Development of angiotensin-converting in fetal rat lungs K. B. WALLACE, M. D. BAILIE, AND Departmknts of Physiology, Pharmacology Michigan State University, East Lansing,
J. B. HOOK and Toxicology, and Human Michigan 48824
WALLACE, K.B.,M. D~AILIE, AND J. B. HOOK. Deuelopment of angiotensin-converti?lg enzyme in fetal rat lungs. Am. J. Physiol. 236(l): R57-R60, 1979 or Am. J. Physiol.: Regulatory Integrative Comp. Physiol. 5(l): R57-R60, 1979. -Angiotensin-converting enzyme (ACE) catalyzes rapid hydrolytic cleavage of angiotensin I to form angiotensin II (AII). Inasmuch as converting enzyme activity is present at birth and increases postnatally to adult values it was of interest to determine the prenatal development of ACE. Converting enzyme activity was determined in the 20,000 x g supernatant fraction of lung homogenates using hippuryk-histidyk-leutine (HHL) as substrate. Hippuric acid liberated by the hydrolysis of HHL was quantified spectrophotometrically, ACE activity was first detectable at 18 days of gestation and increased fourfold prior to birth (21 days gestation). Pulmonary ACE activity of l-day-old animals was twice that of fetuses at day 20 of gestation; however, this increase did not appear to result from ventilation alone. The Michaelis-Menten constant for fetal ACE (2.0 mM HHL) was not different from that calculated for ACE of adult rat lungs (2.6 mM). These data were interpreted to indicate that the age-related increase in ACE activity was due to greater ACE content as opposed to further activation of preexisting enzyme. This increase in fetal ACE activity may play an important role in preparing the renin-angiotensin system for postnatal function.
Sprague-Dawley strate-dependence
rats;
renin;
Eadie-Hofstee
plot;
K,,;
sub-
ENZYME (ACE) (peptidyldipeptide hydrolase, EC 3.4X5.1) catalyzes hydrolytic cleavage of the carboxy terminus dipeptide (His”-Leul”) from angiotensin I (AI). Although ACE was first isolated from plasma (26) the activity of this enzyme was found to be insufficient to account for AI conversion in vivo (18, 19). Angiotensin-converting enzyme has since been detected in nearly every tissue examined (6, 24). Subsequent localization of converting enzyme on the vascular endothelium of nearly every tissue suggests direct apposition of this enzyme to circulating AI (4). However, only lung is capable of liberating significant quantities of angidtensin II (AII) into the venous effluent following addition of AI to the perfusing medium (1, 18-20). It therefore appears that pulmonary ACE is responsible for the generation of AI1 destined for the systemic circulation, whereas, in tissues other than lung, rapid uptake and/or metabolism of AI1 accounts for the small quantities of this peptide in the venous effluent (21). ANGIOTENSIN-CONVERTING
036%6119/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
enzyme
Development,
Age-related differences in plasma renin, angiotensinogen, angiotensinase, and ACE activity have been reported in rats following birth (23, 29). In addition, the renin-angiotensin system is functional and responsive during intrauterine life (2, 3, 28). However, fetal ACE activity is very low compared to that of a.dult rats (29). The purpose of the present in vestigation was to determine the appearance and development of ACE in fetal rat lungs. METHODS
Pregnant female Sprague-Dawley rats of known gestational age were purchased from Spartan Research Animals, Inc. (Haslett, MI), housed separately, maintained on a normal laboratory chow diet (Wayne Lab Blocks), and allowed free access to tap water. On the day of the experiment, the pregnant mothers were killed and the fetuses decapitated in utero. Fetal lungs were immediately removed and washed in cold NaCl (0.9%) then weighed and homogenized (Polytron, Brinkmann Instruments) in 4 vol of cold 100 mM potassium phosphate-300 mM sodium chloride btier (pH 8.3). The homogenate was then centrifuged (Sorvall RCZ-B automatic refrigerated centrifuge) for 20 min at 20,000 x g and 4°C and the supernatant fraction was assayed for ACE activity. Under standard conditions, the enzyme preparations were assayed the same day for ACE activity, whereas the kinetic studies were performed after storage of the supernatant fraction at 4°C for 24 h. Lungs of littermates were pooled to represent one determination. Converting enzyme activity was determined by a modification of the method described by Cushman and Cheung (7). In brief, 0.1 ml of the 20,000 x g supernatant of fetal lung homogenates was incubated at 37°C with 0.15 ml hip&$-Zhistidyl-L-leucine (HHL; Vega Fox Biochemicals). The final concentration was 5.0 mM HHL under standard assay conditions. After a 30-min incubation period (Dubnoff Metabolic Shaking Incubator), the reaction was stopped by addition of 0.25 ml 1 N HCl, The hippuric acid formed from the ACE catalyzed hydrolysis of HHL was extracted into 1.5 ml ethyl acetate and centrifuged at 2,000 x g for 10 min (International Equipment Co.). A l.O-ml aliquot of the ethyl acetate Phase was then evaDorated at 40°C under nitrogen. Thi residual hippuric= acid was dissolved in 1 M NaCl (3.0 ml) and the optical density measured at 228 Society
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
R57
R58
WALLACE,
BAILIE,
AND
HOOK
nm (Beckman DB-GT spectrophotometer). ACE activity was expressed as nanomoles hippuric acid formed per minute per milligram 1ung protein. Protein was determined by th .e method of Low ry et al. (15). Statistical significance was determined by analysis of variance and comparison of regression lines (27). The 0.05 level of probability was used as the criterion of significance l
ACE activity was first measurable in fetal rat lungs at 18 days of gestation (0.34 2 0.1 0 nmol hippuri c acid/ min mg protein) (Fig. 1). It was not possible to detect ACE-catalyzed HHL hydrolysis in younger lungs by the spectrophotometric method employed in these experiments. Converting enzyme activity increased twofold between days 18 and 20 of gestation. Lung weight also increased with advancing gestational age (Fig. 2); however, the concentration of protein in lung remained relatively constant. Pulmonary ACE activity in rats less than 24 h old was twofold greater than that of near-term fetuses (Fig. I). Protein concentration was also greater in newborn (63 mg/g lung) as compared to fetal lungs (40-45 mg/g lung). This increase in converting enzyme activity immediately after birth was not attributed to ventilation of the lungs: allowing near-term fetal rats to breathe room air for 30 min prior to decapitation did not, alter lung weight, protein concentration, or ACE activity cornDared to fetuses decapitated in utero (Table 1). l
19
18 Gestational
20 Age
1
t
(days)
Term FIG.
mates mean plotted
2. Fetal lung wet weight at various gestational ages. Litterwere pooled for each measurement. Each point represents * SE of 4 determinations. Lung weight of I-day-old rats is to right of vertical dashed line representing day of birth.
TABLE 1. Effect of room air breathing on pulmonary angiotensin-converting enzyme activity of near-term fetal ruts ~~ -~_~__ -~--~
.~--
Lung wt, g Protein, mg/g lung ACE activity, nmol hippuric acid/min * mg protein ----~
.-.-. ---
Nonventilated*
0.14
_+ 0.01
35.14 Ik 1.72 1.20 * 0.23
-._--
Ventilated+
0.14
+ 0.01
34.54 2 1.82 1.65 + 0.45
. .----~ Values represent mean + SE of 4 determinations. Each litter of fetuses represented one determination. * Near-term fetal rats were decapitated in utero and ventilation of the lungs prevented prior to enzyme assay. -k Fetuses were delivered by cesarean section and allowed to breathe room air for 30 min prior to decapitation and enzyme assay.
When fetal lung ACE was incubated with HHL, the rate of production of hippuric acid increased with increasing substrate concentrations (Fig. 3). The hyperbolic substrate dependence curve was suggestive of firstorder reaction kinetics. When these data were plotted by the method of Eadie-Hofstee, a straight line was generated (r2 > 0.9) with a slope similar to that obtained from plotting the substrate dependence of adult rat lung ACE (Fig. 4). The points of intersection of the ordinate indicated a fourfold greater maximal velocity (V,,,,,) of converting enzyme from adult lung than that of fetal lung. However, the slopes (K,,, ) of the two lines were not statistically different (fetal, K,,, = 2.0 mM HHL; adult, K, = 2.6 mM HHL). I8 Gestational
19 Age
20 (days)
t
1
Term
FIG. 1. Angiotensin-converting enzyme activity in fetal of various gestational ages. Littermates were pooled sufficient enzyme for one determination. Data points mean + SE of 4 enzyme preparations. Circles without indicate SE was within radius of circle. Pulmonary enzyme activity of l-day-old rats is plotted to right side &shed line representing day of birth I day 21).
rat lungs to obtain represent error bars converting of uertical
DISCUSSION
ACE has been detected in lungs of fetal sheep (ll), rabbits (12), and rats (29). An age-related increase in pulmonary converting enzyme activity, which was attributed to increased enzyme content, has been described in rats following birth (29). However, the prenatal appearance and development of ACE activity-has not been previously examined.
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
FETAL
ANGIOTENSIN-CONVERTING
R59
ENZYME
.
Adult
18
8
1
2 3 4 5 HHL (mM) FIG. 3. Angiotensin-converting enzyme activity of fetal (dny 19 of gestation) rat lung as a function of substrate concentration. Each point represents mean activity of 3 enzyme preparations. Incubation was for 30 min at 37°C in presence of varying concentrations of HHL.
Broken cell preparations of lung tissue possess large amounts of nonspecific proteases that may account for a portion of the observed peptidase activities (14). However, Cushman and Cheung (7) have demonstrated that neither HHL nor hippuric acid is destroyed by contaminating peptidases of crude lung homogenates. Therefore, liberation of hippuric acid during the hydrolysis of HHL is specific to the catalytic action of ACE. Converting enzyme has been detected in both soluble and particulate subcellular fractions of lung homogenates (9). Sander and Huggins (25) found the majority of ACE of rabbit lung to sediment between 1,000 and 25,000 x g, whereas Erdos and Yang (10) localized converting enzyme to the microsomal fraction of hog kidneys. In view of this subcellular distribution of ACE, we measured ACE activity in the 20,000 x g fraction of lung homogenates containing both cytosolic and microsomal substituents. Assuming that the enzyme-membrane complex sediments with the 20,000 x g pellet leaving the membrane dissociated enzyme in the supernate, age-related differences in the ease of membrane dispersion may lead to erroneous comparisons of ACE activity in the 20,000 x g supernate fraction. However, we have previously reported that the fraction of total enzyme appearing in the 20,000 x g supernate is constant during postnatal development (29). The relatively low activity of ACE in fetal lungs prevented a similar determination of the fraction of the enzyme present in the assay sample. It must therefore be assumed that the fraction of converting enzyme dissociated from fetal membranes during homogenization is independent of gestational age.
2
1
2
3
4
5
6
7
8
v/s FIG. 4. Eadie-Hofstee plot of fetal I&y 19 of gestation) and adult rat lung converting enzyme activity. Each point represents mean of 3 determinations. ACE activity (V) is expressed as nmol hippuric acidlmin. mg protein and S depicts the concentration of HHL (mM) present in incubation medium.
Pulmonary ACE activity in l-day-old rats was twice that of near-term fetuses. Concomitantly, lung protein concentration increased immediately after birth. This increase in protein concentration was probably due to the ventilatory-induced clearance of alveolar fluid at birth, which would also account for the slight reduction in lung wet weight of these animals. This is implicated by the greater-water content of 19-day-old fetal rat lungs compared to that of rats of 1-8 wk postnatal age (95.7% vs. 85.6%). Serum and lung ACE activity has been found to be elevated in mice exposed to chronic hypobaric alveolar hypoxia (16, 17). The fact that converting enzyme activity was not altered by allowing near-term fetuses to breathe room air prior to death implies the absence of an oxygen-induced activation of pulmonary ACE. The comparable K, values obtained for fetal and adult lung ACE imply that the similarity in converting enzyme persists from the initial appearance of ACE in utero throughout both prenatal and postnatal development to adulthood. The K,, values obtained in these experiments agree with those previously reported (8, 13, 29). The age-related difference in pulmonary ACE activity in the absence of a change in K, suggests the increase in enzyme activity was due to greater content of converting enzyme rather than further activation of preexisting enzyme. The physiological implications of these data can best
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
R60
WALLACE,
be interpreted when viewed in context of the development of the renin-angiotensin system. In contrast to the low pulmonary ACE activity, plasma renin activity (PRA) and angioknsin II are elevated in near-term fetuses as compared to adults (2, 3, 5). Because fetal PRA also increases with advancing gestational age (5), the prenatal rise in pulmonary ACE activity may reflect a specific maturation of the renin-angiotensin system. Alternatively, the increasing PRA and converting enzyme activity may be secondary to the functional development of fetal renal and pulmonary tissue, respectively. O’Hare and Townes (22) have found that fetal rat lungs between 16 and 20 days of age undergo rapid cellular division, whereas between day 20 and term, cell division decreases and cellular differentiation rap-
BAILIE,
AND
HOOK
idly ensues. The observed increase in lung protein and/ or ACE activity may reflect this rapid cellular proliferation during fetal development. Inasmuch as prenatal differences in circulating angiotensin II have not been adequately defmed, the influence of the age-related increase in fetal ACE activity on plasma AI1 concentrations remains obscure. We thank Mr. Bruce G. Hook for his excellent technical assistance and Miss Diane K. Hummel for preparation of the manuscript. This investigation was supported in part by Public Health Service Grants AM-10913 and HD-06290. Address reprint requests to: M. D. Bailie, Dept. of Human Development, B342 Life Sciences, Michigan State University. Received
3 February
1978; accepted
in final
form
2 August
1978.
REFERENCES 1. AIKEN, J. W., AND J. R. VANE. Renin-angiotensin system: inhibition of converting enzyme in isolated tissues. Nature London 228: 30-34, 1970. 2. BROUGHTON PIPKIN, F., S. M. L. KIRKPATRICK, E. R. LUMBERS, AND J. C. MOTT. Renin and angiotensin-like levels in foetal, new-born and adult sheep. J. Physiol. London 241: 575-588, 1974. 3. BROUGHTON PIPKXN, F., E. R. LUMBERS, AND J. C. Mom. Factors influencing plasma renin and angiotensin II in the conscious pregnant ewe and its foetus. J. Phkysiol. London 243: 619-636, 1974. 4. CALDWELL, P. R. B., B. C. SEEGAL, K. C. Hsu, M. DAS, AND R. L. SOFFER. Angiotensin-converting enzyme: vascular endothelial localization. Science 191: 1050-1051, 1976. 5. CARVER, J. G., AND J. C. MOTT. Plasma renin, (Na’) and (K’) in immature fetal lambs with indwelling catheters. J. Physiol. London 245: 73P-75P, 1975. 6. CUSHMAN, D. W., AND H. S. CHEUNG. Concentrations of angiotensin-converting enzyme in tissues of the rat. Biochim. Biophys. Acts 250: 261-265, 1971. 7. CUSHMAN, D. W., AND H. S. CHEUNG. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20: 1637-1648, 1971. 8. DAS, M., J. L. HARTLEY, AND R. L. SOFFERS. Serum angiotensinconverting enzyme: isolation and relationship to the pulmonary enzyme. J. Biol. Chem. 252: 1316-1319, 1977. 9. DEPIERRE, D., AND M. ROTH. Activity of a dipeptidyl carboxypeptidase (angiotensin converting enzyme) in lungs of different animal species. Experientia 28: 154-155, 1972. 10. ERD~S, E. G., AND H. Y. T. YANG. An enzyme in microsomal fraction of kidney that inactivates bradykinin. Life Sci. 6: 569574, 1967. 11. HUBERT, F., J. C. FOURON, J. C. BOILEAU, AND P. BIRON. Pulmonary fate of vasoactive peptides in fetal, newborn and adult sheep. Am. J. PhysioZ. 223: 20-23, 1972. 12. KOKUBU, T., E. UEDA, K. NISHIMURA, AND N. YOSHIDA. Angiotensin I converting enzyme activity in pulmonary tissue of fetal and newborn rabbits. Experientia 33: 1137-1138, 1977. 13. LANZILLO, J. J., AND B. L. FANBURG. Angiotensin I-converting enzyme from guinea pig lung and serum: a comparison of some kinetic and inhibition properties. Biochim. Biophys. Acta 445: 161-168, 1976. 14. LEE, H. J., J. N. LARUE, AND I. B. WILSON. Angiotensinconverting enzyme from guinea pig lung and hog lung. Biochim. Biophys. Acta 250: 549-557, 1971.
15. LOWRY,
0.
H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193: 265-275, 1951. MATTIOLI, L., R. M. ZAKHEIM, K. MULLIS, AND A. MOLTENI. Angiotensin-I-converting enzyme activity in idiopathic respiratory distress syndrome of the newborn infant and in experimental alveolar hypoxia in mice. J. Pediatr. 87: 97-101, 1975. MOLTENI, A., R. M. ZAKHEIM, K. B. MULLIS, AND L. MATTIOLI. The effect of chronic alveolar hypoxia on lung and serum angiotensin I converting enzyme activity. Proc, Sot. Exp. BioZ. Med. 147: 263-265, 1974. NG, K. K. F., AND J. R. VANE. The conversion of angiotensin I to angiotensin II. Nature London 216: 762-766, 1967. NG, K. K. F., AND J. R. VANE. Fate of angiotensin I in the circulation. Nature London 218: 144-150, 1968. NG, K. K. F., AND J. R. VANE. Some properties of angiotensin converting enzyme in the lung in viva. Nature London 225: 1142- . 1144, 1970, OATES, H. F., AND G. S. STOKES. Role of extrapulmonary conversion in mediating the systemic pressor activity of angiotensin I. J. Exp. Med. 140: 79-86, 1974, O’HARE, K. H., AND P. L. TOWNES. Morphogenesis of albino rat lung: an autoradiographic analysis of the embryological origin of type I and type II pulmonary epithelial cells. J. Morphol. 132: 69-87, 1970. POHLOV& I., AND J. JEL~NEK. Components of the renin-angiotensin system in the rat during development. Pfluegers Arch. 351: 259-270, 1974. ROTH, M., A. F. WEITZMAN, AND Y - PIQUILLOD. Converting enzyme content of different tissues of the rat. Experientia 25: 1247, 1969. SANDER, G, E,, AND C. G. HUGGINS. Subcellular localization of angiotensin I converting enzyme in rabbit lung. Nature New Biol. 230: 27-29, 1971. SKEGGS, L. T., J. R. KAHN, AND N. P. SHUMWAY. The purification of hypertensin II. J. Ep. Med. 103: 295-299, 1956. SOKAL, R. R., AND F. J. ROHLF. Biometry. San Francisco, CA: Freeman, 1969, p. 448-458. TRIMPER, C., AND E. LUMBERS. The renin-angiotensin system in foetal lambs. Pfluegers Arch. 336: l-10, 1972. WALLACE, K. B., M. D. BAILIE, AND J. 8. HOOK. Angiotensinconverting enzyme in developing lung and kidney. Am. J. Physiol. 234: R141-R145, 1978 or Am. J. Physiol.: Regulatory Integrative Camp. Physiol. 3: R141-R145, 1978. RANDALL.
16.
17.
18. 19. 20.
21.
22,
23.
25.
26. 27. 28. 29.
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
J. B. HOOK and Toxicology, and Human Michigan 48824
WALLACE, K.B.,M. D~AILIE, AND J. B. HOOK. Deuelopment of angiotensin-converti?lg enzyme in fetal rat lungs. Am. J. Physiol. 236(l): R57-R60, 1979 or Am. J. Physiol.: Regulatory Integrative Comp. Physiol. 5(l): R57-R60, 1979. -Angiotensin-converting enzyme (ACE) catalyzes rapid hydrolytic cleavage of angiotensin I to form angiotensin II (AII). Inasmuch as converting enzyme activity is present at birth and increases postnatally to adult values it was of interest to determine the prenatal development of ACE. Converting enzyme activity was determined in the 20,000 x g supernatant fraction of lung homogenates using hippuryk-histidyk-leutine (HHL) as substrate. Hippuric acid liberated by the hydrolysis of HHL was quantified spectrophotometrically, ACE activity was first detectable at 18 days of gestation and increased fourfold prior to birth (21 days gestation). Pulmonary ACE activity of l-day-old animals was twice that of fetuses at day 20 of gestation; however, this increase did not appear to result from ventilation alone. The Michaelis-Menten constant for fetal ACE (2.0 mM HHL) was not different from that calculated for ACE of adult rat lungs (2.6 mM). These data were interpreted to indicate that the age-related increase in ACE activity was due to greater ACE content as opposed to further activation of preexisting enzyme. This increase in fetal ACE activity may play an important role in preparing the renin-angiotensin system for postnatal function.
Sprague-Dawley strate-dependence
rats;
renin;
Eadie-Hofstee
plot;
K,,;
sub-
ENZYME (ACE) (peptidyldipeptide hydrolase, EC 3.4X5.1) catalyzes hydrolytic cleavage of the carboxy terminus dipeptide (His”-Leul”) from angiotensin I (AI). Although ACE was first isolated from plasma (26) the activity of this enzyme was found to be insufficient to account for AI conversion in vivo (18, 19). Angiotensin-converting enzyme has since been detected in nearly every tissue examined (6, 24). Subsequent localization of converting enzyme on the vascular endothelium of nearly every tissue suggests direct apposition of this enzyme to circulating AI (4). However, only lung is capable of liberating significant quantities of angidtensin II (AII) into the venous effluent following addition of AI to the perfusing medium (1, 18-20). It therefore appears that pulmonary ACE is responsible for the generation of AI1 destined for the systemic circulation, whereas, in tissues other than lung, rapid uptake and/or metabolism of AI1 accounts for the small quantities of this peptide in the venous effluent (21). ANGIOTENSIN-CONVERTING
036%6119/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
enzyme
Development,
Age-related differences in plasma renin, angiotensinogen, angiotensinase, and ACE activity have been reported in rats following birth (23, 29). In addition, the renin-angiotensin system is functional and responsive during intrauterine life (2, 3, 28). However, fetal ACE activity is very low compared to that of a.dult rats (29). The purpose of the present in vestigation was to determine the appearance and development of ACE in fetal rat lungs. METHODS
Pregnant female Sprague-Dawley rats of known gestational age were purchased from Spartan Research Animals, Inc. (Haslett, MI), housed separately, maintained on a normal laboratory chow diet (Wayne Lab Blocks), and allowed free access to tap water. On the day of the experiment, the pregnant mothers were killed and the fetuses decapitated in utero. Fetal lungs were immediately removed and washed in cold NaCl (0.9%) then weighed and homogenized (Polytron, Brinkmann Instruments) in 4 vol of cold 100 mM potassium phosphate-300 mM sodium chloride btier (pH 8.3). The homogenate was then centrifuged (Sorvall RCZ-B automatic refrigerated centrifuge) for 20 min at 20,000 x g and 4°C and the supernatant fraction was assayed for ACE activity. Under standard conditions, the enzyme preparations were assayed the same day for ACE activity, whereas the kinetic studies were performed after storage of the supernatant fraction at 4°C for 24 h. Lungs of littermates were pooled to represent one determination. Converting enzyme activity was determined by a modification of the method described by Cushman and Cheung (7). In brief, 0.1 ml of the 20,000 x g supernatant of fetal lung homogenates was incubated at 37°C with 0.15 ml hip&$-Zhistidyl-L-leucine (HHL; Vega Fox Biochemicals). The final concentration was 5.0 mM HHL under standard assay conditions. After a 30-min incubation period (Dubnoff Metabolic Shaking Incubator), the reaction was stopped by addition of 0.25 ml 1 N HCl, The hippuric acid formed from the ACE catalyzed hydrolysis of HHL was extracted into 1.5 ml ethyl acetate and centrifuged at 2,000 x g for 10 min (International Equipment Co.). A l.O-ml aliquot of the ethyl acetate Phase was then evaDorated at 40°C under nitrogen. Thi residual hippuric= acid was dissolved in 1 M NaCl (3.0 ml) and the optical density measured at 228 Society
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
R57
R58
WALLACE,
BAILIE,
AND
HOOK
nm (Beckman DB-GT spectrophotometer). ACE activity was expressed as nanomoles hippuric acid formed per minute per milligram 1ung protein. Protein was determined by th .e method of Low ry et al. (15). Statistical significance was determined by analysis of variance and comparison of regression lines (27). The 0.05 level of probability was used as the criterion of significance l
ACE activity was first measurable in fetal rat lungs at 18 days of gestation (0.34 2 0.1 0 nmol hippuri c acid/ min mg protein) (Fig. 1). It was not possible to detect ACE-catalyzed HHL hydrolysis in younger lungs by the spectrophotometric method employed in these experiments. Converting enzyme activity increased twofold between days 18 and 20 of gestation. Lung weight also increased with advancing gestational age (Fig. 2); however, the concentration of protein in lung remained relatively constant. Pulmonary ACE activity in rats less than 24 h old was twofold greater than that of near-term fetuses (Fig. I). Protein concentration was also greater in newborn (63 mg/g lung) as compared to fetal lungs (40-45 mg/g lung). This increase in converting enzyme activity immediately after birth was not attributed to ventilation of the lungs: allowing near-term fetal rats to breathe room air for 30 min prior to decapitation did not, alter lung weight, protein concentration, or ACE activity cornDared to fetuses decapitated in utero (Table 1). l
19
18 Gestational
20 Age
1
t
(days)
Term FIG.
mates mean plotted
2. Fetal lung wet weight at various gestational ages. Litterwere pooled for each measurement. Each point represents * SE of 4 determinations. Lung weight of I-day-old rats is to right of vertical dashed line representing day of birth.
TABLE 1. Effect of room air breathing on pulmonary angiotensin-converting enzyme activity of near-term fetal ruts ~~ -~_~__ -~--~
.~--
Lung wt, g Protein, mg/g lung ACE activity, nmol hippuric acid/min * mg protein ----~
.-.-. ---
Nonventilated*
0.14
_+ 0.01
35.14 Ik 1.72 1.20 * 0.23
-._--
Ventilated+
0.14
+ 0.01
34.54 2 1.82 1.65 + 0.45
. .----~ Values represent mean + SE of 4 determinations. Each litter of fetuses represented one determination. * Near-term fetal rats were decapitated in utero and ventilation of the lungs prevented prior to enzyme assay. -k Fetuses were delivered by cesarean section and allowed to breathe room air for 30 min prior to decapitation and enzyme assay.
When fetal lung ACE was incubated with HHL, the rate of production of hippuric acid increased with increasing substrate concentrations (Fig. 3). The hyperbolic substrate dependence curve was suggestive of firstorder reaction kinetics. When these data were plotted by the method of Eadie-Hofstee, a straight line was generated (r2 > 0.9) with a slope similar to that obtained from plotting the substrate dependence of adult rat lung ACE (Fig. 4). The points of intersection of the ordinate indicated a fourfold greater maximal velocity (V,,,,,) of converting enzyme from adult lung than that of fetal lung. However, the slopes (K,,, ) of the two lines were not statistically different (fetal, K,,, = 2.0 mM HHL; adult, K, = 2.6 mM HHL). I8 Gestational
19 Age
20 (days)
t
1
Term
FIG. 1. Angiotensin-converting enzyme activity in fetal of various gestational ages. Littermates were pooled sufficient enzyme for one determination. Data points mean + SE of 4 enzyme preparations. Circles without indicate SE was within radius of circle. Pulmonary enzyme activity of l-day-old rats is plotted to right side &shed line representing day of birth I day 21).
rat lungs to obtain represent error bars converting of uertical
DISCUSSION
ACE has been detected in lungs of fetal sheep (ll), rabbits (12), and rats (29). An age-related increase in pulmonary converting enzyme activity, which was attributed to increased enzyme content, has been described in rats following birth (29). However, the prenatal appearance and development of ACE activity-has not been previously examined.
Downloaded from www.physiology.org/journal/ajpregu at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
FETAL
ANGIOTENSIN-CONVERTING
R59
ENZYME
.
Adult
18
8
1
2 3 4 5 HHL (mM) FIG. 3. Angiotensin-converting enzyme activity of fetal (dny 19 of gestation) rat lung as a function of substrate concentration. Each point represents mean activity of 3 enzyme preparations. Incubation was for 30 min at 37°C in presence of varying concentrations of HHL.
Broken cell preparations of lung tissue possess large amounts of nonspecific proteases that may account for a portion of the observed peptidase activities (14). However, Cushman and Cheung (7) have demonstrated that neither HHL nor hippuric acid is destroyed by contaminating peptidases of crude lung homogenates. Therefore, liberation of hippuric acid during the hydrolysis of HHL is specific to the catalytic action of ACE. Converting enzyme has been detected in both soluble and particulate subcellular fractions of lung homogenates (9). Sander and Huggins (25) found the majority of ACE of rabbit lung to sediment between 1,000 and 25,000 x g, whereas Erdos and Yang (10) localized converting enzyme to the microsomal fraction of hog kidneys. In view of this subcellular distribution of ACE, we measured ACE activity in the 20,000 x g fraction of lung homogenates containing both cytosolic and microsomal substituents. Assuming that the enzyme-membrane complex sediments with the 20,000 x g pellet leaving the membrane dissociated enzyme in the supernate, age-related differences in the ease of membrane dispersion may lead to erroneous comparisons of ACE activity in the 20,000 x g supernate fraction. However, we have previously reported that the fraction of total enzyme appearing in the 20,000 x g supernate is constant during postnatal development (29). The relatively low activity of ACE in fetal lungs prevented a similar determination of the fraction of the enzyme present in the assay sample. It must therefore be assumed that the fraction of converting enzyme dissociated from fetal membranes during homogenization is independent of gestational age.
2
1
2
3
4
5
6
7
8
v/s FIG. 4. Eadie-Hofstee plot of fetal I&y 19 of gestation) and adult rat lung converting enzyme activity. Each point represents mean of 3 determinations. ACE activity (V) is expressed as nmol hippuric acidlmin. mg protein and S depicts the concentration of HHL (mM) present in incubation medium.
Pulmonary ACE activity in l-day-old rats was twice that of near-term fetuses. Concomitantly, lung protein concentration increased immediately after birth. This increase in protein concentration was probably due to the ventilatory-induced clearance of alveolar fluid at birth, which would also account for the slight reduction in lung wet weight of these animals. This is implicated by the greater-water content of 19-day-old fetal rat lungs compared to that of rats of 1-8 wk postnatal age (95.7% vs. 85.6%). Serum and lung ACE activity has been found to be elevated in mice exposed to chronic hypobaric alveolar hypoxia (16, 17). The fact that converting enzyme activity was not altered by allowing near-term fetuses to breathe room air prior to death implies the absence of an oxygen-induced activation of pulmonary ACE. The comparable K, values obtained for fetal and adult lung ACE imply that the similarity in converting enzyme persists from the initial appearance of ACE in utero throughout both prenatal and postnatal development to adulthood. The K,, values obtained in these experiments agree with those previously reported (8, 13, 29). The age-related difference in pulmonary ACE activity in the absence of a change in K, suggests the increase in enzyme activity was due to greater content of converting enzyme rather than further activation of preexisting enzyme. The physiological implications of these data can best
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R60
WALLACE,
be interpreted when viewed in context of the development of the renin-angiotensin system. In contrast to the low pulmonary ACE activity, plasma renin activity (PRA) and angioknsin II are elevated in near-term fetuses as compared to adults (2, 3, 5). Because fetal PRA also increases with advancing gestational age (5), the prenatal rise in pulmonary ACE activity may reflect a specific maturation of the renin-angiotensin system. Alternatively, the increasing PRA and converting enzyme activity may be secondary to the functional development of fetal renal and pulmonary tissue, respectively. O’Hare and Townes (22) have found that fetal rat lungs between 16 and 20 days of age undergo rapid cellular division, whereas between day 20 and term, cell division decreases and cellular differentiation rap-
BAILIE,
AND
HOOK
idly ensues. The observed increase in lung protein and/ or ACE activity may reflect this rapid cellular proliferation during fetal development. Inasmuch as prenatal differences in circulating angiotensin II have not been adequately defmed, the influence of the age-related increase in fetal ACE activity on plasma AI1 concentrations remains obscure. We thank Mr. Bruce G. Hook for his excellent technical assistance and Miss Diane K. Hummel for preparation of the manuscript. This investigation was supported in part by Public Health Service Grants AM-10913 and HD-06290. Address reprint requests to: M. D. Bailie, Dept. of Human Development, B342 Life Sciences, Michigan State University. Received
3 February
1978; accepted
in final
form
2 August
1978.
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