**5. Neprilysin**

proximal tubule angiotensinogen, the AT1R, or intracellular Ang II also resulted in a sustained

The predominant pathway of the classical RAS for the conversion of Ang I to the bioactive peptide Ang II is catalyzed by the metallopeptidase ACE [EC 3.4.15.1], a dipeptidyl carboxypeptidase that cleaves two residues from the carboxy terminus of Ang I (**Figure 1)**. The peptidase is a membrane-bound, glycosylated protein (120–180 kDa) ubiquitously expressed in multiple tissues [29]. Soluble forms of the enzyme are present in the circulation, cerebrospinal fluid (CSF), lymph fluid, and urine that fully retain peptidase activity [29]. The soluble form of ACE arises from the hydrolysis of the membrane-anchoring or stalk region of the protein that may reflect the processing by A Disintegrin and Metalloproteinase (ADAM) family of metalloenzymes, although the precise role of ACE shedding in cardiovascular disease is presently unclear. Somatic ACE is characterized by two active sites termed N and C terminal domains that likely arose from the gene duplication of germinal or testicular ACE that contains only the single C terminal-active site. Importantly, in addition to forming Ang II, ACE degrades a number of other peptides that exhibit cardiovascular actions including bradykinin, substance P, acetyl-SDKP, and Ang-(1-7) [29]. Indeed, the cardioprotective effects of ACE inhibitors may reflect the protection of these peptides from metabolism in addition to the blockade of Ang II formation. We showed that ACE hydrolyzes Ang-(1-7) to the pentapeptide Ang-(1-5) and that ACE inhibition markedly reduced the clearance of the peptide [30, 31]. The reduced metabolism of Ang-(1-7) likely contributes to the elevation in circulating levels of Ang-(1-7) following the chronic treatment with ACE inhibitors in experimental animals and in humans [32]. Moreover, treatment with an ACE inhibitor was required to clearly demonstrate the accumulation of Ang-(1-7) derived from either Ang II or Ang I in isolated proximal tubules [33]. These data suggest a pivotal role for ACE to regulate the balance of Ang II and Ang-(1-7), two peptides

within the RAS that exhibit strikingly different actions from one another [7].

Chymases [EC 3. 4.21.39] comprise a family of serine peptidases that may generate Ang II by

chymases) [34–39]. Humans express α-chymase while rodents express primarily β-chymases, as well as other isoforms (mouse MCP-4 and rat MCP-5) that more closely resemble α-chymase in regard to the processing of Ang I to Ang II [35]. The human and mouse enzymes may also play a role in the conversion of the endothelin precursor to the active peptide, as well as the activation of various inflammatory cytokines [40]. Chymases (35 kDa) are synthesized and stored in an inactive proform within mast cells and neutrophils that are released with other proteases (cathepsin G, tryptases, and renin) upon degranulation following injury or inflam-


bond of Ang I and other peptide precursors [Ang-(1-12), Ang-

to form Ang-(1-4) and Ang-(5-8) (β-

increase in blood pressure [23–28].

6 Enzyme Inhibitors and Activators

**4. Chymase**

hydrolysis of the Phe8


(1-25)] (α-chymases) or metabolize Ang II at Tyr4

**3. Angiotensin-converting enzyme**

Neprilysin [EC 3.4.24.11; ~95 kDa] is a metalloendopeptidase that is predominantly anchored to the plasma membrane. The peptidase was initially characterized in brain to hydrolyze the opiate peptide enkephalin, hence the original terminology of the enzyme as "enkephalinase." Neprilysin was subsequently found in a number of peripheral tissues with particularly high expression within the brush border on the apical region of the renal proximal tubules. Interest in the renal expression of neprilysin reflected the potential role of the peptidase to metabolize natriuretic peptides, and selective inhibitors alone or combined with an ACE inhibitor ("vasopeptidase inhibitor") were developed as cardiovascular therapies to prolong the renal actions of these peptides [47–52]. Although the vasopeptidase inhibitor omapatrilat was clinically withdrawn regarding a greater incidence of angioedema that may reflect increased levels of bradykinin, a new generation of agents that combine a neprilysin inhibitor and an AT1R antagonist may be a promising therapeutic approach for the treatment of heart failure [53]. One potential caveat to this approach would be that the neprilysin inhibitor component may reduce circulating levels of Ang-(1-7) and potentially attenuate the cardioprotective effects of the combined antagonist/inhibitor.

Vascular neprilysin is responsible for the extracellular conversion of Ang I to Ang-(1-7) in the circulation, particularly under conditions of chronic ACE inhibition (**Figure 1**) [54]. Neprilysin hydrolyzes the Pro7 -Phe8 bond of Ang I to form Ang-(1-7), as well as the Tyr4 -Ile5 bond to generate Ang-(1-4) that reflects the enzyme's preference for aromatic and hydrophobic residues. We further showed that renal neprilysin on the proximal tubules and the renal cortex readily converted both Ang I and Ang-(1-12) to Ang-(1-7), as well as that a neprilysin inhibitor attenuated the blood pressure-lowering effects of the ACE inhibitor lisinopril in the spontaneously hypertensive rat [33, 55, 56]. Apart from the conversion of Ang I to Ang-(1-7), neprilysin is capable of metabolizing a number of other peptides including adrenomedullin, Ang II [to Ang-(1-4)], endothelin, bradykinin, and the natriuretic peptides ANP and BNP.
