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Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells.
Grobe, Nadja; Di Fulvio, Mauricio; Kashkari, Nada; Chodavarapu, Harshita; Somineni, Hari K; Singh, Richa; Elased, Khalid M.
Afiliação
  • Grobe N; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Di Fulvio M; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Kashkari N; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Chodavarapu H; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Somineni HK; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Singh R; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio.
  • Elased KM; Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio khalid.elased@wright.edu.
Am J Physiol Cell Physiol ; 308(9): C767-77, 2015 May 01.
Article em En | MEDLINE | ID: mdl-25740155
The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Sistema Renina-Angiotensina / Peptidil Dipeptidase A / Proteínas ADAM Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2015 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Sistema Renina-Angiotensina / Peptidil Dipeptidase A / Proteínas ADAM Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2015 Tipo de documento: Article