Ghrelin对豚鼠胃窦平滑肌细胞钙离子浓度的影响及与NO关系研究  

Study on the effects of Ghrelin on intracellular calcium mobilization and its relationship with NO in guinea pig gastric antrum smooth muscle cells

作　　者：信芳杰[1] 徐珞[1] 

机构地区：[1]青岛大学医学院病理生理教研室,山东青岛266021

出　　处：《现代生物医学进展》2010年第2期216-220,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30470642和30670780);山东省科技攻关项目(2008GG10002006);山东省卫生厅项目(2007HZ026);青岛市科技局项目(05-1-JC-93)

摘　　要：目的:探讨Ghrelin对豚鼠胃窦平滑肌细胞内钙离子浓度的影响及其与一氧化氮(NO)的关系。方法:采用荧光免疫组化检测胃窦平滑肌细胞ghrelin受体(GHS-R)的表达;应用钙离子(Ca2+)指示剂Fluo-3/AM作为细胞内Ca2+的荧光探针,对负载培养的平滑肌细胞应用激光共聚焦显微镜技术,检测不同浓度ghrelin对平滑肌细胞内Ca2+荧光强度(FI)的影响,以及ghrelin受体阻断剂D-Lys3-GHRP-6、NO供体硝普钠(SNP),一氧化氮合酶(NOS)抑制剂N-硝基左旋精氨酸甲酯(L-NAME)对ghrelin调控Ca2+荧光强度的影响。结果:(1)豚鼠胃窦平滑肌细胞呈GHS-R免疫反应阳性表达.(2)随着ghrelin浓度升高(10-11,10-10,10-9,10-8,10-7mol/L),平滑肌细胞内Ca2+荧光强度逐渐升高,组间峰值(分别为54.7±11.5,58.1±5.7,64.8±6.6,84.9±7.1,95.7±10.5)和峰高(分别为1.8±0.3,2.1±0.8,5.3±1.3,28.9±4.2,37.6±3.7)均存在显著差异(P<0.05-0.01),即呈明显剂量依赖关系.(3)采用Ghre-lin受体拮抗剂D-Lys3-GHRP-6孵育后,再加入ghrelin,ghrelin可使胞内Ca2+荧光强度升高作用几乎完全被抑制(P>0.05).(4)平滑肌细胞SNP预处理后再加入ghrelin,在SNP作用下胞内Ca2+荧光强度先是明显下降(P<0.01),而后随着ghrelin的加入,Ca2+荧光强度缓慢回升,但与对照组比较仍显著降低(P<0.05).(5)L-NAME可增加细胞内Ca2+荧光强度(P<0.05),并且可显著增加ghrelin促胞内Ca2+浓度增加作用(P<0.01)。结论:豚鼠胃窦平滑肌细胞有GHS-R的表达;Ghrelin通过其特异性受体可增加胃窦平滑肌细胞内Ca2+浓度,该作用可能与NO通路有关。Objective：To investigate the effects of ghrelin on intracellular Ca2＋ mobilization in acutely isolated guinea pig gastric antrum smooth muscle cells and its ralationship with NO pathways. Methods：Expression of the Ghrelin receptor（GHR-S） was identified with immunofluorescence staining technique The effect of ghrelin on intracellular Ca2＋ concentration of gastric antrum smooth muscle cells was detected by a laser confocal microscope using Fluo-3/AM as an the radiometric Ca2＋ indicator. Furthermore,the effects of D-Lys3-GHRP-6,SNP and nitric oxide synthase inhibitor（L-NAME） on Ca2＋ fluorescent intensity-induced by ghrelin were studied. Re-sults：（1） The guinea pig gastric antrum smooth muscle cells showed positive ghrelin receptor immunoreactivity.（2） 10-11,10-10,10-9,10-8,10-7mol/L ghrelin could elavate intracellular [Ca2＋]i to the peak levels of 54.7±11.5,58.1±5.7,64.8±6.6,84.9±7.1,95.7±10.5 and with their relative FI（peak level subtract resting levle） in fluorescent intensity of 1.8±0.3,2.1±0.8,5.3±1.3,28.9±4.2,37.6±3.7 respectively,which existed significant differences（P0.05-0.01） and indicated the dose-dependent effect of ghrelin on [Ca2＋]i.（3） When pretreatment with D-Lys3-GHRP-6,antagonist of ghrelin,the increase of [Ca2＋]i was almost inhibited completely.（4） After pretreatment with SNP,a signficant decrease occurred and then there was a gradual and slow increase of [Ca2＋]i,which compared with normal control were obviously decreased（P0.05） .（5） L-NAME could elavate intracellular [Ca2＋]i（P0.05） ,and which combined use of ghrelin in-creased [Ca2＋]i more than ghrelin single used（P0.01） . Conclusion：The Ghrelin receptor could be expressed by acutely isolated guinea pig gastric antrum smooth muscle cells. Ghrelin could increase [Ca2＋]i in by its specific receptor. The effects of ghrelin could be partial blocked by SNP,and this effect may be mediated by activating NO pathways.

关 键 词：GHRELIN 胃窦平滑肌 钙离子 激光共聚焦 豚鼠 

分 类 号：Q95-3[生物学—动物学] R57[医药卫生—消化系统]