Herbert L. Jackman

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.

Angiotensin 1-9 and 1-7 Release in Human Heart Role of Cathepsin A

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A’s presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B2 receptor in Chinese hamster ovary cells transfected to express human ACE and B2 (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B2 receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC50s) with ACE. They probably induce conformational changes in the ACE/B2 receptor complex via interaction with ACE.

Kininase II-Type Enzymes: Their Putative Role in Muscle Energy Metabolism

Because of the importance of bradykinin in improving heart function in some conditions or in enhancing glucose uptake by skeletal muscle, we investigated kininases in these tissues. In P3 fraction of the heart and skeletal muscles, angiotensin I-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) are the major kininases, as determined first with specific substrates and second with bradykinin. ACE activity was highest in guinea pig heart (2.7 ± 0.07 μmol·h−1 · mg protein−1) but decreased in other species in this order: dog atrium, rat heart, dog ventricle, and human atrium. The specific activity of NEP was lower: 0.45 μmol · h−1 · mg protein−1 in cultured neonatal cardiac myocytes and varying between 0.12 and 0.05 μmol · h−1 · mg protein−1 in human, dog, rat, and guinea pig heart. In the skeletal muscle P3, ACE was most active in guinea pig and rat (1.2 and 1.1 μmol · h−1 · mg protein−1, respectively) but less so in dog (0.09 μmol · h−1 · mg protein−1). NEP activity was higher in dog P3 (0.28 μmol · h−1 · mg protein−1) but lower in rat and guinea pig (0.19 and 0.1 μmol · h−1 · mg protein−1, respectively). Continuous density gradient centrifugation enriched NEP activity in dog and rat (from 0.3 to 1.0 and 0.49 μmol · h−1 · mg protein−1, respectively). Immunoprecipitation with antiserum to purified NEP proved the specificity of the rat enzyme. Bradykinin (0.1 mmol/1) was inactivated in the presence and absence of inhibitors by rat skeletal muscle NEP, as measured by high-performance liquid chromatography. Here, 36% of the activity was caused by NEP and 19% by ACE. In radioimmunoassay (bradykinin 10 nmol/1), 46 and 55% of kininase in rat and dog skeletal muscle P3, respectively, was due to ACE; 36 and 28%, respectively, was due to NEP. Aside from these enzymes, an aminopeptidase in rat P3 also inactivates bradykinin. Thus, in conclusion, heart and skeletal muscle membranes contain kininase II-type enzymes, but their activity depends on the species.

Protamine inhibits plasma carboxypeptidase N, the inactivator of anaphylatoxins and kinins.

Protamine given to neutralize heparin after extracorporeal circulation can trigger a catastrophic reaction in some patients. While searching for a biochemical basis for this reaction, protamine was tested as an inhibitor of human plasma carboxypeptidase N (CPN) or kininase I, the inactivator of anaphylatoxins and kinins. Human plasma and CPN purified from human plasma, (Mr = 280 K) or its isolated active subunit (Mr = 48 K) were the sources of enzyme. The hydrolysis of furylacryloyl (FA)-Ala-Lys was measured in a UV spectrophotometer and that of bradykinin and the synthetic C-terminal octapeptide of anaphylatoxin C3a (C3a8) by high performance liquid chromatography. Protamine inhibited the hydrolysis of FA-Ala-Lys by CPN, (IC50 = 3.2 X 10(-7) M); added human serum albumin (30 mg/ml) increased the IC50 to 7 X 10(-6) M. When plasma was the source of CPN, the IC50 was 2 X 10(-6) M. Protamine more effectively inhibited the hydrolysis of bradykinin and C3a8. The IC50 for protamine was 5 X 10(-8) M with CPN and bradykinin, 7 X 10(-8) M with CPN and C3a8 and with the 48 K subunit and bradykinin it was 7 X 10(-8) M of protamine. Heparin competes with CPN for protamine, because in high concentration (18 U/ml) it reverses the inhibition by protamine. Protamine did not inhibit angiotensin I converting enzyme (kininase II) or the endopeptidase 24.11 (enkephalinase). Kinetic studies showed the mechanism of protamine inhibition to be partially competitive; about 10-20% of the hydrolysis of bradykinin by CPN was not inhibited by protamine. Thus, by blocking the inactivation of mediators released in shock, protamine inhibition of CPN may be partially responsible for the catastrophic reaction observed to occur in some patients.

METABOLISM OF SUBSTANCE P AND BRADYKININ BY HUMAN NEUTROPHILS

The catabolism of substance P and bradykinin, two peptides involved in inflammation, by human neutrophils was investigated. Substance P was cleaved by unstimulated neutrophils, but the rate of hydrolysis increased greatly (about 4-fold) when the cells were lysed by freezing and thawing or stimulated to release with fMet-Leu-Phe and cytochalasin B. The enzyme responsible for cleaving substance P was cathepsin G, hydrolyzing the Phe7-Phe8 bond. Neutral endopeptidase 24.11 (enkephalinase) became the main inactivating enzyme only when neutrophil cytoplasts (containing plasma membrane but no subcellular particles) or washed plasma membrane enriched high speed sediments were tested. Subcellular fractionation showed the highest substance P degrading activity to be in the granules. Purified cathepsin G readily cleaved substance P with a Km of 1.13 MK, a kcat of 6.35 sec-1 and a kcat/Km of 5639 M-1 sec-1, similar to kinetic constants previously reported for the best peptide substrates of cathepsin G. Despite the high Km, purified cathepsin G did hydrolyze SP at a much lower substrate concentration (down to 1 nM) as determined by radioimmunoassay. Bradykinin was also hydrolyzed by intact neutrophils but, in contrast, was not inactivated by cathepsin G, but by neutral endopeptidase at the Pro7-Phe8 bond. The inactivation of bradykinin by intact neutrophils was decreased by phorbol 12-myristate 13-acetate, probably due to down-regulation by endocytosis of the neutral endopeptidase on the plasma membrane. Thus, both bradykinin and substance P are inactivated by human neutrophils, although by different enzymes. In spite of the less favorable kinetics in vitro than with neutral endopeptidase, cathepsin G is the main inactivator of substance P in neutrophils. This may be due to the estimated 300 to 3600-fold higher concentration of cathepsin G in neutrophils than that of the neutral endopeptidase.

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.

Simultaneous determination of mepivacaine, tetracaine, and p-butylaminobenzoic acid by high-performance liquid chromatography

INTRODUCTION The purpose of the present study was to develop a simple method for the simultaneous determination of mepivacaine, tetracaine, and p-butylaminobenzoic acid (BABA) in human plasma using high-performance liquid chromatography (HPLC) with a multiwavelength detector. METHODS Human blood samples containing heparin, as an anticoagulant, and physostigmine (100 microg/ml), as an anticholinesterase, or human plasma containing physostigmine were spiked with various concentrations of mepivacaine, tetracaine and, in some cases, BABA. Blood samples were centrifuged and plasma was deproteinized with trifluoroacetic acid (TFA; 7%). After centrifugation, the pH was adjusted to 4.5 and an aliquot of 20, 50 or 100 microl was injected into the HPLC apparatus. The detection was done simultaneously at wavelengths of 214 and 300 nm. Analytical chromatography was done on a Waters microBondapak C(18) reverse-phase column (3.9 x 300 mm; particle size 10 microm) with a 30-min increasing linear gradient of 20-40% acetonitrile+0.05% TFA in H(2)O+0.05% TFA at a flow rate of 1 ml/min. The Waters HPLC system included two pumps, an automatic injector, a column oven, and a M490 multiwavelength detector. Quantification was done using integration of peak areas with peaks of authentic mepivacaine, tetracaine, and BABA standards. RESULTS Calibration curves for standards of mepivacaine, tetracaine, and BABA were linear in the concentration ranges from 0.1 to 100 microg/ml, and the correlation coefficients exceeded.99 for all compounds. The lower limits of detection were 100 ng/ml for mepivacaine and 50 ng/ml for tetracaine and BABA. The yields for mepivacaine, tetracaine, and BABA were 91+/-2.1%, 82+/-3.3%, and 88+/-2.0% (mean+/-S.E.M., n=6), respectively. Degradation of tetracaine by human plasma at 37 degrees C was inhibited by physostigmine. DISCUSSION The method is sensitive enough to allow blood concentration determinations of mepivacaine and tetracaine or its metabolite, BABA, following local anesthesia induced by these two drugs, especially when their toxic effect may be present. This method also may be useful in forensic medicine for determination of the cause of death after local anesthesia with mepivacaine and/or tetracaine.

Metabolism of angiotensin I in the coronary circulation of normal and diabetic rats.

Formation of metabolites from angiotensin I that passed the coronary vessels in the isolated working rat hearts of normal and streptozotocin-induced diabetes was evaluated. HPLC analysis showed that the levels of angiotensin II and angiotensin 1-7 were unaltered in the diabetic hearts, but the perfusates of the diabetic hearts contained smaller amounts of angiotensin 1-9. It is not clear why the perfusates of diabetic hearts contain less amount of angiotensin 1-9. It is possible that the peptide is metabolized faster or greater internalization takes place in the diabetic heart. The amount of angiotensin II in the perfusates of normal hearts was 5.8 times greater at the perfusion rate of 2 than at 10 ml/min/g wet heart weight. At such conditions, the amount of angiotensin 1-9 and angiotensin 1-7 in the perfusates were increased 2.4 and 1.5 times, respectively. A higher amount of angiotensin II during myocardial hypoperfusion may lead to constriction of the coronary vessels. As a result, myocardial damage may be facilitated.