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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。
AIM: To investigate the effect of Ghrelin on intracellular calcium concentration in guinea-pig gastric smooth muscle cells and its relationship with nitric oxide (NO). Methods: The expression of ghrelin receptor (GHS-R) in gastric antral smooth muscle cells was detected by fluorescent immunohistochemistry. Fluo-3 / AM, a Ca2 + indicator, was used as a fluorescent probe for intracellular Ca2 + The effects of different concentrations of ghrelin on the intracellular Ca2 + fluorescence intensity (FI) of smooth muscle cells were detected by laser scanning confocal microscopy. The effects of ghrelin receptor blocker D-Lys3-GHRP-6, NO donor nitroprusside sodium (SNP) Effect of nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) on ghrelin-mediated Ca2 + fluorescence intensity. Results: (1) The GHS-R immunoreactivity of antral smooth muscle cells in guinea pigs was positive. (2) With the increase of ghrelin concentration (10-11,10-10,10-9,10-8,10-7mol / L ), The fluorescence intensity of Ca2 + in smooth muscle cells increased gradually with peak values (54.7 ± 11.5,58.1 ± 5.7,64.8 ± 6.6,84.9 ± 7.1,95.7 ± 10.5, respectively) and peak height (1.8 ± 0.3,2.1 ± 0.8, 5.3 ± 1.3, 28.9 ± 4.2, 37.6 ± 3.7) (P <0.05-0.01), which showed a significant dose-dependent relationship. (3) The Ghrelin receptor antagonist D-Lys3- After incubation with ghrelin and ghrelin, the increase of intracellular Ca2 + fluorescence intensity was almost completely inhibited (P> 0.05). (4) Ghrelin was pretreated with SNP in smooth muscle cells and intracellular Ca2 + fluorescence (P <0.01). Then, the fluorescence intensity of Ca2 + gradually rose with the addition of ghrelin, but still significantly decreased compared with the control group (P <0.05). (5) L-NAME increased the intracellular Ca2 + fluorescence intensity (P <0.05), and could significantly increase the intracellular Ca2 + concentration increased by ghrelin (P <0.01). CONCLUSION: The expression of GHS-R in guinea pig gastric antral smooth muscle cells was increased. Ghrelin increased the intracellular Ca2 + concentration in antral smooth muscle cells through its specific receptor, which may be related to NO pathway.