dynorphins and Neuroblastoma

Recently, it has been suggested that anesthetic agents may have neuroprotective potency. The notion that anesthetic agents can offer neuroprotection remains controversial. Propofol, which is a short-acting intravenous anesthetic agent, may have potential as a neuroprotective agent. In this study, we tried to determine whether propofol affected the viability of human neuroblastoma SH-SY5Y cells by using the MTT assay. Surprisingly, our results showed that propofol at a dose of 1-10 μM could improve cell proliferation. However, at higher doses (200 μM), propofol appears to be cytotoxic. On the other hand, propofol could up-regulate the expression of key proteins involved in neuroprotection including B-cell lymphoma 2 at a dose range of 1-10 μM, activation of phospho-serine/threonine protein kinase at a dose range of 0.5-10 μM, and activation of phospho-extracellular signal-regulated kinases at a dose range of 5-10 μM. Similarly, we demonstrate that propofol (10 μM) could elevate protein levels of heat shock protein 90 and heat shock protein 70. Therefore, we choose to utilize a 10 μM concentration of propofol to assess neuroprotective activities in our studies. In the following experiments, we used dynorphin A to generate cytotoxic effects on SH-SY5Y cells. Our data indicate that propofol (10 μM) could inhibit the cytotoxicity in SH-SY5Y cells induced by dynorphin A. Furthermore, propofol (10 μM) could decrease the expression of the p-P38 protein as well. These data together suggest that propofol may have the potential to act as a neuroprotective agent against various neurologic diseases. However, further delineation of the precise neuroprotective effects of propofol will need to be examined.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Dynorphins; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; In Situ Nick-End Labeling; Neuroblastoma; Neuroprotective Agents; Neurotransmitter Agents; Propofol; Time Factors

It has been proposed that the volatile anesthetic isoflurane induces neuroprotection and that the endogenous opioid peptide dynorphin induces neurocytotoxicity in cells. The levels of dynorphin are often significantly elevated in neuropathophysiological conditions, and dynorphin can directly induce toxicity. However, the neuroprotective effects of isoflurane on dynorphin-induced cytotoxicity are still unclear.. In order to determine the effect of isoflurane on dynorphin-induced cytotoxicity in neuronal cells, we have designed a device wherein cultured human neuroblastoma SH-SY5Y cells can be exposed to isoflurane. Fully differentiated SH-SY5Y cells were obtained by treating the cells with retinoic acid for 6 days. We examined SH-SY5Y cell survival, apoptosis, and antiapoptotic protein expression by cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling stain, and Western blot analysis, respectively.. After 16 h of dynorphin (10 microM) treatment, the SH-SY5Y cells showed significant cytotoxicity, apoptosis, and downregulation of the antiapoptotic Bcl-2 protein expression. These effects of dynorphin were significantly inhibited by isoflurane exposure for 32 h [pretreatment for 16 h and posttreatment (after dynorphin treatment) for 16 h].. Thus, our results suggest that isoflurane exerts neuroprotective effects in the case of dynorphin-induced pathophysiological disruption.

Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Dynorphins; Humans; Isoflurane; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.

Topics: Animals; Brain; Carcinoma, Small Cell; Carrier Proteins; Cell Line; Cell Line, Tumor; Choriocarcinoma; DDT; Dynorphins; Humans; Hydroxymercuribenzoates; Kinetics; Lung Neoplasms; Mice; Mice, Inbred Strains; Nerve Tissue Proteins; Neuroblastoma; Neurons; Protease Inhibitors

Whereas the role of dopaminergic tone in the cortico-striatal-thalamic system is well-established, the role of endogenous opioids in the function of this system is less understood. We show that Borna disease virus infection of adult rats results in an increase in preproenkephalin transcripts in the striatum of Borna-infected rats, a region important for forming coordinated sequential motor actions and in developing programmes of thought and motivation. Stereotypic behaviours and dyskinesias, the clinical hallmarks of infection in adult Lewis rats (BD rats), are accompanied by a disrupted pattern of immediate early gene c-fos activation in the motor thalamus, with significance for the breakdown in coordinated sequential motor actions. We also find increased preproenkephalin in infected cultured neuroblastoma and rat foetal glial cells. The expression pattern of enkephalin mRNA in vivo and in vitro suggest that increased enkephalin function is one of the neuropharmacological means by which Borna disease virus causes motor disease of animals and possibly cognitive and affective disease in man, and further suggest that enkephalins play a critical role in the maintenance of a balanced tone of activity in the cortico-basal ganglia-thalamo-cortical loops.

Topics: Animals; Astrocytes; Borna Disease; Borna disease virus; Cells, Cultured; Dynorphins; Enkephalins; Gene Expression; Genes, fos; Humans; Male; Neuroblastoma; Protein Precursors; Putamen; Rats; Rats, Inbred Lew; Receptors, Dopamine D2; Tumor Cells, Cultured

Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibody Specificity; Blotting, Western; Brain; Cell Line; Chlorocebus aethiops; Dynorphins; Epitopes; Gene Expression; Guinea Pigs; Immunohistochemistry; Kidney; Male; Microscopy, Confocal; Molecular Sequence Data; Neuroblastoma; Neurons; Organ Specificity; Peptide Fragments; Protein Precursors; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Opioid, mu; Recombinant Proteins; RNA, Messenger; Spinal Cord; Transfection

Serum-free culture of mouse neuroblastoma cells was used as the experimental model for the study of neuronal aging, with flow cytometry of cell cycle, DNA and total cellular protein as the indices of neuronal aging. After addition of dynorphin A (1-8) 10(-7) mol.L-1 into the culture medium, the following general tendencies were obtained: (1) The number of S and G2 + M phase cells was increased and the number of G1 phase cells decreased. (2) The total cellular protein in aged experimental neural cells decreased. The results implies that Dyn A (1-8) Produced effects on cell cycle, DNA and total cellular protein in the direction of delaying neuronal aging. The relation between Dyn A (1-8) and neuronal aging merits further investigation.

Topics: Animals; Cell Cycle; Cellular Senescence; Culture Media, Serum-Free; DNA; Dynorphins; Mice; Neuroblastoma; Peptide Fragments; Protein Biosynthesis; Tumor Cells, Cultured

The modulation of neuropeptide Y (NPY) and peptide YY (PYY) receptors by dynorphin, luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), and cholecystokinin octapeptide has been studied in human neuroblastoma cell lines SK-N-MC and SMS-MSN, which express Y1 and Y2 receptors for NPY/PYY. Dynorphin A and LHRH inhibited the binding of NPY/PYY to SK-N-MC cell membranes at concentrations ranging from 10(-7) to 10(-5) M, whereas dynorphin A and CRF were effective in SMS-MSN cells. The inhibitory effect of dynorphin A on NPY/PYY binding was observed in the presence of guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, as well as H-7 and H-8, novel inhibitors of protein kinases C and A. However, U-50488, the most potent kappa-selective compound did not mimic the dynorphin action. Dynorphin A showed neither effect on the dissociation of NPY/PYY from their receptors nor inhibition on the basal as well as forskolin-stimulated adenosine 3',5'-cyclic monophosphate response. These results indicate that the interaction of dynorphin A with Y1 and Y2 receptors is not mediated by changes in receptor-G protein interaction, receptor phosphorylation, and allosteric binding to NPY/PYY receptors but that dynorphin A binds to NPY/PYY receptors at high concentrations, probably in an antagonistic manner.

Topics: Corticotropin-Releasing Hormone; Dynorphins; Gonadotropin-Releasing Hormone; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Protein Kinase Inhibitors; Receptors, Gastrointestinal Hormone; Receptors, Neuropeptide Y; Sincalide; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Animals; Brain; Cell Line; Dynorphins; Gonadotropin-Releasing Hormone; Mice; Molecular Sequence Data; Neuroblastoma; Neuropeptides; Peptidyl-Dipeptidase A; Rats; Substance P; Substrate Specificity

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.

Topics: Animals; Cell Line; Chloromercuribenzoates; Chromatography, Gel; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Dynorphins; Ethylmaleimide; Mice; Molecular Weight; Neuroblastoma; p-Chloromercuribenzoic Acid; Protease Inhibitors

A trypsin-like enzyme has been purified to apparent homogeneity from neuroblastoma cell membranes by a procedure including extraction with Triton X-100, soybean trypsin inhibitor-immobilized Sepharose 4B affinity chromatography, and gel filtration. SDS-polyacrylamide gel electrophoresis under reducing conditions of the purified enzyme gave a single band corresponding to a molecular weight of 28,000. The molecular weight of the enzyme was also estimated to be 32,000 by gel filtration. The pH optimum of the activity was 8.5-9.0. The purified enzyme was inhibited by diisopropylphosphorofluoridate, p-aminobenzamidine, and leupeptin, and moderately by chymostatin, but not, or only scarcely, by bestatin, phosphoramidon, p-chloromercuribenzoate, and N-ethylmaleimide. The substrate subsite specificity of the purified enzyme was broad toward various peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides, but it cleaved dynorphin(1-17) only at two sites, i.e., between the Arg6-Arg7 and Lys11-Leu12 bonds, both of which correspond to the initial cleavage sites of dynorphin with a membrane preparation of neuroblastoma cells. A trypsin-like enzyme was also purified from a synaptic membrane preparation of rat brain, which shows almost the same properties as those of the enzyme from the neuroblastoma cell membrane. Thus, the trypsin-like enzyme present in the synaptic membrane would participate in the degradation of dynorphin.

Topics: Cell Membrane; Dynorphins; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Molecular Weight; Neuroblastoma; Trypsin; Tumor Cells, Cultured

The membrane of mouse neuroblastoma N-18 cells degraded dynorphin-(1-13), dynorphin-(1-17), and Leu-enkephalin. The degradation of the former two peptides was inhibited strongly by N-ethylmaleimide, moderately by diisopropylphosphorofluoridate and phosphoramidon, and slightly by bestatin. When Leu-enkephalin was the substrate, however, the effects of phosphoramidon and bestatin were marked and those of N-ethylmaleimide and diisopropylphosphorofluoridate were negligibly small. Captopril did not affect the degradation of the two dynorphins and Leu-enkephalin, but inhibited the further cleavage of N-terminal fragments generated from dynorphin-(1-13) by the N-ethylmaleimide-sensitive protease. Thus, a cysteine protease and, probably, a serine protease are responsible to the initial fragmentation of the dynorphins.

Topics: Amino Acids; Captopril; Cell Line; Cysteine Endopeptidases; Dynorphins; Endopeptidases; Enkephalin, Leucine; Ethylmaleimide; Neuroblastoma; Peptide Fragments

Neuroblastoma x glioma hybrid cells (NG 108CC15) and tumors derived thereof were examined for dynorphin- and alpha-neoendorphin-like material. The techniques employed for analyses of opiate-like material were the isolated mouse vas deferens bioassay and gel chromatography and high pressure liquid chromatography in combination with radioimmunoassays. Dynorphin- and alpha-neoendorphin-like material was detected in both the hybrid cells and the corresponding tumors. Immunoreactive dynorphin and alpha-neoendorphin was also in NCB 20 hybrid cells and in tumors thereof assayed. In all samples investigated, the amounts of alpha-neoendorphin-like material was higher than that of dynorphin-like material. The results revealed considerable variability in the amount of dynorphin- and alpha-neoendorphin-activity between particular samples, suggesting the need for studies into the responsible mechanisms.

Topics: alpha-Endorphin; Animals; Biological Assay; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Glioma; Hybrid Cells; Male; Mice; Neuroblastoma; Radioimmunoassay; Rats; Vas Deferens