Affinity chromatography and some properties of the angiotensin-converting enzyme from human heart

Human heart angiotensin-converting enzyme has been purified by affinity chromatography on immobilized N-[1(S)-carboxy-5-aminopentyl]-Gly-Gly. The isolation procedure permitted a 1650-fold-purified enzyme to be obtained. The specific activity of angiotensin-converting enzyme was 38 units per mg protein. The molecular weight of enzyme determined by polyacrylamide gel electrophoresis in denaturing conditions was 150,000. The isoelectric point (5.3) of the enzyme was determined by chromatofocusing. The Km values of the enzyme for Bz-Gly-His-Leu and angiotensin I are 1.2 mM and 10 microM, respectively. Substrate inhibition of heart angiotensin-converting enzyme with a K's of 14 mM has been shown. The human heart enzyme is inhibited by SQ 20881 (IC50 = 40 nM). It was shown that NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- are activators of the heart angiotensin-converting enzyme, whereas CH3COONa and NaNO3 have no effect on a catalytic activity of the enzyme.     

Various properties of angiotensin-converting enzyme from the seal Phoca groenlandica

It was found that the molecular mass of the angiotensin-converting enzyme from seal (Phoca groenlandica) lungs determined by electrophoresis in 7.5% PAAG in the presence of sodium dodecyl sulfate is 150 kD. The enzyme has a pH optimum with respect to hippuryl-L-histidyl-L-leucine at 7.3--7.5; KM is 1.2 mM. The enzyme is inhibited by the substrate to form a nonproductive ES2 complex with the dissociation constant (Ks') of 4.8 mM. The activation of the seal angiotensin-converting enzyme in the presence of NaCl was studied. The bradykinin-potentiating factor (SQ 20881) inhibits the seal enzyme with a high efficiency (IC50 = 2.5.10(-8) M). Monoclonal antibodies to the angiotensin-converting enzyme from human lungs do not interact with its seal lung counterpart, which points to the species specificity of the angiotensin-converting enzyme.     

Metabolic and pharmacokinetic activity of the isolated sheep bronchial circulation

We sought to determine bronchial vascular metabolic and pharmacokinetic activity toward benzoyl-Phe-Ala-Pro (BPAP), ADP, adenosine, and prostaglandin E2 (PGE2) by developing an isolated sheep bronchial circulation preparation. We measured mean transit time (t), uptake, and metabolism by injecting 3H-labeled substrates with [14C]sucrose into the bronchial artery of sheep lungs stripped clean of parenchymal tissue. After [3H]BPAP the t for 3H was the same as for 14C. Thirty-six percent of the injected BPAP was converted to metabolite ([3H]benzoyl-Phe) in a single pass. An inhibitor of angiotensin-converting enzyme, SQ 20,881, depressed BPAP metabolism by 50%, while perfusion of the bronchial circulation with glutaraldehyde reduced metabolism to a basal level. After [3H]ADP the t for 3H was again the same as for 14C. 3H recovery after 40 pmol [3H]ADP was less (58%) than after 400 nmol [3H]ADP (79%). Twenty-two percent of the injected radioactivity emerged in the effluent as metabolites of ADP for either dose. Adenosine and PGE2 uptake was negligible, and most of the recovered radioactivity in each case was unchanged substrate. This study suggests that the bronchial circulation is pharmacokinetically and metabolically active with respect to vasoactive mediators like angiotensin I, bradykinin, and adenine nucleotides, and that the enzymes responsible for this metabolic activity line the vascular lumen.     

Physico-chemical characteristics of angiotensin-converting enzyme from the bovine lung

The angiotensin-converting enzyme from bovine lung was isolated by chromatography with a 25-30% yield and purified 2200-2600-fold. The active molecule concentration in the enzyme preparations was 70-100% as could be judged from titration by inhibitor SQ 20,881. The molecular mass of the enzyme according to electrophoretic data is about 132 kDa; the maximal radius of the enzyme molecule as determined by electron microscopy is 68 +/- 9 A. Five enzyme isoforms with pI of 4.85, 4.7, 4.54, 4.38 and 4.3, respectively, were identified. The kinetic parameters of hydrolysis of three synthetic peptide substrates and the constants of activation of the substrate (Z-Phe-His-Leu) hydrolysis by chloride anions were determined.     

Role of the renin-angiotensin system in drinking of seawater-adapted eels Anguilla japonica: a reevaluation

The role of ANG II, a potent dipsogenic hormone, in copious drinking of seawater eels was examined. SQ-14225 (SQ), an angiotensin-converting enzyme inhibitor, infused intra-arterially at 0.01-1 microgram. kg(-1). min(-1), depressed drinking and arterial blood pressure in a dose-dependent manner. The inhibition was accompanied by a small decrease in plasma ANG II concentration, which became significant at 1 microgram. kg(-1). min(-1). After the infusate was changed back to the vehicle, the depression of drinking and arterial pressure continued for >2 h, although plasma ANG II concentration rebounded above the level before SQ infusion. By contrast, infusion of anti-ANG II serum (0.01-1 microgram. kg(-1). min(-1)) did not suppress drinking and arterial pressure, although plasma ANG II concentration decreased to undetectable levels. Plasma atrial natriuretic peptide and plasma osmolality, which influence drinking rate in eels, did not change during SQ or antiserum infusions. These results suggest that the renin-angiotensin system plays only a minor role in the vigorous drinking observed in seawater eels. The results also suggest that the antidipsogenic and vasodepressor effects of SQ in seawater eels are not due solely to the inhibition of ANG II formation in plasma.     

Study of peptides inhibiting the angiotensin-converting enzyme of human seminal plasma.

Several peptides were investigated for their inhibitory capacity against dipeptidyl carboxypeptidase (angiotensin-converting enzyme) from human seminal fluid. The strongest inhibitor was the nonapeptide SQ 20881. A marked inhibition was also shown by the compounds Phe-Ala-Pro and Boc-Phe-Ala-Pro, which behaved as competitive inhibitors. Among the peptides related to angiotensin, angiotensin III was the strongest inhibitor, followed by angiotensin II and the C-terminal hexapeptide of angiotensin II. The results indicate the dipeptidyl carboxypeptidase of human semen is very similar to pulmonary dipeptidyl carboxypeptidase in its susceptibility to peptide inhibitors. In view of these and other previously reported similarities, it is possible that both enzymes are identical.     

Isolation and properties of the angiotensin-converting enzyme from human lungs

Using chromatofocusing, an angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human lung. The procedure allows for 24 300-fold purification of the enzyme. The enzyme specific activity is 36.3 u. per mg protein; Mr as determined by polyacrylamide gel electrophoresis is 150 000. The lung enzyme after solubilization by trypsin treatment was found to be heterogeneous. Four isoforms of the enzyme with pI 5.3, 4.9, 4.8 and 4.6 were identified. The pH-optimum for the enzyme with respect to hippuryl-L-histidyl-L-leucine hydrolysis lies at 8.3; Km = 2.8 mM. The effect of Cl- on the enzyme activity was studied. It was found that the bradykinin-potentiating factor (SQ 20 881) inhibits the human lung angiotensin-converting enzyme (I50 = 1.6 X 10(-8) M).     

Inhibition of angiotensin-converting enzyme by des-Leu10-angiotensin I: a potential mechanism of endogenous angiotensin-converting enzyme regulation

Des-Leu10-angiotensin I is a nonapeptide generated from angiotensin I by the action of carboxypeptidase-like activities residing in the human platelet and mast cell. This nonapeptide was found to inhibit rabbit lung angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) with a Ki of 3.1 X 10(-7) M. The mechanism of inhibition was competitive. Inhibition of human serum angiotensin-converting enzyme by des-Leu10-angiotensin I was comparable in magnitude to inhibition by bradykinin and angiotensin III. These results suggest that limited proteolysis of angiotensin I by cells resident in vascular tissue may result in the generation of an endogenous inhibitor of angiotensin-converting enzyme. Such pathways may play roles in controlling levels of vasoactive peptides at local vascular sites.     

Differential uptake of endothelin-1 by the coronary and pulmonary circulations.

Substantial removal of the vasoconstrictor peptide endothelin-1 (ET-1) by the pulmonary circulation has been reported to occur in perfused guinea pig and rat lungs. We examined the uptake of ET-1 by coronary and pulmonary circulations of the rabbit by measuring single-pass extraction of ET-1 in the isolated heart and lung. In separate experiments, each organ was perfused at 30 ml/min with Krebs-albumin (3%) solution. A bolus of 125I-ET-1 and [14C]dextran in 0.3 ml Krebs-albumin solution was injected, and extraction of endothelin (EET), relative to that of an intravascular reference indicator, [14C]dextran, was determined by multiple indicator-dilution technique. EET was 5 +/- 2% (SE) in the heart and 49 +/- 4% in the lung. Increasing flow rate in the lung preparation to approximate the mean transit time in the heart preparation did not significantly alter EET. Despite insignificant uptake of ET-1, the coronary circulation extracted an angiotensin-converting enzyme inhibitor (351A) and metabolized a synthetic angiotensin-converting enzyme substrate (benzoyl-phenyl-alanyl-proline), both properties of the normal pulmonary circulation. We therefore conclude that there is no significant ET-1 uptake in the coronary vascular bed.     

Change of the activities of angiotensins I and II on the vascular smooth muscle by crude kallikrein.

Incubation in vitro of angiotensin I (A I) with crude kallikrein induced a potentiation in the response to decapeptide of the isolated continuously superfused rabbit aorta. Crude kallikrein when incubated with angiotensin II (A II) caused a decrease in response to octapeptide of the same assay tissue. Converting enzyme inhibitor, SQ 20881, produced a competitive inhibition in the response to A I preincubated with crude kallikrein but did not alter the inhibitory effect of the enzyme on A II response. Pure kallikrein did not induce any change in the responses to both peptides when used at the same concentrations. The competitive inhibitor of A II (N,N-dimethyl) Gly1-Ile5-Ile8-angiotensin II (DMGIA II), abolished the effects of both A II- and A I-preincubated with crude kallikrein. From these results it was concluded that crude kallikrein-induced potentiation in the response to A I of the aorta is probably due to the conversion of decapeptide to octapeptide by an enzyme fraction in crude kallikrein preparation. These results also indicate that crude kallikrein (Padutin) is not a pure enzyme preparation and probably contains some other enzyme fractions which are responsible from the changes of the vascular activities of angiotensin-peptides.     

Isolation of human liver angiotensin-converting enzyme by chromatofocusing

Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).     

Reduction in hypertension-induced protein synthesis in the rat pulmonary trunk after treatment with teprotide (SQ 20881)

Angiotensin II has been previously implicated as a mediator of vasoconstriction during the development of hypoxic pulmonary hypertension. The effect of angiotensin-converting enzyme inhibition with teprotide (SQ 20881) on development of pulmonary hypertension was determined by measurement of the drug's ability to modify hypertension-induced protein synthetic changes in the rat pulmonary trunk. Rats were injected with either SQ 20881 (2 mg/kg body wt every 8 hr) or saline vehicle during exposure to chronic hypoxia at 0.5 atm for either 3 or 7 days. Comparisons were made of tissue weight, absolute protein content, and in vitro synthesis of collagen and noncollagen protein of the pulmonary trunks of SQ-treated hypoxic, SQ-treated normoxic, saline-treated hypoxic, and saline-treated normoxic rats. Treatment of hypoxic rats with SQ 20881 was found to significantly decrease right ventricular pressure, tissue weight, absolute protein content, and in vitro protein synthesis after 7 days compared to saline-treated hypoxic rats. Neither right ventricular hypertrophy nor the development of polycythemia was decreased by SQ 20881 treatment.     

Effects of enalaprilat on neointimal growth of cultured rabbit aorta following balloon injury

Our objective was to determine if the ability of an angiotensin-converting enzyme (ACE) inhibitor to attenuate neointima formation in balloon-damaged vessel is expressed in an isolated organ culture model of neointimal growth. In vivo balloon angioplasty in combination with in vitro organ culture was used to produce a unique model of vascular neointima formation. Aortic segments were cultured in medium containing a broad concentration range of the ACE inhibitor enalaprilat (0-100 microM). Cell proliferative indices and neointima:media thickness ratios were determined from vessel segments after 1, 4, and 7 days in culture. We observed no significant effect on either parameter at any dose of enalaprilat. Linear regression analysis on the rate of increase in intima to media thickness ratios during the 7 days of culture also showed no effect of enalaprilat at any concentration. We conclude that enalaprilat has no effect on neointimal growth or cell proliferation in this vascular organ culture model, and it is suggested that ACE inhibitors may act by mechanisms other than local converting enzyme inhibition to attenuate neointimal growth in rabbits following vascular ballooning in vivo.     

Titration of active centers of enzymes by means of reversible inhibitors. Dipeptidyl-carboxypeptidase - inhibitor SQ 20881 from venom of the snake Bothrops jararca)

The essentials of estimation of the number of enzyme active sites by reversible inhibition are discussed. The necessity of evaluation of the substrate effect on the equilibrium of the systems with a rapidly dissociating enzyme -- inhibitor complex has been demonstrated. Some procedures for determination of the number of active sites of dipeptidyl-carboxypeptidase (EC 3.4.15.1) from bovine kidney cortex, using the competitive inhibitor SQ 20 881 (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) have been developed. The kinetic and equilibrium constants for the enzyme-inhibitor interaction (ki = 3.2 . 10(6) M-1s-1, k-i = 8 ms-1 and Ki = 2.5 +/- 0.5 nm) have been calculated.     

Purification and Inhibition by Neuropeptides of Angiotensin-Converting Enzyme from Rat Brain

Angiotensin-converting enzyme was solubilized with papain from a particulate fraction of rat brain and purified to apparent homogeneity by a procedure including DEAE-cellulose, hydroxylapatite, Sephadex G-200, Cys(Bzl)-Pro-Sepharose, and ricin-Sepharose chromatography. Bradykinin potentiators, SQ 14,225, and Arg-Pro-Pro strongly inhibited the activity of the purified enzyme, whereas Phe-Ala, phosphoramidon, and pentobarbital exerted little inhibitory effect on the activity. Among neuropeptides investigated, substance P, bradykinin, and Leu-enkephalin (Arg6) exerted strong inhibitory actions on the enzyme. Furthermore, the latter two peptides were shown to be good substrates for the enzyme. Thus, angiotensin-converting enzyme of rat brain is distinct from endogenous enkephalinase and may interact with various neuropeptides located in the brain.     

Role of superoxide and thromboxane receptors in acute angiotensin II-induced vasoconstriction of rabbit vessels

This study explored the hypothesis that a portion of angiotensin II-induced contractions is dependent on superoxide generation and release of a previously unidentified arachidonic acid metabolite that activates vascular smooth muscle thromboxane receptors. Treatment of rabbit aorta or mesentery artery with the thromboxane receptor antagonist SQ29548 (10 μM) reduced angiotensin II-induced contractions (maximal contraction in aorta; control vs. SQ29548: 134 ± 16 vs. 93 ± 10%). A subset of rabbits deficient in vascular thromboxane receptors also displayed decreased contractions to angiotensin II. The superoxide dismutase mimetic Tiron (30 mM) attenuated angiotensin II-induced contractions only in rabbits with functional vascular thromboxane receptors (maximal contraction in aorta; control vs. Tiron: 105 ± 5 vs. 69 ± 11%). Removal of the endothelium or treatment with a nitric oxide synthase inhibitor, nitro-l-arginine (30 μM) did not alter angiotensin II-induced contractions. Tiron and SQ29548 decreased angiotensin II-induced contractions in the denuded aortas by a similar percentage as that observed in intact vessels. The cyclooxygenase inhibitor indomethacin (10 μM) or thromboxane synthase inhibitor dazoxiben (10 μM) had no effect on angiotensin II-induced contractions indicating that the vasoconstrictor was not thromboxane. Angiotensin II increased the formation of a 15-series isoprostane. Isoprostanes are free radical-derived products of arachidonic acid. The unidentified isoprostane increased when vessels were incubated with the superoxide-generating system xanthine/xanthine oxidase. Pretreatment of rabbit aorta with the isoprostane isolated from aortic incubations enhanced angiotensin II-induced contractions. Results suggest the factor activating thromboxane receptors and contributing to angiotensin II vasoconstriction involves the superoxide-mediated generation of a 15-series isoprostane.     

Indomethacin (IND) inhibits an enhanced renin release following the captopril,SQ 14225, administration

The present study was done to investigate the role of endogenous prostaglandins in the mechanism of renin release stimulated by angio-tensin I-converting enzyme inhibitor, SQ 14225 in 9 hypertensive patients and 3 normotensive subjects. Plasma renin activity was significantly i increased after the administration of SQ 14225 and decreased to control levels after the addition of indomethacin. Pre-treatment with indomethacin also inhibited the augmentation of renin release following the SQ 14225 administration. Urinary excretion of prostaglandin E was not significantly increased after the SQ 14225 administration but significantly decreased after the administration of indomethacin. The present data that the augmented renin release due to the suppression of the negative short feedback mechanism of renin release by SQ 14225 was inhibited by indomethacin suggest that endogenous prostaglandin system may contribute to the short feedback mechanism of renin release.     

Drugs inhibiting the renin - angiotensin system

There are several approaches for interfering with the renin-angiotensin system. Antibodies against renin angiotensins I and II (AI and AII) have not been consistently successful in the past, probably because of nonspecific effects; however, recent purification of renin now makes this approach more promising. Renin inhibitors include pepstatin and analogs, lipids and phospholipids, and renin-substrate analogs. Pepstatin and analogs are the most potent and specific but they are not orally active. The phospholipids are the most effective in vivo but their specificity is yet to be established. No renin-substrate analogs have been developed that have biologically significant effects. Some of the most potent and specific agents available for interfering with the renin-angiotensin system are the AII-receptor antagonists. While these compounds effectively prevent the actions of AII, they suffer from several severe deficiencies: partial agonist activity, short duration of action, and lack of oral activity. The recent development of angiotensin-converting enzyme ACE) inhibitors that are orally active has provided the greatest degree of clinical success for inhibitors of the renin-angiotensin system and, consequently, the impetus to develop still better compounds. Captopril (SQ 14,225) is the prototype ACE inhibitor, being highly potent and specific with no other demonstrated pharmacological activity. Captopril is effective in all forms of human and animal models of hypertension except mineralocorticoid hypertension, which requires concomitant diuretic therapy. Because ACE is the same enzyme as kininase II, the enzyme that degrades kinins, the possibility exists that kinins are involved in the cardiovascular action of captopril, although this prospect is unlikely.     

Blockade of thromboxane responses in the airway of the cat by SQ 29,548

The effects of SQ 29,548, a thromboxane receptor antagonist, on airway responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the thromboxane and prostaglandin precursor, arachidonic acid (AA), and the thromboxane mimic, U 46619, produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SQ 29,548 (0.5 mg/kg iv), bronchoconstrictor responses to AA were reduced by approximately 50%, whereas responses to U 46619 were reduced by approximately 90%. The cyclooxygenase inhibitor, sodium meclofenamate (2.5 mg/kg iv), blocked the component of the airway response to AA remaining after treatment with SQ 29,548. The thromboxane receptor antagonist had no significant effect on bronchoconstrictor responses to prostaglandins F2 alpha, and D2, methacholine, 5-hydroxytryptamine, histamine, or BAY K 8644, an agent that promotes calcium entry. Reductions in systemic arterial pressure in response to AA were enhanced by the thromboxane receptor antagonist and abolished by meclofenamate. SQ 29,548 had no effect on terminal enzyme activity in microsomal fractions from cat lung. These data support the hypothesis that AA-induced bronchoconstriction in the cat is mediated in large part by the actions of thromboxane A2. These data also suggest that U 46619 and U 44069 stimulate the same airway receptor as thromboxane A2 and mimic the bronchomotor effects of this hormone, which has not yet been isolated as a pure substance. These data demonstrate that SQ 29,548 is a selective thromboxane receptor antagonist in the airways of the closed-chest cat and may be a useful probe for studying responses to thromboxane A2 in physiological and pathophysiological processes in the lung.     

The Hageman factor-dependent system in the vascular permeability reaction

The mechanism by which the Hageman factor-dependent system induces vascular permeability has been analyzed. The Mr-28,000 active fragment of guinea pig Hageman factor (beta-HFa), injected intradermally, induces an increase in local vascular permeability. Inhibition of vascular permeability resulted from pretreatment of the beta-HFa with immunopurified anti-Hageman factor F(ab')2 antibody at concentrations of 10(-6)-10(-7) M as well as by incubation with corn and pumpkin seed inhibitors of beta-HFa. To determine whether prekallikrein and kallikrein participated in the permeability induced by beta-HFa, circulating prekallikrein was depleted by intra-arterial injections of anti-prekallikrein F(ab')2 antibody. This resulted in about 80% diminution of the vascular permeability response to beta-HFa, without affecting the permeability reaction to bradykinin. Soybean trypsin inhibitor (10(-6) M), injected at the same cutaneous site as the beta-HFa, inhibited the vascular permeability response to beta-HFa by more than 90%. This concentration of soybean inhibitor blocked more than 90% of the activity of guinea pig plasma kallikrein, but did not inhibit the amidolytic capacity of beta-HFa. The permeability activity of beta-HFa (but not its amidolytic activity) was augmented 10-fold by simultaneous injection of a synthetic kinin potentiator, SQ 20,881 (Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), and was almost completely inhibited by the simultaneous injection of a kinin-destroying enzyme, carboxypeptidase B. These results support the hypothesis that the greatest proportion of vascular permeability induced by beta-HFa is produced by the activation of prekallikrein followed by the release of kinin in the cutaneous tissue. These data offer the first in vivo evidence that the Hageman factor-dependent system by itself can induce inflammatory changes.