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β-Adrenergic-induced surfactant synthesis, secretion, and reutilization in fetal rabbit lung and isolated differentiating type II alveolar cells
J3-Adrenergic-induced surfactant synthesis, secretion, and reutilization in fetal rabbit lung and isolated differentiating type II alveolar cells Mary-Gordon Rasmusson, MSc, J. Elliott Scott, PhD, and Margaret R. Oulton, PhD Halifax, Nova Scotia, Canada In vivo and in vitro approaches were used to examine the role of ~-adrenergic agonists in the regulation of surfactant synthesis and secretion in the lung. Rabbit fetuses of either 28 or 30 gestational days were treated with isoxsuprine. Fetuses from half of the does in each group were removed and allowed to breathe for 30 minutes. The others were left in utero. Intracellular and extracellular surfactant pools were isolated. Breathing increased secreted surfactant. On the twenty-eighth day without breathing, isoxsuprine treatment increased secretion of surfactant. The reverse effect was noted in the group that received the drug and also breathed. In contrast, on the thirtieth day, the drug inhibited surfactant release in those fetuses that did not breathe. In in vitro studies, undifferentiated type II alveolar cells were isolated and stimulated to differentiate. Subsequent exposure to isoxsuprine (5 or 10 1-Lmoi/L) stimulated both the. synthesis and secretion of radiolabeled disaturated phosphatidylcholine. Concurrent incubation of those cells exposed to 10 1-Lmoi/L isoxsuprine with either unsaturated or disaturated phosphatidylcholine that was carbon 14 labeled showed a strong preference for incorporation of the latter phospholipid into total cellular phosphatidylcholine. These results suggest that ~-adrenergics may inhibit as well as stimulate secretion of surfactant by type II alveolar cells and that these cells may reincorporate secreted disaturated phospholipid. (AM J 0BSTET GYNECOL 1988;158:373-9.)
Key words: 13-Adrenergic, type II alveolar cell, surfactant
To facilitate gas exchange at birth, the pulmonary tissue spaces that give rise to mature alveoli must be cleared of liquid. The exact mechanism by which this is accomplished is not known. The role of glucocorticoids has been investigated extensively in both tissue maturation in general and in the lung in particular.'· 2 However, their effect has been largely defined as one of induction of differentiation. On the other hand, evidence suggests that 13-adrenergics may regulate the reabsorption of fetal alveolar fluid in late gestation, 3 • 4 as well as stimulate the secretion of the pulmonary surfactant.•-6 Indeed, the fetal lung apparently demonstrates a marked increase in !;S-adrenergic receptors as term approaches. 7 However, the role of these compounds at the cellular level in regulating the synthesis and secretion of the pulmonary surfactant has not been determined. The present study was designed first to establish the effect of 13-adrenergics on both the intra-
From the Departments of Anatomy, Obstetrics and Gynecology, and Physiology, Dalhousie University. Supported fly the Medical Research Council of Canada, Presented at the Forty-third Annual Meeting of The Society of Obstetricians and Gynaecologists of Canada, Ottawa, Ontario, Canada, june 24-27, 1987.. Reprint requests:]. E. Scott, PhD, Department ofAnatomy, Dalhousie University, Halifax, Nova Scotia, Canada BJH 4H7.
cellular and extracellular surfactant levels and how these phospholipid pools vary with breathing. Second, we wanted to correlate these findings with the ability of isoxsuprine to regulate the synthesis and secretion of disaturated phosphatidylcholine, the major constituent of the surfactant, by isolated type II alveolar cells and to assess the potential for these cells to use exogenously supplied phospholipid.
Material and methods Timed-pregnant New Zealand white rabbits were purchased from Rieman's Fur Ranch, St. Agatha, Ontario. The number of fetuses per litter ranged from one to 15, and one to two litters were used per experiment. Isoxsuprine hydrochloride (Vasodilan, 5 mg/ml) was purchased from Bristol-Myers, Candiac, Quebec. Culture materials obtained from Gibco Laboratories (Mississauga, Ontario). Radioactive choline chloride ([3H-methyl] choline chloride), l-palmitoyl-2[ 14 C]oleoyl phosphatidylcholine, and dipalmitoyl-[ dipalmitoyl-' 4 C]phosphatidylcholine were from New England Nuclear (Lachine, Quebec). Metrizamide was from Sigma Chemical Co. (St. Louis, Missouri). Other chemicals were obtained from Fisher Scientific, Dartmouth, Nova Scotia. On either the twenty-eighth or thirtieth day of ges-
373
374 Rasmusson, Scott, and Oulton
tation (day 0 is day of mating), a laparotomy was performed on the pregnant does, and the fetal rabbits were treated with an intraperitoneal dose of isoxsuprine (0.5 mg/fetus, 0.1 ml) by injection through the uterine wall. Fetuses were then either left in utero for 30 minutes or removed to a warming tray and allowed to breathe for 30 minutes. Concurrent controls consisted of animals on which a laparotomy was performed, and the uterus was exteriorized and maintained in this condition with warming for 30 minutes. Control animals that received a saline solution injection were also used. No detectable differences were observed in the parameters measured in the present study (i.e., 30 minutes after injection) between the untreated and saline-treated control animals, and therefore these data have been pooled. All does were maintained under anesthesia (sodium pentobarbital, 30 mg/kg, intravenously to the lateral ear vein) during this period. Fetuses were killed by an intraperitoneal lethal dose of sodium pentobarbital. Fetuses were weighed, the trachea was cannulated via a midline neck incision, and the lungs were l~vaged with 0.15 mol/L sodium chloride in 7 X 1.00 ml aliquots. Lavage was pooled and centrifuged at 10,000 g for 30 minutes to collect "extracellular surfactant." This was stored at - 20° C until analyzed. Lungs from each fetus were excised, and a representative portion was removed and dried to constant weight at 120° C. The remainder was pooled for preparation of subcellular fractions. Fractions were prepared according to the method of Oulton et aLB Briefly, this involves homogenization of the tissue in 0.01 mol/L Tris buffer (pH 7.4) and centrifugation at 140 and 10,000 g. The pellet from the latter centrifugation is layered on a discontinuous sucrose gradient (0.25 mol/L over 0.68 mol/L) and centrifuged at 65,000 g for 60 minutes. The material that collects at the gradient interface we termed "intracellularly stored surfactant" and have identified as lamellar body-like in composition and morphologic appearance. 8 The lavage pellet (extracellular surfactant) and the gradient interface were extracted into chloroform:methanol (2: 1); the lower phase was collected and concentrated to dryness under a stream of air at 37° C and resuspended in chloroform:methanol (2: 1). Aliquots were removed for determination of phospholipid content and composition as described previously.8 In vitro examination of the effects of isoxsuprine was undertaken with isolated undifferentiated type II alveolar cells from 24-gestational-day fetal rabbit lung as described previously. 9 Preparation of these cells involved trypsinization of excised fetal lung for 45 minutes. The mixture was filtered through gauze and the cells were collected by gentle centrifugation. Fibroblasts were partially eliminated by a 90-minute in vitro at-
February 1988 Am J Obstet Gynecol
tachment, since these cells attach and grow more rapidly than the epithelial cells. Pretype II alveolar cells were collected at the interface of a discontinuous metrizamide gradient (0.099 mol/L over 0.218 mol/L). Cells are cultivated in minimum essential medium supplemented with 10% fetal bovine serum that had been hormone stripped. 1° Conditioned medium was produced from confluent cultures of fetal lung fibroblasts as described previously. 9 After cultivation of the undifferentiated type II alveolar cells for 2 to 3 days, the cells were incubated with 0, 5, or 10 J.Lmol/L isoxsuprine plus 0.5 J.LCi of 'H choline chloride. After 12 or 24 hours, the medium was removed and saved for analysis. Cells were washed with Hanks' balanced salt solution, released with trypsin/ethylenediaminetetraacetic acid, and an aliquot was counted on a Coulter cell counter (Coulter Electronics, Hialeah, Florida); and the remainder was extracted into chloroform: methanol as described previously. Disaturated phosphatidylcholine was separated on neutral alumina columns as described by Mason et al. 11 Medium disaturated phosphatidylcholine was analyzed in an identical fashion. In a second series of experiments to examine reutilization of secreted phospholipid, these cells were exposed to 20% conditioned medium (v/v) plus 0.5 j.LCi of ['H-methyl]choline chloride for 24 hours. Isoxsuprine was then added at a concentration of 10 J.Lmol/L, together with ['H-methyl]choline chloride plus 1-palmitoyl-2['4C]oleoyl phosphatidylcholine (57 mCi/mmol) or dipalmitoyl-[dipalmitoyl- 14C]phosphatidylcholine (112 mCi/mmol). Phospholipids were sonicated briefly (2 X 5 seconds) in Tris buffer (pH 7.4) before being added to the cultures. Estimation of the secretion of tritium-labeled phosphatidylcholine and disaturated phosphatidylcholine into the culture medium at the time of addition of the phospholipids (i.e., after 24 hours with tritiated choline) enabled the ratios of 14C-labeled phosphatidylcholine or [14C] disaturated phosphatidylcholine to medium phosphatidylcholine or disaturated phosphatidylcholine to be initiated at approximately 0.175 and 0.214, respectively. After the addition of the drug and phospholipid, the medium and cells were removed at 12 and 24 hours and extracted as previously described. Phosphatidylcholine was separated on LK5D thin layer chromatographic plates in a solvent system of chloroform : methanol: acetic acid: water (65: 25: 3: 1) modified from Skipski. 12 Radioactivity in phosphatidylcholine was determined and measured in a Beckman LS 5801 scintillation counter. Quench compensation was done by the method of H# (Beckman Instruments, Palo Alto, California). Disintegrations per minute of dual-labeled samples was determined with an efficiency of 65% for isotope 1 (tritium labeled) and 80% for isotope 2 (' 4 C) and < 1% spill.
~-Adrenergics,
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surfactant, and lung cell function 375
Table I. Effect of isoxsuprine administration on body and lung weights in breathing or nonbreathing preterm-delivered rabbits. After isoxsuprine administration the fetuses were either left in utero or allowed to breathe for 30 minutes, results are expressed as the mean ± SD for the nuinber of determinations shown Lung weight (gm)
No. of Treatment
de~inations*
28 day, no breathing Control Isoxsuprine 28 day, 30 min breathing Control Isoxsuprine 30 day, no breathing Control lsoxsuprine 30 day, 30 min brea~ing Control Isoxsuprine
Body weight (gm)
Fetus
7 7
34.65 ± 2.69 30.26 ± 4.98
1.33 ± 0.17 1.06 ± 0.14t
7 5
33.78 ± 4.65 31.71 ± 3.55
1.25 ± 0.15 1.08 ± 0.16
5 5
44.57 ± 10.40 45.17 ± 3.03
1.29 ± 0.34 1.37 ± O.o7
13
42.96 ± 5.64 44.40 ± 5.50
1.32 ± 0.43 1.30 ± 0.17
9
*Each determination consisted of a group of four to eight fetuses from three to five litters. tlndicates significantly different from corresponding control value (p < 0.05).
Table II. Effect of isoxsuprine administration on surfactant pool size in breathing or nonbreathing preterm-delivered rabbits, isoxsuprine administration was as described in Table I, results are expressed as the mean ± SD for five to 13 determinations
Treatment
Intracellular pool size (mg phospholipidlgm dry lung)
28 day, no breathing Control* lsoxsuprine 28 day, 30 min breathing Control Isoxsupri~e
30 day, no breathing Control Isoxsuprine 30 day, 30 min breathing Control lsoxsuprine
Extracellular pool size (mg phospholipidlgm dry lung)
Extracellular Intracellular pool size
1.84 ± 0.39 3.05 ± 1.56
0.12 ± 0.03t 0.19 ± 0.08
6.63 ± 1.93 6.68 ± 1.93
2.71 ± 1.17 2.04 ± 0.97
0.37 ± 0.19tt 0.17 ± 0.09
13.71 ± 2.95t 7.96 ± 0.99
12.11 ± 3.75 8.50 ± 0.94
1.65 ± 0.68t 0.58 ± 0.19
17.50 ± 4.45t 6.98 ± 2.44
9.92 ± 2.57 10.06 ± 1.91
2.42 ± 0.95* 2.59 ± 0.82
22.30 ± 5.70 26.14 ± 7.71
X
100
*All control values on the twenty-eighth gestational day were significantly different (JJ < 0.05) compared with the corresponding control values on the thirtieth gestational day. tlndicates significantly different (JJ < 0.05) from corresponding values of isoxsuprine-treated animals. Undicates significantly greater (JJ < 0.05) than corresponding nonbreathing control values.
Statistical analysis was qone with the Student's t test after transformation of the data to accommodate unequal variances. Significance was taken at 5% after Bonnferroni adjustment of the significance level. u Results In vivo studies. The mean body and lung weights from fetuses on the twenty-eighth or thirtieth gestational day and prematurely delivered newborns of the same gestational ages are shown in Table I. No significant alterations were ·detected after isoxsuprine administration on either day with or without breathing except in animals that received the drug on day 28. In
this instance, a combination of 0.5 mg of isoxsuprine without breathing significantly reduced (p < 0.05) the lung weight/fetus. The effect of isoxsuprine administration to fetuses that either did not breathe (fetuses) or were allowed to breathe for 30 minutes (newborns) on intracellular and extracellular surfactant pool sizes and the ratio of extracellular to intracellularly stored phospholipid are shown in Table II. Significantly greater (p < 0.05) amounts of phospholipid were observed to be present in both the intracellular and extracellular surfactant pools of the thirtieth gestational day animals compared with twenty-eighth gestational day animals. The intra-
376
Rasmusson, Scott, and Oulton
February 1988 Am J Obstet Gynecol
Table III. Effect of isoxsuprine administration on intracellularly stored surfactant composition in breathing or nonbreathing preterm-delivered rabbits, isoxsuprine administration was as described in Table I, results are expressed as the mean ± SD for five to 13 determinations
% of total phospholipid
PS
Treatment
28 day, no breathing Control 4.15 ± 1.91 Isoxsuprine 4.19 ± 2.52 28 day, 30 min breathing Control 4.12 ± 0.80* Isoxsuprine 5.27 ± 1.50 30 day, no breathing Control 1.93 ± 0.77 Isoxsuprine 1.13 ± 0.77* 30 day, 30 min breathing Control 2.69 ± 0.49 Isoxsuprine 2.91 ± 0.53
PI
SM
PC
PG
PE
Unknown
11.96 ± 2.25 11.80 ± 2.13
3.68 ± 0.79* 2.95 ± 0.71
66.40 ± 3.62* 68.70 ± 2.33
ND 0.06 ± 0.11
13.80 ± 1.47* 12.30 ± 1.67
ND ND
11.08 ± 0.80 10.31 ± 0.69
2.58 ± 0.64* 3.47 ± 0.78
72.44 ± 4.50* 68.20 ± 3.05
0.29 ± 0.27 0.22 ± 0.30
10.39 ± 0.63* 11.38 ± 2.22
0.45 ± 0.52 1.15 ± 1.07
13.17 ± 1.68 15.93 ± 0.45*
0.89 ± 0.13 1.87 ± 1.40
76.35 ± 1.09 77.44 ± 5.21
0.92 ± 0.47t 0.18 ± 0.13*
6.66 ± 0.38 9.21 ± 3.73
0.08 ± 0.15 0.09 ± 0.19
11.72 ± 0.67 10.94 ± 0.77
1.17 ± 0.57 1.43 ± 0.45
77.27 ± 1.65 77.09 ± 1.44
0.69 ± 0.46 1.04 ± 0.85
6.45 ± 0.49 6.63 ± 0.91
0.01 ± 0.03 ND
PS = Phosphatidylserine; PI = phosphatidylinositol; SM = sphingomyelin; PC = phosphatidylcholine; PG = phosphati· dylglycerol; PE = phosphatidylethanolamine; ND = not detected. *Indicates significantly differeqt (p < 0.05) compared with the corresponding control value on the thirtieth gestational day. tindicates significantly different (p < 0.05) from the isoxsuprine-treated animals. Undicates significantly different from the corresponding thirtieth gestational day isoxsuprine-treated animals that were allowed to breathe.
Table IV. Effect of isoxsuprine on the synthesis and secretion of tritiated choline-labeled desaturated phoshatidylcholine. Results are based on a minimum of six replicate determinations over three preparations pmol disaturated phosphatidylcholinel10' cells Isoxsuprine ( p.mol/L)
12 hr
0 5 10
2.17 ± 0.11 2.36 ± 0.22 2.66 ± 0.38*
I
pmol disaturated phosphatidylcholine/2 ml of medium
24 hr 10.03 ± 1.05 6.06 ± 0.89* 3.27 ± 0.31*
12 hr
7.98 ± 0.64 7.45 :±: 0.16 21.35 ± 9.29*
I
24 hr 33.45 ± 3.76 99.86 ± 11. 78* 103.67 ± 14.81*
*Indicates significantly different from corresponding controls (p < 0.05) as determined by the Student t-test.
cellular pool sizes were not altered in those groups of animals that received either isoxsuprine or were allowed to breathe for 30 minutes. On the other hand, extracellular pool sizes were significantly increased (p < 0.05) in both groups of animals that breathed. Administration of isoxsuprine to twentyeighth gestational day animals depressed the release of intracellular surfactant but only in those prematurely delivered newborns that breathed. In contrast, in the thirtieth gesta~onal day fetuses, isoxsuprine treatment significantly depressed (p < 0.05) the extracellular surfactant pool size but only in those fetuses that did not breathe after drug injection. The drug did not significantly affect the extracellular surfactant pool size in thirtieth gestational day newborns that were delivered and allowed to breathe for 30 minutes. Isoxsuprine diq not influence the ratio of extracellular to intracellular surfactant pool size in twenty-eighth gestational day
fetuses that did not breathe. In contrast, treatment with isoxsuprine on the twenty-eighth gestational day and subsequent premature delivery and breathing for 30 minutes produced a significant depression (p < 0.05) in the ratio of extracellular to intracellular phospholipid. In the thirtieth gestational day animals, the drug again significantly decreased (p < 0.05) the ratio of extracellular to intracellular phospholipid; however, this occurred only in those fetuses that did not breathe. No effect was observed in thirtieth gestational day newborns that were delivered prematurely and allowed to breathe for 30 minutes. Pho~pholipid analysis of the lamellar body fraction (intracellular surfactant) that had been isolated on the sucrose gradient is shown in Table III. Several significant changes were observed in the relative percentages of the phospholipids of particular interest, phosphatidyli~ositol, phosphatidylcholine, and phosphatidyl-
Volume 158 Number2
glycerol, especially in animals of 30 days' gestational age. Isoxsuprine treatment in animals that did not breathe induced two significant changes: phosphatidylinositol levels were higher whereas phosphatidylglycerol levels were reduced compared with the corresponding control values. Breathing negated these effects. In vitro studies. The effect of isoxsuprine on synthesis and secretion of sfi choline-labeled disaturated phosphatidylcholine is shown in Table IV. Twelve hours after the initial exposure to the drug and radioactive choline, the labeling of intracellular disaturated phosphatidylcholine was significantly increased (p < 0.05) but only at the highest dosage, 10 J.Lmol/L isoxsuprine. Similarly, a significantly higher level (p < 0.05) of sH disaturated phosphatidylcholine was detected in the medium of those cultures exposed to 10 J.Lmol/L isoxsuprine at this time. After 24 hours, the medium content of labeled disaturated phosphatidylcholine was four to five times greater, and both 5 and 10 J.Lmol/L isoxsuprine had significantly increased (p < 0.05) the medium content of radioactively labeled disaturated phosphatidylcholine. In contrast, the cellular radioactive disaturated phosphatidylcholine was significantly depressed (p < 0.05) after 24 hours of exposure to either 5 or 10 J.Lmol/L isoxsuprine. Since the highest dosage of the ~-adrenergic produced the most dramatic turnover of cellular radioactive disaturated phosphatidylcholine between the extracellular and intracellular compartments, the incorporation of p•q phospholipid into total cellular phosphatidykholine by differentiating type II alveolar cells in the presence of 10 J.Lmol/L isoxsuprine was examined (Table V). The type II alveolar cells incorporated only a small percentage of the unsaturated phosphatidylcholine that was present in the medium. In contrast, a significant percentage of the disaturated phospholipid supplied in the medium was incorporated into cellular phosphatidylcholine. After 12 hours of exposure to the [' 4 C]-labeled phosphatidylcholine, between 15% and 25% of the radioactivity had appeared in cellular phosphatidylcholine. This radioactivity declined to approximately a third of this value after 24 hours.
Commerit Dramatic changes occur in the fetal lung as -term gestation approaches and the lung prepares to meet the sudden adaptive challenge required when faced with the extrauterine environment. Inability to successfully adapt to this new setting cari precipitate neonatal respiratory distress syndrome. Indeed, this syndrome is the leading cause of morbidity and death in the _premature human 'nfant in whom the pulmonary
13-Adrenergics, surfactant, and lung cell function 377
Table V. Isoxsuprine effect on reutilization of exogenous phospholipid after stimulation for 24 hours with fetal lung fibroblast-conditioned medium*
% of label incorporated into ceUular phoshatidylcholine Label
1-palmitoyl-2[14C]oleoyl phoshatidylcholine dipalmitoyl-[dipalmitoyl- 14C] phoshatidylcholine 1-palmitoyl-2[14C]oleoyl phoshatidylcholine + 20% conditioned medium dipalmitoyl-[dipalmitoyl-' 4C] phoshatidylcholine + 20% conditioned medium
12 hr
24 hr
0.33 ± 0.10 0.60 ± 0.10 15.71 ± 3.07 4.83 ± 1.89 0.23 ± 0.05 0.30 ± 0.01 23.51 ± 5.28 7.77 ± 2.97
*Incorporation of ['4C]-labeled phospholipid into total cellular phoshatidylcholine by differentiating type II alveolar cells in the presence of 10 IJ.mol/L isoxsuprine. Results are expressed as a percentage of the radioactive phospholipid that was supplied and are based on a minimum of triplicate determinations over three culture sequences.
tissues have not yet completed maturation.'• As a result of this immaturity, an essential component, the pulmonary surfactant, that prevents pulmonary collapse at maximal expiration is not produced in sufficient quantity. 15 Numerous hormones and factors have been implicated iri inducing maturation of the lung and thus of the surfactant-producing type II alveolar cells. 2 Among these, ~-adrenergic ag~>nists are of particular interest since their clinical administration in an attempt to delay premature labor appears to result in a decreased incidence of neonatal respiratory distress in the premature infant." Several lines of evidence, eg, observations that ~-adrenergic receptors in the fetal lung increase near term, and this increase may be regulated by glucocorticoidS'· 7 that in turn are instrumental in the induction of pulmonary maturation,2 indicate that adrenergic stimulation may be important in surfactant synthesis and secretion, as well as reduction of fetal lurig wate~· 16 that occurs at birth. The results of the present study are somewhat at odds with these previous observations5· 16 that ~-adrenergics indeed reduce lung water. In fact, only minor changes in fetal lung weight were observed after isoxsuprine treatment, whieh can be interpreted as lung fluid loss. This discrepancy may be related to the route of administration, since the drug was not administered directly to the fetus in either of the studies cited previously.5· 16 On the other hand, McDonald et al. 17 have recently found that lung water was not altered in fetuses treated directly with bromacetylalprenolomenthane, an irreversible ~-blocker.
378
Rasmusson, Scott, and Oulton
The present observations also indicate that breathing augmented the airway surfactant pool in control animals on both the twerity-eighth and thirtieth gestational days. No corresponding pattern was observed in the size of the intracellular surfactant pool. Furthermore, the effect of isoxsuprine appeared to be dependent on both the gestational stage and the breathing status of the animal. On the twenty-eighth gestational day, the drug augmented airway phospholipid, but once the animal was allowed to breathe, the ~-adrenergic inhibited the breathing-induced increase in the lavage surfactant pool size. The reason for this apparent inhibition in the level of extracellular surfactant in these fetuses is not readily apparent, but the observation that inflationinduced surfactant release is mediated by a different mechanism and may not be directly under ~-adrenergic controP' suggests that several mechanisms may be involved at different levels in the regulation of surfactant secretion. In contrast to the ~-adrenergic effect observed on the twenty-eighth day, in thirtieth gestational day fetuses isoxsuprine again depressed the airway pool sizes, but only in those animals that did not breathe. Furthermore, this depression of the airway surfactant correlated with high phosphatidylinositol and low phosphatidylglycerol levels in the intracellular pool. Since anionic phospholipid synthesis switches during the perinatal period from phosphatidylinositol to phosphatidylglyceroP 8 arid the latter phospholipid has been found to be a useful clinical predictor of fetal lung maturity, 19 these results suggest that ~-adrenergic administration without concomitant breathing delays lung maturation. Breathing appeared to overcome this inhibition. Further experiments will be conducted to elucidate this effect and the underlying mechanisms. The effects of ~-agonist stimulation on type II alveolar cells that are in the process of differentiating indicated that adrenergic stimulation can potentially alter both the synthetic and secretory rates of these cells. Furthermore, several studies have documented the substantial capacity and rapidity of Uptake of endogenous surfactant, surfactant subfractions, and labeled disaturated phosphatidylcholine in both in vivo lung models and isolated adult type II alveolar cells. 20-22 We have attempted to determine if differentiating type II cells exhibit a potential for. uptake or selective incorporation of phospholipid. The fact that a substantially higher per<:entage of the 14C-labeled disaturated phosphatidylcholine was incorporated compared with 2-unsaturated phosphatidylcholine inay indicate some specifiCity in this process, at least in these cells. In vivo studies by Jacobs et aP" demonstrated smile degree of speCific uptake of disaturated phosphatidylcholine as opposed to lyso phosphatidylcholine but failed to detect any preferential reutilization with various disaturated phosphatidylcholine analogs. No unsaturated phos-
February 1988 Am J Obstet Gynecol
phatidyicholine was used. On the other hand, in cultures of mature rat type II alveolar cells, Chander et aP3 using disaturated phosphatidylcholine and biosynthesized 1-palmitoyl-2-oleoyl phosphatidylcholine, both of which were radioactively labeled, found evidence that type II cells could resynthesize phosphatidylcholine from degradation products. However, their results did not support a role for preferential transfer of disaturated phosphatidylcholine to type II cell lamellar bodies. Therefore the present results may suggest that under appropriate conditions as used here, that is, under conditions of stimulated secretion via the ~-adrenergic, some preferential uptake can occur. Clearly, further examination of the control of the flux of reutilized phospholipids is required.
REFERENCES l. Ballard PL, Benson BJ, Brehier A. Glucocorticoid effects
in the fetallur~g. Am Rev Respir Dis 1977;115:29-36. 2. Smith BT. Pulmonary surfactant during fetal development and neonatal adaptation: hormonal controL In: Robertson B, Van Golde LMG, Batenburg JJ, eds. Pulmonary surfactant; Amsterdam: Elsevier, 1984:357-81. 3. Roberts PL, Jacobs MM, Cheng JB, Barnes PJ, O'Brien AT, Ballard PJ. Fetal pulmonary ~-adrenergic receptors: characterization in the human and in vitro modulation by glucocorticoids in the rabbit. Pediatr Pulmonol 1985; I (suppl):S69-S76. 4. Lawson EE, Brown ER, Torday JS, Madansky DL, Taeusch HW. The effects of epinephrine on tracheal fluid and surfactant efflux in fetal sheep. Arri Rev Respir Dis 1978;118:1023-6. 5. Kanjanapone V, Hartig-Beechem I, Epstein MF. Effects of isoxsuprine on fetal lung surfactant in rabbits. Pediatr Res 1980;14:278-81. 6. Bergman B. Beta-mimetics and the preterm neonatal lung. Acta Physiol Scand l981;113(suppl497):1-52. 7. ChengJB, Goldfien A, Ballard P,Janes R. Glucocorticoids increase pulmonary beta-adrenergic receptors in fetal rabbit. Endocrinology 1980;107:1646-8. 8. Oulton M, Fraser M, Dolphin M, Yoon R, Faulkner G. Quantification of surfactant pool sizes in rabbit lung during perinatal development. J Lipid Res 1985;27:602-12. 9. ScottJE, Possmayer F, Quirie MA, Tanswell AK, Harding PGR. Alveolar pre-type II cells from the fetal rabbit lung: characterization of the production of disaturated phospholipid during ~;ellular differentiation. Biochim Biophys Acta 1980;879:292-300.. 10. Tanswell AK, Joneja MG, Lindsay J, Vreeken E. Differentiation-arrest rat fetal lung in primary culture monolayer cell culture. I. DevelopiJlent of a differentiationarrested and growthcsupporting culture system using carbon-stripped bovine fetal calf serum. Exp Lung Res 1983;5:37-48. 11. Mason RJ, Nellanbogan J, Clements JA. Isolation of disaturated phospha~idylcholine with osmium tetroxide. J Lipid Res 1976;17:281-4. 12. Skipski VP, Barclay M. Thin-layer chromatographic separation of lipids. Methods Enzymoll969;14:530-98. 13. Wilks SS. Mathematical statistics. New YoJ
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electasis and hyaline membrane disease. Am J Dis Child 1959;97:517-23. Enhorning G, Chamberlain D, Contreras C, Burgoyne R, Robertson B. Isoxsuprine infusion to the pregnant rabbit and its effect on fetal lung surfactant. Bioi Neonate 1979;35:43-51. McDonald JV, Gonzales LW, Ballard PL, Pitha J, Roberts JM. Lung-adrenoreceptor blockade affects perinatal surfactant release but not lung water. J Appl Physiol 1986;60(5): 1727-33. Batenburg JJ. Biosynthesis and secretion of pulmonary surfactant. In: Robertson B, Van Golde LMG, Batenburg JJ, eds. Pulmonary surfactant. Amsterdam: Elsevier, 1984;237-270. • Oulton M, Bent AE, Gray JH, Luther ER, Peddle LJ. Assessment of fetal pulmonary maturity by phospholipid analysis of amniotic fluid lamellar bodies. AM J 0BSTET GYNECOL 1982;142:684-91.
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20. Jacobs H, Jobe A, Ikegami M, Miller D, Jones S. Reutilization of phosphatidylcholine analogues by the pulmonary surfactant system. The lack of specificity. Biochim Biophys Acta 1984;793:300-9. 21. Chander A, Claypool WD, StraussJF, Fisher AB. Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am J Physiol 1983;245:C397C404. 22. Oguchi K, Ikegami M, Jacobs H, Jobe A. Clearance of large amounts of natural surfactants and liposomes of dipalmitoyl-phosphatidylcholine from the lungs of rabbits. Exp Lung Res 1985;9:221-35. 23. Chander A, Reicherter J, Fisher A. Degradation of dipalmitoyl phosphatidylcholine by isolated rat granular pneumocytes and reutilization for surfactant synthesis. J Clin Invest 1987;79:1133-8.
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Key words: 13-Adrenergic, type II alveolar cell, surfactant
To facilitate gas exchange at birth, the pulmonary tissue spaces that give rise to mature alveoli must be cleared of liquid. The exact mechanism by which this is accomplished is not known. The role of glucocorticoids has been investigated extensively in both tissue maturation in general and in the lung in particular.'· 2 However, their effect has been largely defined as one of induction of differentiation. On the other hand, evidence suggests that 13-adrenergics may regulate the reabsorption of fetal alveolar fluid in late gestation, 3 • 4 as well as stimulate the secretion of the pulmonary surfactant.•-6 Indeed, the fetal lung apparently demonstrates a marked increase in !;S-adrenergic receptors as term approaches. 7 However, the role of these compounds at the cellular level in regulating the synthesis and secretion of the pulmonary surfactant has not been determined. The present study was designed first to establish the effect of 13-adrenergics on both the intra-
From the Departments of Anatomy, Obstetrics and Gynecology, and Physiology, Dalhousie University. Supported fly the Medical Research Council of Canada, Presented at the Forty-third Annual Meeting of The Society of Obstetricians and Gynaecologists of Canada, Ottawa, Ontario, Canada, june 24-27, 1987.. Reprint requests:]. E. Scott, PhD, Department ofAnatomy, Dalhousie University, Halifax, Nova Scotia, Canada BJH 4H7.
cellular and extracellular surfactant levels and how these phospholipid pools vary with breathing. Second, we wanted to correlate these findings with the ability of isoxsuprine to regulate the synthesis and secretion of disaturated phosphatidylcholine, the major constituent of the surfactant, by isolated type II alveolar cells and to assess the potential for these cells to use exogenously supplied phospholipid.
Material and methods Timed-pregnant New Zealand white rabbits were purchased from Rieman's Fur Ranch, St. Agatha, Ontario. The number of fetuses per litter ranged from one to 15, and one to two litters were used per experiment. Isoxsuprine hydrochloride (Vasodilan, 5 mg/ml) was purchased from Bristol-Myers, Candiac, Quebec. Culture materials obtained from Gibco Laboratories (Mississauga, Ontario). Radioactive choline chloride ([3H-methyl] choline chloride), l-palmitoyl-2[ 14 C]oleoyl phosphatidylcholine, and dipalmitoyl-[ dipalmitoyl-' 4 C]phosphatidylcholine were from New England Nuclear (Lachine, Quebec). Metrizamide was from Sigma Chemical Co. (St. Louis, Missouri). Other chemicals were obtained from Fisher Scientific, Dartmouth, Nova Scotia. On either the twenty-eighth or thirtieth day of ges-
373
374 Rasmusson, Scott, and Oulton
tation (day 0 is day of mating), a laparotomy was performed on the pregnant does, and the fetal rabbits were treated with an intraperitoneal dose of isoxsuprine (0.5 mg/fetus, 0.1 ml) by injection through the uterine wall. Fetuses were then either left in utero for 30 minutes or removed to a warming tray and allowed to breathe for 30 minutes. Concurrent controls consisted of animals on which a laparotomy was performed, and the uterus was exteriorized and maintained in this condition with warming for 30 minutes. Control animals that received a saline solution injection were also used. No detectable differences were observed in the parameters measured in the present study (i.e., 30 minutes after injection) between the untreated and saline-treated control animals, and therefore these data have been pooled. All does were maintained under anesthesia (sodium pentobarbital, 30 mg/kg, intravenously to the lateral ear vein) during this period. Fetuses were killed by an intraperitoneal lethal dose of sodium pentobarbital. Fetuses were weighed, the trachea was cannulated via a midline neck incision, and the lungs were l~vaged with 0.15 mol/L sodium chloride in 7 X 1.00 ml aliquots. Lavage was pooled and centrifuged at 10,000 g for 30 minutes to collect "extracellular surfactant." This was stored at - 20° C until analyzed. Lungs from each fetus were excised, and a representative portion was removed and dried to constant weight at 120° C. The remainder was pooled for preparation of subcellular fractions. Fractions were prepared according to the method of Oulton et aLB Briefly, this involves homogenization of the tissue in 0.01 mol/L Tris buffer (pH 7.4) and centrifugation at 140 and 10,000 g. The pellet from the latter centrifugation is layered on a discontinuous sucrose gradient (0.25 mol/L over 0.68 mol/L) and centrifuged at 65,000 g for 60 minutes. The material that collects at the gradient interface we termed "intracellularly stored surfactant" and have identified as lamellar body-like in composition and morphologic appearance. 8 The lavage pellet (extracellular surfactant) and the gradient interface were extracted into chloroform:methanol (2: 1); the lower phase was collected and concentrated to dryness under a stream of air at 37° C and resuspended in chloroform:methanol (2: 1). Aliquots were removed for determination of phospholipid content and composition as described previously.8 In vitro examination of the effects of isoxsuprine was undertaken with isolated undifferentiated type II alveolar cells from 24-gestational-day fetal rabbit lung as described previously. 9 Preparation of these cells involved trypsinization of excised fetal lung for 45 minutes. The mixture was filtered through gauze and the cells were collected by gentle centrifugation. Fibroblasts were partially eliminated by a 90-minute in vitro at-
February 1988 Am J Obstet Gynecol
tachment, since these cells attach and grow more rapidly than the epithelial cells. Pretype II alveolar cells were collected at the interface of a discontinuous metrizamide gradient (0.099 mol/L over 0.218 mol/L). Cells are cultivated in minimum essential medium supplemented with 10% fetal bovine serum that had been hormone stripped. 1° Conditioned medium was produced from confluent cultures of fetal lung fibroblasts as described previously. 9 After cultivation of the undifferentiated type II alveolar cells for 2 to 3 days, the cells were incubated with 0, 5, or 10 J.Lmol/L isoxsuprine plus 0.5 J.LCi of 'H choline chloride. After 12 or 24 hours, the medium was removed and saved for analysis. Cells were washed with Hanks' balanced salt solution, released with trypsin/ethylenediaminetetraacetic acid, and an aliquot was counted on a Coulter cell counter (Coulter Electronics, Hialeah, Florida); and the remainder was extracted into chloroform: methanol as described previously. Disaturated phosphatidylcholine was separated on neutral alumina columns as described by Mason et al. 11 Medium disaturated phosphatidylcholine was analyzed in an identical fashion. In a second series of experiments to examine reutilization of secreted phospholipid, these cells were exposed to 20% conditioned medium (v/v) plus 0.5 j.LCi of ['H-methyl]choline chloride for 24 hours. Isoxsuprine was then added at a concentration of 10 J.Lmol/L, together with ['H-methyl]choline chloride plus 1-palmitoyl-2['4C]oleoyl phosphatidylcholine (57 mCi/mmol) or dipalmitoyl-[dipalmitoyl- 14C]phosphatidylcholine (112 mCi/mmol). Phospholipids were sonicated briefly (2 X 5 seconds) in Tris buffer (pH 7.4) before being added to the cultures. Estimation of the secretion of tritium-labeled phosphatidylcholine and disaturated phosphatidylcholine into the culture medium at the time of addition of the phospholipids (i.e., after 24 hours with tritiated choline) enabled the ratios of 14C-labeled phosphatidylcholine or [14C] disaturated phosphatidylcholine to medium phosphatidylcholine or disaturated phosphatidylcholine to be initiated at approximately 0.175 and 0.214, respectively. After the addition of the drug and phospholipid, the medium and cells were removed at 12 and 24 hours and extracted as previously described. Phosphatidylcholine was separated on LK5D thin layer chromatographic plates in a solvent system of chloroform : methanol: acetic acid: water (65: 25: 3: 1) modified from Skipski. 12 Radioactivity in phosphatidylcholine was determined and measured in a Beckman LS 5801 scintillation counter. Quench compensation was done by the method of H# (Beckman Instruments, Palo Alto, California). Disintegrations per minute of dual-labeled samples was determined with an efficiency of 65% for isotope 1 (tritium labeled) and 80% for isotope 2 (' 4 C) and < 1% spill.
~-Adrenergics,
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surfactant, and lung cell function 375
Table I. Effect of isoxsuprine administration on body and lung weights in breathing or nonbreathing preterm-delivered rabbits. After isoxsuprine administration the fetuses were either left in utero or allowed to breathe for 30 minutes, results are expressed as the mean ± SD for the nuinber of determinations shown Lung weight (gm)
No. of Treatment
de~inations*
28 day, no breathing Control Isoxsuprine 28 day, 30 min breathing Control Isoxsuprine 30 day, no breathing Control lsoxsuprine 30 day, 30 min brea~ing Control Isoxsuprine
Body weight (gm)
Fetus
7 7
34.65 ± 2.69 30.26 ± 4.98
1.33 ± 0.17 1.06 ± 0.14t
7 5
33.78 ± 4.65 31.71 ± 3.55
1.25 ± 0.15 1.08 ± 0.16
5 5
44.57 ± 10.40 45.17 ± 3.03
1.29 ± 0.34 1.37 ± O.o7
13
42.96 ± 5.64 44.40 ± 5.50
1.32 ± 0.43 1.30 ± 0.17
9
*Each determination consisted of a group of four to eight fetuses from three to five litters. tlndicates significantly different from corresponding control value (p < 0.05).
Table II. Effect of isoxsuprine administration on surfactant pool size in breathing or nonbreathing preterm-delivered rabbits, isoxsuprine administration was as described in Table I, results are expressed as the mean ± SD for five to 13 determinations
Treatment
Intracellular pool size (mg phospholipidlgm dry lung)
28 day, no breathing Control* lsoxsuprine 28 day, 30 min breathing Control Isoxsupri~e
30 day, no breathing Control Isoxsuprine 30 day, 30 min breathing Control lsoxsuprine
Extracellular pool size (mg phospholipidlgm dry lung)
Extracellular Intracellular pool size
1.84 ± 0.39 3.05 ± 1.56
0.12 ± 0.03t 0.19 ± 0.08
6.63 ± 1.93 6.68 ± 1.93
2.71 ± 1.17 2.04 ± 0.97
0.37 ± 0.19tt 0.17 ± 0.09
13.71 ± 2.95t 7.96 ± 0.99
12.11 ± 3.75 8.50 ± 0.94
1.65 ± 0.68t 0.58 ± 0.19
17.50 ± 4.45t 6.98 ± 2.44
9.92 ± 2.57 10.06 ± 1.91
2.42 ± 0.95* 2.59 ± 0.82
22.30 ± 5.70 26.14 ± 7.71
X
100
*All control values on the twenty-eighth gestational day were significantly different (JJ < 0.05) compared with the corresponding control values on the thirtieth gestational day. tlndicates significantly different (JJ < 0.05) from corresponding values of isoxsuprine-treated animals. Undicates significantly greater (JJ < 0.05) than corresponding nonbreathing control values.
Statistical analysis was qone with the Student's t test after transformation of the data to accommodate unequal variances. Significance was taken at 5% after Bonnferroni adjustment of the significance level. u Results In vivo studies. The mean body and lung weights from fetuses on the twenty-eighth or thirtieth gestational day and prematurely delivered newborns of the same gestational ages are shown in Table I. No significant alterations were ·detected after isoxsuprine administration on either day with or without breathing except in animals that received the drug on day 28. In
this instance, a combination of 0.5 mg of isoxsuprine without breathing significantly reduced (p < 0.05) the lung weight/fetus. The effect of isoxsuprine administration to fetuses that either did not breathe (fetuses) or were allowed to breathe for 30 minutes (newborns) on intracellular and extracellular surfactant pool sizes and the ratio of extracellular to intracellularly stored phospholipid are shown in Table II. Significantly greater (p < 0.05) amounts of phospholipid were observed to be present in both the intracellular and extracellular surfactant pools of the thirtieth gestational day animals compared with twenty-eighth gestational day animals. The intra-
376
Rasmusson, Scott, and Oulton
February 1988 Am J Obstet Gynecol
Table III. Effect of isoxsuprine administration on intracellularly stored surfactant composition in breathing or nonbreathing preterm-delivered rabbits, isoxsuprine administration was as described in Table I, results are expressed as the mean ± SD for five to 13 determinations
% of total phospholipid
PS
Treatment
28 day, no breathing Control 4.15 ± 1.91 Isoxsuprine 4.19 ± 2.52 28 day, 30 min breathing Control 4.12 ± 0.80* Isoxsuprine 5.27 ± 1.50 30 day, no breathing Control 1.93 ± 0.77 Isoxsuprine 1.13 ± 0.77* 30 day, 30 min breathing Control 2.69 ± 0.49 Isoxsuprine 2.91 ± 0.53
PI
SM
PC
PG
PE
Unknown
11.96 ± 2.25 11.80 ± 2.13
3.68 ± 0.79* 2.95 ± 0.71
66.40 ± 3.62* 68.70 ± 2.33
ND 0.06 ± 0.11
13.80 ± 1.47* 12.30 ± 1.67
ND ND
11.08 ± 0.80 10.31 ± 0.69
2.58 ± 0.64* 3.47 ± 0.78
72.44 ± 4.50* 68.20 ± 3.05
0.29 ± 0.27 0.22 ± 0.30
10.39 ± 0.63* 11.38 ± 2.22
0.45 ± 0.52 1.15 ± 1.07
13.17 ± 1.68 15.93 ± 0.45*
0.89 ± 0.13 1.87 ± 1.40
76.35 ± 1.09 77.44 ± 5.21
0.92 ± 0.47t 0.18 ± 0.13*
6.66 ± 0.38 9.21 ± 3.73
0.08 ± 0.15 0.09 ± 0.19
11.72 ± 0.67 10.94 ± 0.77
1.17 ± 0.57 1.43 ± 0.45
77.27 ± 1.65 77.09 ± 1.44
0.69 ± 0.46 1.04 ± 0.85
6.45 ± 0.49 6.63 ± 0.91
0.01 ± 0.03 ND
PS = Phosphatidylserine; PI = phosphatidylinositol; SM = sphingomyelin; PC = phosphatidylcholine; PG = phosphati· dylglycerol; PE = phosphatidylethanolamine; ND = not detected. *Indicates significantly differeqt (p < 0.05) compared with the corresponding control value on the thirtieth gestational day. tindicates significantly different (p < 0.05) from the isoxsuprine-treated animals. Undicates significantly different from the corresponding thirtieth gestational day isoxsuprine-treated animals that were allowed to breathe.
Table IV. Effect of isoxsuprine on the synthesis and secretion of tritiated choline-labeled desaturated phoshatidylcholine. Results are based on a minimum of six replicate determinations over three preparations pmol disaturated phosphatidylcholinel10' cells Isoxsuprine ( p.mol/L)
12 hr
0 5 10
2.17 ± 0.11 2.36 ± 0.22 2.66 ± 0.38*
I
pmol disaturated phosphatidylcholine/2 ml of medium
24 hr 10.03 ± 1.05 6.06 ± 0.89* 3.27 ± 0.31*
12 hr
7.98 ± 0.64 7.45 :±: 0.16 21.35 ± 9.29*
I
24 hr 33.45 ± 3.76 99.86 ± 11. 78* 103.67 ± 14.81*
*Indicates significantly different from corresponding controls (p < 0.05) as determined by the Student t-test.
cellular pool sizes were not altered in those groups of animals that received either isoxsuprine or were allowed to breathe for 30 minutes. On the other hand, extracellular pool sizes were significantly increased (p < 0.05) in both groups of animals that breathed. Administration of isoxsuprine to twentyeighth gestational day animals depressed the release of intracellular surfactant but only in those prematurely delivered newborns that breathed. In contrast, in the thirtieth gesta~onal day fetuses, isoxsuprine treatment significantly depressed (p < 0.05) the extracellular surfactant pool size but only in those fetuses that did not breathe after drug injection. The drug did not significantly affect the extracellular surfactant pool size in thirtieth gestational day newborns that were delivered and allowed to breathe for 30 minutes. Isoxsuprine diq not influence the ratio of extracellular to intracellular surfactant pool size in twenty-eighth gestational day
fetuses that did not breathe. In contrast, treatment with isoxsuprine on the twenty-eighth gestational day and subsequent premature delivery and breathing for 30 minutes produced a significant depression (p < 0.05) in the ratio of extracellular to intracellular phospholipid. In the thirtieth gestational day animals, the drug again significantly decreased (p < 0.05) the ratio of extracellular to intracellular phospholipid; however, this occurred only in those fetuses that did not breathe. No effect was observed in thirtieth gestational day newborns that were delivered prematurely and allowed to breathe for 30 minutes. Pho~pholipid analysis of the lamellar body fraction (intracellular surfactant) that had been isolated on the sucrose gradient is shown in Table III. Several significant changes were observed in the relative percentages of the phospholipids of particular interest, phosphatidyli~ositol, phosphatidylcholine, and phosphatidyl-
Volume 158 Number2
glycerol, especially in animals of 30 days' gestational age. Isoxsuprine treatment in animals that did not breathe induced two significant changes: phosphatidylinositol levels were higher whereas phosphatidylglycerol levels were reduced compared with the corresponding control values. Breathing negated these effects. In vitro studies. The effect of isoxsuprine on synthesis and secretion of sfi choline-labeled disaturated phosphatidylcholine is shown in Table IV. Twelve hours after the initial exposure to the drug and radioactive choline, the labeling of intracellular disaturated phosphatidylcholine was significantly increased (p < 0.05) but only at the highest dosage, 10 J.Lmol/L isoxsuprine. Similarly, a significantly higher level (p < 0.05) of sH disaturated phosphatidylcholine was detected in the medium of those cultures exposed to 10 J.Lmol/L isoxsuprine at this time. After 24 hours, the medium content of labeled disaturated phosphatidylcholine was four to five times greater, and both 5 and 10 J.Lmol/L isoxsuprine had significantly increased (p < 0.05) the medium content of radioactively labeled disaturated phosphatidylcholine. In contrast, the cellular radioactive disaturated phosphatidylcholine was significantly depressed (p < 0.05) after 24 hours of exposure to either 5 or 10 J.Lmol/L isoxsuprine. Since the highest dosage of the ~-adrenergic produced the most dramatic turnover of cellular radioactive disaturated phosphatidylcholine between the extracellular and intracellular compartments, the incorporation of p•q phospholipid into total cellular phosphatidykholine by differentiating type II alveolar cells in the presence of 10 J.Lmol/L isoxsuprine was examined (Table V). The type II alveolar cells incorporated only a small percentage of the unsaturated phosphatidylcholine that was present in the medium. In contrast, a significant percentage of the disaturated phospholipid supplied in the medium was incorporated into cellular phosphatidylcholine. After 12 hours of exposure to the [' 4 C]-labeled phosphatidylcholine, between 15% and 25% of the radioactivity had appeared in cellular phosphatidylcholine. This radioactivity declined to approximately a third of this value after 24 hours.
Commerit Dramatic changes occur in the fetal lung as -term gestation approaches and the lung prepares to meet the sudden adaptive challenge required when faced with the extrauterine environment. Inability to successfully adapt to this new setting cari precipitate neonatal respiratory distress syndrome. Indeed, this syndrome is the leading cause of morbidity and death in the _premature human 'nfant in whom the pulmonary
13-Adrenergics, surfactant, and lung cell function 377
Table V. Isoxsuprine effect on reutilization of exogenous phospholipid after stimulation for 24 hours with fetal lung fibroblast-conditioned medium*
% of label incorporated into ceUular phoshatidylcholine Label
1-palmitoyl-2[14C]oleoyl phoshatidylcholine dipalmitoyl-[dipalmitoyl- 14C] phoshatidylcholine 1-palmitoyl-2[14C]oleoyl phoshatidylcholine + 20% conditioned medium dipalmitoyl-[dipalmitoyl-' 4C] phoshatidylcholine + 20% conditioned medium
12 hr
24 hr
0.33 ± 0.10 0.60 ± 0.10 15.71 ± 3.07 4.83 ± 1.89 0.23 ± 0.05 0.30 ± 0.01 23.51 ± 5.28 7.77 ± 2.97
*Incorporation of ['4C]-labeled phospholipid into total cellular phoshatidylcholine by differentiating type II alveolar cells in the presence of 10 IJ.mol/L isoxsuprine. Results are expressed as a percentage of the radioactive phospholipid that was supplied and are based on a minimum of triplicate determinations over three culture sequences.
tissues have not yet completed maturation.'• As a result of this immaturity, an essential component, the pulmonary surfactant, that prevents pulmonary collapse at maximal expiration is not produced in sufficient quantity. 15 Numerous hormones and factors have been implicated iri inducing maturation of the lung and thus of the surfactant-producing type II alveolar cells. 2 Among these, ~-adrenergic ag~>nists are of particular interest since their clinical administration in an attempt to delay premature labor appears to result in a decreased incidence of neonatal respiratory distress in the premature infant." Several lines of evidence, eg, observations that ~-adrenergic receptors in the fetal lung increase near term, and this increase may be regulated by glucocorticoidS'· 7 that in turn are instrumental in the induction of pulmonary maturation,2 indicate that adrenergic stimulation may be important in surfactant synthesis and secretion, as well as reduction of fetal lurig wate~· 16 that occurs at birth. The results of the present study are somewhat at odds with these previous observations5· 16 that ~-adrenergics indeed reduce lung water. In fact, only minor changes in fetal lung weight were observed after isoxsuprine treatment, whieh can be interpreted as lung fluid loss. This discrepancy may be related to the route of administration, since the drug was not administered directly to the fetus in either of the studies cited previously.5· 16 On the other hand, McDonald et al. 17 have recently found that lung water was not altered in fetuses treated directly with bromacetylalprenolomenthane, an irreversible ~-blocker.
378
Rasmusson, Scott, and Oulton
The present observations also indicate that breathing augmented the airway surfactant pool in control animals on both the twerity-eighth and thirtieth gestational days. No corresponding pattern was observed in the size of the intracellular surfactant pool. Furthermore, the effect of isoxsuprine appeared to be dependent on both the gestational stage and the breathing status of the animal. On the twenty-eighth gestational day, the drug augmented airway phospholipid, but once the animal was allowed to breathe, the ~-adrenergic inhibited the breathing-induced increase in the lavage surfactant pool size. The reason for this apparent inhibition in the level of extracellular surfactant in these fetuses is not readily apparent, but the observation that inflationinduced surfactant release is mediated by a different mechanism and may not be directly under ~-adrenergic controP' suggests that several mechanisms may be involved at different levels in the regulation of surfactant secretion. In contrast to the ~-adrenergic effect observed on the twenty-eighth day, in thirtieth gestational day fetuses isoxsuprine again depressed the airway pool sizes, but only in those animals that did not breathe. Furthermore, this depression of the airway surfactant correlated with high phosphatidylinositol and low phosphatidylglycerol levels in the intracellular pool. Since anionic phospholipid synthesis switches during the perinatal period from phosphatidylinositol to phosphatidylglyceroP 8 arid the latter phospholipid has been found to be a useful clinical predictor of fetal lung maturity, 19 these results suggest that ~-adrenergic administration without concomitant breathing delays lung maturation. Breathing appeared to overcome this inhibition. Further experiments will be conducted to elucidate this effect and the underlying mechanisms. The effects of ~-agonist stimulation on type II alveolar cells that are in the process of differentiating indicated that adrenergic stimulation can potentially alter both the synthetic and secretory rates of these cells. Furthermore, several studies have documented the substantial capacity and rapidity of Uptake of endogenous surfactant, surfactant subfractions, and labeled disaturated phosphatidylcholine in both in vivo lung models and isolated adult type II alveolar cells. 20-22 We have attempted to determine if differentiating type II cells exhibit a potential for. uptake or selective incorporation of phospholipid. The fact that a substantially higher per<:entage of the 14C-labeled disaturated phosphatidylcholine was incorporated compared with 2-unsaturated phosphatidylcholine inay indicate some specifiCity in this process, at least in these cells. In vivo studies by Jacobs et aP" demonstrated smile degree of speCific uptake of disaturated phosphatidylcholine as opposed to lyso phosphatidylcholine but failed to detect any preferential reutilization with various disaturated phosphatidylcholine analogs. No unsaturated phos-
February 1988 Am J Obstet Gynecol
phatidyicholine was used. On the other hand, in cultures of mature rat type II alveolar cells, Chander et aP3 using disaturated phosphatidylcholine and biosynthesized 1-palmitoyl-2-oleoyl phosphatidylcholine, both of which were radioactively labeled, found evidence that type II cells could resynthesize phosphatidylcholine from degradation products. However, their results did not support a role for preferential transfer of disaturated phosphatidylcholine to type II cell lamellar bodies. Therefore the present results may suggest that under appropriate conditions as used here, that is, under conditions of stimulated secretion via the ~-adrenergic, some preferential uptake can occur. Clearly, further examination of the control of the flux of reutilized phospholipids is required.
REFERENCES l. Ballard PL, Benson BJ, Brehier A. Glucocorticoid effects
in the fetallur~g. Am Rev Respir Dis 1977;115:29-36. 2. Smith BT. Pulmonary surfactant during fetal development and neonatal adaptation: hormonal controL In: Robertson B, Van Golde LMG, Batenburg JJ, eds. Pulmonary surfactant; Amsterdam: Elsevier, 1984:357-81. 3. Roberts PL, Jacobs MM, Cheng JB, Barnes PJ, O'Brien AT, Ballard PJ. Fetal pulmonary ~-adrenergic receptors: characterization in the human and in vitro modulation by glucocorticoids in the rabbit. Pediatr Pulmonol 1985; I (suppl):S69-S76. 4. Lawson EE, Brown ER, Torday JS, Madansky DL, Taeusch HW. The effects of epinephrine on tracheal fluid and surfactant efflux in fetal sheep. Arri Rev Respir Dis 1978;118:1023-6. 5. Kanjanapone V, Hartig-Beechem I, Epstein MF. Effects of isoxsuprine on fetal lung surfactant in rabbits. Pediatr Res 1980;14:278-81. 6. Bergman B. Beta-mimetics and the preterm neonatal lung. Acta Physiol Scand l981;113(suppl497):1-52. 7. ChengJB, Goldfien A, Ballard P,Janes R. Glucocorticoids increase pulmonary beta-adrenergic receptors in fetal rabbit. Endocrinology 1980;107:1646-8. 8. Oulton M, Fraser M, Dolphin M, Yoon R, Faulkner G. Quantification of surfactant pool sizes in rabbit lung during perinatal development. J Lipid Res 1985;27:602-12. 9. ScottJE, Possmayer F, Quirie MA, Tanswell AK, Harding PGR. Alveolar pre-type II cells from the fetal rabbit lung: characterization of the production of disaturated phospholipid during ~;ellular differentiation. Biochim Biophys Acta 1980;879:292-300.. 10. Tanswell AK, Joneja MG, Lindsay J, Vreeken E. Differentiation-arrest rat fetal lung in primary culture monolayer cell culture. I. DevelopiJlent of a differentiationarrested and growthcsupporting culture system using carbon-stripped bovine fetal calf serum. Exp Lung Res 1983;5:37-48. 11. Mason RJ, Nellanbogan J, Clements JA. Isolation of disaturated phospha~idylcholine with osmium tetroxide. J Lipid Res 1976;17:281-4. 12. Skipski VP, Barclay M. Thin-layer chromatographic separation of lipids. Methods Enzymoll969;14:530-98. 13. Wilks SS. Mathematical statistics. New YoJ
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electasis and hyaline membrane disease. Am J Dis Child 1959;97:517-23. Enhorning G, Chamberlain D, Contreras C, Burgoyne R, Robertson B. Isoxsuprine infusion to the pregnant rabbit and its effect on fetal lung surfactant. Bioi Neonate 1979;35:43-51. McDonald JV, Gonzales LW, Ballard PL, Pitha J, Roberts JM. Lung-adrenoreceptor blockade affects perinatal surfactant release but not lung water. J Appl Physiol 1986;60(5): 1727-33. Batenburg JJ. Biosynthesis and secretion of pulmonary surfactant. In: Robertson B, Van Golde LMG, Batenburg JJ, eds. Pulmonary surfactant. Amsterdam: Elsevier, 1984;237-270. • Oulton M, Bent AE, Gray JH, Luther ER, Peddle LJ. Assessment of fetal pulmonary maturity by phospholipid analysis of amniotic fluid lamellar bodies. AM J 0BSTET GYNECOL 1982;142:684-91.
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20. Jacobs H, Jobe A, Ikegami M, Miller D, Jones S. Reutilization of phosphatidylcholine analogues by the pulmonary surfactant system. The lack of specificity. Biochim Biophys Acta 1984;793:300-9. 21. Chander A, Claypool WD, StraussJF, Fisher AB. Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am J Physiol 1983;245:C397C404. 22. Oguchi K, Ikegami M, Jacobs H, Jobe A. Clearance of large amounts of natural surfactants and liposomes of dipalmitoyl-phosphatidylcholine from the lungs of rabbits. Exp Lung Res 1985;9:221-35. 23. Chander A, Reicherter J, Fisher A. Degradation of dipalmitoyl phosphatidylcholine by isolated rat granular pneumocytes and reutilization for surfactant synthesis. J Clin Invest 1987;79:1133-8.
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