Total Angiotensin-converting Enzyme Research Articles

Evaluation of Radio Frequency-Assisted Enzymatic Extraction of Non-Anthocyanin Polyphenols from Akebia trifoliata Flowers and Their Biological Activities Using UPLC-PDA-TOF-ESI-MS and Chemometrics.

A new radio frequency heating-assisted enzymatic extraction (RF-E) method is applied for the determination of phenolic compounds in Akebia trifoliata flowers, compared with hot water, acidified ethanol (EtOH), and enzymatic-assisted (EA) extractions. Non-anthocyanin polyphenol profiles, antibacterial, angiotensin-converting enzyme (ACE) inhibitory, anti-inflammatory activities, and structures of extracts are evaluated. Results show no significant differences in the extraction of total flavonoid content (15.85-16.63 mg QEs/g) and ACE inhibitory activity (51.30-52.86%) between RF-E and EA extracts. RF-E extract shows the highest anti-inflammatory activities. FTIR and UV spectra reveal that acidified EtOH treatment has a significant effect on the structure of the extract due to its highest flavonoid content (20.33 mg QEs/g), thus it has the highest antibacterial activity against Staphylococcus aureus and Escherichia coli. Sixteen non-anthocyanin polyphenols are identified by UPLC-PDA-TOF-ESI-MS and RF pre-treatment did not cause significant compound degradation. The chemometric analysis shows that enzymatic hydrolysis significantly increased biological activities, and the presence of non-anthocyanin polyphenols correlates well with ACE inhibitory and anti-inflammatory activities. Accordingly, A trifoliata flowers have potential as reagents for the food and pharmaceutical industries due to their abundant polyphenols that could be extracted efficiently using RF-E.

Endothelial nitric oxide synthase, angiotensin-converting enzyme and angiotensinogen gene polymorphisms in hypertensive disorders of pregnancy

We investigated the variations in genes encoding endothelial nitric oxide synthase (NOS3), angiotensin-converting enzyme (ACE) and angiotensinogen (AGT) in hypertensive disorders of pregnancy and the relationship between the polymorphisms and circulating nitric oxide (NO) and ACE levels in pregnant north Indian women. Frequencies of NOS3 G894T, 4b/a and T(-786) --> C, AGT T704C and ACE ins/del polymorphisms were studied in 342 subjects: 120 with preeclampsia (PE), 104 with gestational hypertension and 118 normotensive pregnant women. Variations were evaluated by polymerase chain reaction-restriction fragment length polymorphism. NO and ACE levels were determined using ELISA. There was no difference in the distribution of individual NOS3 and ACE polymorphisms in the study groups. Haplotype analysis showed a global difference in the NOS3 haplotype distribution between the PE and non-PE subjects (P=0.03). The presence of AGT 704C allele was associated with a reduced risk of developing PE (odds ratio: 0.33, 95% CI: 0.19-0.59 in recessive mode). Circulating total NO and ACE levels were similar in three groups. No relationship was found between circulating NO levels and any of the NOS3 polymorphisms, but the circulating ACE levels were higher in those with DD genotype (P<0.05). In conclusion, there was no association between individual NOS3 and the ACE gene polymorphisms and hypertensive disorders of pregnancy in north Indian women. The presence of minor alleles at all the three sites in NOS3 seemed to increase the risk of PE, and AGT 704C allele was associated with a reduced PE risk. The complexity of interaction between these genetic abnormalities requires further studies.

Inhaled activated protein C protects mice from ventilator-induced lung injury

IntroductionActivated Protein C (APC), an endogenous anticoagulant, improves tissue microperfusion and endothelial cell survival in systemic inflammatory states such as sepsis, but intravenous administration may cause severe bleeding. We have thus addressed the role of APC delivered locally by inhalation in preventing acute lung injury from alveolar overdistention and the subsequent ventilator-induced lung injury (VILI). We also assessed the effects of APC on the activation status of Extracellular- Regulated Kinase 1/2 (ERK) pathway, which has been shown to be involved in regulating pulmonary responses to mechanical stretch.MethodsInhaled APC (12.5 μg drotrecogin-α × 4 doses) or saline was given to tracheotomized C57/Bl6 mice starting 20 min prior to initiation of injurious mechanical ventilation with tidal volume 25 mL/Kg for 4 hours and then hourly thereafter; control groups receiving inhaled saline were ventilated with 8 mL/Kg for 30 min or 4 hr. We measured lung function (respiratory system elastance H), arterial blood gases, surrogates of vascular leak (broncho-alveolar lavage (BAL) total protein and angiotensin-converting enzyme (ACE)-activity), and parameters of inflammation (BAL neutrophils and lung tissue myeloperoxidase (MPO) activity). Morphological alterations induced by mechanical ventilation were examined in hematoxylin-eosin lung tissue sections. The activation status of ERK was probed in lung tissue homogenates by immunoblotting and in paraffin sections by immunohistochemistry. The effect of APC on ERK signaling downstream of the thrombin receptor was tested on A549 human lung epithelial cells by immunoblotting. Statistical analyses were performed using ANOVA with appropriate post-hoc testing.ResultsIn mice subjected to VILI without APC, we observed hypoxemia, increased respiratory system elastance and inflammation, assessed by BAL neutrophil counts and tissue MPO activity. BAL total protein levels and ACE activity were also elevated by VILI, indicating compromise of the alveolo-capillary barrier. In addition to preserving lung function, inhaled APC prevented endothelial barrier disruption and attenuated hypoxemia and the inflammatory response. Mechanistically, we found a strong activation of ERK in lung tissues by VILI, which was prevented by APC, suggestive of pathogenetic involvement of the Mitogen-Activated Kinase pathway. In cultured human lung epithelial cells challenged by thrombin, APC abrogated the activation of ERK and its downstream effector, cytosolic Phospholipase A2.ConclusionsTopical application of APC by inhalation may effectively reduce lung injury induced by mechanical ventilation in mice.

Relationship between genetic polymorphisms of angiotensin-converting enzyme and methylenetetrahydrofolate reductase as risk factors for type 2 diabetes in Tunisian patients

Background/aims The role of methylenetetrahydrofolate reductase (MTHFR) and angiotensin-converting enzyme (ACE) gene polymorphisms as being risk factors for diabetes is still controversial. The aim was to investigate the distribution of ACE and MTHFR genotypes as well as to evaluate the role of plasmatic total homocysteine levels (tHcy) and ACE activity in Tunisian patients with type 2 diabetes mellitus (T2DM). Design and methods 115 T2DM patients compared to 116 healthy volunteers. Results The ACE I/D polymorphism was significantly associated with diabetes ( p < 0.0001). The DD genotype and D allele were more frequent in patients compared to control group [DD: OR = 4.93; p < 0.0001; 95 % CI: 2.71–8.97; D: OR = 3.08, 95% CI: 2.09–4.51 p < 0.0001]. MTHFR allele and genotype frequencies did not differ between patients and controls. The susceptibility to diabetes in individuals with genotypes DD +vTT was 13.39 and in the individuals with DD + CT was 6.57 times that of the controls. However, individuals with genotypes ID + CC or II + CT have a protective effect against diabetes. The DD and TT genotypes were associated with significantly higher ACE activity and tHcy levels in diabetics. Conclusion Our data suggest that ACE ID polymorphism may act synergistically with MTHFR C677T polymorphism to assess diabetes risk.

Inhibition of angiotensin converting enzyme (ACE) by normal solutes of human plasma

Human plasma ACE concentration is ~4 nM, yet net ACE activity is <1 nM. Inhibition is evident on assaying ACE of increasing dilutions of plasma. Here, means are defined for computing net ACE activity of undiluted plasma, total ACE and % inhibition. Under first order kinetics, the Michaelis-Menten equation is v/s = Vmax/(Km (1 + i/Ki)), where i is inhibitor concentration; v/s is called apparent (app) (Vmax/Km). The inverted equation describes a straight line: app(Km/Vmax) = Km/VmaxKi(i) + Km/Vmax. When i, fractional concentration (fr conc) of plasma, is plotted against app(Km/Vmax), the y-intercept is true (Km/Vmax). The x-intercept is the fr conc of plasma for inhibition of ACE by half. Net ACE activity of undiluted plasma is computed for i = 1.0; 1/x is units of inhibitory activity. Plasmas of 37 healthy adults, 23 – 57 y.o., were assayed in fr conc of 0.2 – 0.00625 using [3H]hippuryl-His-Leu, 40 nM, in Hepes buffer, pH 8.0, plus NaCl and sodium sulfate, at 37oC. Results (mean±SD): Vmax/Km 0.3755±0.098/min, app(Vmax/Km) 0.0442±0.003/min, inhibitor units 7.29±2.0, inhibition 88.2±5.0%. Thus, <12% of plasma ACE is active. Inhibition, relieved by dialysis, is attributable to arginine, lysine, ornithine, cysteine, Cys-Gly and glutathione. Following a meal, their concentrations can double and increase inhibition, which raises the question, Is ACE activity physiologically modulated by food intake?

Role of Tyrosine Phosphorylation in the Regulation of Cleavage Secretion of Angiotensin-converting Enzyme

Both germinal (gACE) and somatic (sACE) isozymes of angiotensin-converting enzyme (ACE) are type I ectoproteins whose enzymatically active ectodomains are cleaved and shed by a membrane-bound protease. Here, we report a role of protein tyrosine phosphorylation in regulating this process. Strong enhancements of ACE cleavage secretion was observed upon enhancing protein Tyr phosphorylation by treating gACE- or sACE-expressing cells with pervanadate, an inhibitor of protein Tyr phosphatases. Secreted gACE, cell-bound mature gACE and its precursors were all Tyr-phosphorylated, as was the endoplasmic reticulum protein, immunoglobulin heavy chain-binding protein, that co-immunoprecipitated with ACE. The enhancement of cleavage secretion by pervanadate did not require the presence of the cytoplasmic domain of ACE, and it was not accomplished by enhancing the rate of intracellular processing of the protein. The observed enhancement of cleavage secretion of ACE in pervanadate-treated cells was specifically blocked by an inhibitor of the p38 mitogen-activated protein (MAP) kinase but not by inhibitors of many other Ser/Thr and Tyr protein kinases, including a specific inhibitor of protein kinase C that, however, could block the enhancement of cleavage secretion elicited by phorbol ester. These results indicate that ACE Tyr phosphorylation, probably in the endoplasmic reticulum, enhances the rate of its cleavage secretion at the plasma membrane using a regulatory pathway that may involve p38 MAP kinase.

Maps of end-stage renal disease and amounts of angiotensin-converting enzyme inhibitors prescribed in Japan

We recently found regional differences in the incidence of end-stage renal disease (ESRD) in Japan, which is generally ethnically homogeneous, suggesting that factors other than genetic may contribute to the difference. Here, we examined regional differences in the amounts of expenses spent on antihypertensives, especially angiotensin-converting enzyme (ACE) inhibitors, in our search for an explanation. Annually, the Japanese Society for Dialysis Therapy reports the numbers of patients entering maintenance dialysis in each prefecture of Japan since 1982. We used the findings for 1995 to 2000 to calculate the annual incidence of ESRD in each of the 11 regions of Japan. In addition, regional differences in annual amounts paid for antihypertensive drugs, presumably corresponding to the amounts used, during the same 6 years, corrected for population, were estimated. As in our 1982 to 1998 study, the incidence of ESRD was high in Okinawa, Kyushu, and Shikoku, while low in Hokuriku, Koshinetsu, and Tohoku (P < 0.0001) [one-way repeated measures analysis of variance (ANOVA)]. We found regional differences in the corrected sum paid for total antihypertensive drugs, ACE inhibitors and calcium antagonists. Only ACE inhibitors were negatively correlated with the incidence of ESRD by linear and multiple regression analyses. The renal protective effects of ACE inhibitors have been established by results with animal models of progressive nephropathy and large-scale clinical trials. Our epidemiologic results for Japan as a whole show the same protective effects still more convincingly from a different approach.

Angiotensin-converting enzyme activity in ovine bronchial vasculature.

Angiotensin-converting enzyme (ACE) plays a major role in the metabolism of bradykinin, angiotensin, and neuropeptides, which are all implicated in inflammatory airway diseases. The activity of ACE, which is localized on the luminal surface of endothelial cells (EC), has been well documented in pulmonary EC; however, few data exist regarding the relative activity of ACE in the airway vasculature. Therefore, we measured ACE activity in cultured EC from the sheep bronchial artery and bronchial mucosa (microvascular) and compared it with pulmonary artery EC. The baseline level of total ACE activity (cellular plus secreted) was significantly greater in bronchial microvascular EC (1.24 +/- 0.24 mU/106 cells) compared with bronchial artery EC (0.59 +/- 0.15 mU/106 cells; P < 0.05) and comparable to pulmonary artery EC (1.12 +/- 0.14 mU/106 cells; P > 0.05). Measured ACE activity secreted into culture medium for each cell type was 64-74% of total activity and did not differ among the three EC types (P = 0.17). Hydrocortisone (10 microg/ml; 48-72 h) treatment resulted in a significant increase in ACE activity in bronchial EC. Likewise, TNF-alpha (0.1 ng/ml) treatment markedly increased ACE activity in all cell lysates (P < 0.05). We confirmed the importance of ACE activity in vivo since, at the highest dose of bradykinin studied (10-8 M), bronchial artery pressure at constant flow showed a greater decrease after captopril treatment (36% before vs. 60% after; P = 0.05). These results demonstrate high ACE expression of the bronchial microvasculature and suggest an important regulatory role for ACE in the metabolism of kinin peptides known to contribute to airway pathology.

Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa.

Testicular angiotensin converting enzyme (ACE) isozyme is likely to play important functional roles in male reproduction. Several studies have shown that ACE is released from human spermatozoa during capacitation and that ACE is associated with reduced sperm motility. Recently, we established an assay to detect testicular ACE activity in human spermatozoa. The purpose of this study was to determine if testicular ACE activity is related to sperm motility in human ejaculates. Semen samples were collected from 80 infertile patients. According to the semen characteristics, they were divided into four (WHO) categories. Enzyme activities of ACE in spermatozoa (testicular ACE) and seminal plasma (somatic ACE) were spectrophotometrically determined. Total testicular ACE activity in spermatozoa was measured by solubilization of spermatozoa with Triton X-100. Membrane testicular ACE activity was measured in a sperm : PBS suspension. Sperm concentration and sperm motility were 136.6 +/- 154.1 x 10(6)/mL and 58.6 +/- 23.4%, respectively (mean +/- SD). Enzyme activities of membrane testicular ACE, total testicular ACE and somatic ACE were 0.273 +/- 1.219 microU/10(6) spermatozoa, 0.35 +/- 1.34 microU/10(6) spermatozoa and 684.7 +/- 226.6 mU/mL, respectively. A negative correlation was observed between sperm motility and membrane testicular ACE activity (p < 0.05). Membrane testicular ACE activity in 44 normal semen samples was 0.04 +/- 0.02 microU/10(6) spermatozoa, whilst that in 36 abnormal semen samples was 0.24 +/- 0.42 microU/10(6) spermatozoa. There was a significant difference between these two groups (p < 0.01). Membrane testicular ACE in sperm samples from normozoospermic men was significantly lower than that from oligoasthenozoospermic men (p < 0.05). These findings suggest that testicular ACE is released from normal functional spermatozoa for them to have fertilizing ability.

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.

Assay of testicular angiotensin-converting enzyme activity in human spermatozoa.

The testicular isozyme of angiotensin-converting enzyme (ACE) is associated with male fertility. Spermatozoa from mice lacking ACE showed defects in transport within the oviducts and in binding to zonae pellucidae although the animals had normal sperm count, morphology and motility. In fact, unexplained infertility is difficult to be predicted by conventional parameters such as sperm count. We measured membrane testicular ACE activity in a sperm suspension in PBS and total testis ACE activity in spermatozoa by solubilization with Triton X-100. Total testis ACE activity and membrane testis ACE activity of the same subject were compared in 12 control subjects. We demonstrated that testicular ACE is stable in spermatozoa and the assay of testicular ACE activity is possible. Total testicular ACE activity was approximately twice the membrane testicular ACE activity in all of the subjects tested. The assay of testicular ACE activity in human spermatozoa could be a new method for the assessment of sperm function.

Antiatherogenic effect of angiotensin converting enzyme inhibitor (benazepril) and angiotensin II receptor antagonist (valsartan) in the cholesterol-fed rabbits

The purpose of this study was to determine whether an angiotensin converting enzyme (ACE) inhibitor, benazepril, and an angiotensin receptor antagonist, valsartan, would decrease atherosclerotic severity in cholesterol-fed rabbits. Male rabbits were fed either: (a) normal rabbit chow; (b) 2% cholesterol diet; (c) 2% cholesterol diet supplemented by benazepril (3 mg/kg per day, subcutaneous injection); or (d) 2% cholesterol diet supplemented by valsartan (1 mg/kg per day, subcutaneous injection). After 12 weeks, the arteries were harvested for histomorphometry and immunohistochemistry. We observed that decreases in serum triglyceride (TG) and total cholesterol (TC) and ACE activity with benazepril-treatment were more than 60, 30, and 84%, respectively, in comparison with the cholesterol group; with valsartan-treatment, TG levels were 53% lower than in the cholesterol group, however, there was no significant difference in TC and ACE activity. The percentage of aortic surface atherosclerotic area, intimal thickness and the ratio of aortic intimal area to medial area were about 40% lower in the benazepril-treated group in comparison with those of the cholesterol group; the difference was more than 60% in the thoracic aorta. The valsartan-treated group had 23% less atherosclerotic area, less effective than benazepril treatment. The percent of PCNA-positive cells and the number of intimal proliferative cells/mm 2 were significantly less in the benazepril-treated group compared with the cholesterol group (by 55 and 63%); these parameters were 35 and 17% lower, respectively, with valsartan. The ratio of proliferating macrophages to smooth muscle cells (SMCs) was 3:1 in the cholesterol group, 1:1 in the benazepril and 2:1 in the valsartan-treated group. These results indicate that benazepril could reduce atherosclerotic progression by decreasing macrophage proliferation and accumulation in the arterial wall. The mechanisms for reducing atherosclerotic progression by benazepril and valsartan may be related to reduction of TG and blockade of the angiotensin II action.

Testicular atrophy, zinc concentration, and angiotensin-converting enzyme activity in the testes of vitamin A-deficient rats.

Angiotensin-converting enzyme (ACE) as a part of the renin angiotensin system (RES) regulates blood pressure and fluid and electrolyte homeostasis, and the enzyme is considered to have a function in reproduction. Reduced enzyme activities have been observed in atrophied testes as a results of zinc and pituitary deficiencies. Vitamin A deficiency causes atrophy of testes. The present study was conducted on three groups of male, 3-wk-old, Wistar rats. After 54 d of the experimental period, testicular weights of the vitamin A-deficient rats (A- group, allowed free access to vitamin A- deficient diet) was significantly lower than its pair-fed, PF (given restricted amount control diet) and A+ (allowed free access to control diet) groups. Zinc concentrations and both soluble and particulate ACE activities in the testes of vitamin A- deficient rats (A- group) were significantly lower than the other two groups. No significant differences were observed regarding zinc concentration, particulate ACE, and total ACE activities in the testes of PF and A+ groups. Vitamin A deficiency did not significantly affect the enzyme activity in the lung. From the observations of the present study, we speculate that testicular atrophy in vitamin A deficiency may have resulted from lower zinc concentration and decreased ACE activity in that organ.

Development of a new assay for measurement of total angiotensin-converting enzyme after inhibition.

1. We set out to develop a simple, rapid, highly sensitive and reproducible assay to quantify total angiotensin-converting enzyme (ACE) in serum and tissues after ACE inhibition. 2. Total ACE was measured by dissociating ACE inhibitor from the enzyme using zinc chelation (EDTA). The enzyme reconstituted with zinc ion (ZnSO4) was then measured by enzymatic fluorimetric assay. Angiotensin-converting enzyme inhibition was produced in vitro by incubation of enalaprilat or perindoprilat with human serum or cell membranes from rat heart. To achieve the highest recovery rate, we studied concentrations of EDTA from 1 to 300 mmol/L with a pH range from 4 to 13, where the incubation times of EDTA were between 1 and 12 h. After washing off ACE inhibitor and excess EDTA, the resulting concentrates and pellets were then resuspended with Tris buffer containing ZnSO4 to restore ACE activity. 3. The optimal assay conditions to dissociate the drug from the enzyme were 300 mmol/L EDTA in Tris buffer at pH 11 for 12 h with cell membranes and 23 mmol/L EDTA at the same pH incubated for 4 h with serum. The recovery of ACE activity was 81.7 +/- 15.8 and 97.3 +/- 2.9% in tissues and serum, respectively. Intra- and interassay variability coefficients were 5.6 and 12.8% in tissue, respectively, 3.2 and 13.0% in serum, respectively. The method allows quantification of ACE in 100 microliters or less serum and 20 mg or less tissue. 4. The total ACE concentration after ACE inhibition can be determined by this assay, which is suitable for micro-sampling studies of both tissue and serum ACE levels.

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells.

Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.

Antenatal glucocorticoid therapy suppresses angiotensin-converting enzyme activity in rats with nitrofen-induced congenital diaphragmatic hernia

Background/Purpose: Neonates with congenital diaphragmatic hernia (CDH) have a high morbidity and mortality rate caused by pulmonary hypoplasia associated with pulmonary hypertension (PH). In experimental CDH, antenatal glucocorticoid therapy improves surfactant biochemical immaturity, enhances lung compliance, and induces morphological maturation in CDH rats. The effects of steroid therapy on preventing or treating PH in this condition have not been studied. Angiotensin converting enzyme (ACE), which is produced by the vascular endothelium, is implicated in the pathogenesis of pulmonary hypertension. The aim of this study was to evaluate the effect of antenatal glucocorticoid therapy on ACE activity and expression in CDH rat lungs. Methods: CDH was induced in fetal rats by the maternal administration of 100 mg nitrofen on day 9.5 of gestation (term, day 22). Dexamethasone (Dex) (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 before delivery of the fetuses by cesarean section on day 21.5. Control animals received olive oil (OO) by gavage and normal saline (NS) as vehicle injection. ACE activity was measured spectrophotometrically in the lungs of rats from four treatment groups: CDH-NS, non-CDH-NS, CDH-Dex, and OO-NS controls. Total lung ACE activity (mU per lung) was significantly lower in CDH-NS ( P = .002) and CDH-Dex ( P = .004) rats compared with non-CDH-NS and OO-NS controls (9.1 ± 1.0 and 10.7 ± 1.3 v 16.2 ± 1.6 and 15.4 ± 1.7). When specific ACE activity (mU/mg protein) was derived by expressing ACE activity per milligram of lung protein, CDH-nS animals showed elevated specific ACE activity ( P = .05) compared compared with CDH-NS and Non-CDH-NS rats (4.0 ± 0.4 v 6.31 ± 1.1 and 5.83 ± 0.54). The specific ACE activity levels of CDH-Dex rats were equivalent to those seen in the lungs of OO-NS controls ( P = .24). Conclusion: Antenatal steroid therapy, by suppressing pulmonary ACE activity, may reduce the risk of pulmonary hypertension developing in human newborns with antenatally diagnosed CDH.

Angiotensin I-converting enzyme genotype influences arterial response to injury in normotensive rats.

Two normotensive strains of rat, the Lou and Brown Norway (BN) strains, have contrasting levels of plasma angiotensin-converting enzyme (ACE). To investigate the degree of genetic determination of ACE expression, a polymorphic marker of the ACE gene was analyzed in inbred rats of the two strains. The two inbred strains were shown to bear different alleles for a polymorphic marker at the ACE gene. The segregation of the alleles of this marker and the plasma ACE levels were studied in a group of F2 rats issued from a cross between Lou and BN rats. The degree of genetic determination of plasma ACE activity was estimated to be 94% in the F2 cohort. The ACE locus accounts for 74% of total plasma ACE variance. ACE activity and mRNA expression in lungs were also genetically determined. The difference observed in ACE mRNA accumulation in the lungs between the two strains was due to a difference in the transcriptional rate of the ACE gene, as shown in nuclear run-on experiments. No differences were observed in arterial blood pressure of homozygous F2 progeny. In these animals, ACE genotype did not interfere with the pressor or the depressor responses to ACE-dependent vasoactive peptides. There was a significant effect of strain on constitutive or inducible membrane or soluble ACE activity in primary cultures of vascular cells. Neointima formation in the carotid artery 14 days after balloon injury was also influenced by the genotype in F2 homozygous progeny, whereas the medial area was not. These results demonstrate that there is a close relationship between the genetically determined ACE expression and the inducibility of the ACE gene. The degree of genetic determination of ACE expression in inbred rat strains offers a unique opportunity to study the interaction between genetic and environmental determinants of ACE expression and its involvement in response to experimental cardiovascular and renal injury.

Acute and chronic in vivo inhibition of angiotensin-converting enzyme by perindopril in the endothelium and adventitia of large arteries and organs of the rabbit.

Angiotensin-converting enzyme (ACE) inhibitors are widely used in treating hypertension and chronic heart failure, but their precise sites and mechanisms of the actions are not completely understood. In this study, we evaluated the acute and chronic in vivo inhibition of ACE by perindopril in both the endothelium and adventitia of large blood vessels including the aorta, carotid, and femoral arteries, heart, lung, and kidney by using in vitro autoradiography with [(125)I]351A as a ligand. After short-term (0.1, 0.3, and 1 mg/kg) or long-term oral administration (0.3 mg/kg), perindopril significantly inhibited plasma ACE (p < 0.001), the plasma angiotensin II (Ang II)/Ang I ratio (p < 0.01), and decreased mean arterial pressure (p < 0.001) in a dose-related manner. In the aorta, carotid, and femoral arteries, free ACE was inhibited to a similar extent in both the endothelium and adventitia by perindopril, in a dose-dependent manner, whereas total ACE in both layers of these vessels was unaltered. Similar short- and long-term ACE inhibition by perindopril was observed in the lung and heart, with somewhat greater inhibition of kidney and plasma ACE. Vascular and tissue ACE inhibition correlated highly with both plasma ACE and the plasma Ang II/Ang I ratio (r = 0.63-0.89; p < 0.001). Whereas the effects of perindopril on blood pressure, plasma Ang II/Ang I ratio, plasma and vascular ACE were all highly dose dependent, there were no significant differences on the degree of ACE inhibition observed between the three large blood vessels or between their adventitial and endothelial layers. These results demonstrate that perindopril readily penetrates the vascular wall after short- or long-term oral administration, and in a dose-dependent manner, potently inhibits both endothelial and advential vascular ACE to a comparable degree. Therefore ACE inhibitors may be beneficial in inhibiting both circulating Ang II and its local formation in the vascular wall.

Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

Previous studies suggest that while lung angiotensin converting enzyme (ACE) activity is reduced during chronic hypoxia, inhibitors of ACE attenuate hypoxic pulmonary hypertension. In an attempt to explain this paradox we investigated the possibility that whole lung ACE activity may not reflect local pulmonary vascular ACE expression. The experimental approach combined in vivo hemodynamic studies in control and chronically hypoxic rats, measurement of whole lung ACE activity, and evaluation of local pulmonary vascular ACE expression by in situ hybridization and immunohistochemistry. Total lung ACE activity was reduced to 50% of control activity by 5 d of hypoxia and remained low for the duration of the study. Immunohistochemistry showed a marked reduction of ACE staining in alveolar capillary endothelium. However, an increase in ACE staining was observed in the walls of small newly muscularized pulmonary arteries at the level of alveolar ducts and walls. In situ hybridization studies showed increased signal for ACE mRNA in the same vessels. Inhibition of ACE by captopril during chronic hypoxia attenuated pulmonary hypertension and markedly reduced distal muscularization of small pulmonary arteries. In addition, we demonstrated marked longitudinal variation in ACE expression along the normal pulmonary vasculature with the highest levels found in small muscular arteries associated with terminal and respiratory bronchioles. We conclude that local ACE expression is increased in the walls of small pulmonary arteries during the development of hypoxic pulmonary hypertension, despite a generalized reduction in alveolar capillary ACE expression, and we speculate that local arteriolar ACE may play a role in the vascular remodeling associated with pulmonary hypertension.

Angiotensin-converting enzyme activity is increased in lungs of rats with pulmonary hypoplasia and congenital diaphragmatic hernia.

Lung hypoplasia (LH) and pulmonary hypertension are responsible for the high mortality rate in congenital diaphragmatic hernia (CDH) patients. Angiotensin-converting enzyme (ACE) plays a role in the regulation of pulmonary vascular resistance in the postnatal period and might be involved in the development of pulmonary hypertension of the newborn. A study was made of the development of ACE activity spectrophotometrically in a rat model of LH and CDH. It was previously shown that the lungs in this model are hypoplastic and the muscularization of the pulmonary vascular bed is increased. CDH was induced in fetal rats by oral administration of 115 mg/kg Nitrofen to the mother on day 10.5 of pregnancy. Fetuses were delivered by hysterotomy on days 19, 20, 21, and 22. Nitrofen-exposed rats showed significantly lower lung weights and not statistically significant lower total ACE activities than in controls. ACE activity expressed per milligram lung wet weight and per milligram protein was significantly increased compared to controls. ACE converts angiotensin I to the vasoconstrictor angiotensin II, and it inactivates the vasodilator bradykinin. Increased ACE activity may therefore contribute to pulmonary hypertension. Whether ACE and angiotensin II levels are increased in human newborns with a diaphragmatic defect and whether they contribute to the development of persistent pulmonary hypertension has not been studied up till now.