**Author details**

be assessed to identify the peptidase activities involved in the RAS [101]. One advantage is that the contribution of various peptidases for a given peptide is directly comparable to determine the predominant pathway in a particular tissue or treatment condition. Peptidase activities derived by different synthetic substrates are not comparable unless standardized to the enzyme concentration. Moreover, the use of endogenous peptide substrates may reveal novel peptidase activities involved in angiotensin processing [102, 103]. Peptidase assays developed in our laboratory typically utilize 125I-radiolabled peptides coupled to highperformance liquid chromatography (HPLC)-based separation and automated in-line γdetection. Advantages of this are that only microliter amounts of serum or microgram quantities of tissue are normally required that reflects detection sensitivity in the fmol range

More recent studies have incorporated mass spectroscopy (MS) detection of angiotensin metabolism in tissues, cells, and plasma, as well as the derivation of processing networks [104]. Velez and colleagues applied HPLC-MS analysis of Ang I processing in rat glomeruli to reveal the predominant processing of Ang I to Ang-(1-7) catalyzed by neprilysin [104]. Interestingly, the authors could not demonstrate an Ang I to Ang II pathway even following the blockade of the Ang-(1-7) pathway with a neprilysin inhibitor. Hildesbrand et al. [103] utilized a HPLCtandem quadrupole system (HPLC-MS/MS) to reveal multiple metabolism pathways from Ang I to its N-terminal metabolites Ang-(5-10) and Ang-(4-10), as well as Ang II and Ang-(1-7) in immobilized proteins from human plasma. Suski et al. [105] reported that Ang I was primarily converted to Ang-(1-7) in vascular smooth muscle cells (VSMCs) as characterized by HPLC-MS/MS and confirms our earlier study that thimet oligopeptidase directly processed Ang I to Ang-(1-7) in rat VSMC [106]. Grobe and colleagues have applied "in situ" MALDI to characterize both renal and cardiac metabolism of exogenous Ang II [107, 108]. Ang-(1-7) was the primary product from Ang II in the renal cortex while Ang III was the major metabolite in the medulla [108]. In the heart, Ang III and Ang-(1-7) were products of Ang II metabolism catalyzed by APA and ACE2, respectively [107]. These data confirm earlier HPLC-based studies on the contribution of ACE2 to Ang-(1-7) formation in the mouse and human heart [66, 109]. Although this approach cannot distinguish intracellular versus membrane or extracellular processing and requires relatively high-substrate concentrations, it is likely that these systems will develop the required sensitivity and resolution to detect peptides *in situ*, as well

Portions of this chapter are reproduced from the author's recent publication [54]. These studies were supported in part by grants from the National Institute of Health grants (HL-56973, HL-51952, HD084227, HD-047584, and HD-017644) and the American Heart Association (AHA-151521 and AHA-355741). An unrestricted grant from the Farley-Hudson Foundation (Jacksonville, NC), Groskert Heart Fund, and the Wake Forest Venture Fund is also acknowl-

and the lack of detector interference or quenching [33].

14 Enzyme Inhibitors and Activators

as characterize the extent of enzymatic processing.

**Acknowledgements**

edged.

Nildris Cruz-Diaz1 , Bryan A. Wilson2 and Mark C. Chappell1\*

\*Address all correspondence to: mchappel@wakehealth.edu

1 The Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA

2 McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
