The Role Of Glioma Cell In Bradykinin Selective Modulation Of The Blood Tumor Barrier

In our previous in vitro study, we had showed that bradykinin (BK) could trigger intracellular calcium elevation in astrocyte and C6 glioma cell, and physiological concentration of BK could not induce intracellular calcium elevation in brain Microvascular Endothelial cells (BMECs). This indicated that BMECs are not the direct target for regular dose of BK. Because BK could modulate the permeability of BTB in vivo, some intercellular messenger or other mechanisms must account for the BK modulation of the permeability in BMECs. Several lines of evidence have elucidated that activation of B₂ receptors upon binding bradykinin, elicits a cascade of signal transduction reactions involving transient increase in intracellular Ca²⁺ concentration, activation of constitutive nitric oxide synthase, generation of Nitric Oxide, as a common intracellular and ideal intercellular messenger, NO may play a major role in BK modulation of permeability in BMECs. In this study, Relying on cell permeable DAF-2/DA, we would visualize the NO release and transmission, First we would study whether Nitric Oxide could generated in C6 glioma cell after BK was applied;second, we would test whether it could diffuse into its adjacent BMECs.Our previous study has showed that BK could trigger intracellular calcium response in astrocyte and C6 glioma cell, but their calcium response pattern is much different: when treated with low dose of BK (10^-5M), the intracellular calcium level in C6 glioma cells elevated rapidly, and form a peak after 30sec, there is an apparent peak-after plateau and shoulder in calcium response. Repeated stimulation could cause desensitization of BK, which lacks of calcium plateau and shoulder, just like the pattern of astrocyte under treatment of 10^-4M,to discover whether plateau formation is tumor cell specific and its significance, Beside the intracellular calcium response under the range of BK from 10"7M to 10'3M, we further investigate the relationship between extracellular calcium influx and intracellular calcium stores, discovered that calcium induced calcium release (CICR) participated in the intracellular calcium elevation after BK treatment and it contributes to the secondary NO release.Materials and MethodsBMECs, Astrocyte were from the primary culture of neonatal Wistar rat, C6 glioma cell line was supported by the neurobiology department of China Medical University.Measurement of [Ca2+];and NO change with BK treatmentNO was monitored by labeling with 4,5-diaminofluorescein diacetate (DAF-2 DA) that was de-esterified intracellular to DAF-2. NO provided the third nitrogen to form a triazo ring from the two amino groups of the nonfluorescent DAF-2 and converted it to diaminotriazolofluorescein (DAF-2T) that could be monitored at 490 nm excitation and 520 nm emission. The intracellular Ca was labeled by Fluo-3. Cells were incubated with lOumol/L DAF-2 DA or 5umol/L Fluo-3/AM at 37 °C for 30 min. Both Fluo-3 and DAF-2 were excited at 490 nm laser and emissions between 515-560 nm were obtained. Images of 512x512 pixels were acquired with a 20x objective.The artificial cerebrospinal fluid (ACSF):(126 mmol/L NaCl, 3.75 mmol/L KC1, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 26 mmol/L NaHCO3, 1.25 mmol/L KH2PO4, 10 mmol/L glucose, pH 7.4) with distilled water (1:1) was employed as the extracelluar balance solution. With 2mM EDTA add to obtain no extracellular calcium ACSF. The cells were designed into 4 groups: (1) control group;(2) ryanodine (which could block CICR) group;(3) nNOS inhibitor 7-NI group;(4) iNOS inhibitor aminoguanidine group.Under the coculture of BMECs with C6 glioma cell and in vitro Transwell blood-tumor barrier model, the transmission of NO between cells was visually studied by DAF-2/DA incubation.Permeability of HRP in Transwell BTB modelRat BMECs were seeded on micro-pore membrane of gelatin-coated cell culture insert and cultured to confluence. The establishment of BTB was preliminary judged by a 4h water-leaking test. The permeability of HRP through the BTB was analyzed and the effect of BK on permeability of the BTB was investigated.ResultsBK causes CICR and NO elevationWhen small dose of BK (10'5M) was applied, the intracellular calcium in C6 elevated suddenly, and form a peak after 30sec (F/F0=1.68±0.5), then it decreased and finally restored to a normal calcium level. Most importantly, we found that the intracellular calcium in C6 glioma cell is elevated slowly, and reach a peak after 30 sec;the fluorescence record showed that there is peak-after plateau and shoulder after the first peak, Ryanodine, a kind of CICR inhibitor, could block Ca2+-sensitive ryanodine receptors (RyRs) thus limit passage of Ca2+ from smooth endoplasmic reticulum into the cytoplasm, could disrupt the peak-after plateau and shoulder, which indicated this long lasting intracellular calcium wave was resulted from calcium release from intracellular calcium stores which is also called calcium-induced calcium release (CICR). With the concentration of bradykinin elevated, the percentage of responsive cell elevated largely and CICR occurred more frequently. Different to rapid intracellular calcium elevation after BK was applied;there is a slower and long lasting elevation of intracellular NO, which would maintain more than 20 min. The generation of NO was significantly reduced during treatment with ryanodine, which indicated that CICR contributes lots to the secondary NO generation after BK was applied. When concentration of BK elevated, the responsive astrocyte and CICR phenomenon also increased, but the percentage of the response cell is much less than the correspondent C6 glioma cell group, especially the CICR group. Although the intracellular calcium peak value of astrocyte is similar to C6 glioma cell, the secondary NO elevation in astrocyte is much more moderate,possibly due to lack of CICR in astrocyte.NO contributes to BK modulation permeability of BMECsWe showed that NO generation in C6 glioma cell could diffuse into its adjacent BMECs. BK could enhance permeability of BMECs under in vitro BTB model, and nNOS participated in the process. BK could not modulate the permeability of sole monolayer of BMECs, which indicated glioma cell are direct trigger point of BK that could selectively enhance permeability of BMECs.Conclusions1 > There are function Ryanodine receptor in C6 glioma cell and Astrocyte, BK could trigger Calcium induced calcium release (CICR).2> BK triggered CICR contribute lots to the secondary NO release, the NO released in C6 glioma cell could diffused into its adjacent BMECs.3> The difference of calcium response especially CICR may largely account for the selectiveness of small dose of BK to glioma cell.4? The glioma cell is the direct target of BK in vitro, NO released in glioma cell after BK treatment play an import role in BK modulation of the permeability of primary BMECs.