Kallidin and bradykinin metabolism by isolated cerebral microvessels

Although kinins have been reported to affect cerebral vascular tone and permeability, their actions are not potentiated by angiotensin converting enzyme inhibitors. To investigate cerebral vascular kinin metabolism, porcine cerebral microvessels were isolated by differential sieving and centrifugation and characterized by microscopic examination and marker enzyme enrichment. Purified microvessels contained a membrane-bound carboxypeptidase which hydrolyzed the C-terminal Phe-Arg bond of both kallidin and bradykinin. Hydrolysis was optimal at pH 7.0, was activated more than 300% by 0.1 mM CoCl2, and was inhibited by o-phenanthroline and the carboxypeptidase N (EC 3.4.17.3) inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGETPA) (IC50 = 2 microM). Conversely, inhibitors of angiotensin I converting enzyme (captopril), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme (Z-Pro-prolinal), dipeptidyl(amino)peptidase IV (diprotin A) and amino-peptidase M (amastatin) had no effect. When the rates of C-terminal hydrolysis of kallidin by detergent-solubilized cerebral microvasculature were determined over a range of substrate concentrations (16.6 to 250 microM), the Km and Vmax values obtained were 26.0 +/- 3.0 microM and 14.7 +/- 1.3 nmol/min/ml (N = 4) respectively. These data suggest that a cerebral microvascular carboxypeptidase may play a role in vivo in modulating the effects of kinins on cerebral blood flow and permeability and in preventing circulating kinins from crossing the blood-brain barrier.     

Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M

The cellular localization of vascular plasma membrane aminopeptidase M (AmM; EC3.4.11.2) was examined in cultured porcine aorta endothelium and smooth muscle cells. AmM was 14-fold higher on smooth muscle (117 +/- 16 units/mg) than on endothelium (8.4 +/- 0.2). Proportional to its cellular distribution, AmM hydrolyzed the N-terminus of kallidin to produce bradykinin, and degraded des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and Met5-enkephalin. In contrast, bradykinin, angiotensin II and substance P were resistant to AmM-mediated hydrolysis. Peptide metabolism was optimal at pH 7.0 and was inhibited by o-phenanthroline, bestatin (Ki = 2.2 +/- 0.1 microM) and amastatin (Ki = 25 +/- 5 nM). Des(Asp1)angiotensin I and angiotensin III had the highest affinity (lowest Km) for AmM (Km = 2.2 +/- 0.5 and 2.0 +/- 0.4 microM respectively), followed by hepta(5-11)substance P (53.9 +/- 1.7 microM) and Met5-enkephalin (75.7 +/- 3.5 microM). In contrast, maximal velocities of hydrolysis were higher for Met5-enkephalin (313 +/- 2 nmol/min/mg) than for hepta(5-11)substance P (109 +/- 18 nmol/min/mg) or angiotensin III (26.5 +/- 1.0 nmol/min/mg). As expected for hydrolysis by a common enzyme, AmM-mediated enkephalin degradation was inhibited competitively by angiotensin III (Ki = 0.34 +/- 0.04 microM), hepta(5-11)substance P (43.7 +/- 6.3 microM) and kallidin (62 microM). These data suggest that vascular AmM may modulate vasoactive peptide levels in vivo, particularly within the microenvironment of endothelial and smooth muscle cell surface receptors.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.     

N-terminal degradation of low molecular weight opioid peptides in human cerebrospinal fluid

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.     

Kinin and angiotensin metabolism by purified renal post-proline cleaving enzyme

Post-proline cleaving enzyme (PPCE; EC 3.4.21.26) is a proline specific endopeptidase capable of hydrolyzing biologically active peptides. The present studies examined the hydrolysis of kinin- and angiotensin-related peptides by cytosolic PPCE purified from porcine kidney. PPCE hydrolysis of the synthetic substrate Z-Gly-Pro-MCA (30.7 +/- 0.3 mumol . min-1 . mg-1) was competitively inhibited by saralasin, bradykinin, des(Arg9)bradykinin, [Leu8], des(Arg9)bradykinin and angiotensin II (IC50 = 0.5 to 7.0 microM). Qualitative TLC studies demonstrated that each peptide was degraded by hydrolysis on the carboxyl side of proline residues (positions 7 or 8). Quantitative HPLC studies established that peptide degradation was optimal at pH 8.2 to 8.7 and was inhibited by the specific PPCE inhibitor Z-Pro-prolinal (IC50 = 0.8 +/- 0.1 nM). Conversely, degradation was unaffected by inhibitors of aminopeptidases (amastatin), neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin I converting enzyme (captopril). Apparent Km values, obtained from Lineweaver-Burk analysis, were comparable for all kinin and angiotensin peptides (Km = 5.5 to 12.8 microM), whereas Vmax values ranged from 1.7 mumol . min-1 . mg-1 for angiotensin II to 0.44 mumol . min-1 . mg-1 for saralasin. These data are consistent with a role for PPCE in the degradation of kinins and angiotensin in vivo.     

Vascular, plasma membrane aminopeptidase M. Metabolism of vasoactive peptides

Aminopeptidase M (EC 3.4.11.2), an enzyme present on the cell surface of vascular endothelium and/or smooth muscle, rapidly hydrolyzes leucyl- and arginyl-2-naphthylamides and a number of vasoactive peptides at physiologic pH. Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was found that vascular aminopeptidase M converted kallidin to bradykinin and inactivated des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and hexa(6-11)substance P. Aminopeptidase M did not, however, hydrolyze bradykinin, angiotensin I, angiotensin II, saralasin, vasopressin, oxytocin or any form of substance P containing a component of the Arg-Pro-Lys-Pro sequence. Both the naphthylamidase and peptidase activities were inhibited similarly by known amino-peptidase M inhibitors including o-phenanthroline, amastatin, bestatin and puromycin. However, inhibitors of angiotensin I converting enzyme (captopril), carboxypeptidase N (MERGETPA), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme and dipeptidyl(amino)peptidase IV (diisopropylphosphofluoridate, DFP) were without effect. These results demonstrate that vascular, cell surface aminopeptidase M can selectively metabolize vasoactive peptides and may play a role in modulating their levels in the circulation and/or within the vessel wall.     

Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M

Aminopeptidase M (AmM; EC 3.4.11.2) is a membrane-bound peptidase present on renal brush border and vascular plasma membrane. In the present study, AmM, purified from rabbit kidney cortex, produced a single immunoprecipitin line against AmM antisera, hydrolyzed alanyl-, leucyl- and arginyl-beta-naphthylamides at rates of 5.1 +/- 0.5, 3.9 +/- 0.5 and 2.6 +/- 0.3 mumol/min/mg, respectively, exhibited little or no alpha-glutamyl-, aspartyl- or glycyl-prolyl-naphthylamidase activities (less than or equal to 0.14 mumol/min/mg), and was inhibited by o-phenanthroline, amastatin (IC50 = 400 nM) and bestatin (IC50 = 6 microM). The alanyl-naphthylamidase activity of unfractionated rabbit plasma was found to be identical to purified AmM regarding relative rates of hydrolysis of alanyl-, leucyl- and arginyl-naphthylamides (100:79:42), pH optimum, and inhibition profile. In comparative studies with the purified enzyme, immunoreactive AmM accounted for essentially all of the alanyl-2-naphthylamidase activity of rabbit plasma. N-Terminal metabolism of (Met5)enkephalin by purified renal AmM was 3.92 +/- 0.69 mumol/min/mg, followed by somatostatin (1.25 mumol/min/mg), hepta(5-11)substance P (1.14 +/- 0.13 mumol/min/mg), (Asn1)angiotensin II (1.11 +/- 0.06 mumol/min/mg), angiotensin III (0.45 +/- 0.04 mumol/min/mg) and des(Asp1)-angiotensin I (0.36 +/- 0.04 mumol/min/mg). In contrast, substance P, bradykinin, (Sar1,Ala8)angiotensin II and neurokinin analogs containing modified N-termini (e.g. Ac-Arg) were resistant to hydrolysis by AmM. Peptide degradation was optimal at neutral pH and was inhibited by amastatin (IC50 = 200 nM) and bestatin (IC50 = 5 microM). Apparent Km values ranged from 15.7 +/- 0.4 microM for angiotensin III to 102 +/- 2 microM for (Met5)enkephalin. These data support a significant role for vascular and plasma AmM in the metabolism of circulating vasoactive peptides.     

The bradykinin B2 receptor antagonist icatibant (Hoe 140) blocks aminopeptidase N at micromolar concentrations: off-target alterations of signaling mediated by the bradykinin B1 and angiotensin receptors

The N-terminal sequence of icatibant, a widely used peptide antagonist of the bradykinin B(2) receptors, is analogous to that of other known aminopeptidase N inhibitors. Icatibant competitively inhibited the hydrolysis of L-Ala-p-nitroanilide by recombinant aminopeptidase N (K(i) 9.1 microM). In the rabbit aorta, icatibant (10-30 microM) potentiated angiotensin III, but not angiotensin II (contraction mediated by angiotensin AT(1) receptors), and Lys-des-Arg(9)-bradykinin, but not des-Arg(9)-bradykinin (effects mediated by the bradykinin B(1) receptors), consistent with the known susceptibility of these agonists to aminopeptidase N. At concentrations possibly reached in vivo (e.g., in kidneys), icatibant alters physiological systems different from bradykinin B(2) receptors.     

Carboxyl-terminal tripeptidyl hydrolysis of substance P by purified rabbit lung angiotensin-converting enzyme and the potentiation of substance P activity in vivo by captopril and MK-422

The hydrolysis of substance P is catalyzed by purified rabbit lung angiotensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1). The kcat/Km for the reaction at 37 degrees is 3.3 +/- 0.3 X 10(3) M-1 sec-1, which is 60 times less than that which has been reported for the hydrolysis of angiotensin I. The initial site of hydrolysis is the antipenultimate peptide bond, which generates the tripeptide amide (Gly-Leu-Met-NH2). This hydrolysis is inhibited by the angiotensin-converting enzyme inhibitors captopril, MK-422, and EDTA, and is dependent on the concentration of chloride ion. Both captopril and MK-422 potentiate the substance P-induced stimulation of salivation in rats. Thus, angiotensin-converting enzyme may be one of the enzymes that degrade substance P in vivo.     

Effect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblasts.

OBJECTIVE: The purpose of this investigation was to determine whether the aminopeptidase inhibitor with broad specificity, bestatin, affects angiotensin I (Ang I)-, angiotensin II (Ang II)- or angiotensin III (Ang III)-stimulated collagen gel contraction in cardiac fibroblasts. DESIGN AND METHODS: Cardiac fibroblasts (from normal male adult rats) were cultured to confluency in Dulbeccos modified Eagles medium (DMEM) with 10% foetal bovine serum (FBS). These fibroblasts (100,000 cells) were then further incubated in a floating collagen gel lattice with the test products Ang I (1 micromol/L), Ang II (100 nmol/L), Ang III (100 nmol/L) and bestatin (100 micromol/L) for three days in DMEM without FBS. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. RESULTS: Ang I, II and III stimulated (p<0.05) collagen gel contraction by 30.4+/-4.8 (SEM)%, 27.1+/-3.1% and 15.4+/-3.6% respectively. Ang I- and II-induced stimulation of collagen gel contraction was of the same order but more pronounced (p<0.05) than Ang III- stimulated collagen gel contraction. The Ang I-, II- and III-stimulated collagen contraction was reduced by bestatin. Bestatin, however, did not affect basal collagen gel contraction in cardiac fibroblasts. Bestatin dose-dependently inhibited the hydrolysis of arginine- and alanine-p-nitroanilide in cardiac fibroblasts. When a neutralising antibody to transforming growth factor TGF-b1 was added to the collagen gel simultaneously with the angiotensins, the stimulated collagen contraction was not affected. Beta-aminoproprionitrile, an inhibitor of lysyl oxidase, completely abolished basal as well as Ang I-, II- and III-stimulated collagen contraction in cardiac fibroblasts. RESULTS: Our data suggest that aminopeptidases are involved in the Ang I-, II- and III-induced stimulation of collagen contraction in cardiac fibroblasts.     

A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.

Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.     

Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme.

The effects of a range of metallopeptidase inhibitors on the activities of the porcine kidney cell surface zinc aminopeptidases, aminopeptidase A (AP-A; EC 3.4.11.2), aminopeptidase N (AP-N; EC 3.4.11.7) and aminopeptidase W (AP-W; EC 3.4.11.16), have been directly compared. Amastatin and probestin were effective against all three aminopeptidases, with the concentration of inhibitor required to cause 50% inhibition (I50) in the low micromolar range (I50 = 1.5-20 microM), except for probestin with AP-N which displayed an I50 of 50 nM. Actinonin failed to inhibit significantly either AP-A or AP-W, and thus can be considered a relatively selective inhibitor (I50 = 2.0 microM) of AP-N. In contrast, bestatin was a relatively poor inhibitor of AP-N (I50 = 89 microM) and failed to inhibit AP-A, but was more potent towards AP-W (I50 = 7.9 microM). Thus, some of the observed chemotherapeutic actions of bestatin may be due to inhibition of cell-surface AP-W. A number of other metallopeptidase inhibitors, including inhibitors of endopeptidase-24.11 (EC 3.4.24.11) and membrane dipeptidase (EC 3.4.13.11), and the carboxylalkyl and phosphoryl inhibitors of angiotensin converting enzyme (EC 3.4.15.1) failed to inhibit significantly AP-A, AP-N or AP-W. However, AP-W was inhibited with I50 values in the micromolar range by the sulphydryl converting enzyme inhibitors rentiapril (I50 = 1.6 microM), zofenoprilat (I50 = 7.0 microM) and YS 980 (I50 = 17.7 microM). Neither AP-A nor AP-N were affected by these sulphydryl compounds. Inhibition of AP-W may account for some of the side effects noted with the clinical use of the sulphydryl converting enzyme inhibitors. The availability of compounds which are totally selective for AP-W over any of the other mammalian cell surface zinc aminopeptidases may aid in identifying endogenous substrates, and thus physiological or pathophysiological role(s) of AP-W.     

Ovarian hormones in man: their effects on resting vascular tone, angiotensin converting enzyme activity and angiotensin II-induced vasoconstriction

AIMS: Oestrogens in women have been shown to cause vasodilation which may reflect alterations in the activity of vascular angiotensin converting enzyme (ACE) and/or sensitivity to angiotensin II. The aim of this study was to assess the effect of ovarian hormones on vascular tone, vascular ACE activity and vasoconstriction to angiotensin II in males. METHODS: Eight volunteers were randomised in a crossover design to oestradiol, medroxy-progesterone, and placebo. Vasoconstriction to angiotensin I and angiotensin II was assessed by forearm plethysmography. RESULTS: Although baseline forearm flow was increased with oestradiol, suggesting generalized vasodilation, there were no changes in the vasoconstrictor responses to angiotensin I or angiotensin II. Medroxy-progesterone affected neither baseline flow nor vasoconstrictor responses. The results expressed as percentage reduction in flow (mean +/- s.d.) were: angiotensin I 48 pmol ml-1: placebo -48 +/- 14%; oestradiol -42 +/- 16%; medroxyprogesterone -43 +/- 8% and for angiotensin II 16 pmol ml-1: placebo -42 +/- 10%; oestradiol -39 +/- 11%; medroxyprogesterone -46 +/- 13%. CONCLUSIONS: Acute administration of oestradiol caused vasodilation in males, the effect was not due to alterations in vascular ACE activity or to altered sensitivity to angiotensin II.     

Effect of hypoxia on the conversion of angiotensin I to II in cultured porcine pulmonary endothelial cells

Earlier studies by other investigators have shown that acute exposure of cultured endothelial cells to hypoxic atmospheres inhibits the activity of the angiotensin converting enzyme in situ, resulting in severe but reversible depression of the rate of degradation of bradykinin. We exposed primary cultures of endothelial cells from the pulmonary artery of the pig to a range of hypoxic gas mixtures and measured the activity of the angiotensin converting enzyme in situ using angiotensin I as substrate. Each cell flask was exposed in random sequence to both hypoxic gas mixtures (PO2 29-69 torr) and room air for 40 min in Dulbecco's medium containing angiotensin I at concentrations of 1000 (N = 7), 500 (N = 8) or 100 ng/ml (N = 4). Angiotensin I disappearance rates and angiotensin II generation rates were linear. Recovery of immunoreactive peptide as either angiotensin I or II following 40 min of incubation was 86 +/- 17% (S.D.). The rate of increase in angiotensin II concentration in surface medium in room air experiments was 91 +/- 51 (S.D.) ng x ml-1 x hr-1. During hypoxia it was 85 +/- 42 ng x ml-1 x hr-1. The difference in rates was not significant by paired t analysis. The results of this study are consistent with earlier observations by the authors which suggest that hypoxia-induced depression of angiotensin I conversion in vivo is due to hemodynamic phenomena. Further studies are needed to clarify the role of cellular mechanisms in hypoxia-induced depression of angiotensin metabolism.     

Effect of chronic enalapril treatment on enzymes responsible for the catabolism of angiotensin I and formation of angiotensin II

We have investigated the effect of chronic administration of enalapril on the carboxypeptidases responsible for the formation of angiotensin II from angiotensin I and other peptidases known to recognize angiotensin I as a substrate in the rat. These studies have shown an increase in activity in rate of formation of des-Leu-angiotensin I in both kidney S2 and P2 centrifugal fractions as well as a decrease in the rate of degradation of angiotensin I substrate. Similar increases in the formation of A(1-8) have been observed in kidney using A(1-9) as substrate. These two enzyme activities have been named carboxypeptidase K1 and K2, respectively to reflect their presence in rat kidney. These changes were accompanied by significant decreases in the activity of an amastatin-sensitive aminopeptidase and endopeptidase 24.11 in the kidney P2 fraction. These data suggest that chronic treatment with ACE inhibitors may differentially affect the activity of other enzymes capable of degrading angiotensin causing a substantial re-direction of angiotensin metabolism.     

OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.     

Reversal of angiotensin II-stimulated collagen gel contraction in cardiac fibroblasts by aminopeptidase inhibition

The purpose of this investigation was to determine whether aminopeptidase inhibition could affect the angiotensin II-stimulated collagen gel contraction in basal (control) and TGF-beta1-treated cardiac fibroblasts (or myofibroblasts). The tested aminopeptidase inhibitors were the broad range aminopeptidase inhibitor bestatin, the specific inhibitor of alanine aminopeptidase leuhistin, and the specific inhibitor of arginine aminopeptidase arphamenine A. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated with(out) 400 pmol/L TGF-beta1 in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). These fibroblasts were then further incubated in a floating collagen gel lattice with the tested products (angiotensin II, bestatin, leuhistin, or arphamenine A) for 3 days in DMEM without FBS. The contraction of the collagen gel lattice by cardiac fibroblasts was determined by measuring the gel volume with tritiated water. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Angiotensin II (100 nmol/L) reduced the gel volume in control and TGF-beta1-treated cardiac fibroblasts. The angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated fibroblasts was almost completely reversed by leuhistin and arphamenine A (100 micromol/L). Bestatin (100 micromol/L) only partially inhibited the angiotensin II-stimulated collagen gel contraction in control fibroblasts, although it did not affect the angiotensin II-induced contraction in TGF-beta1-treated fibroblasts. In control and TGF-beta1-treated cardiac fibroblasts, 100 micromol/L leuhistin or arphamenine A only partially inhibited alanine aminopeptidase activity, whereas bestatin (100 micromol/L) completely inhibited the alanine aminopeptidase activity. Arginine aminopeptidase activity was only partially inhibited by leuhistin and arphamenine A at 100 micromol/L in control and TGF-beta1-treated fibroblasts. Bestatin, however, completely blocked the arginine aminopeptidase activity in control fibroblasts and only partially in TGF-beta1-treated fibroblasts at 100 micromol/L. Our data suggest that both alanine and arginine aminopeptidases are involved in the reversal of the angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated cardiac fibroblasts or myofibroblasts.     

Differences between angiotensin-converting enzyme inhibition and angiotensin II-AT1 antagonism on angiotensin-mediated responses in human internal mammary arteries

The current study aimed to demonstrate differences between angiotensin (Ang)-converting enzyme (ACE) inhibition and Ang II-AT1 receptor antagonism on full concentration-contraction responses to Ang I. Contraction responses to increasing concentrations of Ang I (1 nM-1 microM) were evaluated in organ baths in the presence of captopril (10 microM-1 mM) with or without a chymase inhibitor (1 microM soybean trypsin inhibitor), or irbesartan (0.1 nM-microM), in internal mammary arteries from 25 patients undergoing coronary bypass surgery. Responses were expressed as a percentage of the control response to 10 microM phenylephrine. Captopril did not change the maximum response to Ang I (control: 46.3 +/- 6.3%, captopril: 43.0 +/- 4.6%). In contrast, 0.1 microM irbesartan completely blocked the maximum response to Ang I (from 45.8 +/- 6.7% to 1.9 +/- 1.9%, p < 0.001). However, addition of soybean trypsin inhibitor to captopril more effectively shifted -log pD2 than captopril alone (0.47 +/- 0.06 vs 0.95 +/- 0.14 log units, p = 0.007). Ang I-mediated effects are much more effectively inhibited by Ang II antagonism than by ACE inhibition. The incomplete effects of captopril on the inhibition of Ang II formation might be caused by alternative Ang II forming enzyme(s), as was demonstrated by the additive effects of soybean trypsin inhibitor added to captopril.     

Interactions of kinins with angiotensin I converting enzyme (kininase II)

Angiotensin I converting enzyme (ACE) was purified to homogeneity from porcine kidney in order to determine whether iodobradykinins bind to the enzyme and, if so, whether pGlu-Trp-Pro-Arg-Pro-Gin-Ile-Pro-Pro, SQ20881, a competitive ACE inhibitor, changes the conformation of the enzyme in such a way that it binds kinins with an affinity and specificity expected of a bradykinin (BK) receptor, i.e. where the BK potentiating action of SQ20881 involves an increase in the number of BK receptors due to a conformational change in ACE. 125I-Labeled derivatives of [Tyr1]-kallidin and [Tyr-8]-bradykinin bound to the EDTA-inhibited enzyme, and binding was inhibited by nonradioactive BK. [125I-Tyr5]-BK was not bound by the enzyme. Specificity of [125I-Tyr5]-kallidin (T1K) binding was tested with forty-eight BK analogs, and the concentrations of analogs that inhibited 50% of T1K binding were determined. BK at 1.6 +/- 0.3 X 10(-8) M inhibited 505 of T1K binding. In addition, the concentrations of analogs that decreased by 50% the rate of [3H]-Hip-Gly-Gly ([3H]-HGG) hydrolysis by ACE were assessed. BK at 1.2 +/- 0.2 X 10(-6) M decreased the rate of [3H]-HGG hydrolysis by 50%. A comparison between these concentrations of analogs for inhibition of T1K binding and [3H]-HGG hydrolysis yielded a high correlation coefficient (r = 0.85). The specificity of ACE binding was clearly different from that expected of a BK receptor. Compounds structurally unrelated to BK, such as 5Q20881, pGlu-Lys-Trp-Ala-Pro-OH (BPP5a) and angiotensin I, inhibited T1K binding and [3H]-HGG hydrolysis by ACE.     

Potentiation of the depressor responses to atrial natriuretic peptides in conscious SHR by an inhibitor of neutral endopeptidase

In previous studies, neutral endopeptidase (NEP) hydrolyzed the Cys105-Phe106 bond of atrial natriuretic peptides (ANP) in vitro. Three such ring-opened peptides derived from ANP 99-126, 103-126, and 103-123 were inactive in conscious rats. In conscious spontaneously hypertensive rats (SHR) in the present study, 100 mumol/kg, intravenously (i.v.) of the NEP inhibitor, SQ 29,072 (7-[[2-(mercaptomethyl)-1-oxo-3-phenyl-propyl]amino]heptanoic acid), significantly increased the area over the curve (AOC) of the depressor response to 3 nmol/kg of ANP 103-126 from 165 +/- 36 to 792 +/- 350, 1,515 +/- 374, and 828 +/- 164 mm Hg.min at 15, 30, and 60 min after inhibitor treatment. Thirty minutes after 3, 10, 30, and 100 mumol/kg of SQ 29,072, the AOC of 3 nmol/kg of ANP 99-126 increased from 175 +/- 59 mm Hg.min in vehicle-treated rats to 296 +/- 100, 318 +/- 34, 632 +/- 194 (p less than 0.05) and 656 +/- 151 (p less than 0.05) mm Hg.min. Furthermore, 100 mumol/kg of SQ 29,072 potentiated the AOC of human ANP 99-126 and 105-126 and rat ANP 99-126, 103-126, and 103-123, suggesting that the exocyclic N-terminal residues and the C-terminal tripeptide did not influence ANP potentiation by SQ 29,072. In contrast, inhibitors of aminopeptidase, angiotensin-converting enzyme (ACE), and serine protease and an arginine vasopressin (AVP) antagonist did not substantially affect the AOC of 3 nmol/kg ANP 99-126. Finally, SQ 29,072 did not alter the activities of bradykinin, AVP, or angiotensin I or II. In conclusion, NEP may inactivate ANP in vivo by cleavage of susceptible bonds within the ANP ring.