omega-conotoxin-(conus-magus) and fura-2-am

[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca(2+)-free medium + 0.5 mM ethylene glycol-bis(beta-aminoethylether)N,N,N',N'-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 microM modified [3H]purine outflow whereas 0.1 microM omega-conotoxin and 0.03-0.1 microM nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 microM omega-conotoxin + 0.1 microM nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor omega-conotoxin influenced the uptake of 45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the 45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 microM Fura-2/AM. 10-30 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 microM thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC, seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Corpus Striatum; Egtazic Acid; Electric Stimulation; Fetus; Fluorescent Dyes; Fura-2; Kinetics; Neuroglia; Nitrendipine; omega-Conotoxins; Peptides; Purines; Radioisotope Dilution Technique; Rats; Rats, Sprague-Dawley; Time Factors; Tritium

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channel Blockers; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Nimodipine; omega-Conotoxins; PC12 Cells; Peptides, Cyclic; Terpenes; Thapsigargin