Angiotensin-converting Enzyme Binding Research Articles

Increases in brain and cardiac AT1 receptor and ACE densities after myocardial infarct in rats.

In the brain, ouabain-like compounds (OLC) and the reninangiotensin system (RAS) contribute to sympathetic hyperactivity in rats after myocardial infarction (MI). This study aimed to evaluate changes in components of the central vs. the peripheral RAS. Angiotensin-converting enzyme (ACE) and angiotensin type 1 (AT1) receptor binding densities were determined by measuring 125I-labeled 351A and 125I-labeled ANG II binding 4 and 8 wk after MI. In the brain, ACE and AT1 receptor binding increased 8-15% in the subfornical organ, 14-22% in the organum vasculosum laminae terminalis, 20-34% in the paraventricular nucleus, and 13-15% in the median preoptic nucleus. In the heart, the greatest increase in ACE and AT1 receptor binding occurred at the infarct scar (approximately 10-fold) and the least in the right ventricle (2-fold). In kidneys, ACE and AT1 receptor binding decreased 10-15%. After intracerebroventricular infusion of Fab fragments to block brain OLC from 0.5 to 4 wk after MI, increases in ACE and AT1 receptors in the subfornical organ, organum vasculosum laminae terminalis, paraventricular nucleus, and medial preoptic nucleus were markedly inhibited, and ACE and AT1 receptor densities in the heart increased less (6-fold in the infarct scar). In kidneys, decreases in ACE and AT1 receptor binding were absent after treatment with Fab fragments. These results demonstrate that ACE and AT1 receptor binding densities increase not only in the heart but also in relevant areas of the brain of rats after MI. Brain OLC appears to play a major role in activation of brain RAS in rats after MI and, to a modest degree, in activation of the cardiac RAS.

Intrarenal AT(1) receptor and ACE binding in ANG II-induced hypertensive rats.

The intrarenal expression of angiotensin II (ANG II) type 1 (AT(1)) receptors and angiotensin-converting enzyme (ACE) was determined in ANG II-induced hypertensive rats (80 ng/min; 2 wk). Systolic blood pressure averaged 184 +/- 3 and 125 +/- 1 mmHg in ANG II-infused compared with Sham rats on day 12. Total kidney AT(1) receptor protein levels were not altered significantly. AT(1) receptor binding mapped by quantitative in vitro autoradiography was significantly decreased in glomeruli (172 +/- 25 vs. 275 +/- 34 disintegrations. min(-1). mm(-2)) and the inner stripe of the outer medulla (121 +/- 17 vs. 178 +/- 19 disintegrations. min(-1). mm(-2)), but not proximal convoluted tubules (48 +/- 9 vs. 58 +/- 6 disintegrations. min(-1). mm(-2)) of ANG II-infused compared with Sham rats. Proximal tubule ACE binding was significantly augmented (132 +/- 4 vs. 97 +/- 3 disintegrations. min(-1). mm(-2)) in ANG II-infused rats. In summary, during ANG II-induced hypertension, glomeruli and inner stripe of the outer medulla have reduced AT(1) receptor binding. Proximal convoluted tubules exhibit maintained AT(1) receptor density and increased ACE binding, which together with the elevated ANG II levels suggest that ANG II exerts a sustained influence on tubular reabsorption and consequently contributes to the development and maintenance of ANG II-dependent hypertension.

Localization of angiotensin-converting enzyme in the human prostate: pathological expression in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common hyperplastic disease in man and it is characterized by increased cellular growth (stromal and epithelial hyperplasia) and enhanced local sympathetic tone, both of which are known to be augmented by activation of the renin-angiotensin system (RAS) in other tissues. Angiotensin-converting enzyme (ACE) is an integral component of the RAS that is responsible for the production of the active peptide angiotensin II from the inactive precursor angiotensin I. The present study was undertaken to map the anatomical localization of ACE protein and messenger ribonucleic acid (mRNA) in the normal human prostate and to establish whether their expression is pathologically altered in BPH. Human prostate samples were obtained at post-mortem and histologically defined as normal or hyperplastic. ACE protein binding/expression was determined by in vitro autoradiography and immunohistochemistry using the ACE-specific radioligand [125I]-MK351A and a mouse anti-ACE polyclonal antibody, respectively, whereas the spatiotemporal distribution of ACE mRNA was determined by in situ hybridization using 35S-labelled oligonucleotide probes. ACE protein was localized to the glandular epithelium in the human prostate. ACE binding and immunostaining were increased in BPH compared with normal (non-hyperplastic) prostate specimens [X-ray film autoradiography: normal 873+/-48 dpm/mm2 (n=8) vs. BPH 1631+/-274 dpm/mm2 (n=6), p<0.05; emulsion autoradiography: normal 3.1+/-0.5 grains/mm2 (n=6) vs. BPH 32.8+/-8.6 grains/mm2 (n=5), p<0.01]. ACE mRNA was also localized to glandular epithelial cells in the human prostate with a significant increase in ACE mRNA expression in BPH compared with the normal prostate [normal 11.04+/-2.03 grains/cell (n=220 cells total) vs. BPH 22.29+/-1.34 grains/cell (n=198 cells total), p<0.05]. The findings of the present study suggest that ACE is localized to the glandular epithelium of the human prostate and that its expression, at both protein and mRNA level, is aberrantly increased in BPH. These data support the concept that hyperactivity of the local RAS in the prostate may be involved in the pathogenesis of BPH.

Increased angiotensin‐converting enzyme and type 1 angiotensin receptors in cortical vasculature and tubulointerstitium of chronically rejected human kidney allografts

SUMMARY: Chronic rejection of human renal allografts after transplantation is characterized by interstitial infiltration, arteriosclerosis and glomerulosclerosis in the grafts. Apart from tissue HLA compatibility, angiotensin II (AII) may be implicated in accelerating the processes of chronic rejection. In the present study, the cellular distribution of angiotensin‐converting enzyme (ACE) and type 1 AII (AT1) receptors was mapped and compared in non‐rejected human kidneys (n = 8) and chronically rejected renal allografts (n = 9) using complementary immunohistochemistry and quantitative in vitro autoradiography. Chronically rejected allografts showed typical histopathological characteristics of tissue rejection, including concentric intimal thickening of intrarenal arteries, extensive or focal glomerulosclerosis, tubular atrophy and severe tubulointerstitial fibrosis. In rejected allografts, total ACE binding in the cortex was decreased to 46% of that in non‐rejected kidneys (P &lt; 0.01), whereas AT1 receptor binding in the glomeruli and the inner stripe of the outer medulla was maintained. However, ACE and AT1 receptor binding were increased in the cortical tubulointerstitium of chronically rejected allografts. In non‐rejected kidneys, strong ACE immunostaining occurred in proximal tubules and vascular endothelium, whereas AT1 receptors occurred in vascular smooth muscle cells, as expected. In rejected allografts, intense ACE and AT1 receptor immunostaining were detected not only in the same sites as those non‐rejected kidneys, but also in cortical tubulointerstitium between atrophied tubules and surrounding the glomeruli. AT1 receptors were markedly up‐regulated in vascular smooth muscle cells of thickened atherosclerotic vessels. These results provide novel morphological evidence that increased expression and/or altered distribution of ACE and AT1 receptors in cortical tubulointerstitium and in the neointima of intrarenal arteries may play an important role in the progression of chronic rejection of human renal allografts after transplantation.

Vasopeptidase inhibition attenuates the progression of renal injury in subtotal nephrectomized rats

Vasopeptidase inhibitors are a new class of cardiovascular compounds that inhibit both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The aim of the present study was to explore the effects of omapatrilat, a vasopeptidase inhibitor, on renal function and pathology in subtotally nephrectomized (STNx) rats. STNx rats were randomized to four groups and treated for 12 weeks: no treatment (N = 14); omapatrilat at a low dose of 10 mg/kg (L, N = 12) and at a high dose of 40 mg/kg (H, N = 10); or an ACE inhibitor, fosinopril, at a dose of 10 mg/kg (N = 12). Sham-operated rats were used as control animals (N = 12). Elevated blood pressure in STNx rats (174 +/- 9 mm Hg) was reduced by omapatrilat in a dose-dependent manner (L, 121 +/- 3 mm Hg; H, 110 +/- 3 mm Hg) and by fosinopril (149 +/- 5 mm Hg). Proteinuria in STNx rats (246 +/- 73 mg/day) was reduced by treatment with fosinopril (88 +/- 21 mg/day) and was normalized by treatment with omapatrilat (L, 30 +/- 4 mg/day; H, 20 +/- 2 mg/day vs. control 25 +/- 1 mg/day). Decreased glomerular filtration rates, elevated plasma urea and creatinine and glomerulosclerosis, and tubulointerstitial fibrosis were ameliorated by omapatrilat and fosinopril to a similar degree. Compared with fosinopril, omapatrilat treatment was associated with increased plasma renin activity and decreased renal ACE and NEP binding in a dose-dependent manner. These findings suggest that vasopeptidase inhibition may provide a useful strategy for the treatment of progressive renal disease.

Local renin-angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells.

NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.Key words: angiotensin-converting enzyme (ACE), ACE binding protein, ACE affinity column, human serum protein.

Angiotensin II, Transforming Growth Factor-β1and Repair in the Infarcted Heart

Tissue repair following myocardial infarction (MI) eventuates in fibrous tissue formation at the site of myocyte necrosis. Following a large transmural MI, fibrosis appears remote to the infarct site. This is associated with extensive tissue remodeling that adversely affects ventricular diastolic function. Substances involved in promoting fibrous tissue formation at MI and remote sites are under investigation. Angiotensin II (AngII), generated at sites of repair, has been implicated. However, its regulatory role on fibrous tissue formation remains uncertain. In the present study we sought to determine whether AngII is correlated to transforming growth factor beta 1 (TGF-β1) expression, a regulator of fibrous tissue formation, at these sites of tissue repair. We studied: (1) localization and expression of angiotensin converting enzyme (ACE), AngII receptors, TGF-β1mRNA and its receptors in the infarcted rat heart; and (2) effect of AngII on TGF-β1synthesis by chronic blockade of AT1receptors began at the time of surgery by losartan in rats with MI. Hearts were studied at 4 weeks post-MI. We found: (1) low-density ACE, AngII and TGF-β1receptor binding and low mRNA for type I collagen and TGF-β1in the normal heart; (2) fibrosis at sites of MI and remote to it, including endocardium and fibrosis of intraventricular septum, interstitial fibrosis of non-infarcted myocardium and fibrosis of visceral pericardium; (3) markedly increased (P<0.01) and colocalized ACE, AngII and TGF-β1receptor binding, type I collagen and TGF-β1mRNA at MI and remote sites of repair; (4) increased TGF-β1concentration (P<0.01) at these sites; and (5) attenuated TGF-β1and type I collagen gene expression (P<0.01) at these sites in rats receiving losartan. These observations suggest locally generated AngII via ATireceptor binding is correlated to TGF-β1expression and synthesis at sites of repair and remote sites in the infarcted rat heart. The mechanism responsible for the role of AngII in TGF-β1remains to be elucidated.

Angiotensin-converting enzyme and wound healing in diverse tissues of the rat

Autoradiographic binding density of angiotensin-converting enzyme (ACE), an indirect measure of ACE activity, is markedly increased at sites of fibrous tissue that appear in the injured heart. This includes myocardial infarction (MI) caused by left coronary artery ligation; endocardial fibrosis of the interventricular septum and perivascular fibrosis of intramyocardial coronary arterioles of the right ventricle, each of which appear remote to MI; and pericardial fibrosis after pericardiotomy (without MI). Expressed in fibroblast-like cells found at each site of tissue repair, ACE may be common to tissue repair in the rat heart, irrespective of the etiologic basis of injury. To address this hypothesis and to determine whether this also applies to other tissues (skin and kidney), the present study was undertaken. ACE binding density was measured by quantitative in vitro autoradiography ( 125I-351A) in injured rat heart, skin, and kidney. Experimental observations included foreign-body fibrosis after placement of silk ligature in skin or myocardium, endomyocardial myocyte necrosis and fibrosis that accompanied isoproterenol administration (1 mg/kg sc × 2 days), and embolic infarction of the kidney as a result of mural thrombus of the left ventricle that appeared after anterior MI. Fibrosis was identified by collagen-specific staining with picrosirius red. Hematoxylin-eosin staining and immunohistochemical labeling with α-smooth muscle actin (α-SMA) antibody were used to address cell morphology and phenotype, respectively. We found (1) endomyocardial fibrosis 2 weeks after isoproterenol; (2) fibrosis surrounding silk suture in heart and skin 1 week after placement; (3) renal infarction 1 week after left coronary artery ligation; (4) numerous fibroblast-like cells containing α-SMA, as well as macrophages, at sites of repair in all tissues studied; and (5) markedly increased ACE binding density at each of these sites. Thus ACE is integral to tissue repair in the heart, skin, and kidney of the rat, irrespective of the etiologic basis of injury. At these sites ACE may serve to regulate local concentrations of substances involved in tissue repair.

Fosinopril treatment of pregnant rats: developmental toxicity, fetal angiotensin-converting enzyme inhibition, and fetal angiotensin II receptor regulation

Pregnant women are advised against using angiotensin-converting enzyme (ACE) inhibitors due to reports of adverse effects on human fetuses. This study examined ACE binding and angiotensin II (Ang II) receptor binding in fetuses of rats treated with the ACE inhibitor fosinopril (16 mg/kg/day fosinopril, p.o. in 4 divided doses, from gestational day (gd) 13 to gd 18). Binding of the potent radiolabeled ACE inhibitor 125I-351A to ACE in the lung and aorta of gd 19 fetuses of fosinopril-treated dams was reduced by 56 and 44%, respectively, compared to fetuses from vehicle-treated dams, indicating that fosinopril or its active metabolite, fosinoprilat, crosses the placental barrier and inhibits fetal ACE. Fetal Ang II receptor binding of 125I-Sar 1,Ile 8 Ang II was not altered in most of the tissues examined, although reductions in binding in the adrenal of fetuses of fosinopril-treated dams approached statistical significance.

Connective tissue and repair in the heart. Potential regulatory mechanisms.

The heart is composed of highly differentiated cardiac myocytes, which constitute parenchyma, and stroma or connective tissue. Fibrillar collagen turnover in the heart and its valve leaflets, in particular, is dynamic and essential to tissue repair. Emerging evidence further suggests connective tissue is a metabolically active entity, where peptide hormones are generated and degraded and, in turn, these peptides regulate collagen turnover. This concept arose from quantitative in vitro autoradiography using an iodinated derivative of lisinopril (125I-351A) as ligand to localize angiotensin converting enzyme (ACE) binding density within the heart. A heterogeneous distribution was found: low-density ACE binding within atria and ventricles; high ACE binding density at sites of high collagen turnover, such as valve leaflets, adventitia, and fibrous tissue of diverse etiologic origins. ACE-producing cells at these latter sites were identified by monoclonal ACE antibody. They included valvular interstitial cells (VIC) and fibroblast-like cells each of which also contained alpha-smooth muscle actin and the transcript for type I collagen (in situ hybridization). Substrate utilization in cultured VIC was found to include angiotensin I and bradykinin. Angiotensin II and bradykinin receptor-ligand binding was observed in VIC and at fibrous tissue sites. Connective tissue ACE is independent of circulating angiotensin II. In vivo, fibrous tissue formation is attenuated by ACE inhibition or antagonism of AT1 receptor. Angiotensin II and bradykinin are stimulatory and inhibitory, respectively, to cultured adult cardiac fibroblast collagen synthesis suggesting a paradigm of reciprocal regulation to fibroblast collagen turnover. Stroma and its cellular constituents represent a dynamic metabolic entity that regulates its own peptide hormone composition and turnover of fibrillar collagen. These findings may provide insights that could be used to advantage to either promote or forestall fibrous tissue formation depending on the nature of cardiovascular disease.

Bradykinin receptor and tissue ACE binding in myocardial fibrosis: Response to chronic angiotensin II or aldosterone administration in rats

Y. Sun, A. Ratajska and K. T. Weber. Bradykinin Receptor and Tissue ACE Binding in Myocardial Fibrosis: Response to Chronic Angiotensin II or Aldosterone Administration in Rats. Journal of Molecular and Cellular Cardiology (1995) 27, 813–822. High density angiotensin converting enzyme (ACE) binding is present in the perivascular fibrosis involving intramyocardial coronary arteries and the microscopic scarring of the myocardium that accompanies chronic elevations in circulating angiotensin II (AngII) and/or aldosterone (ALDO). As a kininase II, ACE degrades bradykinin. Herein we sought to determine whether bradykinin (BK) receptor binding was associated with ACE binding in each of these experimental models. BK receptor binding was localized and quantified by in vitro quantitative autoradiography, using [ 125I-Tyr 8]BK. In serial sections of the same heart hematoxylin and eosin (H&E) and picrosirius red (PSR) staining were utilized to address cardiac myocyte injury and fibrosis respectively. Four experimental groups were examined: unoperated, untreated, age/sex matched controls; age/sex matched uninephrectomized control rats receiving a high sodium diet; animals that received AngII (9 μg/h sc) for 2, 4 or 6 weeks; and uninephrectomized rats on a high sodium diet that received ALDO (0.75 μg/h sc) for similar periods of time. We found: (a) myocardial fibrosis, including perivascular fibrosis and microscopic scarring, at week 2 of AngII, but not until week 4 or more of ALDO treatment; (b) low BK receptor binding in normal ventricles that was increased in scars and markedly increased in perivascular fibrosis at week 2 of AngII and each increased further at week 4 and 6 of AngII; (c) low BK receptor binding at week 2 and 4 weeks of ALDO treatment which became markedly increased at fibrous tissue sites at week 6. BK receptor and ACE binding were anatomically coincident and localized to each site of fibrosis in both models. The co-location of BK receptor and ACE binding in these models raises the prospect that fibrous tissue ACE may utilize BK as substrate and BK, in turn, may play a role in fibrous tissue formation.

Kidney angiotensin II receptors and converting enzyme in neonatal and adult Wistar-Kyoto and spontaneously hypertensive rats

The aim of the present study was to correlate the development of the renin angiotensin system (RAS) in the kidney of the rat with the development of genetic hypertension. Immature (1-week-old) and adult (12-week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive kidney rats (SHR) were used for quantification of angiotensin II (ANG II) receptors and angiotensin converting enzyme (ACE) binding sites using quantitative autoradiography. In both neonatal and adult animals of either strain, ANG II receptors were of AT1 subtype. In all kidney areas of 1-week-old rats. ANG II receptor density was higher in SHR than WKY. Binding density increased with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer medulla of 12-week-old WKY, binding was significantly higher than that present in age-matched SHR. [125I]351A binding to ACE was highest in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to ACE was higher in WKY than in SHR strain. No differences in ACE binding were found between adult SHR and WKY rats, with the exception of the inner stripe of the outer medulla, where no binding was detected in SHR. Our results support the hypothesis that the RAS in kidney is developmentally regulated and is involved in the development and maintenance of genetic hypertension in SHR.

Connective tissue: a metabolic Entity?

The heart is composed of parenchyma (cardiac myocytes) and stroma (connective tissue). Stroma is presumed inert and therefore little attention has been paid to its regulation. Contrary to this notion, evidence presented here raises the possibility that connective tissue is a metabolically active entity capable of regulating peptide hormone generation and degradation and these hormones, in an autocrine manner, regulate collagen turnover. This concept has evolved from quantitative in vitro autoradiography (using 125I-351A), which localized angiotensin converting enzyme (ACE) binding density within the heart. A heterogenous distribution was found. Low-density ACE is present within atria and ventricles. At sites of high collagen turnover, such as valve leaflets, adventitia and fibrous tissue of diverse etiologic origins. ACE binding density is high and independent of circulating angiotensin II. ACE-producing cells at these sites, identified by monoclonal ACE antibody and 125I-351A binding, include fibroblast-like alpha actin-containing cells that express the transcript for type I collagen (in situ hybridization). Receptor-ligand binding for angiotensin II and bradykinin is found in fibrous tissue, where these peptides may provide for a reciprocal regulation of fibroblast collagen turnover. Connective tissue formation is attenuated by ACE inhibition or antagonism of type I angiotensin II receptor. Thus, emerging evidence raises the possibility that stroma and its cellular constituents is a dynamic, metabolically active entity regulating its own peptide hormone composition and, in turn, its turnover of fibrillar collagen.

Fibrosis and myocardial ACE: Possible substrate and independence from circulating angiotensin II

In order to determine the relation between myocardial fibrosis and (1) angiotensin converting enzyme (ACE) binding density, (2) receptor binding of ACE-related peptides, angiotensin II (AngII) and bradykinin (BK), and (3) the regulation of myocardial ACE by circulating Ang II, we used in vitro quantitative autoradiography to localize and assess ACE ([ 125I]351A), AngII receptor ( 125I[Sar 1, IIe 8]AngII), and BK receptor ([ 125I]Tyr 8) binding densities in the rat myocardium. The experimental groups were (1) AngII (9 μg/h subcutaneously) or aldosterone (0.75 μg/h subcutaneously in uninephrectomized rats on a high-sodium diet) administered by implanted minipump to chronically increase or decrease circulating AngII, respectively, (2) unoperated, untreated age- and sex-matched control rats, and (3) age- and sex-matched uninephrectomized control rats on a high-sodium diet. In the same heart studied at 2, 4, and 6 weeks of hormone treatment, serial sections were assessed for myocardial (hematoxylin and eosin) and fibrillar collagen (picrosirius red) morphology. The authors found that (1) marked ACE binding was coincident with sites of fibrous tissue that appeared in both ventricles as either a perivascular fibrosis of intramyocardial coronary arterioles or microscopic scarring in response to AngII or aldosterone infusion, (2) ACE binding was independent of circulating AngII, (3) BK, not AngII, receptor binding density was markedly increased at fibrous tissue sites, and (4) high-density ACE and BK receptor binding were anatomically coincident. Thus, in these experimental models, ACE is related to fibrous tissue formation where it may use BK, not angiotensin I, as a substrate, and its binding density is independent of circulating AngII.

Cardiac angiotensin converting enzyme and myocardial fibrosis in the rat.

The aim was to test the hypothesis that cardiac angiotensin converting enzyme (ACE) is related to the accumulation of fibrous tissue in the heart. A model of tissue repair (pericardiotomy with left coronary artery ligation) was used, together with the following: quantitative in vitro autoradiography (125I-351A) to determine ACE binding density; immunohistochemistry (monoclonal ACE antibody, 9B9) to identify cells expressing ACE; and in situ hybridisation to localise cells expressing type I collagen mRNA. Age and sex matched rats receiving this operative procedure without subsequent infarction (sham operated) served as controls to those with left ventricular myocardial infarction. Serial heart sections obtained from each group at 3 days and at 1, 2, 4, and 8 weeks following operation were examined for morphological evidence of injury and inflammatory cells (haematoxylin/eosin) and fibrillar collagen (picrosirius red). Following myocardial infarction: (a) on day 3, neutrophils and macrophages were present at the site of infarction, while fibrillar collagen and ACE binding were not increased compared with control; (b) at week 1, fibrillar collagen and ACE binding were present at the site of infarction and became progressively more advanced at 2, 4, and 8 weeks; (c) at week 2, there was increased ACE binding in the right ventricle and interventricular septum, when perivascular fibrosis of intramural coronary arteries and microscopic scars appeared, together with endomyocardial fibrosis of the septum; (d) there was marked ACE binding in the fibrosed visceral pericardium two weeks after operation in both myocardial infarction and sham operated groups; (e) there was type I collagen mRNA expression on postoperative week 1, localised within fibroblasts or fibroblast-like cells found at infarct and fibrous tissue sites in the right ventricle, septum, and pericardium; and (f) ACE-labelled cells included fibroblast-like cells, as well as macrophages and endothelial cells. Thus in this model of tissue repair, marked ACE binding density is associated with fibrillar collagen formation that appears within and remote to the site of myocardial infarction, including the pericardium. Cardiac ACE, originating from type I collagen producing cells, therefore represents an integral component of fibrous tissue formation in this rat model of tissue injury.

Trandolapril

The pharmacokinetics and dose proportionality of trandolapril, a new angiotensin-converting enzyme (ACE) inhibitor, were investigated in 12 healthy male volunteers in a four-way randomized crossover study over the therapeutic dose range, 0.5-4 mg. Trandolapril is rapidly absorbed, with a single elimination half-life (t1/2) of 0.72 h, irrespective of dose. Peak plasma levels (Cmax) occurred at 0.5 h and were proportional to the dose, as was the area under the plasma concentration-time curve (AUC). Concentration of the active metabolite (trandolaprilat) increased with increasing doses but in a nonlinear fashion, probably owing to saturable plasma ACE binding. However, the Cmax and AUC values for trandolaprilat were directly proportional to the highest doses, 2 and 4 mg, suggesting linear kinetics for the trandolaprilat, which is not bound to ACE. Trandolapril showed linear kinetics but trandolaprilat showed some features of nonlinear kinetics, particularly at low doses.

Distribution and functional significance of cardiac angiotensin converting enzyme in hypertrophied rat hearts.

The intracardiac conversion rate of angiotensin (Ang) I to Ang II and the expression of angiotensin converting enzyme (ACE) mRNA are amplified in rat hearts with left ventricular hypertrophy (LVH). To examine whether the accelerated intracardiac Ang II generation in LVH is related to an induction of cardiac ACE, we studied localization and function of cardiac ACE in hypertrophied rat hearts using specific ACE inhibitors. Cardiac ACE was localized and quantified in hearts from male Wistar rats with LVH due to chronic experimental aortic stenosis and from control rats. With the ACE inhibitor 125I-351A, a derivative of lisinopril, as a radioligand on coronal sections of LVH and control hearts, in vitro autoradiography demonstrated ACE binding in aorta, coronary arteries, atria, and ventricles of both groups. Quantitative analyses revealed that ACE density (counts per minute per cross-sectional area of tissue) was twofold higher within the myocardium of hypertrophied left ventricles compared with controls (p < 0.005). Quantitative morphometry demonstrated a modest increase in the fractional volume of myocytes as well as capillary volume without an increase in the fractional volume of endothelial cells in left ventricular tissue from aortic stenosis rats. These data suggest that an increase in endothelial cell volume per se cannot alone account for the observed doubling of ACE density and support an upregulation of ACE production in hypertrophied tissue. The role of cardiac ACE in intracardiac conversion of Ang I to Ang II and its specific inhibition was studied in isolated, isovolumic beating, buffer-perfused LVH and control hearts. Biochemical conversion rates as well as functional changes in response to 3 x 10(-7) M Ang I were examined in the absence or presence of the ACE inhibitor enalaprilat (4 x 10(-6) M). After a brief stabilization period, groups of LVH and control hearts were subjected to the following infusion protocols: 15 minutes of vehicle followed by 30 minutes of Ang I plus vehicle, 15 minutes of enalaprilat followed by 30 minutes of Ang I plus enalaprilat (enal/Ang I), or 45 minutes of vehicle only to allow comparison with a time control. Intracardiac Ang I-to-Ang II conversion rate was fourfold higher in LVH than in control hearts (p < 0.05). Infusion of enalaprilat reduced the intracardiac Ang I-to-Ang II conversion rate in LVH hearts by 70% (p < 0.05 versus Ang I). At similar levels of constant coronary flow per gram, Ang I increased coronary perfusion pressure by 23 +/- 5 mm Hg (p < 0.01 versus vehicle) in LVH hearts and by 36 +/- 10 mm Hg (p < 0.005 versus vehicle) in control hearts. When enalaprilat was infused with Ang I, the increase in perfusion pressure was limited to 5 +/- 5 mm Hg (NS versus vehicle) in LVH hearts and 12 +/- 3 mm Hg (p < 0.05 versus vehicle) in control hearts and was significantly lower than in hearts infused with Ang I only (p < 0.05 in LVH and p < 0.05 in control hearts, respectively). Systolic function was not affected by either infusion protocol. In contrast, Ang I infusion was associated with diastolic dysfunction. In LVH hearts, left ventricular end-diastolic pressure (LVEDP) increased from 10 +/- 1 mm Hg at baseline to 25 +/- 2 mm Hg at the end of the Ang I infusion (p < 0.001 versus vehicle), which was inhibited by infusion of enalaprilat. In control hearts, there was a lesser increase in LVEDP from 10 +/- 1 mm Hg to 15 +/- 1 mm Hg in response to Ang I (p < 0.05 versus LVH). Control hearts treated with enalaprilat with Ang I displayed no increase in LVEDP: These observations indicate that ACE protein is increased within the myocardium of LVH hearts, extending recent findings of increased cardiac ACE activity and mRNA levels in this model of pressure-overload LVH in the rat. Blockade of the enzyme by an ACE inhibitor decreases intracardiac Ang I-to-Ang II conversion rate and prevents the functional changes of Ang I-to-Ang II activation

Estrogens regulate angiotensin-converting enzyme and angiotensin receptors in female rat anterior pituitary.

We studied the effects of the estrous cycle, ovariectomy and estrogen replacement on angiotensin-converting enzyme (ACE) (kininase II, EC 3.4.15.1) and angiotensin II (AT) receptors in the pituitary gland of the female rat. Quantitative autoradiography, with the use of consecutive pituitary sections, allowed for simultaneous determination of changes in binding and in the potential AT synthetic ability of individual pituitaries, and for a correlation between these two phenomena. In the anterior pituitary, ACE activity and binding of the ACE inhibitor [125I]-351A were not changed during the estrous cycle. Ovariectomy produced a significant increase in ACE activity and binding, and both of these parameters returned to normal after estrogen replacement. There were no changes in ACE activity or binding in the posterior pituitary during the estrous cycle or after ovariectomy or hormone replacement. AT receptors were characterized as of the AT1 type, since they were displaced by the selective AT1 antagonist DuP 753 and not by the AT2 competitor PD 123177. There were marked changes in the concentration of AT1 receptors during the estrous cycle, with highest numbers in metestrus, lower in estrus and diestrus, and lowest during proestrus. Estrogen replacement in ovariectomized rats decreased AT1 receptor number in the anterior pituitary. Our results indicate a dual effect of estrogen on anterior pituitary AT, physiologically on AT receptor expression and pharmacologically on ACE activity.