表皮生长因子（EGF）用于刺激大鼠泪腺蛋白质分泌的信号转导途径。

Signal transduction pathways used by EGF to stimulate protein secretion in rat lacrimal gland.

作者信息

Tepavcevic Vanja, Hodges Robin R, Zoukhri Driss, Dartt Darlene A

机构信息

Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.

出版信息

Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1075-81. doi: 10.1167/iovs.02-0794.

DOI:10.1167/iovs.02-0794

PMID:12601032

Abstract

PURPOSE

To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components.

METHODS

Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37 degrees C. Acini were incubated with EGF (10(-7) M), the cholinergic agonist carbachol (10(-4) M), or the alpha(1)-adrenergic agonist phenylephrine (10(-4) M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium (Ca(2+)), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22 degrees C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca(2+) was chelated with KRB-BSA without CaCl(2) and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10(-6) M), and peroxidase secretion was measured.

RESULTS

EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10(-7) M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased Ca(2+), whereas chelation of extracellular Ca(2+) inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion.

CONCLUSIONS

EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase Ca(2+) and activate PKC.

摘要

目的

确定表皮生长因子（EGF）对泪腺蛋白质分泌的影响，并对其信号转导成分进行表征。

方法

从雄性Sprague-Dawley大鼠身上摘除双侧眶外泪腺。在37℃下，用胶原酶在Krebs-Ringer碳酸氢盐（KRB）缓冲液中消化分离出分散的腺泡。将腺泡与EGF（10⁻⁷M）、胆碱能激动剂卡巴胆碱（10⁻⁴M）或α₁-肾上腺素能激动剂去氧肾上腺素（10⁻⁴M）一起孵育，并用荧光测定法测量过氧化物酶分泌。为了测量细胞内钙（[Ca²⁺]i），将腺泡在fura-2四乙酰氧基甲酯中于22℃孵育60分钟，并在发射波长为505nm时在340和380nm处测量荧光。在测量过氧化物酶分泌之前，用不含CaCl₂和2mM乙二醇双（2-氨基乙基醚）四乙酸（EGTA）的KRB-牛血清白蛋白螯合细胞外Ca²⁺。通过将腺泡与佛波酯、佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA；10⁻⁶M）一起或不一起孵育过夜来下调蛋白激酶C（PKC），并测量过氧化物酶分泌。

结果

EGF以浓度依赖性方式刺激过氧化物酶分泌，在10⁻⁷M时显著增加。EGF刺激的分泌受到EGF受体（EGFR）抑制剂AG1478的抑制，但不受磷酸肌醇-3激酶抑制剂LY292004或丝裂原活化蛋白激酶激酶（MEK）抑制剂U0126的抑制。EGF增加了[Ca²⁺]i，而细胞外Ca²⁺的螯合抑制了EGF诱导的过氧化物酶分泌达90%。PKC的下调也抑制了EGF刺激的过氧化物酶分泌。