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Insulin effect on some biochemical and biophysical characteristics of lung surfactant
I‘NSULIN EFFECT ON SOME BIOCHEMICAL AND BIOPHYSICAL CHARACTERISTICS OF LUNG SURFACTANT N.
I-~ADJWANOVA’,
K. KWJMANOV IL Iv
‘C‘en~ral ILaboratorq of Biophysics. Phyrical Chemistry.
Bulgarian
PANAJOTOV’
and M.
Slate University
IVANOVA’
of Sciences. I I I3 Sofia and of Sofia, Bulgaria [Tc/. ‘?-M-IO]
Academy
‘Department
01‘
Abstract-1. The it~c(~rp~~r~iti~n of [‘~C]~dlmitic acid into rat alveolar wash total ph(~spholipids ;md ph(~sph~~lipid frnctions has been followed for 6. 8, IO and 12 hr after insulin adrninistr~~tl(~Il. indicating a consider-able enhancement. phosphatidylcthanolamines and phos-7. The fatty acid profiles of phosphatidylcholines, phatidylglyccrols were found changed after the hormone administration. 3. Elghl! hours post insulin treatment the precursor incorporation was highest in all phospholipid fractions :,tudicd, us well as the contribution of long chain fatty acids. 4. Dynamic monoluycr studies of the lung wash lipid extracts indicated a maximally expanded lipid film corresponding to the highly unsaturated phospholipids present.
INTRODf WTION
The atveoli of the mammalian iung are lined with a hpid-rich substance known as surfactant (Goerke, 1074). Surfactant has the capacity to lower the surf,~ce tension in lthe alveoli at end-expiration, thus stabilizing the lung and preventing atelectasis (Scarpclli. 1968). Decreased amounts of pulmonary surfactant. due either to premature birth or to a maternal disease state. is thought to be the immediate cause of the Respiratory Distress Syndrome (RDS) of the newborn (Gluck CI (I/., 1972). The RDS itself is a ma,jor cause of neonatal mortality. particularly in the premature infant Pulmonary surfactant is known to be synthesized in the type II alveolar epithelial cells (Batenburg and v,~n Golde, 1979). Characteristic components of these chills are the osmiophillic lamellar inclusion bodies, which are the storage-site of lung surfactant (Baranska and van Golde. 1977: Chevalier and Collet, 1<)71). The most ~~hund~~nt surfactant constituent in lamallar bodies and alveolar surfactant is a phosphatidylcholine (1.2-diacyl .m-glycero-3-phosphocholine). enriched, in saturated fatty acids, particularly palmitic acid (Frosolono. 1977; van Golde. 1’176). There are numerous hormones currently being investigated which may influence lung phospholipid metabolism, though the mechanisms by which they act have by large not been delineated. There is ample e\ idence that cjrctIlating glucocorticoids are involved in the regulation and acceleration of lung maturation, enhancing as well the production of surfactant in a variety of mammalian species (Avery, 1975: Ballard 1’1 cd., 1977). Several reports (Hit&cock. 1979: Wu PI ul., 1973) suggest that thyroid hormones exert similar stimuIatory effects on lung development in fetal animals. Recent data indicate that prolactin and estrogen may stimulate surfactant formation (Khosla et (I/.. 19X0; Smith
<‘I r/i..
1979).
Adrenaline.
too.
has been
found to influence sign~~cantly the content and fatty acid composition of lung tissue phospholipids (Had1979). whereas, P-adrenergic agonists Jnvanova, specifically stimulated the release of disaturated phosphatidylcholine (Dobbs and Mason, 1978). There are relatively few investigations on the role of insulin in this aspect. Studies from several laboratories have demonstrated the possible signifcance of this hormone in regulating lipid synthesis in the lung (Morishige rt d., 1977; Stubhs rt a/.. 1977). Diabetic animals have been found to incorporate 60 -X@‘,,less gfucose into total tissue and surfactant phospholipids than normal animals (Moxley and Longmore, 1975. 1977), and have reduced activities of lung acetyl-CoA carboxylase and fatty acid synthetase (Das and Kumar. 1975). Insulin administration restored both glucose incorporation and enzyme activities to normal levels. Insulin was shown, as well. to influence the biosynthesis and fatty acid composition of lung tissue ~Hadjiivanova PI ul., 1978) and subcellui~~r fraction phospholipids (Had.iiivanova CI ii/.. 198 I ). Few attempts, however. have been made to correlate surfactant composition with surface activity by altering lipid composition in I.&O (Burnell and Ralint. 1975; Suzuki and Tabata, 1980). Such an approach is required for the clarification of the significance of the constituents to surface activity ill sitzc. No data at present are available on the role of insulin in this This report deals with the extension of our aspect. previous studies, its objective being to investigate insulin effect on the phospholipid and &tty acid composition of alveolar washes and the changes found in their surface activity. MATERIALS
Male
Wistar
strain
rats.
(Mann Res. Lab..
.&ND METHODS ISO-200g
were
administered
U.S.A.) intravenously (v. saphena), 8U/kg of body weight and sacrificed 6. 8. IO and i 2 hr. respectively. after injection. For ~he incorp~~r~ltit~n studies of labelled precursor rats of all groups received insuiin
I96
N. HAIXIIVANOVA rr ~1.
simultaneously with insulin 5 nCi [I-‘JC]palmttic actd (Amersham, En_eland). specitic activity 256 /lCi;mmol. complexed with albumin. Appropriate animals for controls were administered 0.5 ml sahne (0.15 M NaCl solution). containing the labelled palmitate. All rats were fasted I5 hr prior to treatment. but had access to water. The groups consisted of 8 rats each. For the fatty acid composition studies samples from 25 animals were pooled for each group. Following anaesthesia with intraperitoneally administered amobarbital (Merck. FRG). 50 mg/kg of body weight, lungs were lavagcd in .ciru five times, by the procedure using 5ml of SET (0. IS M NaCI. 0.001 M EDTA, 0.01 M Tris-HCI). Lung washes were centrifuged at 600~ for IOmin to rcmovc cells and cell debris. Liptds were extracted according to Bligh and Dyer (1959). Phosphorus content of total phospholiptds was determined in aliquots of the original lipid extract (Kahovkova and Odavic, 1969) and phospholipids were fractionated into indivtdual components by one dimcnsionnl two-phase thin layer chromatography on silica gel G prrcoated glass plates (Merck. FRG). As developing solvents were used chloroform-methanol-acetic aciddwater \“‘I ) (70:35:X:4. and chloroformmethanol-aatcr (60: 35: 5. vvvv). respectively. for the separation of phosphatidyl@ycerol. The specific radioacttvjtty of lung wash phospholipids was determined hy unalysing their radioactivity and concentration after sepamtion by thin htyer chromntography. The radioactivity was measured in IO ml scintillation solution. The concentration of phospholipid components was determined by measuring their phosphorus content (Kyriakides rl ~1.. 1976). Statistical analysis. where :tppropriate. was carried out using Studcnt’c r-test. The fatty acid composition of the three major phospholipids isolated (phosphatidylcholine. phosphatidylglycerol and phosphatidylethanolamine) was determined by gas-liquid chromatograph,y of their methyl esters. Upon hydrolysis of the phosphohpldh. fatty acids were methylatcd according to the procedure of Hartman and Lago (1973). The methyl ester5 were then applied to the gas (Carlo-Erba). equipped with chromatograph Ramc-ionization detector, isothermally at I90 C. with a 2 m column. coated with lo”,, DEGS on Chromosorb W,,, x,, mesh (Pharmacia, Sweden). nitrogen ns gas-carrier. The molar percentage of fatty acids was calculated by adoption of the mode described by Kates ( 1972). Because of the small amounts of phospholipids present. lipid cxtracta for each group of 25 rats wcrc pooled for analysi< of their fatty acid moiety. Dynamic propertic!, studies of monolaycra ot‘ lung wash lipid extracts were performed on a Beckman microbalance. Surface pressure x uas measured ,by the Wilhelmy method using a roughened glass plate. LIpId extracts dissolved in chloroform (I mg of lipid per ml) were spread as films on 0.01 M NaCl solution. using a microplpettc. Experiments were carried out at 20 C. REScII.1 S Previous investigations in this laboratory have demonstrated that insulin significantly enhanced lung phospholipid biosynthesis, raising the incorporation of labelled precursors into whole tissue homogenate and subcellular fraction phospholipids (Hadjiivanova rf u/., 197X. 1981). Results from this study, as presented in Table I. clearly indicate that with alveolar surfactant phospholipids insulin exerted a similar effect. Already at the sixth hour after treatment total phospholipids in the alveolar wash were found to increase (I l7’!,,). their specific activity being maximally raised X hr after the hormone administration (127’:,, as compared with the control values). As is obvious from Table I similar changes were observed
Insulin Table
2. Fatty
GKMJp Fatty acid 14:o 16:O 16:l l8:O l8:l l8:2 IS:3 20:4 S/U C,,.,&w,,
effect on characteristics
of lung surfactant
acid composition of phosphatidylcholines from alveolar insulin-treated rats (molar percentage)
197
washes
of control
and
Control
l-p6 hr
I -8hr
I- 10 hr
IL12 hr
9.06 55.64 8.02 8.57 6.87 6.65 5.19 trace 2.74 2.67
3.16 60.40 16.23 1.87 8.79 6.70 I.81 I .07 I .89 3.95
3.91 49.39 15.43 7.40 12.04 6.93 4.x9 trace 1.54 2.20
4.05 52.03 12.00 7.07 8.75 945 5.05 1.60 I.71 2.13
4.22 57.01 12.94 4.46 6.41 10.97 3.98 I.91 2.87
I--6, 8. IO and I2 hr after insulin treatment; 14:&miristic fatty acid; 16:&palmitic fatty acid; 16: I-palmitoleic fatty acid; 18:Gstearic fatty acid; 18: I-oleic fatty acid; 18:2-linoleic fatty acid; 18: 3Pmlinolenic fatty acid; 20:karachidonic Pdtty acid: S/U-saturated to unsaturated fatty acid ratio; C,,,,,/C,,,20-C,,,,, to C,,,, Fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals.
in the main surfactant phosphohpids, phosphatidylcholine and phosphatidylglycerol, their specific activities being raised up to 125 and 120”/,, respectively. Ten and twelve hours post treatment, the content and specific activities of both total phospholipids and phosphohpid fractions tended towards the control values. The fatty acid composition changes of alveolar surfactant phosphatidylcholines, phosphatidylglycerols and phosphatidylethanolamines observed after insulin administration were of considerable interest. Six and eight hours post treatment the horTable
3. Fatty
mone caused a significant decrease in the percentage contribution of the saturated miristic (14:0), palmitic (16:0) and stearic (18: 0) fatty acids in almost all phospholipid fractions studied (Tables 2 and 3) with the exception of miristic (14:O) fatty acid in the phosphatidylethanolamines (Table 4), where it was found markedly elevated (209%). The content of the unsaturated palmitoleic (16: I), oleic (18: 1) and linoleic (18:2) fatty acids was raised in phosphatidylcholine and phosphatidylglycerol fractions as compared with the controls. Worthwhile to denote is the pronounced contribution of linolenate (I 8 : 3) to
acid composition of phosphatidylglycerols from alveolar insulin-treated rats (molar Dercentaee)
washes of control
and
GrOUp
Fatty acid l4:O l6:O l6:l 18: I 18: I l8:2 l8:3 20:4 SjU CW&W”
Control
1-6 hr
I.38 35.65 9.22 17.24 23.97 7.68 4.84 trace I I9 0.86
I.18 16.X6 II.32 12.18 26.88 15.79 3.90 Il.88 0.43 0.42
I-8
hr
1.35 40.46 6.1 I 9.03 20.43 8.78 5.65 8.20 I .09 0.92
IL10 hr 0.62 32.26 7.68 21.13 15.75 8.27 8.49 6.79 I I5 0.67
I-12
hr
0.61 36.34 6.33 8.99 12.11 14.96 16.72 3.94 0.85 0.76
I-6. 8, IO and I2 hr after insulin treatment; l4:&-miristic fatty acid; l6:O palmitic fatty acid; 16: I--palmitoleic fatty acid; 18:&stearic fatty acid; 18: I-oleic fatty acid; I8:2-linoleic fatty acid; 18:3Plinolenic fatty acid; 20:Garachidonic fatty acid; S/U-Saturated to unsaturated fatty acid ratio: C,,,,,iC,,,,,--C,,,,, to C,,,,, fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals. Table 4. Fatty acid composition
of phosphatidylethanolamines from alveolar insulin-treated rats (molar percentage)
washes of control and
Group Fatty acid
Control
I---6 hr
I--X hr
I-- IO hr
I---l2 hr
l4:O 16:O 16: I l8:O 18: I IS:2 l8:3 20:4 s/u (7,” ,JC,“,”
0.75 24.94 14.93 19.77 17.18 10.53 4.30 7.61 0.83 0.68
1.56 22.95 8.70 19.44 21.51 10.69 8.01 7.14 0.78 0.50
1.87 20.01 12.50 13.60 16.00 9.28 20.48 6.25 0.55 0.52
0.73 36.42 IO.41 8.14 14.46 9.33 8.5X I? 54 0.82 0.90
0.75 30.51 8.34 Il.88 Il.99 6.05 10.63 19.85 0.76 0.66
l-6. 8. IO and I2 hr after insulin treatment; 14:&miristic fatty acid: l6:O palmitic fatty acid; 16: I--palmitoleic fatty acid; 18:sstearic fatty acid; 18: I-oleic fatty acid; l8:2-linoleic fatty acid; l8:3-lmolenic fatty acid: 20:4--arachidonic fatty acid; S/U-saturated to unsaturated fatty acid ratio: C,,,,,/C,,,20-C,,,,, to C,,,,, fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals.
N.
0
2
I -
HADJIIVANOVA
A (rn’ /mg)
Fiz. I. Equilibrium curves surface pressure (n) vs molecular art% (A ) of alveolar surlbctant lipid extracts from control (~-~) and insulin treated (0 -0 6, m--m 8, A-- A IO and n -~-r, I? hr. respectively) rats. as compared with the cquillbri~irn curve of synthetic L-r-dipalmitoylphosph~~tidyich(~ii~~e(0 0,. Each point indicates the mean of 4 determinations. the fatty acid pattern of phosphatidylglycerol and phosphatidylethanolamine fractions. The latter was considerably enriched, as well, in arachidonate IO and I2 hr ifter treatment. Following insulin administration a decrease of the saturated unsatL]~~ted (S/U) fatty acid ratio was observed for the three phospholipid components investigated, with phos~hatidylglyceroi having its lowest value already at the sixth hour. The fatty acid ratio C,,,,, C,,,,,, was also reduced for phosphatidylglycerols and phosphatidylethanolamines, as shown in Tables 3 and 4. Figure I represents the monolayer study results of alveolar wash total lipid extracts from control and insuiill-treated animals, as compared with the equilibrium curve of synthetic I.-~-dipalm~toyiph~~sp~l~ltidylch~line. Data are presented as surface prcssure rr (dynicm) vs molecular area A (m’img). The main results, evident from this study, were the density changes in the monolayer, which occurred after insulin administration. The lipid film monolayer was found maximally expanded 8 hr post hormone treotment. which correlated well with the data of phospholipid content and fatty acid composition of the ph~~sph~~lipidcomponents studied. The most compact and dense monolayer, as indicated by the equilibrium curIre of the WLiS the synthetic diOX palmitoylphosphatidylcholine, where both fatty acid residues were saturated. DISCUSSION
Recent studies have greatly increased our understanding of lung surfactant synthesis and secretion,
ci trl
by defining its composition. by d~~cl~rnciititlg changes in the concentration of sur~ictarlt c~~t~~p~~nctlts induced by diKerent agents and by identifying the role of potential metabolic regulatory mechanisms governing lung phospholipid synthesis and turnover. The production of lung surfactant. a:, well as the maturation 01‘ the fetal lung al-e known to kc undct multiple hormonal control (Avery, 1975: Ballard P( tri., 1977; Das and Kutnar. 1975: lladjiivanova ~i cl/.. 1979: Hitchcock, 1979). Recent studies have cmphz sized a possible role of insulin in this respect. It IS non known that glucose can serve as ;I aubstratr for pulmonary lipid synthesis and can ho utilired for both the glyceride backbone and the fatty acid moiet) 01 surfactant phospholipids; it may also be utilized 17) the lung to provide the energy reducing cqui~~alent~ necessary to support phospholipid fatty acid \\IIthesib. Even though insulin is recognized a\ an inportant regulatory of c~~rb~~hydratcand lipid metahohsm in other tissues, the inf~rll~~~ti~~n availahlc on its role in lung is still insufficient and contradictory. The present investigation indicates the elkct 01‘the hormone on the composition and surface acticit! 01 alveolar phospholipids. As previously demonstr;lted (Hadjiivanova (‘1 t/l., 19XI ) the hormone enhanced phospholipid biosynthesis in lung ‘rubcellular fractions. showing the highest values I and 3 hr post treatment. In this study aIvcola~- surfactant phosph~lipi~is were found rn~~~imally raised. with the tahelled precursor being maximally incorporated into them, from the sixth up to the eighth hour after the hormone administration. It is now well known (Bur-anska and van Goldc. 1977: Chevalier and C‘ollc‘t. 1973) that surfactant phospholipids synthczl/cd 111 the endoplasmic reticulum are stored in the lamellar bodies of pulmonary type II epithclial cells. from where they are secreted on to the alvcolnr surface. Evidently. the lay period of time (h-8 hr). ohser~d in this study. is the time needed fol- the aynthcsi\ and transportation of these surfactant component\ to the site of their physiologic activity. This is known to occur via the Golgi apparatus, the multiccsicular bodies and the cell membrane, prohablt either by fusion of the lipids or as a phospholipid cotnplcx facilitated by phospholipid transfer proteins (Post ct rd.. 1980). Recently insulin has been indicated to afrect the fatty acid c~~rnp~?siti[~n of lung s~lbc~l~u~~~rphospholipids (Hadjiivanova of ~ii.. 1081 ). diminishintl the content of the saturated fatty acids in the f&ions studied. As it is obvious from the present investigation fatty acid composition of alveolar surfactant phospholipids is similarly affected by insulin treatment. Six and X hours after ~idministlatiori the percentage contribution of the saturated fatty acids in all phospholipid fractions markedly decreased with a simuitan~ous elevation of uils~~tur~lted fatty acids. suggesting enhanced desaturase activities in the lung. A considerably less pronounced increase was observed with the long chain fatty acids. Since insulin is known to stimulate the activities of lung tissue fatty acid synthesizing cnlymes (Das and Kumar. 1975). and as frequently fatty acid synthesis and desaturase activities are closely correlated and regulated together (Jeffcoat and James, 197X). the hormone probably acts by direct provision of ticaNO~.O
Insulin
effect on characteristics
synthesized fatty acids which are readily desaturated and/or elongated. A great deal of evidence has accumulated lately, indicating there is a defect in fatty acid desaturation in both experimental and human diabetes. Tissues of diabetic animals have shown alteration in their fatty acid composition, consistent with a depressed desaturase activity (Brenner ct rrl.. 1968; Eck rt d., 1979). The abilities to desaturate stearic (18:O) to oleic (IX: I) fatty acid and linoleic (18:2) to arachidonic (20:4) fatty acid, were markedly reduced (f’elulfo rt (I/., 1970). Insulin treatment corrected the desaturase defect and it was presumed that this effect i\ a protein syntlhetic effect. Sofar, rapid repair of desaturase defect occurring with alloxan diabetes. as well as insulin induction of desaturase activities have been reported (Gelhorn and Bsn.jamin. 1966; Peluffo ct al.. 1971). However, time periods shorter than 34 hr for repair of induction of dzsaturase activity suggest that some metabolic factl)r sensitive to insulin might be responsible, rather than stimulation of enzyme-protein synthesis. Results from this study as presented in Fig. I demonstrated good correlation between fatty acid cl,mposition and surface-activity characteristics of lung wash lipid extracts. Decreased contribution of saturated fatty acids in alveolar surfactant phospholipids. found most pronounced 8 hr post insulin tteatmcnt. caused a considerable diminution of surface pressure (1~11.with a significant expansion of molecular area (~1). as indicated by the equilibrium curve. Data were consistent with the less dense p.tcking of unsaturated fatty acids in the monolayer, Mhercas dipalmitoylphosphtidylcholine. with both fatty acid residues saturated, showed highest values for surface pressure and lowest for molecular area. Reduction of palmitic acid content in surfactant lecithin was found to make this lecithin less effective in lowering minimal surface tension on compression in a surface balance (Kyriakides ct cd.. 1975. 1976). The data of the present investigation. as well, indicate that the quality of lung phospholipids, especially their fatty acid composition, is an important determinant of’ their elTectiveness as surface-active material. Alterattons of alveolar surfactant lipid constituents could significantly affect. the surface characteristics of the alveolar surface-active complex (Colacicco CI cl/., 1975; Tabac and Notter, 1975). and hence its physiological role and significance. Since insulin is lone of the hormones under current in\,estigation that may regulate pulmonary surfactant formation. secretion and turnover, this study is to contribute to the present understanding and controlling the prevention of the Respiratory Distress Syndt-
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of lung surf&ant
199
Batenberg J. J. and of pulmonary
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cells. In Rrrieir.v m Pwinrrrrrl ,Mrc/kk E. M.). Vol. 3, pp. 73-l 14. Raven
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Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. CL/U. J. Biodwm. 37. 91 I-917. Brenner R. R.. Peluffo R. 0.. Mercuri 0. and Restclli M. A. (1968) Effect of arachidonic acid in the alloxandiabetic rat. Am. J. Ph)sio/. 215, 63-70. Burnell J. M. and Balint J. A. (1975) Erects ofesscntial fatty acid (EFA) deficiency on lung mechanics in rats. Li>t/n Proc. F& Am. Sots rq. Bid. 34, 426-427. Chevalier G. and Collet A. J. (1972) In riro incorporation ofcholine-‘H leucine-‘H and galactose-‘H in alveolar type II pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse hy electron microscopy. Am/. Rec. 174, 289 -3 IO. Colacicco G., Basu M. K. and Scarpelli E. M. (1975) Influence of cholesterol and cholesteryl ester on the dynamic force/area curves of dipalmitoyl Iccithin. FM/U Pmt. Am. SOCY carp. Bid 34, 426. Das D. R. and Kumar S. (I 975) Nutritional and hormonal variations after de nono fatty acid synthesis in mammalian lung. F&I Proc. Fedn Am. .%cc v~p. Bid. 34, 673 (Abst.). Dobbs L. and Mason R. (1978) The effect of /j-adrenergic agonists on the release of disaturated phosphatidylcholinc from isplated pulmonary alveolar type II cells. .4!it. Rw. wsp. Dis. 117 (suppl). 331 (Abst). Eck M. G., Wynn J. 0.. Carter W. J. and Faas F. H. ( 1979) Fatty acid desaturation in experimental diabetes mellitu\. Diuhel~5 28, 4799485. Frosolono M. F. (I 977) Lung. In Lipid h’c~/trholi.\m m ,M~~mmulv (Edited by Sneyder F.). Vol. 2. p. I. Plenum Press. New York. Gelhorn A. and Benjamin W. (I 966) The effect of insulin on monounsaturated fatty acid synthesis in diabetic rats. The stability of the informational RNA and of the enqmc svstem concerned with fatty acid dcsaturation. Bicdr/t~~. h;.oplii~.,. .4(,/o 116, 460-466. Gluck L.. Kulovich M. V.. Eidelman A. 1.. Cordero L. and Khazin A. F. (1972) Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin in the human fetus and newborn and ethiology of the re\pira tory distress syndrome. Pcdicrr. Re.s. 6, XI&XX. Goerke J. (1974) Lung surfactant. Bioc~hitn. hropl~~,.t. dc,/o 344, 241.-261. Golde L. M. G. van (I 976) Metabolism of phospholipid\ in the lung. Am. Rrr. resp. LX.\. 114, 97771000. Hadjiivanova N. B.. Koumanov K.. Dimitrov G. and Georgiev G. (1978) Insulin effect on the biosvnthesis ol lung phospholipids and their fatty acid compo
200
N. HAUJI~VAYOVAet ~1
analysis of phospholipids separated by thin layer chromatography. J. Chromat. 40, 90-96. Kates M. (1972) Techniques o#‘Lipidology (Edited by Work T. C. and Work E.), pp. 158-160. Etsevier, New York. Khosia S. S.. Gobrdn L. I. and Rooney S. A. (1980) synthesis by Stimulation of phosphatidylch~line 17-a-estradiol in fetal rabbit lung. Biochirn. Biophlx Actu 617, 282-290. Kyriakides E. C., Burnell J. M. and Balint J. A. (1975) Reduction of disaturated lecithins and impairment of surface activity of lung lavage lipid in essential htty acid deficiency. Ciitz. Res. 23, 598A. Kyriakides E. C., Beeler D. A., Edmonds R. H. and Balint J. A. (1976) Alterations in phosphatidyl~hoiine species and their reversal in pulmonary surfactant during essential fatty acid deficiency. Biochim. hiophys. Acta 431, 399-417. Morishige W. K., Wetake C. A., Greenwood F. C. and Akaka J. (1977) Pulmonary insulin responsitivity: in riro effects of insulin on the diabetic rat lung and specific insulin binding to lung receptors in normal rats. &nJocrinolog~ 100, 17 1o- I7 16. Moxley M. A. and Longmore W. J. (1975) Studies on the effects of alloxan and streptozotocin induced diabetes on lipid metabolism in the isolated perfused rat lung. Lifk Sri. 17, 921-926. Moxley M. A. and Longmore W. J. (1977) Effect of experimental diabetes and insulin on lipid ~netabolism in the isolated perfused rat lung. Biochinz. hiophys. Arfu 488, 2 188224.
Peluffo R., Ayala S. and Brenner R. R. (1970). Metabolism of fatty acids of the linoleic acid series in testicles of diabetic rats. Am. J. Physiol. 218, 669-673. Peluffo R.. de Gomez Dumm N. T., de Alaniz J. T. and Brenner R. R. (1971) Effect of protein and insulin on linoleic acid desaturation of normal and diabetic rats. J. N&r. 101, 107551084. Post M., Batenburg J. J., Schuurmans E. A. J. M. and Golde L. M. G. van (1980) Phospholipid-transfer activity in type II cells isolated from adult rat lung. Biochim. hiophys. Acta 620, 317.-321. Scarpelli Em. (1968) The ~~~~ucfu~f System q/the Lung. Lea & Febiger, Philadelphia. Smith Y. F., Mullon D. K., Hamosh M., Scanlon J. W. and Hamosh P. (1979) Serum prolactin and respiratory distress syndrome in the newborn. P&U. Res. 14, 93-95. Stubbs W. A., Morgan I., Lloyd B. and Alberti K. G. M. M. (1977) The effect of insulin on lung metabolism. Clin. Endocr. 7, 18 1-186, Suzuki Y. and Tabata R. (1980) Selective reduction of phos~hatidyl~lycerol and phosphatidylchoiine in pulmonary surfactant by 4-aminopyrazolo(3,4)pyrimidine in the rat. J. Lipid Res. 21, 109@-1096. Tabac S. H. and Notter R. H. (1975) Factors affecting pure and mixed films of pulmonary surfactant components. Fedn Proc. Fedn Am. Sots elrp. Eiol. 34, 426. Wu B.. Kikkawa Y.. Orzalesi M. M.. Motoyama E. K., Kaibara M., Zigos C. L. and Cook C. D. (1973) The effect of thyroxine on the maturation of fetal rabbit lungs. Bin/. N~wnarr 22, i61- 168.
I-~ADJWANOVA’,
K. KWJMANOV IL Iv
‘C‘en~ral ILaboratorq of Biophysics. Phyrical Chemistry.
Bulgarian
PANAJOTOV’
and M.
Slate University
IVANOVA’
of Sciences. I I I3 Sofia and of Sofia, Bulgaria [Tc/. ‘?-M-IO]
Academy
‘Department
01‘
Abstract-1. The it~c(~rp~~r~iti~n of [‘~C]~dlmitic acid into rat alveolar wash total ph(~spholipids ;md ph(~sph~~lipid frnctions has been followed for 6. 8, IO and 12 hr after insulin adrninistr~~tl(~Il. indicating a consider-able enhancement. phosphatidylcthanolamines and phos-7. The fatty acid profiles of phosphatidylcholines, phatidylglyccrols were found changed after the hormone administration. 3. Elghl! hours post insulin treatment the precursor incorporation was highest in all phospholipid fractions :,tudicd, us well as the contribution of long chain fatty acids. 4. Dynamic monoluycr studies of the lung wash lipid extracts indicated a maximally expanded lipid film corresponding to the highly unsaturated phospholipids present.
INTRODf WTION
The atveoli of the mammalian iung are lined with a hpid-rich substance known as surfactant (Goerke, 1074). Surfactant has the capacity to lower the surf,~ce tension in lthe alveoli at end-expiration, thus stabilizing the lung and preventing atelectasis (Scarpclli. 1968). Decreased amounts of pulmonary surfactant. due either to premature birth or to a maternal disease state. is thought to be the immediate cause of the Respiratory Distress Syndrome (RDS) of the newborn (Gluck CI (I/., 1972). The RDS itself is a ma,jor cause of neonatal mortality. particularly in the premature infant Pulmonary surfactant is known to be synthesized in the type II alveolar epithelial cells (Batenburg and v,~n Golde, 1979). Characteristic components of these chills are the osmiophillic lamellar inclusion bodies, which are the storage-site of lung surfactant (Baranska and van Golde. 1977: Chevalier and Collet, 1<)71). The most ~~hund~~nt surfactant constituent in lamallar bodies and alveolar surfactant is a phosphatidylcholine (1.2-diacyl .m-glycero-3-phosphocholine). enriched, in saturated fatty acids, particularly palmitic acid (Frosolono. 1977; van Golde. 1’176). There are numerous hormones currently being investigated which may influence lung phospholipid metabolism, though the mechanisms by which they act have by large not been delineated. There is ample e\ idence that cjrctIlating glucocorticoids are involved in the regulation and acceleration of lung maturation, enhancing as well the production of surfactant in a variety of mammalian species (Avery, 1975: Ballard 1’1 cd., 1977). Several reports (Hit&cock. 1979: Wu PI ul., 1973) suggest that thyroid hormones exert similar stimuIatory effects on lung development in fetal animals. Recent data indicate that prolactin and estrogen may stimulate surfactant formation (Khosla et (I/.. 19X0; Smith
<‘I r/i..
1979).
Adrenaline.
too.
has been
found to influence sign~~cantly the content and fatty acid composition of lung tissue phospholipids (Had1979). whereas, P-adrenergic agonists Jnvanova, specifically stimulated the release of disaturated phosphatidylcholine (Dobbs and Mason, 1978). There are relatively few investigations on the role of insulin in this aspect. Studies from several laboratories have demonstrated the possible signifcance of this hormone in regulating lipid synthesis in the lung (Morishige rt d., 1977; Stubhs rt a/.. 1977). Diabetic animals have been found to incorporate 60 -X@‘,,less gfucose into total tissue and surfactant phospholipids than normal animals (Moxley and Longmore, 1975. 1977), and have reduced activities of lung acetyl-CoA carboxylase and fatty acid synthetase (Das and Kumar. 1975). Insulin administration restored both glucose incorporation and enzyme activities to normal levels. Insulin was shown, as well. to influence the biosynthesis and fatty acid composition of lung tissue ~Hadjiivanova PI ul., 1978) and subcellui~~r fraction phospholipids (Had.iiivanova CI ii/.. 198 I ). Few attempts, however. have been made to correlate surfactant composition with surface activity by altering lipid composition in I.&O (Burnell and Ralint. 1975; Suzuki and Tabata, 1980). Such an approach is required for the clarification of the significance of the constituents to surface activity ill sitzc. No data at present are available on the role of insulin in this This report deals with the extension of our aspect. previous studies, its objective being to investigate insulin effect on the phospholipid and &tty acid composition of alveolar washes and the changes found in their surface activity. MATERIALS
Male
Wistar
strain
rats.
(Mann Res. Lab..
.&ND METHODS ISO-200g
were
administered
U.S.A.) intravenously (v. saphena), 8U/kg of body weight and sacrificed 6. 8. IO and i 2 hr. respectively. after injection. For ~he incorp~~r~ltit~n studies of labelled precursor rats of all groups received insuiin
I96
N. HAIXIIVANOVA rr ~1.
simultaneously with insulin 5 nCi [I-‘JC]palmttic actd (Amersham, En_eland). specitic activity 256 /lCi;mmol. complexed with albumin. Appropriate animals for controls were administered 0.5 ml sahne (0.15 M NaCl solution). containing the labelled palmitate. All rats were fasted I5 hr prior to treatment. but had access to water. The groups consisted of 8 rats each. For the fatty acid composition studies samples from 25 animals were pooled for each group. Following anaesthesia with intraperitoneally administered amobarbital (Merck. FRG). 50 mg/kg of body weight, lungs were lavagcd in .ciru five times, by the procedure using 5ml of SET (0. IS M NaCI. 0.001 M EDTA, 0.01 M Tris-HCI). Lung washes were centrifuged at 600~ for IOmin to rcmovc cells and cell debris. Liptds were extracted according to Bligh and Dyer (1959). Phosphorus content of total phospholiptds was determined in aliquots of the original lipid extract (Kahovkova and Odavic, 1969) and phospholipids were fractionated into indivtdual components by one dimcnsionnl two-phase thin layer chromatography on silica gel G prrcoated glass plates (Merck. FRG). As developing solvents were used chloroform-methanol-acetic aciddwater \“‘I ) (70:35:X:4. and chloroformmethanol-aatcr (60: 35: 5. vvvv). respectively. for the separation of phosphatidyl@ycerol. The specific radioacttvjtty of lung wash phospholipids was determined hy unalysing their radioactivity and concentration after sepamtion by thin htyer chromntography. The radioactivity was measured in IO ml scintillation solution. The concentration of phospholipid components was determined by measuring their phosphorus content (Kyriakides rl ~1.. 1976). Statistical analysis. where :tppropriate. was carried out using Studcnt’c r-test. The fatty acid composition of the three major phospholipids isolated (phosphatidylcholine. phosphatidylglycerol and phosphatidylethanolamine) was determined by gas-liquid chromatograph,y of their methyl esters. Upon hydrolysis of the phosphohpldh. fatty acids were methylatcd according to the procedure of Hartman and Lago (1973). The methyl ester5 were then applied to the gas (Carlo-Erba). equipped with chromatograph Ramc-ionization detector, isothermally at I90 C. with a 2 m column. coated with lo”,, DEGS on Chromosorb W,,, x,, mesh (Pharmacia, Sweden). nitrogen ns gas-carrier. The molar percentage of fatty acids was calculated by adoption of the mode described by Kates ( 1972). Because of the small amounts of phospholipids present. lipid cxtracta for each group of 25 rats wcrc pooled for analysi< of their fatty acid moiety. Dynamic propertic!, studies of monolaycra ot‘ lung wash lipid extracts were performed on a Beckman microbalance. Surface pressure x uas measured ,by the Wilhelmy method using a roughened glass plate. LIpId extracts dissolved in chloroform (I mg of lipid per ml) were spread as films on 0.01 M NaCl solution. using a microplpettc. Experiments were carried out at 20 C. REScII.1 S Previous investigations in this laboratory have demonstrated that insulin significantly enhanced lung phospholipid biosynthesis, raising the incorporation of labelled precursors into whole tissue homogenate and subcellular fraction phospholipids (Hadjiivanova rf u/., 197X. 1981). Results from this study, as presented in Table I. clearly indicate that with alveolar surfactant phospholipids insulin exerted a similar effect. Already at the sixth hour after treatment total phospholipids in the alveolar wash were found to increase (I l7’!,,). their specific activity being maximally raised X hr after the hormone administration (127’:,, as compared with the control values). As is obvious from Table I similar changes were observed
Insulin Table
2. Fatty
GKMJp Fatty acid 14:o 16:O 16:l l8:O l8:l l8:2 IS:3 20:4 S/U C,,.,&w,,
effect on characteristics
of lung surfactant
acid composition of phosphatidylcholines from alveolar insulin-treated rats (molar percentage)
197
washes
of control
and
Control
l-p6 hr
I -8hr
I- 10 hr
IL12 hr
9.06 55.64 8.02 8.57 6.87 6.65 5.19 trace 2.74 2.67
3.16 60.40 16.23 1.87 8.79 6.70 I.81 I .07 I .89 3.95
3.91 49.39 15.43 7.40 12.04 6.93 4.x9 trace 1.54 2.20
4.05 52.03 12.00 7.07 8.75 945 5.05 1.60 I.71 2.13
4.22 57.01 12.94 4.46 6.41 10.97 3.98 I.91 2.87
I--6, 8. IO and I2 hr after insulin treatment; 14:&miristic fatty acid; 16:&palmitic fatty acid; 16: I-palmitoleic fatty acid; 18:Gstearic fatty acid; 18: I-oleic fatty acid; 18:2-linoleic fatty acid; 18: 3Pmlinolenic fatty acid; 20:karachidonic Pdtty acid: S/U-saturated to unsaturated fatty acid ratio; C,,,,,/C,,,20-C,,,,, to C,,,, Fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals.
in the main surfactant phosphohpids, phosphatidylcholine and phosphatidylglycerol, their specific activities being raised up to 125 and 120”/,, respectively. Ten and twelve hours post treatment, the content and specific activities of both total phospholipids and phosphohpid fractions tended towards the control values. The fatty acid composition changes of alveolar surfactant phosphatidylcholines, phosphatidylglycerols and phosphatidylethanolamines observed after insulin administration were of considerable interest. Six and eight hours post treatment the horTable
3. Fatty
mone caused a significant decrease in the percentage contribution of the saturated miristic (14:0), palmitic (16:0) and stearic (18: 0) fatty acids in almost all phospholipid fractions studied (Tables 2 and 3) with the exception of miristic (14:O) fatty acid in the phosphatidylethanolamines (Table 4), where it was found markedly elevated (209%). The content of the unsaturated palmitoleic (16: I), oleic (18: 1) and linoleic (18:2) fatty acids was raised in phosphatidylcholine and phosphatidylglycerol fractions as compared with the controls. Worthwhile to denote is the pronounced contribution of linolenate (I 8 : 3) to
acid composition of phosphatidylglycerols from alveolar insulin-treated rats (molar Dercentaee)
washes of control
and
GrOUp
Fatty acid l4:O l6:O l6:l 18: I 18: I l8:2 l8:3 20:4 SjU CW&W”
Control
1-6 hr
I.38 35.65 9.22 17.24 23.97 7.68 4.84 trace I I9 0.86
I.18 16.X6 II.32 12.18 26.88 15.79 3.90 Il.88 0.43 0.42
I-8
hr
1.35 40.46 6.1 I 9.03 20.43 8.78 5.65 8.20 I .09 0.92
IL10 hr 0.62 32.26 7.68 21.13 15.75 8.27 8.49 6.79 I I5 0.67
I-12
hr
0.61 36.34 6.33 8.99 12.11 14.96 16.72 3.94 0.85 0.76
I-6. 8, IO and I2 hr after insulin treatment; l4:&-miristic fatty acid; l6:O palmitic fatty acid; 16: I--palmitoleic fatty acid; 18:&stearic fatty acid; 18: I-oleic fatty acid; I8:2-linoleic fatty acid; 18:3Plinolenic fatty acid; 20:Garachidonic fatty acid; S/U-Saturated to unsaturated fatty acid ratio: C,,,,,iC,,,,,--C,,,,, to C,,,,, fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals. Table 4. Fatty acid composition
of phosphatidylethanolamines from alveolar insulin-treated rats (molar percentage)
washes of control and
Group Fatty acid
Control
I---6 hr
I--X hr
I-- IO hr
I---l2 hr
l4:O 16:O 16: I l8:O 18: I IS:2 l8:3 20:4 s/u (7,” ,JC,“,”
0.75 24.94 14.93 19.77 17.18 10.53 4.30 7.61 0.83 0.68
1.56 22.95 8.70 19.44 21.51 10.69 8.01 7.14 0.78 0.50
1.87 20.01 12.50 13.60 16.00 9.28 20.48 6.25 0.55 0.52
0.73 36.42 IO.41 8.14 14.46 9.33 8.5X I? 54 0.82 0.90
0.75 30.51 8.34 Il.88 Il.99 6.05 10.63 19.85 0.76 0.66
l-6. 8. IO and I2 hr after insulin treatment; 14:&miristic fatty acid: l6:O palmitic fatty acid; 16: I--palmitoleic fatty acid; 18:sstearic fatty acid; 18: I-oleic fatty acid; l8:2-linoleic fatty acid; l8:3-lmolenic fatty acid: 20:4--arachidonic fatty acid; S/U-saturated to unsaturated fatty acid ratio: C,,,,,/C,,,20-C,,,,, to C,,,,, fatty acid ratio. Values represent the mean of duplicate analyses of the pooled samples of each group consisting of 25 animals.
N.
0
2
I -
HADJIIVANOVA
A (rn’ /mg)
Fiz. I. Equilibrium curves surface pressure (n) vs molecular art% (A ) of alveolar surlbctant lipid extracts from control (~-~) and insulin treated (0 -0 6, m--m 8, A-- A IO and n -~-r, I? hr. respectively) rats. as compared with the cquillbri~irn curve of synthetic L-r-dipalmitoylphosph~~tidyich(~ii~~e(0 0,. Each point indicates the mean of 4 determinations. the fatty acid pattern of phosphatidylglycerol and phosphatidylethanolamine fractions. The latter was considerably enriched, as well, in arachidonate IO and I2 hr ifter treatment. Following insulin administration a decrease of the saturated unsatL]~~ted (S/U) fatty acid ratio was observed for the three phospholipid components investigated, with phos~hatidylglyceroi having its lowest value already at the sixth hour. The fatty acid ratio C,,,,, C,,,,,, was also reduced for phosphatidylglycerols and phosphatidylethanolamines, as shown in Tables 3 and 4. Figure I represents the monolayer study results of alveolar wash total lipid extracts from control and insuiill-treated animals, as compared with the equilibrium curve of synthetic I.-~-dipalm~toyiph~~sp~l~ltidylch~line. Data are presented as surface prcssure rr (dynicm) vs molecular area A (m’img). The main results, evident from this study, were the density changes in the monolayer, which occurred after insulin administration. The lipid film monolayer was found maximally expanded 8 hr post hormone treotment. which correlated well with the data of phospholipid content and fatty acid composition of the ph~~sph~~lipidcomponents studied. The most compact and dense monolayer, as indicated by the equilibrium curIre of the WLiS the synthetic diOX palmitoylphosphatidylcholine, where both fatty acid residues were saturated. DISCUSSION
Recent studies have greatly increased our understanding of lung surfactant synthesis and secretion,
ci trl
by defining its composition. by d~~cl~rnciititlg changes in the concentration of sur~ictarlt c~~t~~p~~nctlts induced by diKerent agents and by identifying the role of potential metabolic regulatory mechanisms governing lung phospholipid synthesis and turnover. The production of lung surfactant. a:, well as the maturation 01‘ the fetal lung al-e known to kc undct multiple hormonal control (Avery, 1975: Ballard P( tri., 1977; Das and Kutnar. 1975: lladjiivanova ~i cl/.. 1979: Hitchcock, 1979). Recent studies have cmphz sized a possible role of insulin in this respect. It IS non known that glucose can serve as ;I aubstratr for pulmonary lipid synthesis and can ho utilired for both the glyceride backbone and the fatty acid moiet) 01 surfactant phospholipids; it may also be utilized 17) the lung to provide the energy reducing cqui~~alent~ necessary to support phospholipid fatty acid \\IIthesib. Even though insulin is recognized a\ an inportant regulatory of c~~rb~~hydratcand lipid metahohsm in other tissues, the inf~rll~~~ti~~n availahlc on its role in lung is still insufficient and contradictory. The present investigation indicates the elkct 01‘the hormone on the composition and surface acticit! 01 alveolar phospholipids. As previously demonstr;lted (Hadjiivanova (‘1 t/l., 19XI ) the hormone enhanced phospholipid biosynthesis in lung ‘rubcellular fractions. showing the highest values I and 3 hr post treatment. In this study aIvcola~- surfactant phosph~lipi~is were found rn~~~imally raised. with the tahelled precursor being maximally incorporated into them, from the sixth up to the eighth hour after the hormone administration. It is now well known (Bur-anska and van Goldc. 1977: Chevalier and C‘ollc‘t. 1973) that surfactant phospholipids synthczl/cd 111 the endoplasmic reticulum are stored in the lamellar bodies of pulmonary type II epithclial cells. from where they are secreted on to the alvcolnr surface. Evidently. the lay period of time (h-8 hr). ohser~d in this study. is the time needed fol- the aynthcsi\ and transportation of these surfactant component\ to the site of their physiologic activity. This is known to occur via the Golgi apparatus, the multiccsicular bodies and the cell membrane, prohablt either by fusion of the lipids or as a phospholipid cotnplcx facilitated by phospholipid transfer proteins (Post ct rd.. 1980). Recently insulin has been indicated to afrect the fatty acid c~~rnp~?siti[~n of lung s~lbc~l~u~~~rphospholipids (Hadjiivanova of ~ii.. 1081 ). diminishintl the content of the saturated fatty acids in the f&ions studied. As it is obvious from the present investigation fatty acid composition of alveolar surfactant phospholipids is similarly affected by insulin treatment. Six and X hours after ~idministlatiori the percentage contribution of the saturated fatty acids in all phospholipid fractions markedly decreased with a simuitan~ous elevation of uils~~tur~lted fatty acids. suggesting enhanced desaturase activities in the lung. A considerably less pronounced increase was observed with the long chain fatty acids. Since insulin is known to stimulate the activities of lung tissue fatty acid synthesizing cnlymes (Das and Kumar. 1975). and as frequently fatty acid synthesis and desaturase activities are closely correlated and regulated together (Jeffcoat and James, 197X). the hormone probably acts by direct provision of ticaNO~.O
Insulin
effect on characteristics
synthesized fatty acids which are readily desaturated and/or elongated. A great deal of evidence has accumulated lately, indicating there is a defect in fatty acid desaturation in both experimental and human diabetes. Tissues of diabetic animals have shown alteration in their fatty acid composition, consistent with a depressed desaturase activity (Brenner ct rrl.. 1968; Eck rt d., 1979). The abilities to desaturate stearic (18:O) to oleic (IX: I) fatty acid and linoleic (18:2) to arachidonic (20:4) fatty acid, were markedly reduced (f’elulfo rt (I/., 1970). Insulin treatment corrected the desaturase defect and it was presumed that this effect i\ a protein syntlhetic effect. Sofar, rapid repair of desaturase defect occurring with alloxan diabetes. as well as insulin induction of desaturase activities have been reported (Gelhorn and Bsn.jamin. 1966; Peluffo ct al.. 1971). However, time periods shorter than 34 hr for repair of induction of dzsaturase activity suggest that some metabolic factl)r sensitive to insulin might be responsible, rather than stimulation of enzyme-protein synthesis. Results from this study as presented in Fig. I demonstrated good correlation between fatty acid cl,mposition and surface-activity characteristics of lung wash lipid extracts. Decreased contribution of saturated fatty acids in alveolar surfactant phospholipids. found most pronounced 8 hr post insulin tteatmcnt. caused a considerable diminution of surface pressure (1~11.with a significant expansion of molecular area (~1). as indicated by the equilibrium curve. Data were consistent with the less dense p.tcking of unsaturated fatty acids in the monolayer, Mhercas dipalmitoylphosphtidylcholine. with both fatty acid residues saturated, showed highest values for surface pressure and lowest for molecular area. Reduction of palmitic acid content in surfactant lecithin was found to make this lecithin less effective in lowering minimal surface tension on compression in a surface balance (Kyriakides ct cd.. 1975. 1976). The data of the present investigation. as well, indicate that the quality of lung phospholipids, especially their fatty acid composition, is an important determinant of’ their elTectiveness as surface-active material. Alterattons of alveolar surfactant lipid constituents could significantly affect. the surface characteristics of the alveolar surface-active complex (Colacicco CI cl/., 1975; Tabac and Notter, 1975). and hence its physiological role and significance. Since insulin is lone of the hormones under current in\,estigation that may regulate pulmonary surfactant formation. secretion and turnover, this study is to contribute to the present understanding and controlling the prevention of the Respiratory Distress Syndt-
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