Cellular and Molecular Neurobiology

, Volume 32, Issue 8, pp 1353–1365 | Cite as

Six Commercially Available Angiotensin II AT1 Receptor Antibodies are Non-specific

  • Julius Benicky
  • Roman Hafko
  • Enrique Sanchez-Lemus
  • Greti Aguilera
  • Juan M. Saavedra
Original Research

Abstract

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.

Keywords

GPCR receptor antibodies Antibody characterization Angiotensin II receptors Renin–angiotensin System AT1 receptor immunocytochemistry AT1 receptor antibodies AT1A receptor knock-out 

References

  1. Bader M (2010) Tissue renin–angiotensin-aldosterone systems: targets for pharmacological therapy. Ann Rev Pharmacol Toxicol 50:439–465CrossRefGoogle Scholar
  2. Bodei S, Arrighi N, Spano P, Sigala S (2009) Should we be cautious on the use of commercially available antibodies to dopamine receptors? Naunyn Schmiedeberg’s Arch Pharmacol 379:413–415CrossRefGoogle Scholar
  3. Burson JM, Aguilera G, Gross KW, Sigmund CD (1994) Differential expression of angiotensin receptor 1A and 1B in mouse. Am J Physiol 267:E260–E267PubMedGoogle Scholar
  4. Chiu AT, Dunscomb J, Kosierowski J, Burton CR, Santomenna LD, Corjay MH, Benfield P (1993) The ligand binding signatures of the rat AT1A, AT1B and the human AT1 receptors are essentially identical. Biochem Biophys Res Commun 197:440–449PubMedCrossRefGoogle Scholar
  5. Gasc JM, Shanmugam S, Sibony M, Corvol P (1994) Tissue-specific expression of type 1 angiotensin II receptor subtypes. An in situ hybridization study. Hypertension 24:531–537PubMedCrossRefGoogle Scholar
  6. Hamdani N, van der Velden J (2009) Lack of specificity of antibodies directed against human beta-adrenergic receptors. Naunyn Schmiedeberg’s Arch Pharmacol 379:403–407CrossRefGoogle Scholar
  7. Häuser W, Jöhren O, Saavedra JM (1998) Characterization and distribution of angiotensin II receptor subtypes in the mouse brain. Eur J Pharmacol 348:101–114PubMedCrossRefGoogle Scholar
  8. Inagami T, Guo DF, Kitami Y (1994) Molecular biology of angiotensin II receptors: an overview. J Hypertens Suppl 12:S83–S94PubMedGoogle Scholar
  9. Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM (1995) Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci USA 92:3521–3525PubMedCrossRefGoogle Scholar
  10. Jensen BC, Swigart PM, Simpson PC (2009) Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. Naunyn Schmiedeberg’s Arch Pharmacol 379:409–412CrossRefGoogle Scholar
  11. Jöhren O, Saavedra JM (1996) Expression of AT1A and AT1B angiotensin II receptor messenger RNA in forebrain of 2-wk-old rats. Am J Physiol 271:E104–E112PubMedGoogle Scholar
  12. Jöhren O, Inagami T, Saavedra JM (1995) AT1A, AT1B, and AT2 angiotensin II receptor subtype gene expression in rat brain. NeuroReport 6:2549–2552PubMedCrossRefGoogle Scholar
  13. Jositsch G, Papadakis T, Haberberger RV, Wolff M, Wess J, Kummer W (2009) Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. Naunyn Schmiedeberg’s Arch Pharmacol 379:389–395CrossRefGoogle Scholar
  14. Kakar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD (1992) Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation. Biochem Biophys Res Commun 183:1090–1096PubMedCrossRefGoogle Scholar
  15. Kasckow J, Mulchahey JJ, Aguilera G, Pisarska M, Nikodemova M, Chen HC, Herman JP, Murphy EK, Liu Y, Rizvi TA, Dautzenberg FM, Sheriff S (2003) Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signaling in an immortalized hypothalamic cell line. J Neuroendocrinol 15:521–529PubMedCrossRefGoogle Scholar
  16. Lenkei Z, Corvol P, Llorens-Cortes C (1995) The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control. Brain Res Mol Brain Res 30:53–60PubMedCrossRefGoogle Scholar
  17. Lenkei Z, Nuyt M, Grouselle D, Corvol P, Llorens-Cortes C (1999) Identification of endocrine cell populations expressing the AT1B subtype of angiotensin II receptors in the anterior pituitary. Endocrinology 140:472–477PubMedCrossRefGoogle Scholar
  18. Liu Y, Kalintchenko N, Sassone-Corsi P, Aguilera G (2006) Inhibition of corticotrophin-releasing hormone transcription by inducible cAMP-early repressor in the hypothalamic cell line, 4B. J Neuroendocrinol 18:42–49PubMedCrossRefGoogle Scholar
  19. Lorincz A, Nusser Z (2008) Specificity of immunoreactions: the importance of testing specificity in each method. J Neurosci 28:9083–9086PubMedCrossRefGoogle Scholar
  20. Lu X, Bartfai T (2009) Analyzing the validity of GalR1 and GalR2 antibodies using knockout mice. Naunyn Schmiedeberg’s Arch Pharmacol 379:417–420CrossRefGoogle Scholar
  21. Mehta PK, Griendling KK (2007) Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292:C82–C97PubMedCrossRefGoogle Scholar
  22. Michel MC, Wieland T, Tsujimoto G (2009) How reliable are G-protein-coupled receptor antibodies? Naunyn Schmiedeberg’s Arch Pharmacol 379:385–388CrossRefGoogle Scholar
  23. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE (1991) Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351:233–236PubMedCrossRefGoogle Scholar
  24. Nikodemova M, Kasckow J, Liu H, Manganiello V, Aguilera G (2003) Cyclic adenosine 3′,5′-monophosphate regulation of corticotropin-releasing hormone promoter activity in AtT-20 cells and in a transformed hypothalamic cell line. Endocrinology 144:1292–1300PubMedCrossRefGoogle Scholar
  25. Oliverio MI, Best CF, Kim HS, Arendshorst WJ, Smithies O, Coffman TM (1997) Angiotensin II responses in AT1A receptor-deficient mice: a role for AT1B receptors in blood pressure regulation. Am J Physiol 272:F515–F520PubMedGoogle Scholar
  26. Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM (1998) Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci USA 95:15496–15501PubMedCrossRefGoogle Scholar
  27. Paul M, Mehr AP, Kreutz R (2006) Physiology of local renin angiotensin systems. Physiol Rev 86:747–803PubMedCrossRefGoogle Scholar
  28. Paxton WG, Runge M, Horaist C, Cohen C, Alexander RW, Bernstein KE (1993) Immunohistochemical localization of rat angiotensin II AT1 receptor. Am J Physiol 264:F989–F995PubMedGoogle Scholar
  29. Pradidarcheep W, Stallen J, Labruyère WT, Dabhoiwala NF, Michel MC, Lamers WH (2009) Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors. Naunyn Schmiedeberg’s Arch Pharmacol 379:397–402CrossRefGoogle Scholar
  30. Rateri DL, Moorleghen JJ, Balakrishnan A, Owens AP III, Howatt DA, Subramanian V, Poduri A, Charnigo R, Cassis LA, Daugherty A (2011) Endothelial cell-specific deficiency of Ang II type 1a receptors attenuates Ang II-induced ascending aortic aneurysms in LDL receptor−/− mice. Circ Res 108:574–581PubMedCrossRefGoogle Scholar
  31. Rhodes KJ, Trimmer JS (2006) Antibodies as valuable neuroscience research tools versus reagents of mass distraction. J Neurosci 26:8017–8020PubMedCrossRefGoogle Scholar
  32. Ruan X, Oliverio MI, Coffman TM, Arendshorst WJ (1999) Renal vascular reactivity in mice: AngII-induced vasoconstriction in AT1A receptor null mice. J Am Soc Nephrol 10:2620–2630PubMedGoogle Scholar
  33. Saavedra JM, Sánchez-Lemus E, Benicky J (2011) Blockade of brain angiotensin II AT1 receptors ameliorates stress, anxiety, brain inflammation and ischemia: therapeutic implications. Psychoneuroendocrinology 36:1–18PubMedCrossRefGoogle Scholar
  34. Sánchez-Lemus E, Benicky J, Pavel J, Saavedra JM (2009) In vivo angiotensin II AT1 receptor blockade selectively inhibits LPS-induced innate immune response and ACTH release in rat pituitary gland. Brain Behav Immun 23:945–957PubMedCrossRefGoogle Scholar
  35. Saper CB (2005) An open letter to our readers on the use of antibodies. J Comp Neurol 493:477–478PubMedCrossRefGoogle Scholar
  36. Saper CB (2009) A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem 57:1–5PubMedCrossRefGoogle Scholar
  37. Saper CB, Sawchenko PE (2003) Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. J Comp Neurol 465:161–163PubMedCrossRefGoogle Scholar
  38. Sasamura H, Hein L, Krieger JE, Pratt RE, Kobilka BK, Dzau VJ (1992) Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem Biophys Res Commun 185:253–259PubMedCrossRefGoogle Scholar
  39. Tsutsumi K, Saavedra JM (1991) Characterization and development of angiotensin II receptor subtypes (AT1 and AT2) in rat brain. Am J Physiol 261:R209–R216PubMedGoogle Scholar
  40. Zhou Y, Chen Y, Dirksen WP, Morris M, Periasamy M (2003) AT1b receptor predominantly mediates contractions in major mouse blood vessels. Circ Res 93:1089–1094PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC (outside the USA)  2012

Authors and Affiliations

  • Julius Benicky
    • 1
  • Roman Hafko
    • 1
  • Enrique Sanchez-Lemus
    • 1
  • Greti Aguilera
    • 2
  • Juan M. Saavedra
    • 1
  1. 1.Section on PharmacologyDIRP, NIMH, NIHBethesdaUSA
  2. 2.Section on Endocrine PhysiologyPDEGEN, NICHD, NIH, CRCBethesdaUSA

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