Relationship Between Drug Treatment Time And Bradykinin B₂ Receptor Internalization In Glioma Cells

ObjectiveBradykinin B2 receptor, an important site of bradykinin, can release blood vessel, increase the permeability of blood vessel and cause some other physiological actions. More and more neurobiological scientists have noticed the characteristic of bradykinin B2 receptors, which can open the blood - brain barrier, deliver the chemotherapeutic agents to tumor tissue and increase the survival rate of patients with the action of the agitation. American and European scientists doing a series phase Ⅱ clinical trials in glioma using receptor - mediated perme-abilizer - 7 (RMP - 7, a bradykinin analogue) combined with carboplatin offer preliminary evidence that RMP - 7 combining with bradykinin B2 receptors can open blood - brain barrier selectively and cure the glioma better. But during the clinic trials, scientists found RMP - 7 can only open the blood - brain barrier for about 10 min. With the administration time extending, the permeability of the blood - brain barrier began to decrease. This made scientists have to study the mechanisms of the opening of blood - brain barrier by bradykinin B2 receptors further.Some studies demonstrated that most G - protein - coupled receptors, such as β -adrenoceptor, angiotensin Ⅱ receptor, muscarinic acetylcholine receptor, showed reactive decrease responding to long time and repeated stimulation by agitation. With the drug treatment time expending, the receptors can experience desensitization, internalization and even down - regulation and other different change course. As one of the G - protein - coupled receptor family members, bradykinin B2 receptor can also be desensitized and internalized by agita-tion, therefore influence the permeability of the blood - brain barrier and the glioma chemotherapeutic effect.This experiment use cultivated rat C6 glioma cells to determinate the brady-kinin B2 receptor expressing levels and changes in membrane during different drug treatment time which confirm the internalization time of the receptors and offer reference for clinical administration program of bradykinin and RMP -7.Materials1. Cells: rat C6 glioma cell strain provided by cytobiological laboratory of China Medical University.2. Experimental reagents: RPMI - 1640, 0. 25% Trypsin ( Gibco) ; fetus cattle serum ( H&Y Company, Tianjin) ; bradykinin (Sigma, U. S. A) ; goat anti rat bradykinin B2 receptor polyclonal antibody (Santa Cruz Biotechnology, U. S. A) ; Alkaline phosphatase tagged rabbit anti goat IgG antibody, SP kit, DAB kit ( Zhongshan Company, Beijing) ; BCIP/NBT kit ( Boster Biological Technology Company, Wuhan).3. Experimental instruments: CO2 incubator; invert microscope; low temperature centrifuger; electrophoresis apparatus; transmark apparatus; transmission electron microscope.MethodsTreated the cultivated rat C6 glioma cells with bradykinin for 10, 15, 20, 30, 60 min separately and then collect them.To determine the bradykinin B2 receptor expressing levels and changes in membrane during different drug treatment time, Western blot and immunoelec-tron microscope methods were used.Integrated Density Value (IDV) of B2 receptors protein bands from scanned immunoblot by Chemilmager 5500 V2. 03 software was quantitative analysis by Fluor Chen 2.0 software.Data were disposed with SPSS10.0 statistic software by One - Way ANOVAand linear regression and they were expressed as mean ± standard deviation (SD). A P - value of <0.05 was significant.Results1. The bradykinin B2 receptor expressing levels in the membrane during different drug treatment time.Western - blot result of bradykinin B2 receptors showed a specific protein band of 42KD, which is coincidence with the report. Compared to B2 receptor expressing level before drug treatment time, the B2 receptor expressing level came to the lowest point after 10 min of drug treatment; then the expressing level increased gradually; after 60 min of administration, the B2 receptor expressing level almost reached to that before drug treatment.2. The IDV differences between groups of bradykinin B2 receptor expressing level and the linear regression between drug treatment time and B2 receptor recovery.The IDV diversity between bradykinin B2 receptor expressing level of different drug treatment time; compared to the IDV before drug treatment 25 767. 33 ±4817.01, (n =6) , there were significant differences of 10 min IDV 3528. 50 ±977.86, n=6, (P<0.01), 15 min IDV 10 233.67 ±3428.60, n=6, (P <0.01), 20 min IDV 14 680.00 ±3326.47, n=6, (P<0.01), 30 min IDV 17 143.17 ± 2984.38, n = 6, ( P < 0.01) . After 60 min drug treatment, the B2 receptor expressing level closing to the 0 min administration, the IDV was 22 528.33 ±6228.29, n=6. Regression analysis showed that there was a linear regression between drug treatment time and bradykinin B2 receptor recovery, y = 4919.23+322. 35x,P<0.01.3. To observe bradykinin B2 receptors changes in the membrane in different drug treatment time by transmission electron microscope.After treated with bradykinin for 10 min, there were more compact electronic things in the cytoplasm near the membrane; after treated with bradykinin for 15 min, 20 min, most of the compact electronic things are in the cytoplasm near the membrane; after treated with bradykinin for 30 min, 60 min, the compact e-lectronic things in the cytoplasm near the membrane decreased, more strips of discontinuous compact electronic things locating in the membrane.DiscussionBradykinin combining with B2 receptors can open the blood - brain barrier selectively. We now consider that on one hand bradykinin can open the blood -brain barrier by forming the transport vesicles in the glioma and its capillary vessels , on the other hand bradykinin can activate the Kca channels in the glioma and its capillary vessels to increase the permeability of blood - brain barrier through NO - cGMP system directly and indirectly. Recently the clinic experiments found that the permeability of blood -brain barrier wasn't correspondent with the drug treatment time: as the drug treatment time extending, the permeability of the blood - brain barrier in the glioma patients decreased gradually. This problem restricted the chemotherapy of selective opening of the blood -brain barrier in glioma by bradykinin. We speculated this may be correlated with the internalization of bradykinin B2 receptor.This study used cultivated rat C6 glioma cells, observe the relation between drug treatment time and bradykinin B2 receptor internalization. We evidenced the extending drug treatment time can cause the B2 receptors in the membrane to internalize definitely; then it effected the selective open of blood - brain barrier by bradykinin. In this demonstration, in 10 min drug treatment time the bradykinin B2 receptor in glioma came to the lowest point, which showed B2 receptor internalization caused by bradykinin occurred mainly in 10 min drug treatment time. There is a linear regression between drug treatment time and B2 receptor expressing level recovery. The internalizated receptors dephosphorylate and recycle to the membrane. The bradykinin B2 receptor internalization was different with the β - adrenoceptor down regulation. The β - adrenoceptor decreased obviously when extending the drug treatment time. We found that the bradykinin B2 receptor expressing level decreased in 10 min drug treatment time, but the expressing level can gradually recover to the level before drug treatment time as the time extending. The numbers of the receptors wasn't decreased obviously.