers were unmyelinated with diameters consistent with C-fiber caliber. Positive axons were in close contact with Schwann cell processes. Positive axons contained 24 nm microtubules, 10 nm neurofilaments, clear vesicles and scattered mitochondria. The number of mitochondria and organelles resembling vesicles appeared to increase in presumptive axon terminals. No synaptic membrane specializations were observed.     

Inverse agonist up-regulates the constitutively active D3.49(164)Q mutant of the rat mu-opioid receptor by stabilizing the structure and blocking constitutive internalization and down-regulation

We demonstrated previously that D3.49(164) mutations resulted in constitutive activation of the rat mu-opioid receptor and abolished receptor expression unless cells were pretreated with naloxone, an inverse agonist. In this study, we investigated the properties of the D3.49(164)Q mutant and the mechanisms underlying the effect of naloxone. Naloxone pretreatment up-regulated [(3)H]diprenorphine binding and protein expression of the D3.49(164)Q mutant in a time- and dose-dependent manner without affecting its mRNA level. After naloxone removal, binding and protein expression of the mutant declined with time with no effect on its mRNA level. Naloxone methiodide (a quaternary ammonium analog) caused a maximal up-regulation about 50% of the naloxone effect, indicating that naloxone acts extracellularly and intracellularly. Expression of the mutant was enhanced by inverse agonists, a neutral antagonist, and agonists, with inverse agonists being most effective. In membranes, the mutant was structurally less stable than the wild type upon incubation at 37 degrees C, and naloxone and [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin stabilized the mutant. Coexpression of the dominant-negative mutants GRK2-K220R, arrestin-2(319-418), dynamin I-K44A, rab5A-N133I or rab7-N125I partially prevented the decline in binding of the mutant after naloxone removal. Chloroquine or proteasome inhibitor I reduced the down-regulation of the mutant. These results indicate that the D3.49(164)Q mutant is constitutively internalized via G protein coupled-receptor kinase-, arrestin-2-, dynamin-, rab5-, and rab7-dependent pathways and probably trafficked through early and late endosomes into lysosomes and degraded by lysosomes and proteasomes. Naloxone up-regulates the D3.49(164)Q mutant by stabilizing the mutant protein and blocking its constitutive internalization and down-regulation. To the best of our knowledge, this represents the first comprehensive analysis of the mechanisms involved in up-regulation of constitutively active mutants by an inverse agonist.     

Immunophenotypic characterization and distribution of dendritic cells in odontogenic cystic lesions

Oral Diseases (2012) 19 , 85–91 Objective: To analyze the expression and distribution patterns of mature dendritic cells (mDCs) and immature DCs (imDCs) in radicular cysts (RCs), dentigerous cysts (DtCs), and keratocystic odontogenic tumors (KCOTs). Materials and methods: Forty‐nine odontogenic cystic lesions (OCLs) (RCs, n = 20; DtCs, n = 15; KCOTs, n = 14) were assessed using the following markers: S100, CD1a and CD207 for imDCs; and CD83 for mDCs. Results: Almost all cases were S100, CD1a, and CD207 positive, whereas 63% were CD83 positive. RCs presented greater number of immunostained cells, followed by DtCs, and KCOTs. The number of S100+ cells was greater than both CD1a+ and CD207+ cells (P < 0.001), which showed approximately similar amounts, followed by lower number of CD83+ cells (P < 0.001) in each OCL type. Different from S100+ cells, both CD1a+ and CD207+ cells on the epithelium (P < 0.05) and CD83+ cells on the capsule (P < 0.05) were preferentially observed. In RCs, significant correlation was found between the thickness epithelium with S100+ and CD1a+ cells, and between the degree of inflammation with CD83+ cells. Conclusions: Dendritic cell populations in OCLs can be phenotypically heterogeneous, and it could represent distinct lineages and/or functional stages. It is suggested that besides DC‐mediated immune cell interactions, DC‐mediated tissue differentiation and maintenance in OCLs should also be considered.