ryanodine and Glioma

To investigate the underlying mechanism for the selective modulation of permeability of the blood-tumor barrier by small dose of BK, we established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary culture from neonatal rats. BMECs, astrocytes, or C6 glioma cells were treated with BK, and changes of intracellular NO and intracellular calcium level were measured with a fluorescent spectrophotometer. Similarly to the observations in astrocytes, although the initial application of BK easily triggered a ryanodine-mediated calcium-induced calcium release (CICR), we also detected a long-lasting intracellular nitric oxide (NO) elevation in C6 glioma cells upon BK treatment. However, BMECs are not the direct target of BK. Further study showed that ryanodine-mediated CICR contributes greatly to the secondary NO elevation induced by BK treatment. With an in vitro blood-tumor barrier (BTB) model, we demonstrated that NO generated in C6 glioma cells might act as an intercellular messenger and play an important role in the selective modulation of permeability of BMECs by BK. In conclusion, BK triggered CICR in C6 glioma cells, and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

Topics: Animals; Astrocytes; Bradykinin; Calcium; Capillary Permeability; Cells, Cultured; Coculture Techniques; Endothelial Cells; Female; Glioma; Horseradish Peroxidase; Nitric Oxide; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel

To investigate the underlying mechanism for the selective modulation of the permeability of blood-tumor barrier (BTB) by small dose of bradykinin (BK).. C6 glioma cells were treated with BK, and changes of intracellular nitric oxide (NO) and intracellular calcium level were measured with fluorescent spectrophotometer.. The initial application of BK easily triggered extracellular calcium influx, which resulted in intracellular calcium store release in C6 glioma cells. The above mechanism was also named ryanodine mediated calcium induced calcium release (CICR). We also detected a long-lasting intracellular NO elevation in C6 glioma cells upon BK treatment. Further study showed that ryanodine mediated CICR contributed greatly to the secondary NO elevation induced by BK treatment.. These results suggested that BK triggered CICR in C6 glioma cells and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

Topics: Animals; Bradykinin; Calcium; Cell Line, Tumor; Glioma; Intracellular Fluid; Nitric Oxide; Rats; Ryanodine; Spectrometry, Fluorescence; Time Factors

Volumes of neuroblastoma x glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl2 or MgCl2. In contrast, the cell volume was poorly dependent on tonicity when the Cl- concentration was changed by adding NaCl or H2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl- current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyloxy-alkanoic acid. When the tonicity was reduced at constant Cl- concentration by replacing NaCl with CaCl2 or MgCl2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours-days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.

Topics: Animals; Caffeine; Cell Size; Chloride Channels; Culture Media; Egtazic Acid; Glioma; Hybrid Cells; Hypotonic Solutions; Ionomycin; Membrane Potentials; Neuroblastoma; Osmolar Concentration; Ryanodine; Water-Electrolyte Balance

1. The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M-like current (IK(M,ng)) by acetylcholine was investigated in m1 muscarinic receptor-transformed mouse neuroblastoma-rat glioma hybrid (NG108-15) cells using patch-clamp techniques and calcium microfluorimetry. 2. Acetylcholine (1-100 microM) decreased IK(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8-amino-cyclic ADP ribose (10-100 microM) and 8-bromo-cyclic ADP ribose (100-1000 microM) reduced this inhibition of IK(M,ng) in a concentration-dependent manner. The half-maximal inhibition concentrations for 8-amino- cyclic ADP ribose and 8-bromo-cyclic ADP ribose were around 40 microM and 1 mM, respectively. 3. Neither of the cyclic ADP ribose antagonists altered the amplitude of IK(M,ng) per se, or the incidence of the concurrent Ca2+-activated K+ current (IIK(Ca)) activation, also mediated by acetylcholine. 4. The ryanodine receptor modulators ryanodine (1-10 microM) and Ruthenium Red (10 microM) did not alter IK(M,ng) amplitude or IK(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red. 5. Intracellular calcium levels measured with fura-2 microfluorimetry were increased with low concentrations of ryanodine (1 microM), more consistently with caffeine (10 mM), and in almost every case with both bradykinin (300 nM) and acetylcholine (100 microM). Caffeine-, but not bradykinin-evoked responses were abolished by preincubation with ryanodine (10 microM). 6. The fast 'rundown rate' of the M-current recorded in rat superior cervical ganglion cells under whole-cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 microM) was applied directly to the cytoplasmic face of inside-out membrane patches excised from rat superior cervical ganglion cells containing M-channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening). 7. These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate IK(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP

Topics: Acetylcholine; Adenosine Diphosphate Ribose; Animals; Bradykinin; Calcium; Cyclic ADP-Ribose; Glioma; Hybrid Cells; Membrane Potentials; Mice; Neuroblastoma; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Receptors, Muscarinic; Recombinant Proteins; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Superior Cervical Ganglion; Transfection

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (omega-agatoxin-IVA, omega-conotoxin MVIIC and omega-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca2+ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.

Topics: Becaplermin; Brain Neoplasms; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Elapid Venoms; Endoplasmic Reticulum; Enzyme Inhibitors; Flufenamic Acid; Glioma; Humans; Imidazoles; Ion Transport; Masoprocol; Neoplasm Proteins; Nicardipine; Nimodipine; Nitrendipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Ryanodine; Thapsigargin; Tumor Cells, Cultured; Verapamil