endothelin-1 and Glioma

Hypoxia and exosomes play important roles in the occurrence and development of glioma. While circRNAs are involved in biological processes of various tumors, the mechanism underlying exosome-dependent regulatory effects of circRNAs on the progression of glioma under hypoxia is unclear. Results suggested that circ101491 was overexpressed in tumor tissues and plasma exosomes of glioma patients, while the overexpression of circ101491 was closely related to the differentiation degree and TNM staging of the patients. Moreover, circ101491 overexpression promoted viability, invasion and migration of glioma cells both in vivo and in vitro; the above regulatory effects can be reversed by inhibition of circ101491 expression. Mechanistic studies revealed that circ101491 upregulated EDN1 expression through sponging miR-125b-5p, thus facilitating glioma progression. In summary, hypoxia could promote circ101491 overexpression in glioma cell-derived exosomes, and circ101491/miR-125b-5p/EDN1 regulatory axis might be implicated in the malignant progression of glioma.

Topics: Cell Line, Tumor; Cell Proliferation; Endothelin-1; Glioma; Humans; Hypoxia; MicroRNAs; RNA, Circular

Topics: Cell Line, Tumor; Endoribonucleases; Endothelin-1; Endothelin-Converting Enzymes; Gene Expression; Gene Knockdown Techniques; Glioma; Glucose; Glutamine; Humans; Protein Serine-Threonine Kinases; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Messenger

The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia.. The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction.. It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells.. Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.

Topics: Brain Neoplasms; Cell Hypoxia; Cell Line, Tumor; Endoribonucleases; Endothelin-1; Endothelin-Converting Enzymes; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Humans; Hypoxia; Protein Serine-Threonine Kinases; Receptor, Endothelin A; Receptor, Endothelin B; Tumor Hypoxia

Endothelin-1 (ET-1) is a 21 amino acids peptide that exerts several biological activities through interaction with specific G-protein coupled receptors. Increased ET-1 expression is frequently associated with pathological situations involving alterations in glutamate levels. In the present study, a brief exposure to ET-1 was found to increase aspartate uptake in C6 glioma cells, which endogenously express the neuronal glutamate transporter EAAC1 (pEC50 of 9.89). The stimulatory effect of ET-1 mediated by ETA receptors corresponds to a 62% increase in the Vmax with no modification of the affinity for the substrate. While protein kinase C activity is known to participate in the regulation of EAAC1, the effect of ET-1 on the glutamate uptake was found to be independent of this kinase activation. In contrast, the inactivation of Go/i type G-protein dependent signaling with pertussis toxin was found to impair ET-1-mediated regulation of EAAC1. An examination of the cell surface expression of EAAC1 by protein biotinylation studies or by confocal analysis of immuno-fluorescence staining demonstrated that ET-1 stimulates EAAC1 translocation to the cell surface. Hence, the disruption of the cytoskeleton with cytochalasin D prevented ET-1-stimulated aspartate uptake. Together, the data presented in the current study suggest that ET-1 participates in the acute regulation of glutamate transport in glioma cells. Considering the documented role of glutamate excitotoxicity in the development of brain tumors, endothelinergic system constitutes a putative target for the pharmacological control of glutamate transmission at the vicinity of glioma cells.

Topics: Amino Acid Transport System X-AG; Animals; Cell Line, Tumor; Endothelin-1; Excitatory Amino Acid Transporter 3; Glioma; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Pertussis Toxin; Rats; Receptor, Endothelin A; Symporters

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. It is possible to discriminate between these channels by using the Ca(2+) channel blockers SK&F 96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) and LOE 908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide]. LOE 908 is a blocker for NSCC-1 and NSCC-2, whereas SK&F 96365 is an inhibitor for NSCC-2. The purpose of the present study was to identify the G-proteins that are involved in ET-1-activated Ca(2+) channels in C6 glioma cells. ET-1 activated only NSCC-1 in C6 glioma cells preincubated with U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C (PLC) inhibitor. Microinjection of the dominant negative mutant of G(12)/G(13) (G(12)G228A/G(13)G225A) abolished activation of NSCC-1 and NSCC-2. In contrast, pertussis toxin did not affect any of the Ca(2+) channels in the ET-1-stimulated C6 glioma cells. These results indicate that G(12)/G(13) may couple with endothelin receptors and play an important role in the activation of NSCCs in C6 glioma cells. Moreover, the activation mechanisms of NSCC-1 and NSCC-2 by ET-1 were different. NSCC-1 activation depended upon a G(12)/G(13)-dependent cascade, whereas NSCC-2 activation depended upon both G(q)/PLC- and G(12)/G(13)-dependent cascades.

Topics: Acetamides; Animals; Calcium; Calcium Channels; Cation Transport Proteins; Endothelin-1; Estrenes; Glioma; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Imidazoles; Isoquinolines; Pertussis Toxin; Pyrrolidinones; Rats; Tumor Cells, Cultured

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca2+ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca2+ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca2+ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca2+ entry, but not for its maintenance; 3) Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca2+ influx through NSCC-2.

Topics: Androstadienes; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cations; Cell Division; Chromones; Endothelin-1; Enzyme Inhibitors; Focal Adhesion Kinase 2; Glioma; Imidazoles; Inositol 1,4,5-Trisphosphate Receptors; Ion Channels; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein-Tyrosine Kinases; Rats; Receptors, Cytoplasmic and Nuclear; Tumor Cells, Cultured; Wortmannin

We have recently shown that endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. These channels can be distinguished by their sensitivity to blockers of the receptor-operated Ca2+ channel, 1-[b-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, whereas NSCC-2 is sensitive to both LOE 908 and SK&F 96365. Moreover, extracellular Ca2+ influx through these channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells. The purpose of the present study was to investigate the effects of extracellular Ca2+ influx on intracellular pathways of endothelin-1-induced mitogenic responses in C6 glioma cells. We focused on extracellular signal-regulated kinase 1 and 2 (ERK1/2) in this context. An inhibitor of mitogen-activated protein kinase, 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059), abolished the endothelin-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenic response. ERK1/2 activation by endothelin-1 was partially suppressed in the absence of extracellular Ca2+. On the basis of the sensitivity to LOE 908 and SK&F 96365, Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in the extracellular Ca2+-dependent component of ERK1/2 activity. In contrast, Ca2+ influx through NSCC-2 is involved in the ERK1/2-independent component of endothelin-1-induced mitogenesis. These results indicate that (1) the endothelin-1-induced mitogenic response involves both ERK1/2-dependent and -independent mechanisms, (2) ERK1/2 activation by endothelin-1 involves an extracellular Ca2+ influx-dependent cascade as well as an extracellular Ca2+ influx-independent cascade, (3) because endothelin-1-induced mitogenesis is completely dependent on extracellular Ca2+ influx, extracellular Ca2+ influx also plays an important role in mitogenic pathways downstream of ERK1/2, (4) extracellular Ca2+ influx through NSCC-1 and NSCC-2 has an important role in the extracellular Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, (5) extracellular Ca2+ influx through NSCC-2 has an important role in ERK1/2-independent mitogenesis, and (6) Ca2+ influx through each Ca2+ channel may play a distinct role in intracellular mitogenic ca

Topics: Acetamides; Animals; Brain Neoplasms; Calcium; Calcium Channel Blockers; Calcium Channels; DNA; Drug Interactions; Endothelin-1; Enzyme Inhibitors; Flavonoids; Glioma; Imidazoles; Isoquinolines; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mitogens; Rats; Thymidine; Tritium; Tumor Cells, Cultured

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.

Topics: Animals; Brain Neoplasms; Calcium; Cell Differentiation; Endothelin-1; Endothelin-3; Fluorescent Dyes; Fura-2; Glioma; Oligopeptides; Piperidines; Rats; Receptor Cross-Talk; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Signal Transduction; Tumor Cells, Cultured; Viper Venoms

Ca(2+) channels activated by endothelin-1 (ET-1) in C6 glioma cells (C6 cells) were characterized using whole-cell patch-clamps and by monitoring the intracellular free Ca(2+) concentration ([Ca(2+)](i)), when administering Ca(2+) channel blockers such as LOE 908 and SK&F 96365. Using this methodology, the Ca(2+) channels involved in ET-1-induced mitogenesis were identified. The patch-clamp study and [Ca(2+)](i) monitoring showed that 10 nM ET-1 activated two types of Ca(2+)-permeable nonselective cation channels (NSCC); one was sensitive to LOE 908 but resistant to SK&F 96365 (NSCC-1) and the other was sensitive to both LOE 908 and SK&F 96365 (NSCC-2). Conversely, 0.1 nM ET-1 activated only NSCC-1.ET-1-induced mitogenesis in a concentration-dependent manner, with the maximum effect arising at concentrations > or =10 nM. LOE 908 completely suppressed the 10 nM ET-1-induced mitogenesis, whereas SK&F 96365 only partially suppressed it. The IC(50) values of these blockers for the ET-1-induced mitogenesis were similar to those for the 10 nM ET-1-induced increase in [Ca(2+)](i). In contrast, LOE 908 completely suppressed 0.1 nM ET-1-induced mitogenesis, whereas SK&F 96365 did not affect it.Collectively, these results demonstrate that the sustained increase in [Ca(2+)](i), via NSCC-1 and NSCC-2, may be essential for ET-1-induced mitogenesis in C6 cells. Moreover, the sensitivity of NSCC-1 to ET-1 is higher than that of NSCC-2 to ET-1.

Topics: Acetamides; Animals; Brain Neoplasms; Calcium; Cell Division; Cell Line; Electrophysiology; Endothelin-1; Glioma; Imidazoles; Ion Channels; Isoquinolines; Mitosis; Patch-Clamp Techniques; Rats; Tumor Cells, Cultured

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

Topics: Animals; Antibodies, Monoclonal; Endothelin-1; G(M3) Ganglioside; Glioma; Kinetics; Neuroglia; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Rats; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

Endothelin-1 (ET-1) plays an important role in the development, physiology and pathophysiology of the cardiovascular system in mammals. ET-1 is mainly expressed in endothelial cells thus making it an attractive model for the study of transcriptional regulation in this cell type. We have previously reported that expression of the human ET-1 gene is positively regulated by a cooperative interaction between GATA-2 and AP-1 transcription factors in cultured endothelial cells, however these factors are not sufficient to mediate cell type-specific expression. In vivo transcription studies of the murine ET-1 gene have demonstrated the presence of important cell-specific DNA elements in the 5.9 kb region upstream of the transcription initiation site. Using reporter gene transfection, site-directed mutagenesis and DNA-protein binding studies of the 5.9 kb region, we have identified a tripartite DNA element that positively regulates the expression of ET-1 specifically in cultured endothelial cells. This complex enhancer element demonstrates an endothelial cell-specific pattern of binding, suggesting that it interacts with cell-restricted regulatory factors. These findings provide important insights into the mechanisms that mediate the expression of ET-1 in the endothelium and a basis for future transgenic and cloning studies aimed at identifying the endothelial cell-specific binding site and transcription factor(s).

Topics: 3T3 Cells; Animals; Cells, Cultured; Endothelin-1; Endothelium, Vascular; Enhancer Elements, Genetic; Gene Expression Regulation; Glioma; Humans; Mice; Mice, Transgenic; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transfection

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.

Topics: Alkaloids; Benzophenanthridines; Calcium-Calmodulin-Dependent Protein Kinases; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Estrenes; Flavonoids; Gene Expression Regulation; Genes, fos; Glioma; Oligopeptides; Phenanthridines; Phosphorylation; Pyrrolidinones; RNA, Messenger; Tumor Cells, Cultured

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; CHO Cells; Consensus Sequence; Cricetinae; Cyclic AMP; Dogs; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Glioma; Indoles; Isoenzymes; Maleimides; Mutagenesis, Site-Directed; Phosphorylation; Protein Kinase C; Rats; Receptor, Adenosine A2A; Receptors, Purinergic P1; Signal Transduction; Tetradecanoylphorbol Acetate; Triazines; Triazoles; Tumor Cells, Cultured

Endothelin induced intracellular Ca(2+)signaling was studied in C6 rat glial cells. Endothelins 1 and 3 increased transiently intracellular Ca(2+)concentration, endothelin 3 being less potent inducer. Dibutyryl-cAMP treated cells responded with less sensitivity. While BQ123, a specific endothelin A receptor antagonist, inhibited both endothelins induced response in proliferating cells, it failed to inhibit in dibutyryl-cAMP treated ones. IRL1620, a specific endothelin B receptor agonist, was devoid of any significant effect. Although re-stimulation by both endothelins after endothelin-1 did not cause any Ca(2+)oscillation, both endothelins evoked new Ca(2+)transient after endothelin-3 stimulation. Our findings suggest that endothelin induced Ca(2+)signaling is mediated probably through the receptor A in proliferating C6 cells. The lack of both BQ123 and IRL 1620 effect in dibutyryl-cAMP treated cells could be caused by an alteration of endothelin A receptor alone, by a change of receptor expression pattern, or by more complex postreceptor mechanism.

Topics: Animals; Bucladesine; Calcium; Calcium Signaling; Cell Differentiation; Endothelin-1; Endothelin-3; Glioma; Rats; Receptors, Endothelin; Tumor Cells, Cultured

Agonist-induced intracellular signal transduction often involves activation of protein kinase C by diacylglycerol (DAG) released from membrane phospholipids by phospholipases. Using either DAG kinase or HPLC assays to quantitatively determine DAG mass, we observed a time-dependent increase in DAG accumulation upon incubation of rat C6 glioma cells with 200 nM endothelin-1 (ET-1). Total cell DAG rapidly increased by 25-35% from a basal level of 4.5 +/- 0.3 nmol/mg protein during one min of ET-1 treatment and remained constant or slightly decreased between 1 and 2 min. Thereafter, DAG increased to a maximum (1.6-fold above basal) by 5-10 min. and remained elevated to 30 min. Resolution of DAG molecular species by HPLC after incubation of cells with ET-1 revealed that accumulation of DAG species differed in total cell lysate and subcellular compartments. In plasma membrane, major DAG species increased at 1 min. followed by a decrease at 10 min. whereas in microsomes DAG species did not change at 1 min. and decreased at 10 min. Although phospholipid sources of DAG species were not identified specifically, there was preferential hydrolysis of molecular species of phospholipid for DAG production. We propose that molecular species of DAG produced at the plasma membrane may be transferred to the endoplasmic reticulum so that phospholipid resynthesis can replenish molecular species initially utilized in signal transduction.

Topics: Angiotensin II; Animals; Bradykinin; Brain Neoplasms; Cell Membrane; Chromatography, High Pressure Liquid; Diglycerides; Endoplasmic Reticulum; Endothelin-1; Fibroblasts; Glioma; Humans; Membrane Lipids; Mice; Microsomes; Neurotensin; Phospholipids; Rats; Serotonin; Signal Transduction; Tumor Cells, Cultured

Because the prominent neovascularization characteristic of high grade primary brain tumors is composed mostly of vascular smooth muscle cells (VSMC), we studied the expression of the potent smooth muscle mitogen endothelin-1 (ET-1) and one of its secretagogues, transforming growth factor beta 1 (TGF-beta 1) in a series of astrocytic tumors. TGF-beta 1 is also of interest due to its known activity as an angiogenic factor. Using immunohistochemical methods, we examined 30 surgical cases: 10 glioblastoma multiforme, 10 anaplastic astrocytomas, and 10 low-grade astrocytomas. Using a monoclonal antibody to TGF-beta 1 and a polyclonal antibody to ET-1, we detected both growth factors in all cases of glioblastoma examined. In cases of anaplastic astrocytoma, 4 tumors were positive for both factors; 2 contained only ET-1; 2 contained only TGF-beta 1; and 2 exhibited no tumor cell immunoreactivity for either factor. In low-grade astrocytoma, 4 of 10 tumors showed weak ET-1 immunoreactivity; 2 of those contained TGF-beta 1 immunopositive tumor astrocytes: 6 tumors were negative for both factors. In all tumors that expressed both factors, serial sections showed that regions of ET-1 immunopositivity also tended to be positive for TGF-beta 1. Endothelial cells within all tumors were positive for ET-1. ET-1 and TGF-beta 1 are present in human astrocytomas and their expression correlates with tumor vascularity and malignancy. These results suggest roles for both ET-1 and TGF-beta 1 in the growth and progressive angiogenesis of the human glioma.

Topics: Blood Vessels; Brain Neoplasms; Endothelin-1; Glioma; Humans; Immunohistochemistry; Staining and Labeling; Transforming Growth Factor beta

The endothelin (ET) peptides, ET-1, ET-2, and ET-3, as well as the ETA and ETB receptor subtypes, are known to occur in brain, but there is a dearth of information on the metabolism of these peptides by the central nervous system (CNS). In this study we have investigated the kinetics of ET-1 binding to and dissociation from the hybrid neuroblastoma x glioma cell line, NG108-15, which is known to contain functional ET receptors, and metabolism of bound ET-1. [125I]ET-1 was incubated with cells for various periods of time up to 6 h, and the nature of the radioactivity in the cell medium and lysate was analyzed by reverse phase high performance liquid chromatography (HPLC). It was found that NG108-15 cells are capable of degrading [125I]ET-1 to [125I]Tyr and several fragments of intermediate hydrophobicity; however, a portion of the cell-associated [125I]ET-1 was protected from degradation for several hours.

Topics: Central Nervous System; Chromatography, High Pressure Liquid; Endothelin-1; Glioma; Hybrid Cells; Linear Models; Neuroblastoma; Radioligand Assay

We have investigated the effects of interleukin (IL)-1 beta and lipopolysaccharide (LPS) on endothelin (ET)-induced intracellular Ca2+ rise in C6 rat glioma cells in order to study the mechanisms of their effects on Ca2+ signaling systems. Pretreatment with IL-1 beta (10(3) U/mL) and LPS (1 microgram/mL) for 24 h significantly inhibited 100 nM ET-1-induced increase in intracellular Ca2+ either in the presence or absence of external Ca2+. Their inhibitory effects were in dosedependent (IL-1 beta; 50-1000 U/mL, LPS; 10-1000 ng/mL) and time-dependent (12-24 h) manners. A tyrosine kinase antagonist genistein (50 microM) but not a protein kinase C inhibitor H7 (30 microM) prevented the inhibition of the ET response by IL-1 beta and LPS. These results suggest that activation of tyrosine kinase may be essential for the inhibition of the ET receptor-mediated Ca2+ signaling systems by IL-1 beta and LPS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Endothelin-1; Enzyme Inhibitors; Genistein; Glioma; Interleukin-1; Isoflavones; Kinetics; Lipopolysaccharides; Protein-Tyrosine Kinases; Rats; Tumor Cells, Cultured

The endothelin (ET) peptides have been identified in the CNS, but there is a paucity of information on their physiological roles. NG108-15 cells, a clonal strain of a neuroblastoma x glioma hybrid cell line, have been widely used in neurobiological research since they retain certain differentiated properties of the non-transformed parental cells. It is known that NG108-15 cells respond to the ET peptides, but only limited information is available on the characterization of the ET receptors that mediate these effects. The present study was designed to identify the type(s) of ET receptors on NG108-15 cells in a proliferative state by competitive binding assays using [125I]ET-1 as the radiolabelled ligand and the receptor-selective ligands. ET-1, ET-3, BQ-123, sarafatoxin-6-c and [Ala1,3,11,15]ET-1. The results suggested the presence of conventional ETA and ETB receptor subtypes, with ETA in excess over ETB. These findings were consistent with the results of Northern analysis in that mRNAs encoding the ETA and ETB receptor subtypes were identified in NG108-15 cells, with a preponderance of ETA to ETB. Of considerable interest was the observation of other ET-binding components with much higher affinities than the conventional receptors. It remains to be demonstrated if these particular binding components are functional and represent differ gene products or arise from association of the conventional ETA and ETB receptor subtypes with themselves or other structures, e.g. proteins or lipids, of CNS origin.

Topics: Animals; Binding, Competitive; Clone Cells; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-3; Endothelins; Glioma; Hybrid Cells; Ligands; Membranes; Mice; Models, Chemical; Neuroblastoma; Peptides, Cyclic; Rats; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Topics: Animals; Calcium; Egtazic Acid; Endothelin-1; Glioma; Hybrid Cells; Intercellular Signaling Peptides and Proteins; Kinetics; Membrane Potentials; Mice; Neuroblastoma; Peptides; Phosphatidylinositols; Rats