摘要
目的观察大鼠肠肌间神经元胃动素受体(MTLR)的表达,并探讨胃动素引起神经元内钙信号的机制。方法采用免疫荧光双标技术观察大鼠肠肌间神经元MTLR的表达;应用激光共聚焦显微镜技术检测不同药物处理组胃动素引起的单个神经元内Ca2+荧光强度的变化。结果大鼠肠肌间神经元呈MTLR免疫反应阳性表达;在Hank's液中,10-6mol/L胃动素可引起神经元内Ca2+浓度的显著升高,其峰高(峰值减去静息值)为30.6±3.7,荧光强度相对变化百分比为(100.8±18.4)%。在D-Hank's液(去除细胞外Ca2+)中或用L型钙离子通道阻断剂维拉帕米预处理细胞后,胃动素可轻度升高胞内Ca2+浓度。与单独应用胃动素组相比差异有统计学意义(P<0.05)。当分别用G蛋白拮抗剂NEM和PLC抑制剂Compound48/80预处理细胞后再加入胃动素,胞内Ca2+浓度升高的程度明显降低,与单独应用胃动素组相比差异有统计学意义(P<0.05)。当用PKC抑制剂D-鞘氨醇预处理细胞后,再加入胃动素,其峰高和荧光强度相对变化百分比同单独应用胃动素组相比差异无统计学意义(P>0.05)。结论大鼠肠肌间神经元能自身表达MTLR。胃动素可明显升高神经元内Ca2+浓度,胞内Ca2+浓度的升高既源于外钙的内流又源于内钙的释放。外钙的内流主要通过L型Ca2+通道,G蛋白偶联型MTLR-PLC-IP3途径参与细胞内钙的释放。
Objective To observe whether the motilin receptor （MTLR） can be expressed in primarily cultured myenteric neurons of rats and investigate the mechanism of motilin induced Ca^2＋ signaling in myenteric neurons of rats. Methods Expression of the motilin receptor was identified with double-immunofluorescence staining technique. Data on the intracellular Ca^2＋ concentration （[Ca^2＋]i） of cultured myenteric neurons with different treatments were collected by measuring Ca^2＋ fluorescent intensity （FI） in each neuron under confocal microscope. Results The cultured myenteric neurons showed positive motilin receptor immunoreactivity. In Hank＇s solution,10^-6 mol/L motilin could elevate [Ca^2＋]i, its height of peak being 30. 6±3.7 and its FI relative change percentage being （100. 8±18. 4）%. In D-Hank＇s solution （after removal of extracellular Ca^2＋, or after treatment with verapamil,an L-type calcium channel blocker）, motilin could induce a small increase of [Ca^2＋ ]i, After pretreatment with NEM,a G protein inhibitor, and Compound 48/80, a PLC inhibitor, in Hank＇s solution respectively, motilin was inhibited and the [Ca^2＋ ]i was significantly lower than that of the group to which was added only motilin （P〈0.05）. After pretreatment with D-sphingosine, a PKC inhibitor, the effect of motilin was not significantly different from that of the group to which was added only motilin（P〉0.05）. Conclusion The motilin receptor could be expressed by cultured myenteric neurons of rats. Motilin could increase [Ca^2＋ ]i. The increase of [Ca^2＋ ]i was caused by release of intracellular stores and influx of extracellular Ca^2＋, mainly through the L-type calcium channel. The motilin receptor-coupled G-protein, PLC and IP3 pathway participated in the release of Ca^2＋ from intracellular stores.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第5期683-686,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30170414)资助
关键词
胃动素
胃动素受体
肠肌间神经元
钙信号
Motilin Motilin receptor（MTLR） Myenteric neuron Ca^2＋ signaling
