血清素（5-HT7）受体刺激牛眼角膜上皮和内皮细胞中环磷酸腺苷-PKA 通路的激活。

Serotonin (5-HT7) receptor-stimulated activation of cAMP-PKA pathway in bovine corneal epithelial and endothelial cells.

机构信息

Department of Ophthalmology, University of Tübingen, Tübingen, Germany.

出版信息

Ophthalmic Res. 2012;48(1):22-7. doi: 10.1159/000334912. Epub 2012 Jan 3.

DOI:10.1159/000334912

PMID:22222787

Abstract

BACKGROUND

5-Hydroxytryptamine (5-HT; serotonin) is a major neurotransmitter, and its receptors are found throughout the whole body. The 5-HT7 receptor subtype was detected in human corneal epithelial and endothelial cells and found to be functionally active in a corneal epithelial cell line. The aim of the present study was to demonstrate that native bovine corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and protein kinase A (PKA) formation.

METHODS

5-HT7 receptors were studied using polyclonal antibodies. cAMP concentration after 5-HT7 receptor stimulation with 5-carboxamidotryptamine (5-CT, a 5-HT7 agonist) was tested by enzyme immunoassay, PKA activity was estimated by kinase consumption of ATP.

RESULTS

Immunocytochemistry and immunofluorescence revealed the presence of 5-HT7 receptors in corneal epithelial and endothelial cells. Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in intracellular cAMP concentration in corneal epithelium (0.01-0.34 pmol/ml) and endothelium (0.01-0.19 pmol/ml) between 10(-10) and 10(-7) mg/ml 5-CT (p = 0.001) with maximal stimulation from 10(-7) to 10(-3) mg/ml 5-CT (0.30 ± 0.03 and 0.18 ± 0.01 pmol/ml, respectively). Incubation with 10(-6) mg/ml SB269970 (a selective 5-HT7 antagonist) blocked 5-CT-induced cAMP increase in corneal epithelial (0.03 pmol/ml) and endothelial cells (0.02 pmol/ml; p = 0.001). Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in PKA activity between 10(-10) and 10(-8) mg/ml 5-CT in corneal epithelium and endothelium (<1 to >99%; p = 0.013 and p = 0.017, respectively) with maximal stimulation from 10(-8) to 10(-4) mg/ml (>99%) 5-CT.

CONCLUSIONS

Our data demonstrate that native corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and PKA formation. However, at the present time, the physiological role of 5-HT receptors and the cAMP-PKA pathway in the cornea remains a matter of speculation.

摘要

背景

5-羟色胺（5-HT；血清素）是一种主要的神经递质，其受体存在于全身各处。5-HT7 受体亚型在人角膜上皮细胞和内皮细胞中被检测到，并在角膜上皮细胞系中发现其具有功能性。本研究旨在证明天然牛角膜上皮细胞和内皮细胞表达一种功能性 5-HT7 受体，该受体与环磷酸腺苷（cAMP）和蛋白激酶 A（PKA）的形成呈正偶联。

方法

使用多克隆抗体研究 5-HT7 受体。通过酶免疫测定法检测 5-羧基色胺（5-CT，一种 5-HT7 激动剂）刺激 5-HT7 受体后 cAMP 浓度，通过激酶消耗 ATP 来估计 PKA 活性。

结果

免疫细胞化学和免疫荧光显示 5-HT7 受体存在于角膜上皮细胞和内皮细胞中。用 5-CT 刺激角膜 5-HT7 受体，在角膜上皮（0.01-0.34 pmol/ml）和内皮（0.01-0.19 pmol/ml）中观察到 cAMP 浓度呈剂量依赖性增加，浓度范围为 10（-10）至 10（-7）mg/ml 5-CT（p=0.001），用 10（-7）至 10（-3）mg/ml 5-CT 进行最大刺激（分别为 0.30±0.03 和 0.18±0.01 pmol/ml）。用 10（-6）mg/ml SB269970（一种选择性 5-HT7 拮抗剂）孵育可阻断角膜上皮细胞（0.03 pmol/ml）和内皮细胞（0.02 pmol/ml）中 5-CT 诱导的 cAMP 增加（p=0.001）。用 5-CT 刺激角膜 5-HT7 受体，在角膜上皮和内皮细胞中观察到 10（-10）至 10（-8）mg/ml 5-CT 范围内 PKA 活性呈剂量依赖性增加（<1 至>99%；p=0.013 和 p=0.017），用 10（-8）至 10（-4）mg/ml 5-CT （>99%）进行最大刺激。