Biol. Neonate 36 : 85-91 (1979)

Developmental Pattern of Pulmonary Lipoprotein Lipase in Growing Rats Eino Hietanen and Jaakko Hartiala Department of Physiology, University of Kuopio, Kuopio and Department of Physiology, University of Turku, Turku

Key Words. Growth • Lipids • Lipoprotein lipase • Lungs

In addition to respiration the mammalian lung is capable of other metabolic functions such as to produce biologically active amines, to metabolize xenobiotics entering the body, and to regulate steroid metabolism (2, 3, 11, 12). In the functional development of the lung the synthesis of the surfactant is of utmost importance to initiate the respiratory function (7, 12). In this synthesis the formation of dipalmitoyllecithin from fatty acids is one of the critical steps in the surfactant formation (12). On the other hand, the lipoprotein lipase (LPL) enzyme hydrolyses chylomicron and VLDL lipoprotein triglycerides to glycerol and

fatty acids providing free fatty acids for this synthesis (6, 10, 12). It is quite well established that corticoste­ roid treatment will increase the synthesis of surfactant and decrease the possibility of respi­ ratory distress syndrome. In adult animals the dexamethasone administration (10, 14, 18, 19) increases pulmonary LPL activity concomitant­ ly with the enhanced surfactant synthesis (10). Although the developmental pattern of the LPL activity in adipose tissue is well documented (12), less is known on the development of pulmonary LPL. In the present study the devel­ opmental pattern of pulmonary LPL activity

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Abstract. The developmental pattern of pulmonary lipoprotein lipase (LPL) activity was measured in growing male and female rats from the neonatal period to the adult age. The pulmonary phospholipid content showed a sharp increase at term from premature low level. At the same time the LPL activity increased markedly showing thereafter a decrease at 1 week of age and increasing thereafter to the adult age. Also the phospholipid content was low at 1 week of age. Thus the pulmonary phospholipid content and LPL activity change in the same way during the development indicating possibly that the LPL by supplying free fatty acids for the phospholipid biosynthesis may be one of the rate-limiting enzymes in the pulmonary surfactant synthesis.

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Materials and Methods Animals. Both male and female Wistar rats were used (n = 96) ranging in age from premature (approxi­ mately 2 days) fetuses to 30 weeks of age. The animals were fed commercial pelleted diet ad libitum or they were suckling (up to 3 weeks old) and had free access to water. The animal rooms were temperature con­ trolled and they had a 12-hour light-dark cycle. The rats were sacrificed at 7.30-8.30 a.m. In addition when the enzyme methodology was established adult (3 months old) male rats were used. Reagents. Disodium EDTA, fatty acid poor bovine serum albumin from fraction V, unlabelcd triolein, Trizma® base and ¿-phosphatidylcholine (egg yolk, Type I, 98% pure) were purchased from Sigma Chem­ ical Co. (St. Louis, Mo.). The radioactive tri(l-,4C) oleylglycerol, specific activity 55 mCi/mmol, was from Amersham/Searle Co. (Amersham, England). LumaGel® (Lumac AG, Basel, Switzerland) was used as the scintillation cocktail and the radioactivity was counted with LKB/Wallac (Turku, Finland) liquid scintillator. Sodium deoxycholate was from Fluka AG (Buchs, Switzerland) and the solvents were from Merck AG (Darmstadt, FRG) of pro analysis purity. Tissue Preparation. After weighing rats were decap­ itated and the lungs were removed and weighed after rinsing in 0.25 M sucrose-1 rn.fi EDTA buffer, pH 7.4, and plotting dry with a piece of paper. A 20% homogenate was made in 0.25 M sucrose-1 mM EDTA buffer, pH 7.4 with an Ultra-Turrax homogenizer whereafter the postmitochondrial (10,000 g for 15 min) supernatant (Sorvall SS-1) was prepared for enzyme assays. Enzyme and Protein Assays. The LPL activity (glycerol ester hydrolase, EC 3.1.1.3) was determined using labeled triolcylglycerol as a substrate by modify­ ing the method developed for adipose tissue (13, 21). The substrate emulsion was prepared by pipetting 3.0 pCi labeled triolein and 10 mg cold triolein in benzene into plastic scintillation vial together with 0.6 mg lysolccithin in heptane. The organic solvents were evaporated under nitrogen flow whereafter 2.55 ml of 0.2 M This-HCl buffer, pH 8.0, 0.45 ml of 1% bovine serum albumin and 3.0 ml of thawed

human serum, taken originally after a 12-hour fast and pooled from several persons, were added into the vial and the mixture was sonicated in ice bath for 4 min at 30-sec intervals by a MSE sonicator with a microtip. In the enzyme assays 2 sets of duplicates were always made; one was made 1 M with NaCl and another was without and the difference was deter­ mined as LPL based on the previous data and prelimi­ nary experiments that NaCl inhibits lipolytic activity not due to the LPL. The enzyme assay was made in plastic test tubes containing 0.1 ml of substrate emul­ sion (0.19 jumol of triolein). All the tubes were prein­ cubated 30 min at 37 °C. The reaction was started by the addition of 0.1 ml of pulmonary postmitochon­ drial supernatant solution. After a 45-min incubation period the reaction was stopped by adding 3.25 ml of the fatty acid extraction mixture, chloroform: methanol:heptane (2.3:2.5:1.8) as described earlier (5). The incubation time and tissue concentration were such to ensure linearity of the reaction. There­ after, 1.05 ml of 0.1 M bicarbonate buffer, pH 10.5, was added to facilitate the separation of the two phases (13, 21). The tubes were mixed with a Vortex mixer and centrifuged at 800 g for 20 min at room temperature. 1-ml aliquots of the upper phase contain­ ing released free fatty acids were pipetted into the glass scintillation vials and 8 ml of Luma-Gel® was added. The recovered radioactivity was counted in a refrigerated Wallac-LKB liquid scintillation spectrom­ eter and the results were converted to micromoles of FFA released per hour with the counting efficiency approximately 85% and recovery of free fatty acids 64%. The protein contents of the postmitochondrial supernatants were determined as described earlier by Gornall et al. (9) in 5% Na-deoxycholatc solubilized preparation. The phospholipid content was deter­ mined as inorganic phosphate (4).

Results

Both sexes were combined in groups of less than 1-week-old rats. The weight gain in female rats was lower than in male rats from 10 weeks onwards as was also the wet weight of the lungs (fig. 1A. B). The relative weight of the lungs did not show any sex difference except at 2 and 3

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has been determined in growing rats from neo­ natal period to adulthood in rats.

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Pulmonary Lipoprotein Lipase

Age, weeks

Fig. 1. A The body weights of the growing rats. The results are given as means ± SEMs in male (•) and female (o) rats. The time of birth is shown by a vertical dotted line and the age of fetuses as days before the term. Up to the age of 1 week both male and female rats were combined (®). The number of rats is shown in parentheses from 5 weeks onwards

and the number of experiments in each group of young rats was 3 and in each experiment 2 or more rats were combined. B The weight of the lungs (as grams) in growing rats. For other explanations see figures 1A. C The relative weights of the lungs as percent of the body weight in growing rats. For other explanations see figure 1A.

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Days

88

Hietanen/Hartiala

Age, weeks

Fig. 2. A The pulmonary postmitochondrial pro­ tein contents in growing rats as milligrams of protein per gram of lung wet weight. For other explanations see figure 1A. B The postmitochondrial phospholipid contents in growing male (■) and female (a) rats. In

the rats, groups of less than 1 week of age both males and females were combined («). The amount of phospholipid is expressed as micromoles inorganic phosphate per gram of lung wet weight. For other explanations see figure 1A.

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Days

Pulmonary Lipoprotein Lipase

Age, weeks

Fig. 3. The pulmonary LPL activity in growing rats expressed as micromoles of free fatty acids (FFA)

released per hour and per gram of tissue wet weight. For other explanations see figure 1A.

weeks of age (fig. 1C). The relative pulmonary wet weights expressed as percent of the body weights were highest in the fetuses and de­ creased to the adult age (fig. 1C). At birth the relative lung weights decreased 50% with a slower decrease to the age of 10 weeks in both sexes. The protein content of the pulmonary postmitochondrial supernatant did not show any uniform sex differences (fig. 2A). It was lowest in immature fetuses while it increased to the age of 1 week having a peak level which was twice that seen in the premature fetuses (fig. 2A). The protein content decreased to adult age and then remained constant in older rats. The postmitochondrial phospholipid con­ tent was at a very low level in fetuses showing an abrupt increase at birth (fig. 2B). At 1 week of age there was a decrease followed by an increase at the age of 2 and 3 weeks (fig. 2B).

In female rats the phospholipid content was lower than in males at these ages (fig. 2B). The LPL activity was lowest at the age of 1 week (fig. 3). In the fetal period the LPL activity rose from 2 days before term to 1 day before term (more than twofold) decreasing abruptly after the birth to the age of 1 week. After 1 week of age the LPL activity began to increase both in the female and male rats (fig. 3). The increase continued in female rats to the age of 5 weeks whereafter a transient decrease in activity took place. The activity was at 16 weeks of age in female rats at the same level as at 5 weeks of age remaining at this level (fig. 3). In the male rats a respective compari­ son cannot be made because no 5-week-old rats were studied. At 10 weeks of age the pulmo­ nary LPL activity was slightly but not signifi­ cantly higher in male than in the female rats

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Days

89

Hictancn/Hartiala

90

Discussion

During the development and maturation of the lungs many metabolic changes take place. The timing of the pulmonary maturation is of paramount importance as the disturbance may lead to the respiratory distress syndrome which is a severe, often fatal disease, hampering the ventilation (7). The synthesis of the surfaceactive agent dipalmitoyllecithin needs fatty acids as the substrate (1), which synthesis is catalyzed by acyl transferases. In this synthesis the LPL may play a key role in providing the free fatty acids (12). The composition and quan­ tity of the pulmonary fatty acids change during the development in the rat (15). Phospholipases may increase the turnover of pulmonary phos­ pholipids (16). The administration of glucocorticoids affects in many ways the development of the lungs (8, 17, 20). It changes the anatomy and physiology of the lungs facilitating the initiation of the pulmonary ventilation. The glucocorticoids also influence the pulmonary biochemical functions, favoring production of surfactant and increas­ ing the maturation rate of the lungs (10, 19). It increases possibly the activities of choline phos­ photransferase in choline synthesis, glycero­ phosphate phosphatidyltransferase in phosphatidylglycerol production and lipoprotein lipase (17, 18). Although recently a few studies have been published on the pulmonary LPL activity, no studies were conducted to find out the develop­ mental pattern of LPL in growing rats (10). In our present study there was a peak in LPL

activity 1 day prior to term in rat fetuses, which was followed by the high level of the pulmonary phospholipids possibly due to the enhanced phospholipid synthesis as LPL provid­ ed more free fatty acids. Thus it is possible that during the normal lung development the LPL activity increases just prior to term to supply free fatty acids in the neonatal period for the surfactant phospholipids. In the LPL activity there is a low activity level during the first week postnatally followed by a notch in the LPL activity which might predict also that this period is critical in the normal development of the lung. Thereafter, the LPL activity reaches the adult levels. The LPL enzyme is active in the capillary endothelium (6). As the lungs have a large vascular bed and a high circulatory capacity this makes the LPL activity in lungs of great interest. During the development of the pulmonary choline phosphotransferase activity increases during the fetal period having its peak 2 days before term in mice, which is in accor­ dance with our data showing the peak LPL activity at this time in rats (18). This enzyme was also very adaptive to the glucocorticoid treatment (18, 19) as LPL was found to be in other reports (10). Thus it seems that the developmental pattern of both choline phos­ photransferase and LPL is quite similar, the peak activity possibly preceding the surfactant synthesis. Moreover, both enzymes are respon­ sive to glucocorticoid treatment suggesting that these enzymes might play a key role in the lung maturation and surfactant synthesis. On the other hand, the decreased activities of these enzymes might disturb the lung maturation.

References 1 Abe, M. and Akino, T.: Comparison of metabolic heterogeneity of glycerolipids in rat lung and liver. Tohoku J. exp. Med. 106: 343-355 (1972).

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and also later no significant difference could be found (fig. 3). The increase in the pulmonary LPL activity from 1 week to adult age was over 20-fold in both sexes.

Pulmonary Lipoprotein Lipase

14 Kauffman, S.L.: Acceleration of canalicular devel­ opment in lungs of fetal mice exposed transplacentally to dexamethasone. Lab. Invest. 36: 395-401 (1977). 15 Kehrer, J.P. and Autor, A.P.: Changes in the fatty acid composition of rat lung lipids during develop­ ment and following age-dependent lipid peroxida­ tion. Lipids 12: 596 -603 (1977). 16 Melin, B.; Maximilien, R.; Friedlander, G.; Etienne, J. et Alcinder, L.G.: Activités phospholipasiques pulmonaires du foetus de rat. Variations au cours du développement. Biochim. biophys. Acta 486: 590-594 (1977). 17 Oldenborg, V. and Golde, L.M.G. van: Activity of cholinephosphotransfcrase, lysolecithin: lysolccithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung. Biochim. biophys. Acta 441: 433-442 (1976). 18 Oldenborg, V. and Golde, L.M.G. van: The en­ zymes of phosphatidylcholine biosynthesis in the fetal mouse lung. Effects of dexametasone. Bio­ chim. biophys. Acta 489: 454-465 (1977). 19 Possmaycr, F.; Duwe, G.; Metcalfe, R.; StewartDeHaan, P.J.; Wong, G ; Las Heras, J., and Harding, P.G.R.: Cortisol induction of pulmonary matura­ tion in the rabbit foetus. Its effects on enzymes related to phospholipid biosynthesis and on marker enzymes for subcellular organelles. Biochem. J. 166:485—494 (1977). 20 Sarzala, M.G. and Golde, L.M.G. van: Selective utilization of endogenous unsaturated phosphati­ dylcholine and diacylgycerols by cholinephosphotransferase of mouse lung microsomes. Biochim. biophys. Acta 441: 423-432 (1976). 21 Schotz, M.A.; Garfinkel, A.; Huebotter, R.J., and Stewart, J.E.: A rapid assay for lipoprotein lipase. J. Lipid Res. 11: 6 8 -6 9 (1970). Eino Hietanen, MD, Department of Physiology, University of Kuopio, PO Box 138, SF-70101 Kuopio 10 (Finland)

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2 Aitio, A.: Glucuronide synthesis in the rat and guinea pig lung. Xenobiotica 3: 13-22 (1973). 3 Aitio, A.; Hartiala, J., and Uotila, P.: Glucuronide synthesis in the isolated perfused rat lung. Biochem. Pharmac. 25: 1919-1920(1976). 4 Bartlett, C.R.: Phosphorus assay in column chro­ matography. J. biol. Chcm. 234: 466-468 1959). 5 Belfrage, P. and Vaughan, M.: Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J. Lipid Res. 10: 341 -344 (1969). 6 Cunningham, V.J. and Robinson, D.S.: Clearingfactor lipase in adipose tissue: distinction of differ­ ent states of enzyme and the possible role of the fat cell in the maintenance of tissue activity. Biochem. J. 112: 203-209 (1969). 7 Fanconi, A.; Stoll, W.; Due, G.; Bossi, E. und Prod’hom, S.: Das Atemnotsyndrom des Neugeborenen in der Schweiz. Schweiz, med. Wschr. 106: 1426-1429 (1976). 8 Farrell, P.M.: Fetal lung development and the influence of glucocorticoids on pulmonary surfac­ tant. J. Steroid Biochem. 8: 463-470 (1977). 9 Gornall, A.G.; Bardawill, C.J., and David, M.M.: Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177: 751-766 (1949). 10 Hamosh, M.; Yeager, H., jr.; Shechter, Y., and Hamosh, P.: Lipoprotein lipase in rat lung. Effect of dexamethasone. Biochim. biophys. Acta 431: 519-525 (1976). 11 Hartiala, J.: Studies on pulmonary testosterone metabolism; diss., University of Turku, pp. 1-65 (1976). 12 Heinemann, H.O. and Fishman, A.P.: Nonrespiratory functions of mammalian lungs. Physiol. Rev. 49: 1-47 (1969). 13 Hietanen, E. and Greenwood, M.R.C.: A compari­ son of lipoprotein lipase activity and adipocyte differentiation in growing male rats. J. Lipid Res. 18: 480-490 (1977).

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Developmental pattern of pulmonary lipoprotein lipase in growing rats.

Biol. Neonate 36 : 85-91 (1979) Developmental Pattern of Pulmonary Lipoprotein Lipase in Growing Rats Eino Hietanen and Jaakko Hartiala Department of...
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