K. B. Wallace

Effect of paraquat treatment of rats on disposition of 5-hydroxytryptamine and angiotensin I by perfused lung.

Abstract Rats were injected with the herbicide, paraquat dichloride (25 mg/kg, i.p.), and their lungs were perfused 2–28 days later. Isolated lungs from rats treated with paraquat (PQ) 3 or 4 days before perfusion removed significantly less perfused 5-hydroxytryptamine (5-HT) than did saline-injected controls. This effect was not caused by PQ directly, since perfusion of lungs from untreated animals with PQ did not alter removal of co-perfused 5-HT. Monoamine oxidase activity of600 g supernatan fractions of homogenates of lungs from PQ-treated rats was also reduced compared to controls. Although removal of perfused angiotensin I (1 ng/ml) by isolated lungs was not altered by PQpretreatment, antgiotensin-converting enzyme activity in 600 g supernatant fractions of lung homogenates was reducedd significantly. These results suggest that PQ damages pulmonary endothelium and impairs the metabolic function of lung.

Angiotensin II metabolism by tissues from developing rats.

Summary: Asp1-125I-Tyr4-angiotensin II (125I-AII) was degraded during incubation with rat plasma or homogenates of liver or kidney. The electrophoretic profile of peptide fragments revealed that the disappearance of 125I-AII was first order and was accompanied by an accumulation of 125I-tyrosine in the incubation medium. The only other metabolites of angiotensin AII detectable by peptide mapping were the amino-terminus tetrapeptide and the carboxy-terminus hexapeptide. The appearance of these fragments was highly variable, suggesting that endopeptidases did not constitute the ultimate cleavage of angiotensin II hydrolysis. The half-life of 125-AII in plasma or liver homogenates did not change with age (approximately 8 to 12 and 6 to 9 min, respectively). In contrast, the rate of disappearance of 125I-AII in homogenates of rat kidney depended upon the age of the rat from which the tissue was obtained. The half-life of 125AII decreased three-fold (from approximately 8.3 to 2.8 min) between 2 wk after birth and adult (approximately 8 wk). This increase in the rate of metabolism of 125I-AII was accompanied by a concomitant two-fold, age-related increase in the rate of appearance of 125I-tyrosine in the reaction mixture containing renal tissue.Speculation: Age-related differences in the activities of angiotensinase enzymes may be of considerable importance in regulating the concentration of angiotensin II in plasma and tissues of developing animals. When viewed in context of changes in renin and converting enzyme activities in rats, the increase in the rate of metabolism of angiotensin II is the only available evidence consistent with the decrease in plasma angiotensin II concentration between 6 and 8 wk of age.

Nonrespiratory metabolic function and morphology of lung following exposure to polybrominated biphenyls in rats

Exposure to polybrominated biphenyls (PBBs) resulted in increased activity of microsomal arylhydrocarbon hydroxylase and ethoxyresorufin-O-deethylase in rat lung. Clearance of 5-hydroxytryptamine (5-HT) and angiotensin 1 by perfused lungs was decreased by PBBs. However, PBBs had no effect on the activity of epoxide hydrolase, monoamine oxidase, or angiotensin-converting enzyme in lung. The only histopathologic change detected in lungs from PBB-treated rats was an increase in alveolar type II cell lamellar bodies. Selective accumulation of certain PBB congeners by lung was not observed in this investigation.

Age-related differences in the stoichiometry of the renin-angiotensinogen reaction in rat plasma

Generation of immunoreactive angiotensin I (AI) in plasma of either adult or 10-day-old rats proceeded via first-order kinetics. Although the concentration (Vmax) of renin was 5-fold greater in neonatal compared to adult plasma, the specific activity (kapp/Vmax) of the enzyme was 2-fold greater in adults. Both enzymes exhibited a similar affinity (Km) for angiotensinogen; and at both ages, the generation of AI was limited by insufficient endogenous substrate to sustain maximum rates. In conclusion, although the catalytic properties of renin do not change with age, subtle differences in the stoichiometry of the reaction may have profound implications in determining compensatory mechanisms involved in renin-dependent regulation of homeostasis in developing animals.

AGE RELATED DIFFERENCES IN ANGIOTENSIN II (A-II) METABOLISM IN RAT TISSUES

In order to attempt to account for age-related differences in plasma angiotensin II concentration, the activity of angiotensinases in developing rat tissues was examined. The rate of degradation of A-II was determined in vitro during incubation of tissue homogenates with 125-I-tyrosine labeled angiotensin II. Peptide fragments were separated electrophoretically and quantified by gamma scintillation counting. Half-life of labeled A-II in plasma or liver homogenates did not change with age. In contrast, the half-life in renal tissue homogenates decreased from 8.4 ± 1.2 minutes in two-week-old rats to 4.7 ± 0.7 minutes in eight-week-old rats and 2.8 ± 1.8 minutes in adults. This change in the rate of disappearance was accompanied by concomitant increase in the rate of appearance of labeled peptide fragments. Peptide mapping revealed that the principal metabolite of 125-AII was tyrosine. The only other detectable metabolites of A-II were the amino-terminus tetrapeptide and the carboxy-terminus hexapeptide. The appearance of these fragments was highly variable, suggesting that endopeptidases did not constitute the ultimate cleavage of All degradation. The increased rate of metabolism of angiotensin II during development is consistent with the age-related increases in the concentration of angiotensin II in plasma of developing rats as demonstrated by previous studies from our laboratory.(Supported by NIH Grants HD06290 & HL22544)

Species differences in the kinetics of the renin-substrate reaction in plasma

In view of the substrate-dependence of renin, it was of interest to examine the kinetics of the renin-angiotensinogen reaction in plasma of various species to establish differences in stoichiometry. The results also provide an indication of assay conditions appropriate for accurate measurement of the reaction velocity. Plasma from hogs, dogs, and rats served as the source of renin for incubation with homologous angiotensinogen. The rate of production of radioimmunoassayable angiotensin I increased with increasing concentrations of angiotensinogen. This substrate-dependence of renin conformed to conventional Michaelis-Menten kinetics. The concentration of angiotensinogen was less than that required for half maximum velocity in dog and rat plasma. In contrast, endogenous substrate in hog plasma was sufficient to sustain near maximal rates of generation of angiotensin I. Purification of angiotensinogen altered the catalytic properties of angiotensinogen making it a poor representative substrate for renin. Hog and rat renin were saturable with high concentrations of unextracted plasma angiotensinogen. In contrast, it was not possible to saturate the dog enzyme with unextracted substrate. The interspecies differences in stoichiometry of the reaction indicate that standardization of assay conditions for various species of renin is not justified.

221 PRENATAL DEVELOPMENT OF ANGIOTENSIN-CONVERTING ENZYME ACTIVITY IN RAT LUNGS

Angiotensin I (AI) is rapidly converted to angiotensin II (AII) during a single transpulmonary passage. The enzyme responsible for this hydrolysis, angiotensin-converting enzyme (ACE), is present in small amounts in lungs of newborn animals. Inasmuch as ACE is the final catalytic component of the renin-angiotensin system, and since fetal plasma renin activity increases with advancing gestational age, it was of interest to determine the prenatal development of converting enzyme activity in lungs of fetal rats. ACE activity was measured in vitro by virtue of its ability to generate hippuric acid by hydrolysis of the AI-homologue hippuryl-L-histidyl-L-leucine (HHL). Hippuric acid was extracted from the reaction mixture and quantitated spectrophoto-metrically. ACE activity was first detectable in fetal lungs at 18 days of gestation and increased thereafter until birth (day 21 of gestation). The in utero development of ACE activity was paralleled by increases in fetal lung weight and protein content. The affinity of converting enzyme from fetal lung for HHL (Km = 2.0 mM) was similar to that of the adult enzyme, suggesting that the increase in ACE activity was due to increased enzyme content rather than further activation of pre-existing enzyme. This antenatal increase in ACE activity may play an important role in the maturation of the renin-angiotensin system which has been implicated in the regulation of body fluid homeostasis during the perinatal period.