Interstrain differences in angiotensin I-converting enzyme mRNA and activity levels. Comparison between stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.

Plasma angiotensin I-converting enzyme (ACE) levels are different between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and the normotensive Wistar-Kyoto (WKYHD) rat. This interstrain variability in plasma ACE levels is independent of blood pressure and is genetically linked to the ACE gene. The present study explored the hypothesis of an interstrain variability of tissue ACE activity and ACE gene expression levels. Tissue ACE levels were studied by enzymic activity measurement in the membrane fraction, and ACE mRNA levels were quantified by solution hybridization-ribonuclease protection assay. In lung, heart, kidney, and duodenum, membrane-bound ACE activity and ACE mRNA amount were significantly higher in WKYHD rats compared with SHRSPHD rats. No difference was observed in the testis where a specific isoform of the enzyme is produced. Our results suggest that in addition to determine differential plasma ACE levels between the WKYHD and SHRSPHD strains, the interstrain genetic variability also determines differential ACE mRNA and membrane-bound enzyme levels in somatic tissues. This likely reflects a difference in the ACE gene expression due to genetically determined regulatory mechanisms operative in all somatic tissues.

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.

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.

Genetics of angiotensin I-converting enzyme.

The ACE gene is constitutively expressed in several types of somatic cells, including vascular cells. A soluble form of the enzyme is secreted in plasma by proteolytic cleavage of the membrane anchor. The interindividual variability in plasma ACE levels is very large, and a family study has indicated that it was under the influence of a major gene polymorphism. An insertion (I) deletion (D) polymorphism in intron 16 of the ACE gene was then found to be associated with plasma and cellular ACE levels. The D allele, which is associated with higher plasma ACE levels, and the level of ACE in plasma, were found in case control studies to be associated with an increased risk of myocardial infarction, an increased risk of diabetic nephropathy in type I diabetic patients, and a faster rate of renal function degradation in glomerular diseases. Although these findings should be confirmed in prospective studies, they can support the concept that ACE level is a critical factor in the determinism of angiotensins and kinins (and perhaps also other peptide substrates) levels in peripheral circulations and in tissue interstitium, especially in the heart and kidney.

Vascular angiotensin-converting enzyme expression regulates local angiotensin II.

We tested the hypothesis that changes in angiotensin-converting enzyme (ACE) gene expression can regulate the rate of local vascular angiotensin II (Ang II) production. We perfused isolated rat hindlimbs with an artificial medium and infused renin and Ang I via the perfusate. Ang I and II were measured by radioimmunoassay. We then increased ACE gene expression and ACE levels in the rat aorta by producing two-kidney, one clip (2K1C) hypertension for 4 weeks. Gene expression was measured by RNAse protection assay, and ACE activity in the vessel wall was measured by the Cushman-Cheung assay. Angiotensin I infusion at 1, 10, 100, and 1000 pmol/mL led to 371 +/- 14 (+/-SEM), 3611 +/- 202, 44,828 +/- 1425, and 431,503 +/- 16,439 fmol/mL Ang II released, respectively, from the hindlimbs (r = .98, P < .001). Thus, the conversion rate did not change across four orders of magnitude, and the system was not saturable under these conditions. In 2K1C hindlimbs, Ang I infusion (0.5 pmol/mL) resulted in increased Ang II generation (157 +/- 16 versus 123 +/- 23 fmol/mL, P = .014 at minute 10) compared with controls. ACE gene expression and ACE activity were increased in 2K1C hindlimbs compared with controls (36 +/- 4 versus 17 +/- 1 mU/mg protein, P < .001). Ang II degradation in the two groups did not differ. To investigate the conversion of locally generated Ang I, we infused porcine renin (0.5 milliunits per mL) into 2K1C and control hindlimbs. Despite markedly higher Ang I release in sham-operated than in 2K1C rats (71 +/- 8 versus 37 +/- 6 pmol/mL, P = .008 at minute 12), Ang II was only moderately increased (36 +/- 3 versus 25 +/- 6 pmol/mL, P = .12 at minute 12). This difference between 2K1C rats and controls reflected a higher rate of conversion in 2K1C rats. Thus, Ang I conversion in the rat hindlimb is linear over a wide range of substrate concentrations and occurs at a fixed relationship. Nevertheless, increased ACE gene expression and ACE activity in the vessel wall lead to an increase in the conversion of Ang I to Ang II. We conclude that local ACE gene expression and ACE activity can influence the local rate of Ang II production.

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)

ACE in three tunicae of rat aorta: expression in smooth muscle and effect of renovascular hypertension.

Angiotensin I-converting enzyme (ACE) is known to be present at the surface of endothelial cells and also in the adventitia in large vessels. The presence of ACE in the vascular smooth muscle remains controversial. We microdissected segments of adventitia and media with or without endothelium from a region devoid of collateral arteries. The membrane-bound ACE activity in the media averaged 41% (pmol [glycine-1-14C]hippuryl-L-histidyl-L-leucine hydrolyzed.g tissue-1.min-1) of the values found in the whole aorta, whereas the adventitia contained only 6%. Immunoreactive ACE in media was characterized by Western blotting. ACE mRNAs were detected and characterized after polymerase chain amplification in isolated media. Angiotensin I and angiotensin II were equally able to contract medial rings, and the response to angiotensin I was blocked by enalaprilat. In aortas of two-kidney, one-clip hypertensive rats, there was an increase in ACE mRNA estimated by ribonuclease protection assay (P = 0.02) and in ACE activity at 15 days and 1 and 3 mo after clipping. This corresponded to a 1.5- to 2-fold increase in the ACE activity of both the media and the adventitia compared with sham-operated rats (P < or = 0.02). Thus ACE gene expression occurs in smooth muscle of rat aorta, which contains roughly the same amount of enzyme as the endothelium and readily converts angiotensin I to angiotensin II. ACE in the medial layer and the adventitia is upregulated in renovascular hypertension.

Plasma level and gene polymorphism of angiotensin-converting enzyme in relation to myocardial infarction.

BACKGROUND: The angiotensin-converting enzyme (ACE) plays an important role in the production of angiotensin II and the degradation of bradykinin, two peptides involved in cardiovascular homeostasy. Presence of a polymorphism in the ACE gene (ACE Ss) has been postulated from segregation analysis of plasma ACE in families. This putative polymorphism, which strongly affects the plasma and cellular levels of ACE, probably by modulating ACE gene transcription, has not yet been identified at the molecular level; however, an insertion/deletion polymorphism is present in the 16th intron of the ACE gene (ACE I/D) and appears to be a very good marker for ACE Ss. The biological role of ACE suggests that the ACE gene polymorphism could affect the predisposition to myocardial infarction (MI). METHODS AND RESULTS: We have recently shown, in a large case-control study (ECTIM), that the marker allele D of the ACE gene, which is associated with higher levels of ACE in plasma and cells, was more frequent in male patients with MI than in control subjects, especially in patients considered at low risk. ACE activity has now been measured from frozen aliquots of plasma in a large subsample of the ECTIM study (n = 1086). Plasma ACE level did not differ between patients and control subjects in the older age group (> or = 55 years) but was higher in patients than in control subjects in the younger age group (< 55 years); P < .005 after adjustment on ACE I/D and other risk factors. In patients, plasma ACE levels decreased with age (R = -.225, P < 10(-4)), but in control subjects no such trend was observed. In the low-risk group (ApoB < 1.25 mg/dL, body mass index < 26 kg/m2, and not treated with hypolipidemic drugs), plasma ACE level was increased in patients when compared with control subjects among homozygotes and heterozygotes for the ACE I allele (P < .015). Analysis of the distribution of plasma ACE by using commingling analysis conditional on the marker genotype ACE I/D enabled us to infer the frequencies and effects of the postulated ACE Ss genotypes. The results suggest that the higher plasma ACE levels in patients than in control subjects in the younger age group were due to a difference in frequency of the postulated S allele (.47 versus .36). CONCLUSIONS: These results extend our previous findings and indicate that plasma ACE level may be a risk factor for MI, independent of the ACE I/D polymorphism.

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.

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.

[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.

The angiotensin I-converting enzyme (kininase II): molecular organization and regulation of its expression in humans.

Protein sequencing and molecular cloning of human endothelial angiotensin I-converting enzyme (ACE; kininase II), have led to a description of the structure of the enzyme and to several questions concerning the intracellular maturation of ACE and the mechanisms of enzyme action. With the help of recombinant ACE expression in mammalian cells and site-directed mutagenesis, a model for the maturation of ACE in endothelial cells has been proposed. This model comprises transmembrane anchoring of the membrane-bound ACE near its carboxyterminal extremity, and post-translational cleavage of the anchor in the secreted form. The endothelial ACE displays a high degree of internal homology between two large peptidic domains that each bears a consensus sequence for zinc binding and therefore a putative active site. The testicular ACE, however, encoded from the same gene by a shorter mRNA, contains only the carboxyterminal half of endothelial ACE and therefore a single active site. Expression of ACE mutants with only one intact homologous domain, however, indicates that in endothelial ACE both domains are enzymatically active. Further characterization of these two active sites of endothelial ACE is in progress. In humans, population studies have indicated that the large interindividual variability in plasma ACE levels is partly genetically determined and under the influence of a major gene effect. This was later confirmed and extended by the observation of an insertion-deletion polymorphism of the ACE gene that is associated with the level of ACE in plasma. The clinical implications of these observations are discussed.