Immunoelectron microscopic localization of the opioid growth factor receptor (OGFr) and OGF in the cornea

This study was conducted to determine the cellular and subcellular location(s) of the opioid growth factor receptor (OGFr), and the opioid growth factor (OGF), [Met(5)]-enkephalin, in the corneal epithelium. Laser scanning confocal microscopy analysis revealed that both OGFr and OGF were colocalized in the paranuclear cytoplasm and cell nuclei in basal, as well as suprabasal, cells of adult rat corneal epithelium. Using a postembedding immunogold procedure for immunoelectron microscopy that included embedding in Unicryl, both single- and double-face labeling studies were performed. Immunogold labeling of OGFr was detected on the outer nuclear envelope, in the paranuclear cytoplasm proximal to the nuclear envelope, perpendicular to the nuclear envelope in a putative nuclear pore complex, and within the nucleus adjacent to heterochromatin. Immunoreactivity for OGF was noted in locations similar to that for OGFr. In addition, aggregates of staining for OGF were found throughout the cytoplasm, including subjacent to the plasma membrane. Double labeling experiments revealed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, extending across the nuclear pore complex, and in the nucleus. Anti-OGFr IgG by itself, but not anti-OGF IgG alone, was associated with the outer nuclear envelope, and uncomplexed OGF immunoreactivity was detected in the cytoplasm in dual labeling experiments. These results based on complementary approaches of confocal microscopy and immunoelectron microscopy, suggest that: (i) OGFr resides on the outer nuclear envelope, (ii) OGF interacts with OGFr at the outer nuclear envelope, (iii) the colocalized receptor and peptide translocates between the cytoplasm and the nucleus at the nuclear pore, and (iv) signal transduction for modulation of cell proliferation necessitates a peptide-receptor complex that interfaces with chromatin in the nucleus.     

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.     

Conserved expression of the opioid growth factor, [Met]enkephalin, and the zeta (ζ) opioid receptor in vertebrate cornea

In addition to neuromodulation, endogenous opioids serve as growth factors. The naturally occurring opioid peptide, [Met⁵]enkephalin, termed opioid growth factor (OGF), has been found to be a potent and tonic inhibitor of processes related to growth and renewal, particularly cell proliferation. OGF mediates its actions through the zeta (ζ) opioid receptor. In order to determine if OGF and/or the ζ receptor are present in human corneal epithelium, immunocytochemistry was utilized. Immunoreactivity with regard to OGF and to the ζ receptor could be detected in the cortical cytoplasm of both basal and suprabasal epithelial cells, but was not associated with the cell nucleus. Investigation of the ubiquity of OGF and ζ receptor in the vertebrate cornea showed that both elements are present in a wide variety of classes of the phylum Chordata, including mammalia, aves, reptilia, amphibia, and osteichthyes. These results suggest that an endogenous opioid system related to growth may have originated as early as 300 million years ago, and that the function of this system in cellular renewal and homeostasis is a requirement of the vertebrate corneal epithelium.     

Production and characterization of polyclonal and monoclonal antibodies to the zeta (ξ) opioid receptor

The opioid growth factor, [Met⁵]-enkephalin, is an inhibitory agent of cell proliferation and maturation that interacts with the zeta (ξ) opioid receptor to modulate growth. In order to learn more about this receptor, polyclonal and monoclonal antibodies were raised against binding subunits identified on two-dimensional gels by ligand blotting. Using Western blotting, the polyclonal antibodies and some of the monoclonal antibodies recognized all 4 binding polypeptides (32, 30, 17, and 16 kDA) in developing rat cerebellum; no reaction was recorded in adult cerebellum. In addition, other monoclonals were able to distinguish only certain subunits (e.g. 17 kDa). The monoclonal antibodies and their F(ab′)₂ fragments, as well as the polyclonal antibodies, blocked the binding of [³H][Met⁵]-enkephalin to preparations of developing cerebellum. Both the polyclonal and monoclonal antibodies immunoprecipitated ξ opioid binding polypeptides from 6-day-old cerebellar homogenates solubilized by the zwitterionic detergent, CHAPS. Immunocytochemistry performed with polyclonal antibodies showed immunoreactivity associated with proliferating and differentiating cerebellar cells, but no specific staining was detected in the adult cerebellum. These results have identified and characterized antibodies to the ξ opioid receptor, and the antibodies were used to localize this receptor; these antibodies will be valuable to further cellular and molecular studies.     

Characterization of zeta (ζ): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [³H]-[Met⁵]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded aK_d of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na⁺ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [³H]-[Met⁵]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin; other ligands selective for μ, δ, κ, , and σ were not highly competitive. Given the functional significance of [Met⁵]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met⁵]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (ζ) (from the Greek ‘Zoe’, life) related to the proliferation of cells and tissues.     

中国人胶质瘤中表皮生长因子受体基因的扩增及对患者预后的影响

目的：了解国人胶质瘤中表皮生长因子受体基因（EGFR）的扩增情况及对患者预后的影响。方法：使用半定量的Differential PCR方法对113例福尔马林固定-石蜡包埋（FFEP）胶质瘤标本内的EGFR基因扩增情况进行了检测；对其中的83例进行了生存分析研究。结果：113例胶质瘤中存在EGFR基因扩增的有26例，其中低度恶性组中仅占3.5％，而在高度恶性组中占42.1％。生存分析显示：EGFR基因扩增对患者预后并无显著影响，而起病年龄、肿瘤病理分级、肿瘤生长部位及起病症状为失语对患者术后生存期有显著影响。结论：恶性胶质瘤中存在着较为广泛的EGFR基因扩增，但EGFR基因的扩增与胶质瘤患者预后之间并不存在显著关联。     

垂体生长激素腺瘤细胞表皮生长因子受体的表达

目的了解大鼠垂体生长激素腺瘤(GH3)细胞的表皮生长因子受体(EGFR)的表达情况.方法应用免疫组化的方法检测GH3细胞及阴性对照U-2OS细胞和阳性对照HO8910PM细胞的EGFR表达情况.结果 GH3细胞和HO8910PM细胞明显黄染,U-2OS细胞无明显染色;GH3、U-2OS和HO8910细胞表达EGFR的阳性细胞率分别为90.57%、4.84%和93.33%.结论垂体生长激素腺瘤细胞中存在着广泛的EGFR表达,而且具有较高的表达强度.     

Chronic treatment with scopolamine and physostigmine changes nerve growth factor (NGF) receptor density and NGF content in rat brain

Nerve growth factor (NGF) and NGF receptors were measured in cortex and hippocampus of rats treated with drugs affecting cholinergic neurotransmission. High (K_d= 0.045nM) and low (K_d= 21nM) affinity¹²⁵I-NGF binding sites were present in both cortical and hippocampal membranes with hippocampus containing higher numbers of both sites than cortex. Chronic treatment of rats with the muscarinic receptor antagonist scopolamine (5 mg/kg, twice daily) decreased the density of high- and low-affinity sites by 50–90% in cortical and hippocampal membranes. These changes were seen after 7 days, but not 3 days, of scopolamine treatment. Chronic infusion of physostigmine (1 mg/kg/day) using minipumps increased the number of high- and low-affinity sites in cortex 3- and 6-fold, respectively. The changes in receptor-binding parameters induced by physostigmine were transient as they were evident after 3 days of treatment, but returned to control levels after 7 days. NGF content in cortex and hippocampus was reduced by about 50% following 7, but not 3, days of chronic physostigmine infusion. In contrast, scopolamine treatment failed to change NGF levels in the cholinergic neuronal target regions but it decreased NGF content in the septal area. The content of NGF mRNA in the cortex measured by Northern blot analysis failed to change following either scopolamine or physostigmine treatment. The results suggest that levels of NGF and NGF receptors in the target regions of cholinergic neurons are regulated by the extent of cholinergic neurotransmitter activity.     

Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve?

Regeneration was measured after the infliction of a crush lesion on rat sciatic nerves which 4 days earlier had been subjected to a distal conditioning transection. Such nerves exhibited an increased outgrowth of nerve fibers as compared to nerves subjected to a single crush lesion. This increased outgrowth could be prevented, if the nerve was locally perfused around the site of the transection during the 4 days conditioning interval, with cycloheximide, actinomycin D and vinblastine, inhibitors of protein-, RNA-synthesis and retrograde axonal transport, respectively. The inhibitory effect of cycloheximide could be overcome by simultaneous perfusion with insulin-like growth factor I (IGF-1). The results suggest that proteins including IGF-1 which are synthesised locally around a nerve lesion and then transported retrogradely could trigger regenerative events in the neuronal cell body.     

Effects of nerve growth factor (NGF), fluoxetine, and amitriptyline on gene expression profiles in rat brain

Evidence suggests that nerve growth factor (NGF) may have antidepressant properties but the pharmacological mechanisms remain unknown. Previously, we found that NGF improved performance in the forced swim test in Flinders Sensitive Line rats, but did not appear to have similar biochemical actions with the antidepressant fluoxetine. Gene expression profiles for neurotransmitter receptors and regulator-related genes in the amygdala/hippocampus were determined in rats treated for 14 days with NGF, fluoxetine, amitriptyline, or saline. Gene expression was measured using an RT² profiler PCR Array System to determine the basis for this effect. Compared with saline, there were numerous genes with significantly altered mRNA levels in the amygdala/hippocampal region. Overlap was found between the mRNA levels of genes altered by NGF and the two antidepressant medications including genes related to the cholinergic and dopaminergic systems. However, decreased mRNA levels of Drd5, Sstr3, Htr3a, and Cckar genes in the amygdala/hippocampus were uniquely regulated by NGF. The results of this study are consistent with a previous conclusion that the antidepressant effects of NGF are mediated through non-traditional receptors for traditionally considered neurotransmitters and may suggest a particular utility of NGF in treating comorbid depression and addiction.     

Immunological determinants of nerve growth factor involved in p140trk (Trk) receptor binding

Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse β-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75^LNGFR and Trk. None of the three antibodies–N60, M15, and 27/21–showed any reactivity toward denatured NGF. Three experimental methods–radioim-munoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots–detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated β-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/12 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the α and γ subunits, competitively inhibited binding of antibodies to β-NGF. Only the γ subunit inhibited phosphorylation of Trk and biological activity of β-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of β-NGF containing residues 60–80 is proposed as important for binding to the Trk receptoe. © 1994 Wiley-Liss, Inc.     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

PDGF-B及其受体蛋白在缺血大鼠脑组织中的表达

目的 探讨血小板源性生长因子B链(PDGF—B)及其受体PDGFR—β在缺血性脑损伤后濒危神经元存活和损伤修复中的作用。方法 采用线栓法制备大鼠大脑中动脉局灶性缺血模型，分别于缺血2h再灌注6h、24h、3d、7d、14d和21d处死，用免疫组化分别检测PDGF—B和PDGFR—β蛋白在脑缺血区及其周围的表达。结果 缺血再灌注后24h，PDGF—B和PDGFR—β在缺血区及其周围的神经元的表达开始增强，并分别于3d和7d出现第一个高峰，其中PDGFR—β的表达还可见于反应性胶质细胞；两者的第二个表达高峰出现在再灌注后14d，主要表达于梗死区增生的胶质细胞、新生血管和胶质疤痕周围的神经元。结论局灶性脑缺血后，PDGF—B及其受体蛋白在时间和空间上的一致性表达，说明它们对濒危神经元的存活、胶质疤痕形成和血管发生均有重要的作用。     

Regulation of human B lymphocyte activation by opioid peptide hormones. Inhibition of IgG production by opioid receptor class ( μ-, κ-, and δ-) selective agonists

Opioid peptides have been reported by many laboratories to modulate in vitro and in vivo cell-mediated and humoral immune responses. However, less attention has been afforded to the class or classes of opioid receptors involved in these immunomodulatory effects. Previous studies by this laboratory indicated that β-endorphin and methionine-enkephalin were potent inhibitors of Staphylococcus aureus, Cowen strain I (SAC)-induced IgG production by human B lymphocytes. Results obtained from the present studies indicate that, at pharmacological concentrations, μ-, δ-, and κ-receptor-selective agonists arc potent inhibitors of SAC-induced IgG-secreting cells (IgG-ISC) by human B lymphocytes. Moreover, the suppression of IgG-ISC formation was reversed by μ-, δ-, and κ-receptor class-selective antagonists, [ Tic]cTAP, ICI 174,864, and nor-BNI, respectively. These findings are in ag showing that more than one class of receptors are involved in opioid peptide-mediated immunoregulation. Additional studies indicated that all three class-selective receptor agonists were found to suppress SAC-induced IL-6 production in intact PBMC cultures. As observed for suppression of IgG-ISC formation, inhibition of IL-6 production was found to be reversed by the appropriate receptor class-selective antagonist. These results support the hypothesis that one mechanism of opioid peptide-mediated inhibition of antibody production is via the down regulation of cytokine synthesis.     

Light microscopy study of low-affinity nerve growth factor receptor and phosphoprotein B-50/neuromodulin in inflammatory myopathies

Phosphoprotein B-50, also termed neuromodulin or growth-associated protein GAP43, is a membrane-bound molecule expressed in neurons. It is particularly abundant during periods of axonal outgrowth in development and regeneration of the central and peripheral nervous systems. Recently it was reported that B-50 plays a role in the growth morphology of regenerating muscle fibers. Moreover, in vitro studies have demonstrated that the expression of B-50 in the pheochromocytoma PC12 cells can be stimulated by the nerve growth factor (NGF). Expression of the low-affinity nerve growth factor receptor (LNGFR) during muscle regeneration has also been reported. Here, we studied the expression of NGF, LNGFR and B-50 in myopathy. To investigate the state of regeneration, we examined serial sections stained to demonstrate neural cell adhesion molecule and desmin. Light microscopy showed that muscle fiber regeneration in idiopathic inflammatory myopathy corresponds closely to NGF, LNGFR and B-50 immunoreactivity. The coexpression of phosphoprotein B-50, NGF and LNGFR in regenerating muscle fiber corroborates the assumption that in muscle there is a trophic pathway concerning phosphorylation or de novo synthesis of B-50 by the NGF via the LNGFR. In conclusion, a simultaneous expression of NGF, LNGFR and B-50 in muscles plays a role in the growth morphology of regenerating muscle fibers. Received: 4 November 1994 / Revised: 4 August 1995 / Revised, accepted: 23 October 1995     

The expression of nerve growth factor receptor on Schwann cells and the effect of these cells on the regeneration of axons in traumatically injured human spinal cord

To investigate the effects of Schwann cells and nerve growth factor receptor (NGFR) on the regeneration of axons, autopsy specimens of spinal cord from 21 patients with a survival time of 2 h to 54 years after spinal cord trauma were studied using immunohistochemistry and electron microscopy. Regenerating sprouts of axons could be observed as early as 4 days after trauma. At 4.5 months after trauma, many regenerating nests of axons appeared in the injured spinal cord. The regeneration nests contained directionally arranged axons and Schwann cells. Some axons were myelinated. In injured levels of the spinal cord, the Schwann cells exhibited an increased expression of NGFR within spinal roots. These results show that an active regeneration process occurs in traumatically injured human spinal cord. The NGFR expressed on Schwann cells could mediate NGF to support and induce the axon regeneration in the central nervous system. Received: 20 June 1995 / Revised, accepted: 18 September 1995     

Reduction of nerve growth factor receptor immunoreactivity in ischaemic gerbil hippocampal CA1 neurons after treatment with L-threo-3,4-dihydroxyphenylserine (DOPS)

Abstract

Nerve growth factor (NGF) synthesis in cultured mouse L-M fibroblast and astroglial cells can be increased after the treatment with L-threo-3,4-dihydroxyphenylserine (DOPS). Since the increase of NGF is not blocked by the treatment with decarboxylase inhibitor, DOPS may have direct effect to increase the NGF content. NGF and its receptor (NGFR) are suggested to play an important role in the neuronal survival and regeneration under pathologic conditions. In this study, we studied a possible protective effect of DOPS against the hippocampal CA1 cell death after transient forebrain ischaemia in gerbils in relation to the change of NGFR immunoreactivity. We found that treatment with DOPS (300 mg kg^–1) in combination with a decarboxylase inhibitor (benserazide, 10 mg kg^–1) protected ischaemic hippocampal CA1 cell against delayed neuronal death (neuronal density = 125 ± 24 mm^–1) as compared to the treatment with vehicle (49 ± 11 mm^–1) (p < 0.01). The immunoreactivity for NGFR was scarcely present in the sham-control CA1 area but was induced from 1 h and markedly expressed at 7 days after recirculation in the vehicle group. However; it was slightly and transiently induced from 8 h to 2 days in the DOPS plus benserazide treated group. These data suggest that the protective role of DOPS on the ischaemic hippocampal CA1 cells may act through the NGF and its receptor system. [Neurol Res 1994; 16: 201–204]     

Activation of phosphoipase D by platelet-derived growth factor (PDGF) in rat C6 glioma cells: Possible role in mitogenic signal transduction

Abstract

The effects of platelet-derived growth factor (PDGF) on phosphoiipase D (PLD) activity and deoxyribonucleic acid (DNA) synthesis in rat C6 glioma cells have been investigated. Pretreatment of serum-starved C6 cells with PDGF results in enhanced choline production and the phosphatidylethanol (PEt) formation in the presence of ethanol’ indicating the activation of PLD acting on phosphatidylcholine (PG). The dose-response curve for choline generation and DNA synthesis were comparable. In addition, the effects of PDGF on both PEt formation and [ H]thymidine incorporation into acid-precipitable material was blocked by the potent protein kinase G (PKG) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) but not by N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA1004), a relatively weak inhibitor of PKC, suggesting that PDGF plays an important role as a positive regulator of glioma cell growth via a PLD-mediated mitogenic signal transduction cascades, which depends largely on the activation of PKG.     

The role of fibroblast growth factor receptor 4 polymorphisms in the susceptibility and clinical features of ischemic stroke

Some polymorphisms in the fibroblast growth factor receptor 4 gene (FGFR-4) have been correlated with coronary artery disease, however, the role of polymorphisms in the FGFR-4 gene in ischemic stroke remain unknown. A total of 270 patients with ischemic stroke and 297 controls were recruited. Stroke subtype was classified and clinical severity of stroke in patients was evaluated. The polymorphisms in the FGFR-4 were genotyped. There were no significant differences of genotype distributions and allele frequencies of rs145302848C/G and rs147603016G/A between stroke patients and controls (all p > 0.05). However, genotype frequencies and allele frequencies at rs351855G/A (Gly388Arg) were significantly different between stroke patients and controls (both p < 0.001). With the rs351855GG genotype as a reference, the presence of rs351855AA homozygote had a significantly increased risk for stroke (adjusted odds ratio 2.663; 95% confidence interval 1.673–4.229, p < 0.001). The polymorphisms at rs145302848C/G and rs147603016G/A did not influence the susceptibility of stroke in this study. All FGFR-4 polymorphisms were not associated with clinical features such as Trial of Org 10172 in Acute Stroke Treatment subtype or stroke severity as indicated by mean National Institutes of Health Stroke Scale scores. Our study suggests a positive association between FGFR-4 gene polymorphism at rs351855G/A and susceptibility to ischemic stroke.