Yasuyoshi H, Kashii S, Zhang S, Nishida A, Yamauchi T, Honda Y, Asano Y, Sato S, Akaike A
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Japan.
Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2273-8.
To identify the localization and expression of bradykinin (BK)-B2 receptors in rat retina and examine the effects of BK on glutamate-induced neurotoxicity using cultured rat retinal neurons.
An immunohistochemical study using a specific antibody against BK-B2 receptor was performed with rat retina. Primary cultures were obtained from the retina of fetal rats (gestation day 17-19). Expression of BK-B2 receptor mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA obtained from cultured retinal neurons. Cultured cells were exposed to glutamate (1 mM) for 10 minutes and followed by incubation in glutamate-free medium for 1 hour. The effects of BK were assessed by simultaneous application of BK with glutamate. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method.
Immunohistochemical study demonstrated that BK-B2 receptors were expressed in the ganglion cell, inner nuclear layers, and outer nuclear layers. Furthermore, BK-B2 receptor mRNA expression was observed in cultured retinal neurons. Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate-free medium. Simultaneous application of BK at concentrations of 0.001 to 1 microM with glutamate demonstrated dose-dependent protection against glutamate neurotoxicity. The protective action of BK (1 microM) was inhibited by simultaneous application of BK-B2 receptor antagonist, Hoe140 (1 microM). Furthermore, 1 microM BK had protective effects on neurotoxicity induced by 1 microM ionomycin, a calcium ionophore, and sodium nitroprusside (SNP, 500 microM), a nitric oxide (NO)-generating agent. However, BK did not inhibit neurotoxicity induced by 3-morpholinosydnonimine (SIN-1, 10 microM), an NO and oxygen radical donor.
These results suggest that BK-B2 receptors were distributed in rat retinas and cultured retinal neurons and that BK had a protective action against glutamate neurotoxicity through BK-B2 receptors in cultured retinal neurons. It is suggested that BK-induced protection against glutamate neurotoxicity took place downstream to NO generation and upstream to oxygen radical generation.
鉴定大鼠视网膜中缓激肽（BK）-B2受体的定位与表达，并利用培养的大鼠视网膜神经元研究BK对谷氨酸诱导的神经毒性的影响。
使用抗BK-B2受体的特异性抗体对大鼠视网膜进行免疫组织化学研究。原代培养物取自胎鼠（妊娠第17 - 19天）的视网膜。利用从培养的视网膜神经元获得的总RNA，通过逆转录-聚合酶链反应（RT-PCR）测定BK-B2受体mRNA的表达。将培养的细胞暴露于谷氨酸（1 mM）10分钟，然后在无谷氨酸的培养基中孵育1小时。通过将BK与谷氨酸同时应用来评估BK的作用。通过台盼蓝排斥法对视网膜培养物的神经毒性作用进行定量评估。
免疫组织化学研究表明，BK-B2受体在神经节细胞层、内核层和外核层中表达。此外，在培养的视网膜神经元中观察到BK-B2受体mRNA的表达。暴露于1 mM谷氨酸10分钟，然后在无谷氨酸的培养基中孵育1小时，细胞活力显著降低。将浓度为0.001至1 microM的BK与谷氨酸同时应用，显示出对谷氨酸神经毒性的剂量依赖性保护作用。同时应用BK-B2受体拮抗剂Hoe140（1 microM）可抑制BK（1 microM）的保护作用。此外，1 microM BK对由1 microM离子霉素（一种钙离子载体）和硝普钠（SNP，500 microM，一种一氧化氮（NO）生成剂）诱导的神经毒性具有保护作用。然而，BK不能抑制由3-吗啉代辛二亚胺（SIN-1，10 microM，一种NO和氧自由基供体）诱导的神经毒性。
这些结果表明，BK-B2受体分布于大鼠视网膜和培养的视网膜神经元中，并且BK通过培养的视网膜神经元中的BK-B2受体对谷氨酸神经毒性具有保护作用。提示BK诱导的对谷氨酸神经毒性的保护作用发生在NO生成的下游和氧自由基生成的上游。
