Peter A. Deddish

N-Domain–Specific Substrate and C-Domain Inhibitors of Angiotensin-Converting Enzyme: Angiotensin-(1–7) and Keto-ACE

We used the isolated N- and C-domains of the angiotensin 1-converting enzyme (N-ACE and C-ACE; ACE; kininase II) to investigate the hydrolysis of the active 1-7 derivative of angiotensin (Ang) II and inhibition by 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline (keto-ACE). Ang-(1-7) is both a substrate and an inhibitor; it is cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentration than N-ACE; the IC50 of C-ACE with 100 micromol/L Ang I substrate was 1.2 micromol/L and the Ki was 0.13. While searching for a specific inhibitor of a single active site of ACE, we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 47-times lower concentration than by N-ACE; IC50 values with C-ACE were 0.5 and 0.04 micromol/L. Furthermore, we investigated how Ang-(1-7) acts via bradykinin and the involvement of its B2 receptor. Ang-(1-7) was ineffective directly on the human bradykinin B2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1-7) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (1 micromol/L), acting on the B2 receptor when the cells were cotransfected with cDNAs of both B2 receptor and ACE and the proteins were expressed on the plasma membrane of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(1-7) can potentiate the actions of a ligand of the B2 receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(1-7).

Enhancement of Bradykinin and Resensitization of Its B2 Receptor

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Replacement of the Transmembrane Anchor in Angiotensin I-converting Enzyme (ACE) with a Glycosylphosphatidylinositol Tail Affects Activation of the B2 Bradykinin Receptor by ACE Inhibitors

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B2 bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B2 antagonist HOE 140. B2 was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B2 and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B2 receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B2 receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.

Potentiation of bradykinin actions by ACE inhibitors

Angiotensin I-converting enzyme (kininase II; ACE) inhibitors, antibodies to ACE and slowly cleaved substrates of ACE potentiate the effect of bradykinin and its analogs on their B2 receptors independently of blocking peptide metabolism. ACE inhibitors also resensitized the receptors desensitized by the ligand (tachyphylaxis). The studies were performed on isolated organs and cells co-transfected with the receptor and the enzyme or constitutively expressing them. This enhancement of the effect of B2 ligands is attributed to a crosstalk between the enzyme and the receptor, and not to a direct action on the receptors. It might reflect some of the local activities of ACE inhibitors.

Carboxypeptidase M in brain and peripheral nerves.

Abstract: Carboxypeptidase M (CPM), a plasma membrane‐bound enzyme, cleaves C‐terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl‐Ala‐Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross‐reaction with anti‐human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and ∼66% in dog brain. CPM copurified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane‐bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin‐forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.

Human ACE and bradykinin B2 receptors form a complex at the plasma membrane

To investigate how angiotensin I‐converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE‐resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2‐GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme‐receptor complex formation. Using ACE‐fused cyan fluorescent protein donor and B2‐fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.—Chen, Z., Deddish, P. A., Minshall, R. D., Becker, R. P., Erdös, E. G., Tan, F. Human ACE and bradykinin B2 receptors form a complex at the plasma membrane. FASEB J. 20, 2261–2270 (2006)

Hydrolysis of Angiotensin Peptides by Human Angiotensin I–Converting Enzyme and the Resensitization of B2 Kinin Receptors

We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I–converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B2 receptors coupled to green fluorescent protein (B2GFP) or to express only coupled B2GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B2 receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18× slower than Ang I and ≈30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although &mgr;mol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B2GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-&agr;, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B2 activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B2 receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Angiotensin I-Converting Enzyme Inhibitors Block Protein Kinase Cϵ by Activating Bradykinin B1 Receptors in Human Endothelial Cells

Angiotensin I-converting enzyme (ACE) inhibitors are widely used to treat patients with cardiovascular and kidney diseases, but inhibition of ACE alone does not fully explain the beneficial effects. We reported that ACE inhibitors directly activate bradykinin B1 receptor at the canonical Zn2+ binding site, leading to prolonged nitric oxide (NO) production in endothelial cells. Protein kinase C (PKC) ϵ, a novel PKC isoform, is up-regulated in myocardium after infarction, suggesting a role in the development of cardiac dysfunction. In cytokine-treated human lung microvascular endothelial cells, B1 receptor activation by ACE inhibitors (enalaprilat, quinaprilat) or peptide ligands (des-Arg10-Lys1-bradykinin, des-Arg9-bradykinin) inhibited PKCϵ with an IC50 = 7 × 10–9 M. Despite the reported differences in binding affinity to receptor, the two peptide ligands were equally active, even when inhibitor blocked the cleavage of Lys1, thus the conversion by aminopeptidase. The synthetic undecapeptide (LLPHEAWHFAR) representing the binding site for ACE inhibitors on human B1 receptors reduced PKCϵ inhibition by enalaprilat but not by peptide agonist. A combination of inducible and endothelial NO synthase inhibitors, 1400W [N-(3(aminomethyl) benzyl) acetamidine dihydrochloride] and Nω-nitro-l-arginine (2 μM), significantly reduced inhibition by enalaprilat (100 nM), whereas the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (100 μM) inhibited PKCϵ activity just as the B1 ligands did. In conclusion, NO generated by B1 receptor activation inhibits PKCϵ.

Differences in the hydrolysis of enkephalin congeners by the two domains of angiotensin converting enzyme.

The hydrolysis of enkephalin (Enk) congeners by the isolated N- (N-ACE) and C-domain of angiotensin I converting enzyme (ACE) and by the two-domain somatic ACE was investigated. Both Leu5- and Met5-Enk were cleaved faster by the C-domain than by N-ACE; rates with somatic ACE were 1600 and 2500 nmol/min/nmol enzyme with both active sites being involved. Substitution of Gly2 by D-Ala2 reduced the rate to 1/3rd to 1/7th of that of the Enks. N-ACE cleaved Met5-Enk-Arg6-Phe7 faster than the C-domain, probably with the highest turnover number of any naturally occurring ACE substrate (7600 min(-1)). This heptapeptide is also hydrolyzed in the absence of Cl-, but the activation by Cl- is unique; Cl- enhances the hydrolysis of the heptapeptide by N-ACE but inhibits it by the C-domain, yielding about a 5-fold difference in the turnover number at physiological pH. This difference may result in the predominant role of the N-domain in converting Met5-Enk-Arg6-Phe7 to Enk in vivo.

Isolation and characterization of a basic carboxypeptidase from human seminal plasma.

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.