Pulmonary Angiotensin-converting Enzyme Activity Research Articles

Alternative Roles of STAT3 and MAPK Signaling Pathways in the MMPs Activation and Progression of Lung Injury Induced by Cigarette Smoke Exposure in ACE2 Knockout Mice.

Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2-/-) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2-/- mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2-/- mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2-/- mice. CS exposure increased the pulmonary p-p38, p-JNK and p-ERK1/2 level in all mice. In ACE2-/- mice, a significant increase p-STAT3 signaling was detected; however, no effect was observed on the p-STAT3 level in WT mice. Our results support the hypothesis that ACE2 deficiency influences MMPs activation and STAT3 phosphorylation signaling to promote more pulmonary inflammation in the development of lung injury.

Effect of chloride and diamide on sheep kidney, lung and serum angiotensin converting enzyme

The effect of chloride and diamide on angiotensin converting enzyme (ACE) activity from sheep kidney, lung and serum was investigated by using Hip-His-Leu as substrate. Optimum chloride concentrations were 400-1000 mM for kidney, 700 mM for lung and 1000 mM for serum. Optimum chloride concentration increased ACE activity of serum and lung 1.70 folds and 2.73 folds respectively of the activity at physiological chloride concentrations, suggesting that the effect of salt on blood pressure may be due to the chloride sensitivity of ACE. The difference in effect of chloride on lung, kidney and serum ACE suggest that each tissue ACE has unique three dimentional structure. Increased pulmonary and serum ACE activity on pretreatment with diamide indicates that tissue oxidation may alter blood pressure.

Possible Modulatory Role of Nitric Oxide in Lung Toxicity Induced in Rats by Chronic Administration of Bleomycin

Background: The present study was undertaken to evaluate whether stimulation or inhibition of nitric oxide (NO) synthesis could affect lung toxicity induced by chronic administration of bleomycin (BLM). L-arginine (ARG) and N^G-nitro-L-arginine methyl ester (L-NAME) were employed as NO precursor and NO synthesis inhibitor, respectively. Methods: BLM was administered intraperitoneally to male Wistar rats at a dose of 15 mg/kg, 3 times a week, for a total period of 4 weeks. ARG (500 mg/kg/day) and L-NAME (100 mg/kg/day) were given in drinking water, the treatments commenced with BLM and continued up to the end of the experiment. Appropriate controls were performed. Results: BLM treatment resulted in a pronounced fall in the average body weight of animals, together with a rise in the lung weight/body weight ratio. In the lung tissue, elevated levels of hydroxyproline (HP) and lipid peroxides (LP) as well as decreased activity of angiotensin converting enzyme (ACE) further evidenced the toxicity. Pulmonary level of NO end products, nitrite and nitrate, tended to rise but did not reach a significant level. Glutathione (GSH) content and GSH-peroxidase activity measured in the lung remained unaltered. In animals given concurrent treatment of BLM and ARG, a remarkable rise in the pulmonary level of nitrite and nitrate was observed. Average body weight was still decreased when compared with the untreated control group, but the decrease was significantly less than that observed in the BLM group. In addition, ARG decreased the extent of BLM-induced elevations of lung HP and LP levels. Meanwhile, ARG failed to significantly affect the BLM-evoked decrease in pulmonary ACE activity and increase in lung weight/body weight ratio. In animals given simultaneous treatment of BLM and L-NAME, noticeable reductions in the pulmonary levels of nitrite/nitrate and GSH were detected. BLM-induced decrease in body weight and increase in lung weight/body weight ratio were accentuated by L-NAME co-treatment. Furthermore, administration of L-NAME led to more profound elevations in lung HP and LP levels, without affecting the decrease in pulmonary ACE activity elicited by BLM. Conclusion: In principle, the present findings indicate that the lung toxicity exerted by chronic administration of BLM is alleviated by ARG, but exacerbated by L-NAME supplementation. This could indicate a possible protective role of NO.

Endothelial function studies in pulmonary vascular disease: Determination of angiotensin converting enzyme activity in humans (Review)

Angiotensin converting enzyme (ACE, kininase II) is an endothelial luminal ectoenzyme. The majority of ACE has been found on the pulmonary capillary endothelium (PCE). Pulmonary capillary endothelium-bound (PCEB)-ACE has been extensively studied by means of indicator dilution techniques in animals and man. We have recently developed and applied methodologies for assaying pulmonary capillary endothelium-bound (PCEB) angiotensin converting enzyme (ACE) activity in man. This article provides a summary of our studies in human subjects utilizing similar methodology. Trace amounts of the specific ACE substrate, (3)H-benzoyl-Phe-Ala-Pro ((3)H-BPAP; 40 microCi or 2 nmol), was injected as a bolus into the subclavian vein of patients and immediately blood was withdrawn from a radial arterial catheter. Plasma concentrations of surviving substrate and product ((3)H-benzoyl-Phe) were estimated and BPAP utilization was calculated during a single transpulmonary passage. To investigate the PCE in this manner we tested the hypothesis that PCEB-ACE is depressed in patients diagnosed with acute lung injury and estimated interaction of an ACE inhibitor (enalaprilat) with PCE in human subjects. An inverse correlation was found between the pulmonary endothelium-bound ACE activity (v) and the lung injury score (r=0.379; p<0.01). Similarly, an inverse correlation was found between the pulmonary capillary perfusion index (CPI) and the lung injury score (r=0.284, p<0.05). PCEB-ACE activity in the group of patients with mild lung injury was significantly different from the group of control patients (0.44 +/- 0.048 vs. 1.15 +/- 0.05; p<0.01). Trace amounts (1.5 microg/kg) of enalaprilat had no significant effect on mean arterial pressure (91 +/- 6 vs. 84 +/- 7 vs. 88 +/- 6 mmHg for T(1), T(2) and T(3), respectively), but significantly decreased PCEB-ACE activities. When normalized to pre-drug (T(1)) activity levels, enalaprilat inhibited PCEB and SE ACE activity at T(2) by 74 +/- 6 and 68 +/- 6%, respectively. PCEB-ACE measurements can be used in clinical practice for estimating PCE functions and can provide new insight into the physiology of the pulmonary circulation.

Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo.

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [(3)H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [(14)C]AMP were estimated, and the first-order reaction parameter A(max)/K(m), where A(max) is the product of enzyme mass and the catalytic rate constant and K(m) is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A(max)/K(m) were reduced in WHHL (1.69 +/- 0.16 vs. 2.9 +/- 0.44 and 599 +/- 69 vs. 987 +/- 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K(m) and A(max) values were estimated separately under mixed-order reaction conditions. No differences in K(m) values were found (9.79 +/- 1 vs. 9.9 +/- 1.31microM), whereas WHHL rabbit A(max) was significantly decreased (5.29 +/- 0.88 vs. 7. 93 +/- 0.8 micromol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.

Endothelin Regulates Angiotensin-Converting Enzyme in the Mouse Kidney

Using the orally active endothelin-A- (ET(A)) receptor antagonist LU135252, we determined whether endothelin-1 (ET-1) and/or dietary fat may be involved in angiotensin-converting enzyme (ACE) regulation in vivo. In C57BL6/J mice, renal and pulmonary tissue ACE activity (nmol/l His-Leu/mg protein) was measured and ACE mRNA expression, tissue ET-1 protein content and nitrite/nitrate level were measured in the kidney. Western-type diet increased renal ACE activity by 70% (55 +/- 4 vs 33 +/- 3 nmol/l His-Leu/mg protein, p < 0.05) and increased renal ET-1 levels (267 +/- 19 pg/g vs 190 +/- 18, p < 0.05). Chronic LU135252 treatment completely prevented activation of renal ACE activity (13.3 +/- 0.3 His-Leu/mg protein nmol/l, p < 0.05) independent of ACE mRNA expression or renal ET-1 protein levels. Thus, dietary fat activates renal ACE activity and ET-1 is involved in regulation of tissue ACE activity in vivo independently of ACE mRNA expression.

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.

L-arginine prevents cyclosporin A-induced pulmonary vascular disfunction

Cyclosporin A is known to alter endothelium-dependent responses to different agonists. Few data are available concerning the effect of cyclosporin A on the pulmonary vascular bed. The endothelium-dependent responses to acetylcholine (20 micrograms), bradykinin (5 micrograms), and substance P (5 micrograms) were investigated in a dog model of left lung autoperfusion at constant flow. The vasodilator response to bradykinin and substance P was significantly decreased with cyclosporin A (20 mg) administration. The average decreases in pulmonary arterial pressure with bradykinin were 5.4 +/- 1.5 mm Hg and 2.4 +/- 0.4 mm Hg before and after cyclosporin A administration, respectively (p = 0.04). The average decreases in pulmonary arterial pressure with substance P were 4.4 +/- 1.0 mm Hg and 1.8 +/- 0.5 mm Hg before and after cyclosporin A administration, respectively (p = 0.04). The responses to acetylcholine and the endothelium-independent relaxing agent nitroglycerin were not significantly affected by cyclosporin A. The effects of cyclosporin A on endothelium-dependent responses to bradykinin and substance P were overcome by the administration of L-arginine (200 mg/kg intravenously). The decreased response to bradykinin and substance P after cyclosporin A administration was not significantly affected by indomethacin, a cyclooxygenase inhibitor. The pulmonary angiotensin-converting enzyme activity was also measured using [3H]benzoyl-phenylalanyl-glycyl-proline, an inactive angiotensin-converting enzyme substrate. There was an average [3H]benzoyl-phenylalanyl-glycyl-proline hydrolysis of 54% +/- 2% and 55% +/- 2% before and after cyclosporin A administration, respectively (not significant). The present study suggests that cyclosporin A selectively decreases endothelium-dependent responses to bradykinin and substance P without affecting the cyclic guanosine monophosphate-dependent pathway in the canine pulmonary vascular bed. The decreased endothelium-dependent responses to bradykinin and substance P are not related to increased angiotensin-converting enzyme activity. The toxic effect of cyclosporin A on endothelium-dependent responses is reversible by the administration of L-arginine, a source of substrate for nitric oxide.

Tissue-specific regulation of angiotensin-converting enzyme by angiotensin II and losartan in the rat.

Angiotensin II (Ang II) may regulate the release of components of the renin-angiotensin system in a tissue-specific manner. In order to study: (1) the effect of Ang II on gene expression and tissue levels of angiotensin-converting enzyme (ACE), and (2) the mechanism of the possible Ang II effect, we treated normal rats with Ang II and Losartan, an angiotensin AT1-receptor antagonist. Forty normal rats received Ang II (n = 20) at a rate of 200 ng kg-1 min-1 or 0.9% NaCl (n = 20) subcutaneously for 3 days using osmotic Alzet minipumps. Ten rats in both groups received Losartan (15 mg kg-1 day-1) in their drinking water, while the rest received tap water. ACE activity and mRNA levels were measured from pulmonary, cardiac, and renal tissue. Ang II treatment resulted in significant increases in blood pressure and heart weight as well as an increase in plasma Ang II concentration and a decrease in plasma renin activity. Simultaneous treatment with Losartan reduced the Ang II-induced effects on blood pressure and heart weight, and attenuated the Ang II-induced decrease in plasma renin activity. Pulmonary ACE activity and mRNA levels decreased during Ang II treatment, and these effects were not modified by simultaneous treatment with Losartan. Cardiac and kidney ACE activities and mRNA levels did not change significantly during Ang II treatment, but Losartan increased cardiac ACE activity (and decreased pulmonary ACE activity). The data indicate that Ang II regulates gene expression and activity of ACE in a tissue-specific manner in the rat, an effect probably involving angiotensin receptor subtype(s) different from the AT1-receptor.

Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets.

1. Possible associations between maternal nutrition in pregnancy and non-communicable diseases of adulthood were assessed using a rat model. Rats were habituated to diets containing a range of protein levels (18, 12, 9 and 6% by weight), over a 14 day period, before mating. The low protein diets were maintained throughout pregnancy. Lactating mothers and their offspring were transferred to a standard chow diet (20% protein). 2. Pregnant rats demonstrated a graded response to the diets, with those fed 9 and 6% protein tending to consume less energy and gain less weight than 18% protein fed controls. Litter size and newborn death rates were not significantly altered by the low protein diets. 3. Offspring of 12 and 9% protein fed dams were grossly normal, gaining weight at a similar rate to those born to 18% protein fed control rats. Offspring of the 6% protein fed dams were smaller than pups from all other groups, over a 21 week period. 4. At 9 weeks of age, systolic blood pressure was determined in the offspring. All offspring from the three low protein groups were found to have significantly elevated blood pressure (15-22 mmHg) relative to the control group. An inverse relationship between maternal protein intake and the systolic blood pressure of the offspring was observed. Blood pressure remained elevated in the offspring of the 9 and 6% protein fed dams until 21 weeks of age. The observed hypertension was associated with increased pulmonary angiotensin-converting enzyme activity in the low protein groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II (AII) and adrenal gland AII receptors in rats with congenital hypothyroidism

Plasma angiotensin II (AII) concentration, plasma renin concentration (PRC), lung angiotensin-converting enzyme (ACE) activity and adrenal gland AII receptor isoforms have been evaluated in the postnatal development of the rat, in order to determine the alterations of the renin-angiotensin system (RAS) which occur in congenital hypothyroidism. The developmental AII profiles observed in control and hypothyroid rats were quite similar. Thus, AII was elevated at birth and declined at week 5 of life to reach adult values. However, AII levels were lower in hypothyroid than in euthyroid animals before their stabilization. On the other hand, PRC in newborn hypothyroid and euthyroid animals was higher than in adulthood, being significantly increased immediately after birth in hypothyroid rats. Pulmonary ACE activity in both experimental groups was low at birth and increased with age. This increase was greater in euthyroid rats than in congenitally hypothyroid animals from day 28 of life. These results confirm that plasma AII levels in rats are predominantly controlled by plasma renin activity, although other factors, such as renin substrate availability, may be responsible for the reduced plasma AII concentration in congenital hypothyroidism during the first weeks of life. The developmental profile of the adrenal gland AII receptor showed four isoforms, with pI values of 6.8, 6.7, 6.5 and 6.3. AII receptor-complex expression increased with age but, in congenitally hypothyroid rats, a higher induction of AII receptor isoforms migrating to pI 6.8, 6.5 and 6.3 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Feedback regulation of angiotensin converting enzyme activity and mRNA levels by angiotensin II.

Although renin and angiotensinogen are known to be subject to feedback regulation, the effects of angiotensin II (Ang II) on the regulation of angiotensin converting enzyme (ACE) gene expression and enzymatic activity have not yet been studied. Therefore, the effects of exogenous Ang II infusion and ACE inhibition on ACE mRNA expression were examined. Ang II was infused intravenously in male Sprague-Dawley rats for 3 days at 100 (low dose), 300 (medium dose), or 1,000 (high dose) ng/kg per minute (n = 8 for each group). Compared with control (vehicle infusion, n = 8), Ang II infusion increased plasma Ang II concentration (62, 101, 126 [p < 0.05], and 187 [p < 0.05] fmol/ml) and mean arterial blood pressure (106, 119 [p < 0.05], 134 [p < 0.05], and 125 mm Hg for control, low, medium, and high doses, respectively). Ang II infusion decreased ACE mRNA levels in the lung (57%, 52%, and 51%; p < 0.05 for each) and testis (49%, 63%, and 53% of control for low, medium, and high doses, respectively; p < 0.05 for each), two major sites of ACE synthesis. There was, albeit less pronounced, a parallel decrease in pulmonary ACE activity (4.38, 3.92, 3.07 [p < 0.05], and 3.48 [p < 0.05] nM/mg per minute for control, medium, and high doses, respectively). In contrast, serum (54, 50, 48, and 38 [p < 0.05] nM/ml per minute) and testicular (2.63, 2.08 [p < 0.05], 2.24, and 2.18 nM/mg per minute for control, low, medium, and high doses, respectively) ACE activities displayed only minimal change in animals infused with Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic administration of platelet-activating factor in rat reduces specific pulmonary uptake of circulating monoclonal antibody to angiotensin-converting enzyme.

The biodistribution of radiolabeled mouse monoclonal antibody (MoAb) to angiotensin-converting enzyme (ACE) and control, nonimmune mouse IgG in platelet activating factor (PAF)-treated rats was studied. The blood level of both preparations was slightly decreased (90% of the control) in PAF-treated rats. Specific pulmonary accumulation of anti-ACE MoAb was reduced to 50% of control in contrast to a doubling in nonspecific pulmonary uptake of non-immune IgG. The changes in anti-ACE MoAb biodistribution were lung-specific and were accompanied by decrease in the pulmonary ACE activity (to 60% of control) and increase in serum ACE activity (to 170% of control). Thus anti-ACE MoAb reveals PAF-induced changes in the status of the pulmonary ACE and therefore can be used for the studies of pathology of the pulmonary endothelium.

A study of ozone-induced edema in the isolated rat lung in relation to arachidonic acid metabolism, mixed-function oxidases and angiotensin converting enzyme activities

In order to elucidate the role of arachidonic acid in the pathogenesis of ozone-induced pulmonary edema, isolated rat lungs were exposed to 14C-arachidonic acid in the presence or absence of ozone and the incorporation of radiolabelled arachidonate into pulmonary cell lipids was studied. The perfusates from these studies were also subjected to differential extraction and thin layer chromatography (t.l.c.) to determine synthesis of both cyclo-oxygenase and lipoxygenase products. In the presence of an edemagenic concentration of ozone, isolated lungs incorporated significantly less exogenous arachidonic acid into phosphatidyl choline and phosphatidyl ethanolamine, whereas incorporation into phosphatidyl inositol or serine was not affected. The edemagenic concentration of ozone also increased production of a variety of arachidonic acid metabolites via cyclo-oxygenase and lipoxygenase pathways. In separate studies, a similar ozone exposure did not affect 14CO 2 production, resulting from the metabolism of 14C-antipyrine by mixed function oxidases (MFO). Similarly, an edemagenic concentration of ozone did not affect pulmonary angiotensin converting enzyme activity (ACE) as determined by the rate of formation of 14C-hippuric acid from 14C-hippuryl-histidyl-leucine ( 14C-HHL). Thus, acute ozone exposure is specifically associated with a reduced incorporation of arachidonate into phospholipids and with an increased conversion of arachidonate into bio-active metabolites.

Resolution of Impaired Pulmonary Function and Pulmonary Hypertension after Phorbol Ester Administration in Rabbits

To assess metabolic functions of the pulmonary circulation during lung injury and subsequent recovery from injury, we measured angiotensin-converting enzyme (ACE) activity by means of benzoyl-phenylalanyl-alanyl-proline (BPAP) hydrolysis and 5-hydroxytryptamine (5-HT) removal in vivo in three groups of anesthetized rabbits. One group was treated with 30 micrograms/kg/day phorbol myristate acetate (PMA) intravenously 10 times over 14 days (PMA group). A second group received the same PMA treatment but was not studied until 14 days after the last treatment (PMA/recovery group). A third group was treated with vehicle alone. At the end of PMA treatment, rabbits had an elevated pulmonary artery pressure and depressed ACE activity, expressed as the ratio Vmax/Km. Decreased Vmax/Km for ACE was due to a significant reduction in apparent Vmax for BPAP (control = 235 +/- 37, PMA = 139 +/- 12 nmol/s). Km was unchanged (control = 25 +/- 4, PMA = 31 +/- 7 microM). Uptake of 5-HT was unaffected by PMA treatment. After 2 wk of recovery (PMA/recovery group), pulmonary hypertension had resolved. In this group, Vmax for BPAP hydrolysis was not significantly different from control (280 +/- 18 nmol/s), but Km was significantly increased (48 +/- 5 microM). We conclude that repeated exposure of rabbits to PMA results in lung injury manifested as depressed pulmonary ACE activity and pulmonary artery hypertension. Although much of these alterations were reversible within 2 wk after discontinuing PMA, an increase in apparent Km of ACE may be a more persistent alteration in vascular endothelial cell dysfunction.

Correlation of pulmonary ACE activity and capillary surface area during postnatal development.

Although a considerable amount of information is available regarding the remodeling and growth of the pulmonary arterial circulation, relatively little is known regarding postnatal development of the pulmonary microcirculation. We hypothesized that the maximal velocity (Vmax) of pulmonary angiotensin-converting enzyme (ACE) activity, measured from indicator-dilution outflow curves using a synthetic substrate, 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), is directly related to the capillary endothelial cell surface area in the lungs of developing lambs. Accordingly we measured apparent kinetics of pulmonary ACE activity in 22 anesthetized ventilated lambs (2-171 days old) and compared our functional assessment to simultaneous in vivo determinations of CO diffusing capacity (DLCO) and postmortem structural assessment of alveolar septal dimensions using stereology and electron microscopy. There was a progressive increase in Vmax of ACE in this age group, with little change in apparent affinity for BPAP. Similar functional manifestation of growth was noted by an age-dependent increase in DLCO. Neither Vmax nor DLCO was significantly affected by an increase in left atrial pressure to 19 Torr (via inflation of a balloon in the left atrium), suggesting little recruitment of vessels under conditions of the present protocol. A close correlation was observed when either Vmax for ACE activity or DLCO was plotted vs. capillary endothelial cell surface area. Double logarithmic transformation of capillary endothelial cell surface area, Vmax-ACE and DLCO vs. lung volume revealed power functions with slopes all greater than that predicted from isotropic growth, suggesting selective differential postnatal development of the endothelium of the alveolar septum in lambs from 2-171 days of age.

Effects of changes in pulmonary perfusion and surface area on endothelial ACE activity.

Measurement of transport or enzymatic processes associated with pulmonary endothelial cells could provide unique information regarding the physiologic function of the microcirculation as well as data describing the biochemical integrity of these cells in acute lung injury. Since an important goal of such measurements is to quantify the kinetics of reactions of substrates (indicators) with the endothelium, it would be highly advantageous to account for (convective) phenomena related to blood flow and its distribution which also influence whole organ metabolism. In the present report, we describe studies that have utilized a nonlinear model of organ metabolism to estimate Vmax, the apparent maximal velocity and Km, concentration at one-half Vmax of angiotensin-converting enzyme (ACE) activity. Our hypotheses have been that (a) there is sufficient data to calculate apparent kinetic constants from indicator-dilution outflow curves after injection into the pulmonary circulation of a radiolabelled synthetic substrate for ACE, and (b) Vmax for ACE is a property related to the amount of enzyme located on the endothelial cells, and as such, should be directly proportional to perfused surface area of an organ. In isolated perfused rabbit lungs, when flow was increased over a two-fold range, but surface area was unchanged, neither Vmax nor Km for ACE activity was significantly altered. When indicator-dilution measurements of pulmonary ACE activity were made in lambs from 1-171 days of age there was a progressive increase in Vmax with no significant change in Km as a function of age. The increase in Vmax was closely correlated with an independent measure of surface area, carbon monoxide diffusing capacity, and a morphometric measure of capillary endothelial cell surface area (stereology at electron microscopic level) made at post-mortem. These experimental observations in whole organs support our hypotheses and predictions regarding the metabolism or removal of substrates from the circulation by means of a saturable process.

Hyperoxia-induced converting enzyme insufficiency in conscious rat: cardiovascular effects.

The effects of exposing rats to hyperoxia (100 percent O2) at normal atmospheric pressure for periods of 24-48 hours on components of the renin-angiotensin system and on blood pressure control were examined. Intrapulmonary conversion of angiotensin I (AI) to angiotensin (AII) was assessed using an isolated lung preparation perfused at constant flow. Exposure of rats to hyperoxia for 44-48 hours reduced single pass conversion of AI to AII in the pulmonary circulation from control levels of 82 +/- 4 to 29 +/- 5 percent (p less than 0.001). AII levels in trunk blood of 44-48 hour O2 exposed animals were 5.2 +/- 1.9 pg/ml, compared to 37.9 +/- 10.0 pg/ml in controls (p less than 0.001). Mean arterial pressure decreased significantly from 117 +/- 4.3 to 103 +/- 6.7 mmHg (p less than 0.05) in the O2 exposed group despite a threefold increase in plasma renin activity. The pressor response to exogenous AI was significantly diminished by O2 exposure, while the pressor response to exogenous AII remained unchanged from control. Pulmonary angiotensin-converting enzyme activity fell to approximately 50 percent of control in O2 exposed animals, but circulating converting enzyme activity was not change in this group. None of these alterations was apparent following 24 hours of hyperoxic exposure. These data suggest that O2 induced impairment in activity of angiotensin-converting enzyme at the endothelial membrane level has functionally significant effects on cardiovascular homeostasis, probably via reduced generation of endogenous AII.

Kinetics of pulmonary angiotensin-converting enzyme activity in conscious developing lambs.

Apparent enzyme kinetics were determined for pulmonary angiotensin-converting enzyme (ACE) as a function of postnatal development and alveolar hypoxia in intact conscious lambs (9 newborn and 7 sheep, 8-23 wk). We injected into the right atrium a mixture of indocyanine green and 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), a synthetic substrate for ACE, and sampled blood from the aorta at 1-s intervals. From this, we quantified the %BPAP metabolism during a single pass through the lungs by use of indicator-dilution techniques. By adding unlabeled BPAP to the injectate, combining outflow data from two to three measurements, and applying a nonlinear model of pulmonary metabolism (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 51:405-414, 1981), we could determine 1) apparent maximum velocity (Vmax); 2) concentration at which velocity was one-half Vmax (Km); and 3) alpha, a measure of perfusion heterogeneity. All animals were studied during normoxia and while breathing 10% O2 for at least 15 min. Seven of the newborn lambs were studied 1 wk later. Km in the newborn was 15 +/- 3 microM and did not change significantly with age or hypoxia. Vmax increased markedly with development (newborn: 14 +/- 2 nmol X s-1 X g dry lung-1; sheep: 38 +/- 5 nmol X s-1 X g dry lung-1; P less than 0.01). Alveolar hypoxia significantly decreased Vmax in the newborn only (P less than 0.05); there was no significant change in Vmax in the same animal studied 1 wk later or in the older group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged reduction in serum angiotensin converting enzyme activity after treatment of rabbits with bleomycin

Previous work from this laboratory ( J. S. Lazo, J. D. Catravas, and C. N. Gillis, Biochem. Pharmacol. 30, 2577–2584, 1981; J. D. Catraveras, J. S. Lazo, and C. N. Gillis, J. Pharmacol. Exp. Ther. 217, 524–529, 1981) demonstrated that subacute bleomycin (BLM) administration (5 mg/kg) to rabbits three times weekly for 4 weeks produced a marked decrease in serum angiotensin converting enzyme (ACE) activity in addition to producing both morphological and biochemical evidence of pulmonary endothelial damage. In this work, the reversibility of the decreased serum ACE activity and the biochemical and morphological status of the pulmonary endothelium after a substantial drug-free period in rabbits was examined. Rabbits were injected sc three times weekly for 4 weeks with vehicle or BLM (5 mg/kg) and then maintained drug free for an additional 6 or 7 weeks. Serum ACE activity decreased over 60% during the BLM treatment but did not return to control levels at any time during the drug-free period. The pulmonary endothelium, however, appeared undamaged. No decrease in pulmonary ACE activity was detected either in vitro or in vivo nor was the single-pass pulmonary removal in vivo of [ 3H]norepinephrine and 5-[ 14C]hydroxytryptamine different between BLM-treated and control rabbits after the drug-free period. In addition, no evidence of pulmonary endothelial damage was observed by ligh and electron microscopy. Thus, reduction in serum ACE activity appears to be a durable reflection of BLM toxicity in rabbits that persists even in the absence of any detectable biochemical or morphologic endothelial injury.