s6c-sarafotoxin and Glioma

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

Endothelin (ET) receptor-binding assays using [125I]ET-1 in C6-glioma cells and in Rat-1 and Swiss 3T3 fibroblasts indicated the presence of two binding sites, one of which binds agonists at the pM range and the other at the nM range. All three cell lines exhibited the same pharmacological profile for agonist binding (ET-1 congruent to sarafotoxin-b > ET-3), which suggests that the receptor is of the ETA type. Binding of ET-1 to the receptor resulted in activation of two phospholipases, phospholipase C (PLC) and phospholipase D (PLD). The activation of PLC or PLD by endothelin in the three cell lines was mediated by the high affinity binding site (nM range) and was not significantly affected by either extracellular or intracellular Ca2+. Measurement of PLD activation by ET-1 and/or phorbol 12-myristate 13-acetate (PMA), in the presence and absence of two potent inhibitors of protein kinase C (PKC), strongly suggests that activation of PLD by ET receptor in C6 glioma cells as well as in Rat-1 and Swiss 3T3 fibroblasts involves both PKC-dependent and PKC-independent mechanisms.

Topics: 3T3 Cells; Animals; Cell Line; Endothelins; Enzyme Activation; Glioma; Mice; Phospholipase D; Radioligand Assay; Rats; Receptors, Endothelin; Tumor Cells, Cultured; Type C Phospholipases; Vasoconstrictor Agents; Viper Venoms

Because increasing evidence indicates that glial cells are a target of endothelin, we have characterized endothelin-induced phosphoinositide (PI) turnover and Ca2+ homeostasis in C6 glioma cells. Endothelin-1 (ET) increased formation of 3H-inositol phosphate (IP) from PI and elicited an increase in cytosolic free Ca2+ ([Ca2+]i) in rat C6 glioma. In the presence of Li+, the increase in 3H-inositol trisphosphate formation was rapid, reaching its peak at 5 min after stimulation. ET also elicited a rapid and sustained increase in [Ca2+]i in a dose-dependent manner (1-100 nM). The rank orders of efficacy for ET-related peptides in increasing [Ca2+]i were ET = ET-2 greater than sarafotoxin greater than ET-3. Both ET-mediated stimulation of IP formation and [Ca2+]i increase were largely inhibited in the absence of external Ca2+ but unaffected by the depletion of external Na+ and the presence of dihydropyridine derivatives or verapamil. Inorganic Ca2+ channel blockers Cd2+, La3+, and Mn2+ at 1 mM inhibited both responses induced by ET. Cross-desensitization and nonadditivity were observed for both events among ET-related peptides tested, but not between ET and ATP. Pretreatment of cells with pertussis toxin (PTX) attenuated the PI response to ET, but had no effect on ET-elicited [Ca2+]i increase. ET-induced Ca2+ mobilization (measured in Ca(2+)-free medium) was only transient and was inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate. Moreover, the intracellular Ca2+ pools mobilized by ET and ATP appeared to overlap, as indicated by their partial heterologous desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Calcium; Cytosol; Endothelins; Glioma; Hydrolysis; Osmolar Concentration; Phosphatidylinositols; Rats; Tumor Cells, Cultured; Viper Venoms