Advertisement

Use of a Fluorescent Substrate to Measure ACE2 Activity in the Mouse Abdominal Aorta

  • Yu Wang
  • Lisa A. Cassis
  • Sean E. Thatcher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1614)

Abstract

The use of fluorogenic substrates to measure enzymatic activity is widely used to understand function within different experimental models. ACE2 is important in understanding the balance between AngII and Ang-(1-7) and how this balance could then in turn influence hypertension or other disease outcomes. Here, we describe a method to measure ACE2 activity in abdominal aorta of hyperlipidemic mice under both saline and AngII infusion.

Key words

Angiotensin converting enzyme 2 Aorta Activity Enzyme Fluorescence 

References

  1. 1.
    Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ (2000) A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem 275(43):33238–33243. doi: 10.1074/jbc.M002615200. M002615200 [pii]CrossRefPubMedGoogle Scholar
  2. 2.
    Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira-dos-Santos AJ, da Costa J, Zhang L, Pei Y, Scholey J, Ferrario CM, Manoukian AS, Chappell MC, Backx PH, Yagil Y, Penninger JM (2002) Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417(6891):822–828. doi: 10.1038/nature00786. nature00786 [pii]CrossRefPubMedGoogle Scholar
  3. 3.
    Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S (2000) A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 87(5):E1–E9CrossRefPubMedGoogle Scholar
  4. 4.
    Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A, Tummino P (2002) Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277(17):14838–14843. doi: 10.1074/jbc.M200581200. M200581200 [pii]CrossRefPubMedGoogle Scholar
  5. 5.
    Gupte M, Boustany-Kari CM, Bharadwaj K, Police S, Thatcher S, Gong MC, English VL, Cassis LA (2008) ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet. Am J Physiol Regul Integr Comp Physiol 295(3):R781–R788. doi: 10.1152/ajpregu.00183.2008. 00183.2008 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Thatcher SE, Zhang X, Howatt DA, Lu H, Gurley SB, Daugherty A, Cassis LA (2011) Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor −/− mice. Arterioscler Thromb Vasc Biol 31(4):758–765. doi: 10.1161/atvbaha.110.221614 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pedersen KB, Sriramula S, Chhabra KH, Xia H, Lazartigues E (2011) Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays. Am J Physiol Regul Integr Comp Physiol 301(5):R1293–R1299. doi: 10.1152/ajpregu.00339.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ye M, Wysocki J, Gonzalez-Pacheco FR, Salem M, Evora K, Garcia-Halpin L, Poglitsch M, Schuster M, Batlle D (2012) Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2. Hypertension 60(3):730–740. doi: 10.1161/HYPERTENSIONAHA.112.198622 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liu J, Ji H, Zheng W, Wu X, Zhu JJ, Arnold AP, Sandberg K (2010) Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17beta-oestradiol-dependent and sex chromosome-independent. Biol Sex Differ 1(1):6. doi: 10.1186/2042-6410-1-6 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wysocki J, Ye M, Soler MJ, Gurley SB, Xiao HD, Bernstein KE, Coffman TM, Chen S, Batlle D (2006) ACE and ACE2 activity in diabetic mice. Diabetes 55(7):2132–2139. doi: 10.2337/db06-0033 CrossRefPubMedGoogle Scholar
  11. 11.
    Lambert DW, Yarski M, Warner FJ, Thornhill P, Parkin ET, Smith AI, Hooper NM, Turner AJ (2005) Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2). J Biol Chem 280(34):30113–30119. doi: 10.1074/jbc.M505111200. M505111200 [pii]CrossRefPubMedGoogle Scholar
  12. 12.
    Wang Y, Shoemaker R, Thatcher SE, Batifoulier-Yiannikouris F, English VL, Cassis LA (2015) Administration of 17beta-estradiol to ovariectomized obese female mice reverses obesity-hypertension through an ACE2-dependent mechanism. Am J Physiol Endocrinol Metab 308(12):E1066–E1075. doi: 10.1152/ajpendo.00030.2015 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Pharmacology and Nutritional SciencesUniversity of KentuckyLexingtonUSA
  2. 2.Department of Pharmacology and Nutritional SciencesUniversity of KentuckyLexingtonUSA

Personalised recommendations