Characterization of adenosine receptors in human kidney proximal tubule (HK-2) cells.

H Thomas Lee, Charles W Emala
{"title":"Characterization of adenosine receptors in human kidney proximal tubule (HK-2) cells.","authors":"H Thomas Lee,&nbsp;Charles W Emala","doi":"10.1159/000065306","DOIUrl":null,"url":null,"abstract":"<p><p>Renal proximal tubule cells are particularly vulnerable to injury following ischemia and reperfusion due to their marginal blood supply and high metabolic demand. Renal adenosine receptor (AR) modulations preserve renal function following ischemic-reperfusion injury in vivo. Numerous intracellular proteins have been shown to be pivotal in the signal transduction of adenosine-mediated protection in vivo. However, characterization of the expression and function of ARs and intracellular proteins mediating protection in human proximal tubular cells is lacking. Therefore, we studied the ARs in an immortalized human renal proximal tubular cell (HK-2) line to determine if this cell line could function as an in vitro model of AR coupling. Immunoblotting with AR subtype specific antibodies detected all 4 subtypes of ARs (A(1), A(2a), A(2b) and A(3)), several isoforms of protein kinase C (alpha, delta, and epsilon and several heterotrimeric G-protein isoforms (G(i)alpha, G(s)alpha and G(q)alpha). The A(1) and A(3) ARs inhibited forskolin- stimulated adenylyl cyclase activity. The A(1) ARs also activated 42/44-kD ERK mitogen-activated protein kinases via G(i)- and tyrosine kinase-dependent pathways. The A(2a) ARs stimulated adenylyl cyclase activity and activated the protein kinase A-->CREB pathway. Chronic (48 h) treatment with a nonselective AR antagonist (8-phenyltheophylline) upregulated A(1), A(2a) ARs and G(i)alpha. Conversely, chronic stimulation of HK-2 ARs with a nonselective AR agonist (N-ethylcarbamoyladenosine) downregulated all 4 subtypes of ARs and G(s)alpha. Based on these findings, HK-2 cells are a useful in vitro model to study the signaling cascades of AR-mediated renal protection.</p>","PeriodicalId":12179,"journal":{"name":"Experimental nephrology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1159/000065306","citationCount":"35","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Experimental nephrology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1159/000065306","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 35

Abstract

Renal proximal tubule cells are particularly vulnerable to injury following ischemia and reperfusion due to their marginal blood supply and high metabolic demand. Renal adenosine receptor (AR) modulations preserve renal function following ischemic-reperfusion injury in vivo. Numerous intracellular proteins have been shown to be pivotal in the signal transduction of adenosine-mediated protection in vivo. However, characterization of the expression and function of ARs and intracellular proteins mediating protection in human proximal tubular cells is lacking. Therefore, we studied the ARs in an immortalized human renal proximal tubular cell (HK-2) line to determine if this cell line could function as an in vitro model of AR coupling. Immunoblotting with AR subtype specific antibodies detected all 4 subtypes of ARs (A(1), A(2a), A(2b) and A(3)), several isoforms of protein kinase C (alpha, delta, and epsilon and several heterotrimeric G-protein isoforms (G(i)alpha, G(s)alpha and G(q)alpha). The A(1) and A(3) ARs inhibited forskolin- stimulated adenylyl cyclase activity. The A(1) ARs also activated 42/44-kD ERK mitogen-activated protein kinases via G(i)- and tyrosine kinase-dependent pathways. The A(2a) ARs stimulated adenylyl cyclase activity and activated the protein kinase A-->CREB pathway. Chronic (48 h) treatment with a nonselective AR antagonist (8-phenyltheophylline) upregulated A(1), A(2a) ARs and G(i)alpha. Conversely, chronic stimulation of HK-2 ARs with a nonselective AR agonist (N-ethylcarbamoyladenosine) downregulated all 4 subtypes of ARs and G(s)alpha. Based on these findings, HK-2 cells are a useful in vitro model to study the signaling cascades of AR-mediated renal protection.

人肾近端小管(HK-2)细胞中腺苷受体的表征。
肾近端小管细胞由于血供不足和代谢需求大,在缺血再灌注后特别容易受到损伤。体内肾腺苷受体(AR)调节可保护缺血-再灌注损伤后的肾功能。许多细胞内蛋白已被证明是关键的信号转导腺苷介导的保护在体内。然而,在人类近端小管细胞中,ARs和介导保护的细胞内蛋白的表达和功能的表征尚缺乏。因此,我们研究了永生化人肾近端小管细胞(HK-2)系的AR,以确定该细胞系是否可以作为AR偶联的体外模型。AR亚型特异性抗体免疫印迹检测到所有4种AR亚型(A(1), A(2a), A(2b)和A(3)),蛋白激酶C的几种亚型(α, δ和epsilon)和几种异源三聚体G蛋白亚型(G(i) α, G(s) α和G(q) α)。A(1)和A(3) ar抑制福斯克林刺激的腺苷酸环化酶活性。A(1) ar还通过G(i)-和酪氨酸激酶依赖途径激活42/44-kD ERK有丝分裂原激活的蛋白激酶。A(2a) ar刺激腺苷酸环化酶活性,激活蛋白激酶A- >CREB通路。非选择性AR拮抗剂(8-苯基茶碱)的慢性(48小时)治疗上调了a(1)、a (2a) AR和G(i) α。相反,非选择性AR激动剂(n -乙基氨基氨酰基腺苷)慢性刺激HK-2 AR可下调所有4种AR亚型和G(s) α。基于这些发现,HK-2细胞是研究ar介导的肾保护信号级联的一个有用的体外模型。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信