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 carboxylterminal 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.Peptidyl-dipeptidase A (EC 3.4.15.1) plays an important role in blood pressure homeostasis by hydrolyzing angiotensin I, the inactive peptide released after cleavage of angiotensin by renin, into angiotensin II (1). Accordingly, this Zn metallopeptidase is designated angiotensin I-converting enzyme (ACE), although being the same enzyme as kininase II, it is also able to hydrolyze bradykinin and various other peptides (2, 3). This enzyme is a widely distributed peptidase, occurring, for example, as a membrane-bound ectoenzyme on the surface of vascular endothelial cells and renal epithelial cells and as a circulating enzyme in plasma (3-5). We report here the amino acid sequence of ACE as deduced from the nucleotide sequence of DNA complementary to the ACE mRNA.t MATERIALS AND METHODSPurification and Sequencing of ACE and Preparation of Oligodeoxyribonucleotide Probe. The cortex offresh postmortem human kidneys (600 g) was homogenized (54:100, wt/vol) in 20 mM potassium phosphate buffer (pH 8) containing 250 mM sucrose and a mixture of protease inhibitors, cells debris was discarded, and the particulate fraction was sedimented by centrifugation at 105,000 x g for 1 hr. The pellet was resuspended in 200 ml of 150 mM potassium phosphate buffer (pH 8; buffer I) and treated for 18 hr with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 8 mM; Serva). The supernatant obtained after centrifugation at 105,000 x g for 1 hr was dialyzed extensively against b...

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.

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/threonineenriched 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.

A human kidney bradykinin (BK) B 2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B 2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 ؋ 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, B 2 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.Bradykinin (BK) 1 is involved in a variety of physiological and pathological processes, including vasodilation and control of vascular tone, ion transfer in epithelia, and pain (1). BK binds to specific receptors that have been classified into B 1 and B 2 receptors according to their relative affinities for des-Arg 9 -BK and BK (2). These two types of receptors belong to the superfamily of seven-transmembrane domain receptors (3, 4). Most of the BK effects described so far are mediated by the B 2 receptor subtype. BK receptors are coupled through pertussis toxin-insensitive G proteins (5, 6) to at least two separate pathways of phospholipid metabolism (7-12), the hydrolysis of inositol phospholipids by phospholipase C (PLC) and the release of arachidonic acid by phospholipase A 2 (PLA 2 ). PLC stimulation produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate is likely responsible for the release of Ca 2ϩ from internal stores (13-15), and the physiological effects of BK are thought to be strongly dependent on its ability to mobilize [Ca 2ϩ ] i . BK-induced production of inositol phosphates, release of arachidonic acid, and elevation of [Ca 2ϩ ] i as well as the in vitro physiological responses to BK are of a smaller magnitude in cells or tissues preexposed to BK (7, 13, 16 -19). Agonist-induced phosphorylation of the receptor, resulting in receptor uncoupling and modulation of receptor affinity (20, 21) and loss of cellsurface binding sites (19,22,23) as a consequence of internalization of the ligand-receptor complex (19,23,24), has been proposed to play a role in the decreased responsiveness to BK. The receptor-mediated desensitization can contribute, together with the action of kininases and the triggering of physiological cou...

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)

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H Ha ae em mo od dy yn na am mi ic cs s aan nd d g ga as s e ex xc ch ha an ng ge e b be ef fo or re e a an nd d a af ft te er r c co oi il l e em mb bo ol li iz za at ti io on n o of f p pu ul lm mo on na ar ry y a ar rt te er ri io ov ve en no ou us s m ma al lf fo or rm ma at ti io on ns s P. Andrivet*, F. Lofaso*, M-F. Carette**, J. Allegrini + , S. Adnot* Haemodynamics and gas exchange before and after coil embolization of pulmonary arteriovenous malformations. P. Andrivet, F. Lofaso, M-F. Carette, J. Allegrini, S. Adnot. ©ERS Journals Ltd 1995. ABSTRACT: 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 (Pa,O 2 ) of 8.53 kPa.Effective occlusion of all visible pulmonary malformations resulted in a rapid and major improvement in exercise tolerance, whilst resting Pa,O 2 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 Pa,O 2 of 10.53 kPa.We conclude that major increases in cardiac output largely contribute to the maintenance of Pa,O 2 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. Eur Respir J., 1995Respir J., , 8, 1228Respir J., -1230 Large or multiple pulmonary arteriovenous malformations (PAVM) may lead to severe and incapacitating hypoxaemia via right-to-left intrapulmonary shunts. Several reports have recently offered important clinical, gasometric and therapeutic insights into this problem [1][2][3][4]. However, a complete description and follow-up of the haemodynamic and gas exchange data, including assessment of the distribution of ventilation-perfusion ratios, has not previously been reported. The present case report describes how alterations of haemodynamics and gas exchange, observed in a 45 year old woman with hereditary haemorrhagic telangiectasia (HHT) and PAVM, were improved by therapeutic coil embolization. Case reportA female patient was investigated for increasing dyspnoea, with major impairment of her daily activities. Questioning revealed that an elder brother underwent balloon embolization in 1985 for multiple PAVM. Physical examination at rest showed a mild tachypnoea (20 breaths·min -1 ), with digital clubbing, and digital cyanosis was present whilst standing. The patient was unable to perform an exercise test of 1 min at a workload of 40 W. No cutaneous telangiectasis was present, but a previous episod...

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