Expression of angiotensin-converting enzyme activity in cultured pulmonary artery endothelial cells

Angiotensin-converting enzyme (EC 3.4.51.1) is a carboxyterminal dipeptidyl peptidase. The enzyme catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II. In addition, the enzyme catabolizes bradykinin. Because of these actions, the enzyme is of pivotal importance in blood pressure homeostasis. Numerous investigators have demonstrated the presence of the enzyme in association with endothelial cells but relatively little is known concerning the factors controlling the expression enzyme activity by endothelial cells in culture. We have demonstrated that endothelial cells in culture do not express significant amounts of enzyme activity until several days after growth ceases due to high cell density. This is important because it demonstrates a change in function with stage of growth in culture and a possible difference in functional capabilities between nondividing endothelial cells and cells that are dividing in response to injury. Since density-dependent expression of differentiated traits does not appear to be unique to endothelial cells an understanding of the mechanisms underlying this phenomenon may provide a general explanation for the expression of differentiated traits by cultured cells.     

Angiotensin converting enzyme inhibitor potentiated the vasorelaxant response of atrial natriuretic peptide in toad aortic rings

1. The vasorelaxant effect of synthetic atrial natriuretic peptide (ANP) on the vascular response to angiotensin II (A II) and norepinephrine (NE) in aortic rings from Bufo arenarum toad was studied. 2. Pretreatment with ANP partially inhibited the vascular response to A II and NE. 3. Angiotensin converting enzyme inhibitor (ACEI) treatment partially inhibited the contractile response of angiotensin I (A I) and did not affect the A II response. 4. The inhibitory effect of ANP on vascular response to A II and NE were potentiated by pretreatment with ACEI. 5. Results suggest that the angiotensin converting enzyme present in the vascular wall from Bufo arenarum toad may be involved in the metabolism of ANP.     

Angiotensin II generated by a human renal carboxypeptidase

Angiotensin II, the potent hypertensive octapeptide, can be generated by a sequential cleavage of the carboxyl-terminal leucine and histidine from angiotensin I by a human renal extract. This extract does not hydrolyze further the resulting octapeptide. The more widely recognized biosynthetic pathway is by the extracellular dipeptide cleavage of angiotensin I by an enzyme which also degrades bradykinin, i.e., angiotensin converting enzyme. The presence of a carboxypeptidase activity capable of generating but not further hydrolyzing angiotensin II was observed in an ammonium sulfate fraction of a human renal extract. This novel enzymatic activity is distinct from angiotensin converting enzyme activity in that it is not dependent upon calcium and is not inhibited by known angiotensin converting enzyme inhibitors.     

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.     

Des-Leu angiotensin I: biosynthesis and drinking response

The crude rat and bovine synaptosomal lysate from brain can hydrolyze angiotensin I (AI) to des-Leu angiotensin I (AI-dL) and no further. This cytosolic enzyme has a specificity for angiotensin-related sequences, R-His-Pro-Phe-His-Leu and therefore named angiotensin-related carboxypeptidase (ARC). These studies led to the biosynthesis and purification of AI-dL in order to determine if it can provoke a drinking response. This nonapeptide is a potent dipsogen when injected into the cerebroventricles of rats. The drinking response probably requires a second hydrolysis to angiotensin II (AII) since both captopril and saralasin can inhibit this response.     

Characterization of a new inhibitor for angiotensin converting enzyme from the venom of Vipera aspis aspis

1. Angiotensin converting enzyme inhibitor has been isolated from the venom of Vipera aspis aspis by gel filtration and reverse phase HPLC. 2. The purified inhibitor is a decapeptide, whose amino-terminal is blocked, with mol. wt 1044 determined by fast atom bombardment mass spectrometry. 3. The peptide inhibited the conversion of angiotensin I to angiotensin II, and Ki values were determined to be 7.54 x 10(-4) and 1.36 x 10(-4) M, respectively, using Hip-His-Leu and Hip-Gly-Gly as substrates 4. The peptide also inhibited the degradation of bradykinin, induced hypotension in spontaneously hypertensive rats and caused an increase in capillary permeability in rabbits, however, it possessed no lethality.     

Identification of serine proteinases with tonin-like activity in the rat submandibular and prostate glands.

Two enzymes with tonin-like activity, designated rSMT3 and rSMT4, were purified from rat submandibular glands and another, rPT1, was obtained from the prostate. The three enzyme fractions hydrolysed angiotensin I, angiotensinogen (AG) and synthetic AG(1-14) to form angiotensin II. With angiotensin I as substrate, pH optima were 6.5 for rSMT3, 6.8 for rSMT4 and 7.5 for rPT1. With AG(1-14), the three enzymes had optimal activity at pH 7.5. The three enzymes had negligible activity upon a kallikrein substrate, Ac-Phe-Arg-Nan. The enzymes were inhibited by aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride but not by two angiotensin converting enzyme inhibitors, ethylenediaminetetracetic acid or enalaprilat. N-tosyl-L-phenylalanine chloromethyl ketone (1 mM) inhibited rPT1 and rSMT4 but not rSMT3. Molecular weights (SDS-PAGE) were 31,700 for rSMT3, 29,800 for rSMT4 and 28,100 for rPT1. Total activity in the prostate is 150-times lower than in the submandibular gland, where 92% of the tonin activity is related to rSMT4. Physical and chemical properties suggest that rSMT4 is tonin, whereas rSMT3 and rPT1 are tonin-like enzymes which can generate angiotensin II from different substrates.     

Tonin, an esteroprotease from rat submaxillary glands

Tonin is an enzyme found in the rat submaxillary glands which liberates angiotensin II from angiotensinogen, the Skeggs tetradecapeptide renin substrate, and angiotensin I. Tonin hydrolyzes benzoyl-arginine ethyl ester, benzoyl-arginine methyl ester, tosyl-arginine methyl ester, benzoyl-arginine p-nitroanilide and other small synthetic substrates at an optimum ph of 9.0. Tonin shows, however, a great specificity with respect to angiotensin I. Tonin is inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride at high concentrations (greater than 10(-2) M) and by soybean trypsin inhibitor and aprotinin. Tonin is thus an esteroprotease of the class of the serine protease with trypsin- and chymotrypsin-like activity. Tonin belongs to the same family of enzyme as glandular kallikrein and the gamma subunit of the nerve growth factor.     

血管紧张素转换酶的结构功能及相关抑制剂

血管紧张素转化酶(angiotensin converting enzyme, ACE, EC 3.4.15.1)是一种位于细胞膜上, 依赖锌离子的羧二肽酶, 催化水解十肽血管紧张素I羧基末端两个氨基酸, 生成具有血管收缩作用的八肽血管紧张素II。ACE在血压调节系统renin - angiotensin system (RAS系统)中具有重要作用, 从ACE的结构功能、基因多态性及其抑制剂等方面进行了详细综述。发现体细胞ACE两个活性中心催化血管紧张素I和缓激肽的机制不同, 因此以体细胞ACE单个活性中心为靶点的研究, 将会为研制开发副作用更少, 安全性更高的ACE抑制剂提供新的途径。     

Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart

Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.     

Effect of des-aspartate-angiotensin I on the actions of angiotensin II in the isolated renal and mesenteric vasculature of hypertensive and STZ-induced diabetic rats

The present study investigated the action of des-aspartate-angiotensin I (DAA-I) on the pressor action of angiotensin II in the renal and mesenteric vasculature of WKY, SHR and streptozotocin (STZ)-induced diabetic rats. Angiotensin II-induced a dose-dependent pressor response in the renal vasculature. Compared to the WKY, the pressor response was enhanced in the SHR and reduced in the STZ-induced diabetic rat. DAA-I attenuated the angiotensin II pressor action in renal vasculature of WKY and SHR. The attenuation was observed for DAA-I concentration as low as 10(-18) M and was more prominent in SHR. However, the ability of DAA-I to reduce angiotensin II response was lost in the STZ-induced diabetic kidney. Instead, enhancement of angiotensin II pressor response was seen at the lower doses of the octapeptide. The effect of DAA-I was not inhibited by PD123319, an AT2 receptor antagonist, and indomethacin, a cyclo-oxygenase inhibitor in both WKY and SHR, indicating that its action was not mediated by angiotensin AT2 receptor and prostaglandins. The pressor responses to angiotensin II in mesenteric vascular bed were also dose-dependent but smaller in magnitude compared to the renal vasculature. The responses were significantly smaller in SHR but no significant difference was observed between STZ-induced diabetic and WKY rat. Similarly, PD123319 and indomethacin had no effect on the action of DAA-I. The findings reiterate a regulatory role for DAA-I in vascular bed of the kidney and mesentery. By being active at circulating level, DAA-I subserves a physiological role. This function appears to be present in animals with diseased state of hypertension and diabetes. It is likely that DAA-I functions are modified to accommodate the ongoing vascular remodeling.     

Heptapeptide analogues induce greater blockade of renal than femoral vascular responses to angiotensin

We characterized blockade induced by 2 octapeptide and 2 heptapeptide analogues of angiotensin in the vascular beds of the kidney and hindlimb. Bolus injections of angiotensin II and its 1-des Asp analogue (angiotensin III) at the dose which reduced blood flow by about 50 percent and graded infusions of the analogue-antagonists were made directly into each artery and flow responses were measured with an electromagnetic flowmeter in the anesthetized dog. With the dose of antagonist which produced 50 percent inhibition of the control angiotensin response (ID 50) as the index, inhibition was slightly greater in the kidney than in the hindlimb for both the potent octapeptide antagonist {1-Sar, 8-Ala angiotensin II: kidney ID 50 = 15.3±1.7 (SD) ng/kg/min; hindlimb ID 50 = 23.3±1.8 (SD) ng/kg/min} and the weak octapeptide antagonist {1-D-Asn, 8-Ala angiotensin II: kidney ID 50 = 178.7±2.0 (SD) ng/kg/min; hindlimb ID 50 = 266.7±1.9 (SD) ng/kg/min}. In contrast, both the potent and weak heptapeptide analogues were much more effective as antagonists in the renal than the femoral vascular bed {1-des Asp, 8-Ile AII: kidney ID 50 = 14.9±1.8 (SD) ng/kg/min; hindlimb ID 50 = 36.2±1.9 (SD) ng/kg/min}; {1-des Asp, 8-Ala angiotensin II: kidney ID 50 = 408.9±1.8 (SD) ng/kg/min; hindlimb ID 50 = 1270±2.8 (SD) ng/kg/min}. The difference in the influence of the analogues in the two vascular beds may reflect either a difference in their angiotensin receptors or in the rate at which heptapeptide analogues are degraded in their transit through the renal and femoral vasculature.     

In vitro renin inhibition to prevent generation of angiotensins during determination of angiotensin I and II

To prevent in vitro generation of angiotensins, the renin inhibitor CGP 29287 (CGP) was added to blood sampling tubes. Plasma immunoreactive angiotensin (ir-ANG) I and II were simultaneously measured by radioimmunoassay after rapid and quantitative extraction from a single plasma sample on phenylsilylsilica (Bondelut PH). True plasma ANG-(1-8)octapeptide was determined after additional separation of the different angiotensins by high performance liquid chromatography. Ir-ANG II/CGP showed the known linear relationship with ANG-(1-8)octapeptide (r = 0.87, n = 23), but - in contrast to studies without addition of CGP - the y-axis intercept which presumably represents cross-reacting angiotensins other than ANG II was very small. Ir-ANG II/CGP concentrations fell below 1 fmol/ml after converting enzyme inhibition. The results suggest that CGP 29287 prevents in vitro generation of ANG I and ANG II as well as the ANG-metabolites. Ir-ANG I/CGP measured after Bondelut PH extraction of the plasma was strongly correlated with ir-ANG I obtained after blood ethanol extraction (r = 0.97, n = 23). Thus, it is now possible to measure reliably both ANG I and ANG II within the same plasma extract after a simple extraction procedure.     

Enalapril in the treatment of hypertension with renal artery stenosis.

The converting enzyme inhibitor enalapril, in single daily doses of 10-40 mg, was given to 20 hypertensive patients with renal artery stenosis. The blood pressure fall six hours after the first dose of enalapril was significantly related to the pretreatment plasma concentrations of active renin and angiotensin II and to the concurrent fall in angiotensin II. Blood pressure fell further with continued treatment; the long term fall was not significantly related to pretreatment plasma renin or angiotensin II concentrations. At three months, 24 hours after the last dose of enalapril, blood pressure, plasma angiotensin II, and converting enzyme activity remained low and active renin and angiotensin I high; six hours after dosing, angiotensin II had, however, fallen further. The rise in active renin during long term treatment was proportionally greater than the rise in angiotensin I; this probably reflects the fall in renin substrate that occurs with converting enzyme inhibition. Enalapril alone caused reduction in exchangeable sodium, with distinct increases in serum potassium, creatinine, and urea. Enalapril was well tolerated and controlled hypertension effectively long term; only two of the 20 patients required concomitant diuretic treatment.     

Influence of intrarenally generated angiotensin II on renal hemodynamics and tubular reabsorption.

There is growing recognition that angiotensin II (ANG II) formed intrarenally exerts direct effects on renal hemodynamics and tubular reabsorption. In vivo micropuncture experiments performed in anesthetized rats have shown that peritubular capillary infusion of either ANG II or angiotensin I (ANG I), at rates that do not markedly influence baseline vascular resistance, can increase proximal tubular reabsorption rate and enhance the responsiveness of the tubuloglomerular feedback mechanism. With higher ANG II or ANG I infusion rates, pronounced preglomerular vasoconstriction occurs, resulting in reduced glomerular capillary pressure and single nephron glomerular filtration rate. The effects of peritubular capillary infusion of ANG I on glomerular function have been shown to be inhibited by the ANG II receptor antagonist, saralasin, indicating that the observed effects of ANG I on proximal tubular reabsorption and glomerular function are not due to direct effects of the decapeptide but are mediated by increases in the interstitial ANG II concentrations resulting from intrarenally generated ANG II. Interestingly, neither peritubular capillary infusion nor systemic administration of large doses of the angiotensin-converting enzyme (ACE) inhibitor, enalaprilat, elicited significant blockade of the single nephron hemodynamic responses to peritubular infusion of ANG I. These findings indicate that intrarenal conversion of ANG I to ANG II occurs, at least in part, at a site which is inaccessible to acutely administered ACE inhibitors, or that there is an alternative pathway for the intrarenal conversion of ANG I to ANG II that is not blocked by ACE inhibitors.     

Role of converting enzyme in the cardiovascular and adrenal cortical responses to (des-Asp1)-angiotensin I.

(Des-Asp1)-angiotensin I, angiotensin II and III were evaluated for pressor activities in conscious nephrectomized rats and for steroidogenic actions in rat adrenal zona glomerulosa. The pressor effect of this angiotensin nonapeptide was similar to that found with mole-equivalent doses of angiotensin III (one-third as active as angiotensin II) and was significantly attenuated by pretreatment with the 0. jararaca nonapeptide converting enzyme inhibitor. Hence, (des-Asp1)-angiotensin I is a substrate for converting enzyme in vivo, and the rapid conversion indicates that an alternate pathway for the formation of angiotensin III could exist. (Des-Asp1)-angiotensin I possessed only 0.1% of the activity of angiotensin III as a steroidogenic agent in cell suspensions of rat adrenal zona glomerulosa. Angiotensin I was a weak steroidogenic agent in vitro (1%) and was not blocked by an inhibitor of converting enzyme. Adrenal cells dispersed from the outer zone of the cortex would appear to be devoid of significant converting enzyme activity.     

Role of lipoxygenase products in the effects of angiotensin II in the isolated aorta and perfused heart of the rat

The objective of this study was to determine whether arachidonate metabolites are involved in the vasoconstrictive effects of angiotensin II in rats. In the isolated perfused heart, dexamethasone (4 mg/kg) significantly suppressed the maximal decreases in coronary flow induced by angiotensin II and vasopressin (reference drug). In the heart, the nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 1 muM) markedly suppressed the angiotensin II-induced decreases in coronary flow. NDGA (10 muM) inhibited both angiotensin II- and methoxamine- (reference drug) induced contractions in aortic rings with (in the presence of L-NAME) and without endothelium. In the heart, the leukotriene synthesis inhibitor MK-886 (0.3 muM) significantly reduced the maximal effects to angiotensin II, but the leukotriene antagonist FPL 55712 (0.1 and 0.3 muM) had no effect. We conclude that in the isolated perfused rat heart angiotensin II-induced decreases in coronary flow are in part mediated by Hpoxygenase products, which might be derived from the 5-Hpoxygenase pathway, but are probably not leukotrienes. Furthermore, endothelium independent Hpoxygenase products mediate part of the contractile responses to angiotensin II in the isolated rat aorta.     

Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats

We report here on the extraction and characterization of angiotensin I (ANG I) and angiotensin II (ANG II) from the brain of rats. High pressure liquid chromatography (HPLC) with different mobile phases combined with specific radioimmunoassays (RIA) proved to be a powerful tool for peptide characterization in biological samples; (Ile5)-ANG I, (Ile5)-ANG II and (Ile5)-ANG III could clearly be identified in cerebrospinal fluid (CSF), incubated in vivo and in vitro with renin, in total brain extracts, as well as in hypothalamus (HT), medulla oblongata (MO), cerebellum (CER) and cortex (CO). Angiotensin cleaved from CSF angiotensinogen and angiotensin extracted from brain showed retention times identical to those of plasma angiotensin and synthetic standard peptides, indicating that their amino acid sequence is probably identical. ANG I and ANG II were highest in the HT and lowest in the CO. Following bilateral nephrectomy (NX) both ANG I and ANG II persisted at control levels. Young 10 week old spontaneously hypertensive rats (SHRSP) showed significantly lower ANG I and ANG II concentrations in the HT compared with Wistar Kyoto rats (WKY). Intracerebroventricular (i.c.v.) administration of the converting enzyme inhibitor captopril caused a significant increase in ANG 1 in nephrectomized SHRSP but not in WKY. These differences were not found in 40 week old SHRSP. The data show that ANG I and ANG II are synthetized in the brain of rats. The lower concentrations and the enhanced accumulation of ANG I after converting enzyme blockade in nephrectomized young SHRSP indicate an increased turnover of angiotensin in hypertensive rats.     

Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.