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钙激活腺苷酸环化酶和蛋白激酶A对低渗条件下Lens Src家族激酶和Na-K ATP酶响应的作用

Shahidullah, M., Mandal, A., Delamere, N.A.

期刊名称:Investigative Ophthalmology & Visual Science

卷期:2017年第58卷第11期

摘要

摘要:目的:晶状体上皮中的NaK-ATP酶活性受Src家族酪氨酸激酶(SFKs)控制。以前,我们显示低渗溶液可导致上皮细胞中NaK-ATP酶活性的SFK依赖性增加。在这里我们探讨了cAMPSFKNaK-ATPase应答信号机制中的作用。方法:将完整的猪晶体暴露于低渗Krebs溶液(200mOsm)中,然后测定上皮细胞的cAMPSFK磷酸化(激活)或NaK-ATP酶活性。结果:暴露于低渗溶液中的晶体上皮中观察到cAMP增加。暴露于低渗溶液的晶体中,上皮中的SFK磷酸化与NaK-ATP酶活性一样大约增加2倍,两种反应均被蛋白激酶A抑制剂H89阻断。通过加入一种TRPV4拮抗剂HC067047以阻断TRPV4介导的钙流入以及一种细胞质Ca 2+螯合剂BAPTA-AM可降低对低渗溶液的SFK应答的等级。暴露于低渗溶液的晶体上皮中的NaK-ATP酶活性反应可被BAPTA-AM消除。作为cAMP依赖性SFK激活的一种直接检测,将完整的晶体暴露于8-pCPT-cAMP(一种细胞可渗透性cAMP类似物)。 8-pCPT-cAMP可引起强大的SFK活化。使用Western印迹,在晶状体上皮中可检测到两种钙激活腺苷酸环化酶ADCY3ADCY8。结论:钙激活的腺苷酸环化酶在晶状体上皮中表达,并且SFK活化与低渗状态下的cAMP升高相关。研究结果指出,cAMPTRPV4通道介导的钙流入,SFK激活和随后的NaK-ATP酶活性升高之间的连接点。

Purpose:Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response.Methods:Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity.Results:An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium.Conclusions:Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.


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