Measurement of urinary kallikrein activity. Species differences in kinin production.

A sensitive, specific radioimmunoassay for kinins has been developed, which is able to detect 1.5 pg bradykinin or 3 pg lysyl-bradykinin (Lys-bradykinin). 50% displacement in the standard curve was obtained with 10 pg bradykinin or 15 pg Lys-bradykinin in 0.6-ml incubates. The antisera, raised against bradykinin, recognized well Lys-bradykinin and methionyl-lysyl-bradykinin (Met-lys-Bradykinin), but cross-reacted 0.4% or less with bradykinin fragments. Kininogen cross-reacted only 0.2%. The radioimmunoassay and kininogen from several species were used in the measurement of human and rat urinary kallikrein activity. The peptide generated by hydrolysis of the substrates by rat or human urines was characterized by radioimmunoassay in two different systems: polyacrylamide gel electrophoresis and carboxymethyl cellulose chromatography. Both urines did not produce the same kinin: the kinin produced by human urine migrated like Lys-bradykinin, whereas the kinin produced by rat urine migrated like bradykinin. This gives evidence of differences in the specificity between kinin-forming enzymes in rat and human urines.

Rat high-molecular-weight kininogen: purification, production of antibodies and demonstration of lack of immunoreactive kininogen in a strain of brown Norway rats.

High-molecular-weight kininogen was purified to apparent homogeneity from Wistar rat plasma by a two-steps chromatographic procedure. 3 mg of kininogen were obtained from 205 ml of plasma. The purified high-Mr kininogen had a bradykinin content of 10.2 micrograms bradykinin equivalents/mg protein. Under denatured and reduced conditions it gave a single band on polyacrylamide gel electrophoresis corresponding to an apparent molecular mass of 110 kDa. Antibodies obtained against rat high-Mr kininogen gave a single precipitation line when tested against rat plasma in double immunodiffusion and in crossed immunoelectrophoresis. Although rat high-Mr kininogen possesses physicochemical properties (molecular mass, kinin content per molecule and amino acid composition) similar to human high-Mr kininogen, its antibodies do not cross-react with human, monkey or rabbit plasma, indicating major interspecies differences in the structure of the molecule. Immunoreactive kininogen of Wistar rats was identical to that of Brown Norway rats from a strain bred in Orleans, France (BN/Orl). However, plasma from a strain of Brown Norway rats bred in Leuven, Belgium (BN/Kat), reported to be deficient in a kinin precursor (Damas, J. and Adam, A. (1980) Experientia 36, 586-587), did not contain immunoreactive material discernible by double immunodiffusion or crossed immunoelectrophoresis.

[Gene transfers of kallikrein, bradykinin receptor B2 and a mutated B2 receptor stimulate neoangiogenesis in peripheral ischemia]. / La surexpression de la kallicréine tissulaire, du récepteur B2 de la bradykinine et d'une forme mutée du récepteur B2, stimule la néoangiogenèse dans un modèle d'ischémie périphérique.

In this work, we evaluated the angiogenic effect of the gene transfer of human tissue kallikrein (TK), bradykinin B2 receptor (B2R) and a mutated form (RB2m) in a rabbit peripheral model of ischaemia. We studied the effects of the transfection of each of these factors and the effects of their co-transfection. In New Zealand anesthetised rabbits we first induced an ischaemia of the left posterior leg by ligation-excision of the superficial femoral artery and its collaterals. Seven days later, we performed i.m. injections in the ischemic tight with transfection solutions containing either the control (pcDNA3 empty backbone) or the pcDNA3-TK, the pcDNA3-TK and the pcDNA3-B2R, the pcDNA3-TK and the pcDNA3-B2Rm. Twenty eight days later, the therapeutic effect was evaluated using ultrasonographic debitmetry of the common iliac artery, perfusion index (PI) = ischemic leg blood flow /non ischemic leg blood flow (%) and capillaries measurements i.e. capillary density: number of vessels/mm2 and the ratio of vessels/muscular fiber, in the adductors and gastrocnemian muscles. The PI was increased in each treated group vs control (32.61 +/- 5.2%), pcDNA3-TK: 59.72 +/- 2.33%; p = 0.001; pcDNA3-RB2: 55.25 +/- 2.29%; p = 0.008; pcDNA3-TK + pcDNA3-RB2: 84.77 +/- 3.15%; p < 0.001; pcDNA3-TK + pcDNA3-RB2m: 103.25 +/- 4.9%; p < 0.001. The capillary density and the vessel/muscular fiber ratio increased in a parallel with the hemodynamic in the ischemic adductors (pcDNA3-TK + pcDNA3-B2Rm > pcDNA3-TK + pcDNA3-RB2 > pcDNA3-TK = pcDNA3-B2R; p < 0.001). There was no angiogenic effect measurable neither in the non ischemic adductors (right) nor in the gastrocnemian muscles. In rabbit peripheral ischaemia, the cotransfection of TK and B2R increases the arterial flow in the treated leg and potentiates the neoangiogenesis. Cotransfection of the B2Rm cDNA enhanced the synergic effect of this therapeutic strategy.

Cardiac angiotensin converting enzyme overproduction indicates interstitial activation in renovascular hypertension.

OBJECTIVES: Angiotensin converting enzyme (ACE) activity in the plasma does not change significantly with hypertension in two-kidney, one-clip hypertensive (2K-1C) rats. However, heart ACE activity and mRNA increase with hypertension. We measured the ACE activity and mRNA in hypertrophied hearts at different times after clipping, and determined the cellular distribution of its increase in the left ventricle of 2K-1C hypertensive rats. METHODS: Cardiac ACE activity was quantified in left and right ventricles using a radiolabeled synthetic ACE substrate, and ACE mRNA steady-state level was quantified by ribonuclease protection assay. Tissue localization of ACE in normal and hypertrophied hearts was determined by measuring ACE activity in isolated ventricular cells. In situ hybridization with a rat ACE cDNA and immunohistochemistry with a monoclonal anti-ACE antibody were used to identify tissue compartments producing ACE mRNA and protein. RESULTS: The left ventricle was hypertrophied 2 weeks after clipping and remained hypertrophied at 12 weeks. Left ventricular ACE activity was significantly increased 2 and 4 weeks (3.2 +/- 0.3 in 2K-1C vs. 1.7 +/- 0.1 pmol/mg prot/min in sham-operated rat) after renal artery clipping, but not at 12 weeks. The right ventricle was slightly hypertrophied 4 weeks after clipping and remained hypertrophied at 12 weeks. Right ventricular ACE activity was significantly increased at 4 (6.7 +/- 0.6 in 2K-1C vs. 3.1 +/- 0.3 pmol/mg prot/min in sham-operated rat) and 12 weeks. ACE activity was not detectable in cardiomyocytes isolated by Percoll gradient. Neither was ACE mRNA detected in isolated cardiomyocytes, even after ACE mRNA amplification by RT-PCR. In contrast, ACE activity and mRNA were detected in pooled non-cardiomyocytic cells. Thus the increase in cardiac ACE activity associated with hypertension must be due to an increase in ACE expression by non-cardiomyocytic cells. In situ hybridization showed an autoradiographic signal for ACE mRNA over the endothelial cells of coronary arteries and over the interstitial spaces including pericoronary and fibrosis areas. Immunohistochemistry confirmed these data, showing ACE on endothelial cells and in pericoronary spaces with an increased signal in pericoronary and fibrosed areas in hypertensive hypertrophied left ventricle. CONCLUSION: Besides its usual endothelial expression, ACE is absent from cardiomyocytes and present in interstitial tissue, in the pericoronary spaces in normal tissue and more markedly in hypertrophied ventricles.

The testicular transcript of the angiotensin I-converting enzyme encodes for the ancestral, non-duplicated form of the enzyme.

The endothelial angiotensin I-converting enzyme (ACE) is organized in two large homologous domains, each bearing a putative active site. However, only one of these sites is probably involved in catalyzing the conversion of angiotensin I into angiotensin II. The testicular form of ACE is equally active, encoded by the same gene, but translated from a shorter mRNA. Molecular cloning of the human testicular ACE cDNA indicates that the mRNA codes for 732 residues (vs 1306 in endothelium). The testicular transcript corresponds to the 3' half of the endothelial transcript and encodes one of the two homologous domains of endothelial ACE, preceded by a short specific sequence. This 5' specific sequence contains 228 nucleotides and encodes 67 amino acids, including the putative signal peptide followed by a serine/threonine-enriched region, presumably glycosylated. The testicular transcript corresponds to the ancestral, non-duplicated form of the ACE gene. Since the carboxyl-terminal domain of the endothelial ACE is expressed in the testicular enzyme, it is likely that it bears the active site in both forms.

Angiotensin I-converting enzyme in foetal membranes and chorionic cells in culture.

Angiotensin I-converting enzyme (ACE) is found in human amniotic fluid and foetal membranes taken during Caesarean sections at term. ACE contents are higher in the chorion than in the amnion. Cells cultured from chorion contain ACE together with renin. Chorionic ACE was inhibited by captopril and by an excess of anti-ACE immuneserum. It is concluded that extravascular ACE is present in the uteroplacental complex during pregnancy.

Angiotensin I-converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in T-lymphocytes.

The expression of angiotensin-I converting enzyme (ACE; EC 3.4.15.1) in human circulating mononuclear cells was studied. T-lymphocytes contained the highest level of enzyme, approx. 28 times more per cell than monocytes. No activity was detected in B-lymphocytes. ACE was present mainly in the microsomal fraction, where it was found to be the major membrane-bound bradykinin-inactivating enzyme. An mRNA for ACE was detected and characterized after reverse transcription and amplification by PCR in T-lymphocytes and several T-cell leukaemia cell lines. We have previously observed that the interindividual variability in the levels of ACE in plasma is, in part, genetically determined and influenced by an insertion/deletion polymorphism of the ACE gene. To investigate the mechanisms involved in the regulation of ACE biosynthesis, the ACE levels of T-lymphocytes from 35 healthy subjects having different ACE genotypes were studied. These levels varied widely between individuals but were highly reproducible and influenced by the polymorphism of the ACE gene. T-lymphocyte levels of ACE were significantly higher in subjects who were homozygote for the deletion than in the other subjects. These results show that ACE is expressed in T-lymphocytes and indicate that the level of ACE expression in cells synthesizing the enzyme is genetically determined.

Angiotensin I-converting enzyme (kininase II) in cardiovascular and renal regulations and diseases.

The angiotensin I-converting enzyme (kininase II, ACE) is the major angiotensin II forming and kinin degrading enzyme in the circulation, and has other physiological peptide substrates. Recent studies have established that the interindividual variability in the levels of ACE in plasma and tissues is under the influence of a genetic polymorphism. The genetic polymorphism of ACE levels has been linked in case-control studies to the susceptibility of developing cardiovascular diseases, especially myocardial infarction and diabetic nephropathy, and to the risk of progression of renal diseases. The new concept that the level of ACE in peripheral circulations and tissue interstitium is an important factor in the determinism of the local concentration of peptides and their putative protective/deleterious effects, especially in the kidney and the heart, will be further appraised.

Regulation of ACE gene expression and plasma levels during rat postnatal development.

Angiotensin I-converting enzyme (kininase II, ACE) is a transmembrane ectoenzyme of vascular endothelial cells that is also secreted in plasma. To understand why plasma ACE levels are elevated in children compared with adults, the age-related changes in ACE mRNA and enzyme levels were studied in 1-day- to 3-mo-old rats. In the lung, a rich source of endothelial ACE, the abundance of ACE mRNA and the microsomal ACE concentration increased progressively and tripled during the first 3 mo. This large increase reflects, at least in part, development of the capillary network. In plasma, ACE levels rose dramatically a few days after birth and decreased toward adult values after the 14th day of life. Because the elevation of ACE in plasma was contemporary to thyroid maturation, the effect of perinatal suppression of thyroid function by propylthiouracil was studied. Hypothyroidism slightly delayed the evolution of ACE in lung but blunted the postnatal rise in plasma ACE levels. A 3,5,3'-triiodothyronine injection to 14-day-old hypothyroid rats failed to alter ACE mRNA levels in the lung. Thus thyroid hormones are involved in the postnatal rise in plasma ACE levels but act probably on the posttranslational proteolytic pathway involved in ACE secretion by endothelial cells or on an unknown extrapulmonary ACE source. ACE gene expression is also developmentally regulated in epithelia and male germinal cells. In the intestine, ACE mRNA levels and ACE activity were very high at birth and then decreased dramatically during the next 2 wk. In the kidney, they were low and decreased further during growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemodynamics and gas exchange before and after coil embolization of pulmonary arteriovenous malformations.

A complete description of haemodynamics and gas exchange before and after percutaneous coil embolization of multiple pulmonary arteriovenous malformations is reported in a 45 year old woman with hereditary haemorrhagic telangiectasis (HHT). Before treatment, whilst the patient complained of severe dyspnoea during daily activities, an intrapulmonary shunt of 31% was measured (inert gas elimination technique), together with a cardiac output (thermodilution technique) of 12.4 L.min-1, resulting in a resting arterial oxygen tension (PaO2) of 8.53 kPa. Effective occlusion of all visible pulmonary malformations resulted in a rapid and major improvement in exercise tolerance, whilst resting PaO2 remained almost unchanged. A second investigation performed 4 months after treatment revealed a persistent intrapulmonary shunt of 19%, a cardiac output of 7.35 L.min-1, and a resting PaO2 of 10.53 kPa. We conclude that major increases in cardiac output largely contribute to the maintenance of PaO2 in patients with multiple pulmonary arteriovenous malformations and intrapulmonary shunt. The benefit of coil embolization is due both to an improvement in arterial oxygenation and a normalization of cardiac output.

The peculiar characteristics of the amino acid sequence of angiotensin I-converting enzyme, as determined by cDNA cloning of the human endothelial enzyme.

The angiotensin-I converting enzyme (ACE) is a membrane bound zinc metallopeptidase of the vascular endothelial cell. Recently, the complete amino-acid sequence of human ACE has been determined by protein sequencing and cDNA cloning in endothelial cell libraries. The ACE is encoded from a 4.3 kb transcript and comprises 1,306 amino acids. The molecule comprises a signal peptide of 29 residues cleaved off during maturation. It is most likely anchored by a short transmembrane domain situated near the carboxyterminal extremity. Interestingly, the molecule presents a high degree of internal homology between two large peptidic domains. Each of these domains contains short sequences identical to zinc binding and active site sequences of other zinc metallopeptidases and therefore bears a putative active site. The ACE gene results probably from duplication and fusion of a more ancestral gene, coding for a putative nonduplicated enzyme. However, despite the duplicated structure of ACE, it has been reported that there is only one zinc atom bound per molecule. Competitive inhibitors seem to interact with a unique high affinity binding site. Therefore, there is only one active site in ACE whose location remains to be determined.

[Angiotensin converting enzyme (kininase II). Molecular and physiological aspects]. / L'enzyme de conversion de l'angiotensine (kininase II). Aspects moléculaires et physiologiques.

The angiotensin I-converting enzyme (kininase II, ECA) is a membrane bound enzyme anchored to the cell membrane through a single transmembrane domain located near its carboxyterminal extremity. Secretion of ACE by the cell occurs most likely as a result of a posttranslational cleavage of the membrane anchor and intracellular region. The ACE molecule is organized into two large highly homologous domains, each bearing consensus sequences for zinc binding in metallopeptidases. Site directed mutagenesis allowed to establish that both domains bear in fact a functional active site, able to convert angiotensin I into angiotensin II and to hydrolyze bradykinin or substance P. The two active sites of ACE, however, do not display the same sensitivity to anion activation (the C terminal active site being more chloride activatable) and also differs in kinetic parameters for peptide hydrolysis. The C terminal active site can hydrolyze faster angiotensin I and substance P and the N terminal active site is able to perform a peculiar endoproteolytic cleavage of an in vitro substrate of ACE, the luteinizing hormone releasing hormone. Both active sites bind with a high affinity, competitive inhibitors but the Kd of the reaction can vary up to 10 between the two active sites. All together, these observations suggest that ACE contains two active sites, whose structure is not exactly identical. They may have a different substrate specificity, however this remains speculative at the present time. Concerning the regulation of ACE gene expression in man, population studies indicated that the large interindividual variability in plasma ACE levels is genetically determined. An insertion/deletion polymorphism located in an intron of ACE gene is associated with differences in the level of ACE in plasma and cells. The physiological and clinical implications of these observations is discussed.

Kallikrein along the rabbit microdissected nephron: a micromethod for its measurement. Effect of adrenalectomy and DOCA treatment.

Active and inactive kallikrein were measured along the rabbit microdissected nephron. A sensitive and specific micromethod for the measurement of kininogenase activity was developed in order to quantify kallikrein in pieces of tubule as small as 0.3-0.5 mm. Our study confirms that active and inactive kallikrein are located to the connecting tubule (CNT). The effects on renal kallikrein of a chronic DOCA treatment and of adrenalectomy were studied. Urinary excretion of kallikrein was also monitored. After DOCA treatment, active kallikrein increased in the tubule and in urine but inactive kallikrein did not significantly change. Adrenalectomy decreased by 50% active and inactive contents of CNT, as well as reduced the excretion of total kallikrein. Kallikrein content in CNT was also measured in adrenalectomized rabbits 3 h after a single injection of either aldosterone (10 micrograms) or dexamethasone (100 micrograms). After either aldosterone or dexamethasone injections, kallikrein activities were not restored, whereas in the same animals Na-K-ATPase activity which was depressed on cortical and medullary collecting tubules after adrenalectomy returned toward normal values. These data indicate that kallikrein synthesis and activation are influenced by adrenal hormones. Renal kallikrein is, however, regulated at a much slower rate than Na+-K+-ATPase. This may suggest an indirect rather than direct action of corticosteroid hormones on kallikrein.

Angiotensin I-converting enzyme inhibition but not angiotensin II suppression alters angiotensin I-converting enzyme gene expression in vessels and epithelia.

The concentration of angiotensin-converting enzyme (ACE) increases during chronic treatment with ACE inhibitors for unknown reasons. We investigated whether alterations in ACE mRNA and ACE concentration occur in the different tissues during ACE inhibition and the role of angiotensins in these regulations by comparing ACE inhibitors with other blockers of the renin-angiotensin system. Enalapril, an ACE inhibitor, in the range of 0.3 to 10 mg/kg/day in rats induced dose- and time-dependant increases in plasma ACE up to two to three times control values. There were significant increases in the steady state ACE mRNA in the lung (32%), duodenum (64%) and aorta (324%) and 40% to 140% increases in membrane-bound enzyme concentration in these tissues and in the heart and kidney. The ACE content of purified duodenal brush border was increased by 80%, but the enzyme and its mRNA in the testis were not altered. The angiotensin II receptor antagonist losartan at several regimens of up to 30 mg/kg twice a day for 14 days produced no change in plasma ACE level or lung ACE mRNA. The human renin inhibitor ciprokiren was tested in guinea pigs, a species sensitive to this compound. Both enalapril and cilazapril induced 2-fold increases in plasma ACE, but ciprokiren (24 mg/kg/day for 12 days) had no effect. Enalapril treatment of BN/Kat rats (lacking circulating kininogens) caused a similar increase in ACE as in other rats. This study documents a general increase in ACE gene expression and enzyme concentration in tissues during ACE inhibition, with the exception of the testis, most probably reflecting an activation of the 5', so-called somatic promoter of the ACE gene. Angiotensins are not involved in this regulation and do not seem to control ACE gene expression in normal rodents.

The angiotensin I-converting enzyme (kininase II): progress in molecular and genetic structure.

The complete amino acid sequence of the human angiotensin I-converting enzyme (ACE) has been determined by protein sequencing of the purified kidney enzyme and cDNA cloning in endothelial cell libraries. The ACE molecule comprises 1,306 amino acids. It possesses a signal peptide of 29 residues cleaved off during maturation. The enzyme is most likely anchored to the plasma membrane by a short transmembrane domain situated near the carboxy-terminal extremity. Interestingly, the molecule presents a high degree of internal homology between two large peptidic domains. Each of these domains contains short sequences identical to zinc binding and active site sequences of other zinc metallopeptidases and therefore bears a putative active site. However, earlier experiments indicate only one zinc atom bound per molecule of ACE. Competitive inhibitors seem to interact with a unique class of high-affinity binding site. These observations may suggest that, despite the duplicated structure of the enzyme, there is only one functional active site per molecule of ACE. The respective role of the two homologous domains in this active site remains to be determined. A single gene coding for ACE is present in humans, transcribed as a 4.3-kilobase mRNA species in endothelial cells. In other studies, evidence for a genetic polymorphism in plasma ACE levels has been obtained by analyzing a large group of "healthy" nuclear families. A familial association of plasma ACE levels was observed. A major gene effect can possibly explain part of the interindividual variability observed in this enzyme.

Negative cooperativity in the human bradykinin B2 receptor.

A human kidney bradykinin (BK) B2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 x 10(6) receptors/cell allowed us to carry out a detailed study of BK-receptor interaction over a wide range of BK concentrations. A model assuming that BK binds to two receptor affinity states (depending on guanine nucleotide-sensitive coupling) was not sufficient to account for the kinetics of BK binding. Equilibrium kinetic analysis and studies of the effects of receptor occupancy by agonists or antagonists on the kinetics of BK-receptor complex dissociation revealed features typical of negative cooperative binding. The negative cooperativity phenomenon was also observed in isolated membranes in both the presence and absence of guanine nucleotide. Thus, following the interaction with BK, B2 receptor molecules likely interact with each other, resulting in an acceleration of bound ligand dissociation and a decrease in the apparent affinity of the receptor for BK. This phenomenon can participate in the desensitization process.

Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning.

The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15.1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxyl-terminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.

Familial resemblance of plasma angiotensin-converting enzyme level: the Nancy Study.

Plasma angiotensin I-converting enzyme (ACE) activity has been measured in a sample of 87 healthy families participating in a study of cardiovascular risk factors. The mean +/- SD levels of plasma ACE were 34.1 +/- 10.7, 30.7 +/- 10.4 and 43.1 +/- 17.2 units/liter in fathers (n = 87), mothers (n = 87) and offspring (n = 169), respectively. Plasma ACE was uncorrelated with age, height, weight, or blood pressure in the parents, but a negative correlation with age was observed in offspring (r = -.32). The age-adjusted familial correlations of plasma ACE were .038, .166, .323 and .303 for spouses, father-offspring, mother-offspring, and siblings, respectively. The results of the genetic analysis suggest that a major gene may affect the interindividual variability of plasma ACE, with different codominant effects in parents and offspring. According to this model, the major gene effect accounts for 4.8, 4.0, and 10.8 units/liter of the overall mean and for 29%, 29% and 75% of the variance of age-adjusted ACE in fathers, mothers, and offspring, respectively. The estimate of the probability of the less frequent allele is .26, and the major gene effect is approximately twice as great in high homozygotes than in heterozygotes and in offspring than in parents. The results of this study demonstrate the occurrence of a familial resemblance of plasma ACE activity in healthy families and suggest that this observation can be explained by the segregation of a major gene.