Low dose of bradykinin selectively increases intracellular calcium in glioma cells

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Abstract

To investigate the underlying basis for the selective modulation of the permeability of the blood–brain barrier by small doses of bradykinin, we first established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary cultures from neonatal rats. BMECs, astrocytes and C6 glioma cells were treated with different concentrations of BK (range from 10– 8 M to 10– 4 M), and changes of intracellular calcium levels were measured by fluorescence spectrophotometry. Expression levels of B2 receptors were analyzed by Western blot analysis. We found that a low dose (10– 6 M) of BK could trigger an elevation of intracellular calcium level in C6 glioma cells, whereas astrocytes responded to a higher concentration of BK (10– 5 M), and the BMECs remained unresponsive to BK. Western blot results showed that C6 glioma cells expressed the highest level of B2 receptors compared with primary astrocytes or BMECs. B2 receptors are highly expressed in glioma cells and a low dose of BK selectively increases the intracellular calcium level in tumor cells which, in turn, could contribute to the selective increase in the permeability of the blood–tumor barrier by small doses of BK.

Introduction

The blood–brain barrier (BBB) protects the brain by restricting the entry of circulating non-lipid molecules that are not recognized by specific transporters on the luminal side of the brain capillaries. Brain tumor capillaries have different structural and functional characteristics from the normal brain capillaries, and constitute the blood–tumor barrier (BTB), which, despite being more ‘leaky’ than the BBB, also limits delivery of therapeutic agents to the tumor [1]. Some studies showed that if the local drug concentration is elevated by two fold, the efficacy to kill the brain tumor cells can be increased by 10 fold [2]. So, efficient delivery of drugs to tumor cells is crucial for effective chemotherapy of glioma. It was found that, in contrast to a large dose of Bradykinin (BK) that breaks down the whole BBB, a small dose of BK can selectively open the blood–tumor barrier therefore enhancing the delivery of large particles which are normally impeded by the blood–tumor barrier [1], [3]. The selective modulation of blood–tumor barrier provides a promising tool for efficient chemotherapy.

Pharmacological and molecular biological studies have proved the existence of two major subtypes of bradykinin receptors, B1 receptor and the G-protein coupled B2 receptor. In vivo studies showed that the selective increase in BTB permeability induced by intracarotid infusion of low doses of bradykinin is mediated solely by B2 receptors because such increased permeability can be significantly inhibited by the B2 receptor antagonists, HOE 140 and d-Arg, but not by the B1 receptor antagonist [1], [4]. In our previous animal study, we have shown that the B2 receptors are expressed mainly in tumor cells, but not in normal or tumor capillaries [4]. But it still cannot clarify why a small dose of BK selectively opens the BTB, while a large dose of BK breaks down the whole BBB. In this study, we show that B2 receptor is highly expressed in glioma cells compared with primary astrocytes and brain microvascular endothelial cells (BMECs), and a low dose BK selectively increases the intracellular calcium level in tumor cells, which might be the mechanism for the selective modulation of blood–tumor barrier by a low dose of BK treatment; and a larger dose of BK triggers similar intracellular responses in astrocytes, which maybe the reason for breaking down the whole BBB.

Section snippets

Cell culture and drug treatment

Rat C6 glioma cells (obtained from the Cell Biology Department of the China Medical University), primary astrocytes, and BMECs were maintained in complete culture media (DMEM, 20% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin), supplemented with 100 μg/ml heparin and 10 μg/ml endothelial cell growth supplement (BD Biosciences).

Female neonatal Wistar rats were used for the isolation of primary BMECs and astrocytes. Animals (1–2 weeks) were sacrificed by CO2 inhalation.

Bradykinin (BK) differentially regulates intracellular calcium level

To further clarify why a small dose of BK selectively enhances the permeability of blood–tumor barrier, we first examined the changes of intracellular calcium level after BK treatment in C6 glioma cells, primary astrocytes and BMECs. On the one hand, as shown in Fig. 2A, when treated with a low dose of BK (10− 6 M), the intracellular calcium level in C6 glioma cells elevated rapidly and formed a peak after 30 s (increased more than 70%), then it decreased and finally restored to a normal level.

Discussions

Bradykinin, a naturally occurring nonapeptide, when used at high doses, induces breakdown of the normal BBB. However, intracarotid infusion of low dose of bradykinin can selectively increase the permeability of capillaries in brain tumors without affecting the permeability of normal brain capillaries. A common explanation for this selectivity to open the blood–tumor barrier is that the unequal distribution of B2 receptors between tumor capillary and normal brain capillary [10]. In our previous

Acknowledgments

This work was supported by the Natural Science Foundation of China, under contract No.30670723, No.30570650, No.30600060 and No.30400145, Natural Science Foundation of Liaoning Province, No.20052102, Special fund for scientific research of doctor-degree subjects in colleges and universities, No.20050159005.

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