肾上腺素对视网膜色素上皮细胞ATP酶活性及细胞内cAMP和钙离子浓度的影响

Epinephrine-induced ATPase Activity,[cAMP]_i and [Ca^(2+)]_i Changes in Human Retinal Pigment Epithelium

目的研究肾上腺素对培养的成人视网膜色素上皮细胞(retinal pigment epithelium,RPE)钠-钾三磷酸腺苷酶(Na+-K+ATP酶)、钙离子三磷酸腺苷酶(Ca2+-ATP酶)和细胞内环腺嘌呤核苷酸(cAMP)[、Ca2+]i的影响与超微结构的变化,以探讨视网膜下液转运和吸收的机制。方法采用不同浓度肾上腺素诱导RPE细胞24 h后,采用ATP酶试剂盒检测细胞Na+-K+ATP酶、Ca2+-ATP酶活力,125I-cAMP放免试剂盒检测胞内cAMP浓度,Fura-2/AM荧光探针检测细胞内[Ca2+]i,并采用透射电镜技术观察细胞超微结构的改变。结果0.1~100 nmol/L肾上腺素能显著降低RPE细胞Na+-K+ATP酶活性,并提升Ca2+-ATP酶活性(均P<0.01)。10 nmol/L肾上腺素可明显升高REP细胞内cAMP水平,加入10 nmol/L肾上腺素30 min可明显引起RPE细胞内[Ca2+]i降低,透射电镜观察发现肾上腺素诱导后的RPE细胞内水肿样改变。结论肾上腺素可能通过β-肾上腺素能受体-cAMP途径影响RPE细胞离子与液体转运。

Objective To define Na^＋-K^＋ ATPase and Ca2^＋-ATPase activity, [cAMP]i and [Ca^2＋ ]i changes in epinephrineinduced human retinal pigment epithelium （RPE）, and the influence on RPE intracellular uhrastructure in order to to investigate the mechanism of subretinal fluids transport. Methods Epinephrine and adrenergic blocking agent （prazosin and propranolol）-induced human RPE ATPase changes were monitored with ATPase kit. Intracellular cAMP levels were determined with ^125I-cAMP radioimmunoassay kit. Fura-2/AM fluorescent probe was used to detect the [Ca^2＋ ]i. Scanning electron microscope （SEM） was used to identify RPE uhrasturcture changes. Results 0.1-100 nmol/L epinephrine increased the 5 % RPE homogenate Ca^2＋-ATPase activity but decreased Na^＋-K^＋ ATPase activity （P〈0.01）. [cAMP]i elevation and [Ca^2＋]i decrease were induced by epinephrine （10 nmol/L）. Electron transmission microscopic examination of epinephrine-treated hRPE revealed intracellular edema, which could only be blocked by propranolol partially. Conclusion Epinephrine response in human RPE ions transport was mediated by β-adrenergic receptors-cAMP pathway mechanism.