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Aminophylline stimulates the incorporation of choline into phospholipid in explants of fetal rat lung in organ culture
263
BBA 57039
AMINOPHYLLIN~ STIMULATES THE INCORPORATION OF CHOLINE INTO PHOSPHOL~~~ IN EXPLANTS OF FETAL RAT LUNG IN ORGAN CULTURE
We have i~~~~~~~~d t&e dire& effect of a~~~~~~l~i~~~ a cyclic AMP phos~hod~esterase inhibitor, and of cyclic AMP on choline ~ncorpo~tion into phosphotipid in exphmts of fetal rat lung in organ culture. Explants from 19 day fetal rat lung (term is 22 days) were cultured for 24 or 48 h in F12 medium containing 1.0 mM aminophylfine or 0.2 mM dibutyryl cyclic AMP. Explants cultured in F12 medium alone served as controls. Exposure to aminophylline resulted in an 88% increase in choline incorporation into phosphatidylcholine (I’ < 0.01) and a 95% increase in choline incorporation into sphingomyelin (P < 0.001~ in the 48-h cultures. Similar changes were noted after 24 h in culture. There was a small increase in the percentage of synthesized ph~spbat~dy~ch~~~ne which was disaturated. The activities of choline kinase and cho~mephosphQ~~sfe~se in the explants were not s~~ific~tly altered by exposure to amin~phy~~e* 71 and 53% st~u~at~on of choline incorporatiun into phos~hat~d~~ch~~jne and sph~~~om~e~~ res~ec~j~e~~ was demonstrated in explants cultured in medium containing cyclic AMP. Am~o~hyll~ne treatment of explants from ‘&I- and 2Xday fetuses resulted in a smaller degree of stimulation of choline incorporation into both phospholipids. These data suggest that aminophylline has a direct stimulatory effect on the incorporation of choline into phospholipid in fetal rat lung and that this effect is probably cyclic AMP mediated.
264
Introduction The alveoli of the lung are lined by surface active material, pulmonary surfactant, which lowers surface tension at the air-alveolar interface, thereby preventing alveolar collapse. The major surface-active components of surfactant are phospholipids; dipalmitoylglycerophosphocholine is thought to be the most important in this regard [ 11. The production of pulmonary surfactant in fetal lung increases toward the end of gestation [Z]. It has been recently suggested by Barrett et al. that the administration of aminophylline to fetal rabbits may enhance pulmonary phosphatidylcholin~ synthesis [3]. Ami~lophylline is the ethyl~ine diamine salt of theophylline, a methyl xanthine compound, which has the property of increasing tissue levels of cyclic AMP through its action as a cyclic AMP phosphodiesterase inhibitor [4]. Karotkin et al. [ 51 have also reported that there is an increase in the phospholipid content of fetal rabbit lung tissue and lung lavage (source of surfactant) after aminophylline administration to the mother. Corticosteroid is another agent that is believed to stimulate fetal lung surfactant production [ 2,6]. Since Barrett et al. [ 31 found that exposure of fetal rabbits to either corticosteroid or aminophylline resulted in increased lung tissue cyclic AMP levels, they have suggested that cyclic AMP may be a mediator of fetal lung phosphatidylcholine synthesis. We have examined the direct effect of ~inophylline and cyclic AMP on phospholipid synthesis in explants of fetal rat lung grown in organ culture, in order to further evaluate the role of these agents in fetal lung maturation. Materials and Methods Culture qinterial. Timed 1% to 21-day pregnant Sprague-Dawley rats (term is 22 days) were used in all experiments. Pregnant rats were killed by a blow to the head and the fetuses removed immediately under sterile conditions. All further procedures were performed in a laminar flow hood and sterility was carefully maintained. The lungs from two to three litters of fetuses were immediately excised, pooled, and chopped into cubes with 0.8 mm sides by means of a McIlwain tissue chopper (Brinkm~n Instruments, Inc., Westbury, N.Y.). A stainless steel grid with a 25 X 25 mm surface (Frankel Company, Philadelphia, Pa.) was placed in a plastic tissue culture dish to which 5.5 ml F12 medium (Grand Island Biological Company, Grand Island, N.Y.) was added so that it just reached the undersurface of the grid. A sterile 25-mm diameter Metricel filter (Gelman Instrument Company, Ann Arbor, Mich.) was placed on the grid and 30 lung explants placed on the filter. The undersurface of the explants was thus exposed to the medium and the upper surface to the air. The explants were then incubated in a humidified atmosphere of 95% O2 and 5% CO:! at 37°C. Control cultures were incubated in F12 medium only. ~xperiment~ cultures were grown in F12 medium containing either aminophylliile, 1.0 mM, or dibutyryl cyclic AMP, 0.2 mM. F12 is an enriched synthetic medium which contains choline chloride, 0.1 mM.
Phospholipid synthesis. After 24 or 48 h in culture the medium was removed and replaced with 5 ml of F12 medium containing 1.25 @Z!i [Me3H]choline chloride, 2.5 Ci/mol. The explants were cultured for 6 h in the presence of the isotope after which time the medium was removed and the explants washed with ice-cold 0.9% NaCl. The explants from each flask were then sonicated in 2 ml of 0.9% NaCl prior to lipid analysis and protein determination. Lipid was extracted from the sonicate with chloroform and methanol by the method of Bligh and Dyer [7]. Phospholipids were identified by thin layer chromatography on silica gel (LQD plates, Quantum Industries, Fairfield, N.J.) in chloroform/methanol/7 M NH40H (60 : 35 : 5, v/v). Lipids were visualized on the chromatograms by exposure to iodine vapor. The phosphatidylcholine and sphingomyelin spots were scraped off the plates into scintillation vials and the radioactivity in these fractions was determined in 0.5 ml H,O and 14.5 ml Aquasol (New England Nuclear, Boston, Mass.). Protein content was determined by the method of Lowry et al. [S] and DNA content by a modification of the method of Burton [9]. Disaturated phosphatidylcholine was determined by the method of Mason et al. [lo]. Enzyme actiuities. After 48 h in culture the explants were sonicated in chilled 0.33 M Tris HCl, pH 7.4, containing 0.001 M EDTA. Choline kinase (EC 2.7.1.32) was assayed in the homogenate by measuring the rate of incorporation of radioactivity from [Me-‘4C]choline into phosphocholine as described previously [ 111. Cholinephosphotransferase (EC 2.7.8.2) was assayed in the homogenate by measuring the rate of incorporation of radioactivity from CDP [Me-14C]choline into lipid as described previously [12], except that the reaction was carried out at pH 8.5 and 19 mM dioleoylglycerol and 0.08 mM CDP choline were used as substrates. Chemicals. All radiochemicals were purchased from New England Nuclear, Boston, Mass. Phospholipids were obtained from Serdary, London, Ont. Aminophylline U.S.P. was obtained from G.D. Searle and Company, Chicago, Ill. and dibutyryl cyclic AMP and all other biochemicals from Sigma Chemical Company, St. Louis, MO. All other chemicals were reagent grade. Results Fetal rat lung explants remain viable under the cnditions described in Materials and Methods. This has been determined previously [13] by examining the histologic appearance by light and electron microscopy and by measuring the rate of glucose oxidation and comparing it to that obtained with fresh fetal lung tissue. As is shown in Table I, there was an 88% increase in choline incorporation into phosphatidylcholine in explants of 19-day fetal lung after exposure to 1.0 mM aminophylline for 48 h. A similar degree of stimulation of choline incorporation into sphingomyelin was also observed. These changes are statistically significant. Varying the dose between 0.5 and 5.0 mM had little effect on the rate of choline incorporation. The rate of choline incorporation into phosphatidylcholine was thirty times greater than that into sphingomyelin. Exposure of the explants to aminophylline for 24 h resulted in a nearly two-fold increase in choline incorporation into phospholipid. Similar results were obtained when
266
TABLE
I
THE
INFLUENCE
AND
ON
RAT
THE
LUNG
OF
AMINOPHYLLINE
ACTIVITY
IN
OF
ORGAN
Explants
were
nificance
was evaluated
for by
48
h.
Values
reported
t test for
Student’s
Choline
INCORPORATION
WITH
INTO
PHOSPHOLIPID
are the means
dependent
protein
of 3-5
variables.
PHOSPHOLIPID
SYNTHESIS
experiments
n.s..
not
Enzyme
incorporation
(pmol/mg ---
_-
CHOLINE
INVOLVED
IN
FETAL
CULTURE
cultured
_
ON
ENZYMES
Statistical
siy-
activity
(pmol/mg
per h) into .._____-.
? S.E.
significant.
protein
per min)
Phosphatidyl-
Sphingo-
Choline
Cholinephosopho-
choline _____________.__~__
my&n
kinase
transferase
_“~_____~______
Control
1813
i 325
58.2
i
11.6
1125
1 142
1319
1 261
Aminophylline
3257
?- 413
106.1
i
10.5
1262
t
1118
t 315
52
Aminophylline: control
1.88
P
1
0.17
1.95
_+
0.17
1.17
_+
0.83
0.22
t
0.14
n.s.
n.s.
the explants were cultured for 48 h in medium containing 0.2 mM dibutyryl cyclic AMP. Under these conditions choline incorporation into phosphatidylcholine was increased by 77% and incorporation into sphingomyelin by 53%. There was a small increase in the percentage of radioactive phosphatidylcholine which was disaturated. When the distribution of radioactivity in phosphatidylcholine was examined, it was found that 27% of the radioactivity in the phosphatidylcholine fraction of the control cultures was in the disaturated species, whereas 32% of that obtained from the aminophylline treated cultures was disaturated (P < 0.01). The DNA:protein ratio in the control cultures was 0.107 pg DNA per pg protein, while that in the aminophylline treated cultures was 0.118 1.18DNA per lug protein. Thus when the data was expressed per pg DNA, the degree of stimulation of choline incorporation into phosphatidylcholine or sphingomyelin was about 10% less than when expressed per protein. Despite the stimulation of choline incorporation into phosphatidylcholine, no significant differences were found in the activities of choline kinase or cholinephosphotransferase after exposure of the explants to aminophyll~e for 48 h. TABLE THE
II INFLUENCE
EXPLANTS The
OF
results
Explants Gestational
are
were
OF
AMINOPHYLLINE
FETAL
RAT
expressed
cultured
for
age (days)
LUNG
as the 48
ON OF
mean
CHOLINE
DIFFERENT of
the
INCORPORATION GESTATIONAL
INTO
aminophylline:control
ratios
Aminophylline:control
ratio
of choline
3-5
incorporation
experiments
18
1.44
t 0.12
*
1.30
+ 0.20
**
1.88
* 0.17
**
1.95
i 0.17
*
20
1.25
* 0.14
1.38
i 0.21
***
21
1.13
r 0.17
1.19
+ 0.23
difference
from
control
into
Sphingomyelin
19
significant
of
h.
Phosphatidylcholine
* Statistically
PHOSPHOLIPID
value
(P <: 0.001).
IN
AGES
** P <
0.01.
***
P < 0.05.
f
S.E.
267
The influence of am~ophylline on choline incorporation into phospholipid was also examined in 18-, 20- and 21day gestation fetal rats to determine whether aminophylline action was dependent on the degree of lung maturity. As is shown in Table II, the degree of stimulation of choline incorporation into both phosphatidylcholine and sphingomyelin was maximal in explants from 19day fetal rats and then declined progressively. Discussion Aminophylline has multiple effects on the organs of the body through its action as an inhibitor of cyclic AMP phosphodies~r~e, the enzyme responsible for the degradation of cyclic AMP [4 1. Barrett et al. 13) have recently reported that the administration of aminophylline to pregnant rabbits results in decreased phosphodiesterase activity, increased cyclic AMP concentrations, and stimulation of in vivo choline incorporation into phosphatidylcholine in the lungs of the fetuses. They also reported that administration of corticosteroid had a similar effect and suggested that the well-described corticosteroidinduced enhancement of lung phosphatidylcholine synthesis [2,6,14] may be cyclic AMP mediated. Karotkin et al. [ 5] have shown that when pregnant rabbits are injected with aminophylline there is an increase in the lung tissue and lung lavage phospholipid content in the fetuses. They did not further analyze the phospholipid composition of the lung tissue or lavage. Associated with the stimulation of phospholipid synthesis there was an increase in lung compliance, as determined by pressure-volume studies. There was, however, no increase in the number of lamellar bodies in the alveolar type II cells of the aminophylline-treated animals. Lamellar bodies are believed to be the site of storage and possibly of synthesis of surfactant [ 151. In contrast to this, corticosteroid administration has been reported to result in increased numbers of lamellar bodies in fetal rabbit lung [ 161. In addition administration of corticosteroids to fetal rabbits results in the specific stimulation of pulmonary phosphatidylcholine production so that the phosphatidylcholine:sphingomyelin ratio of lung lavage fluid is markedly increased [ 61. We have attempted to determine whether ~~ophylline has a specific effect on lung phosphatidylchol~e synthesis by examining its effect on fetal lung in organ culture. Organ culture is a useful model for the study of fetal lung maturation and has been used extensively in the past for studies of morphogenesis 1171 and more recently for studies of biochemical development [18]. Under the conditions described in this paper, fetal rat lung continues morphologic development in short term organ culture with the formation of larger and more numerous alveoli and flatter alveolar lining cells. On electron microscopy there is a progressive decrease in the glycogen content of the alveolar type II cells and an increase in the number of lamellar bodies [ 131. The data reported here indicate that aminophylline may stimulate fetal lung phosphohpid synthesis, but that this stimulation may be non-specific as the incorporation of choline into sphingomyelin was enhanced to the same degree as that into phosphatidylcholine. This aminophylline effect appears to differ from that observed with corticosteroid. Smith et al. (191 have reported that exposure of monolayer
268
cultures of mixed fetal rabbit lung cells to cortisol results in an increase in palmitate incorporation into phosphatidylcholine, but not into sphingomyelin. The administration of corticosteroid to pregnant rabbits has been shown to result in increased activity of cholinephosphate cytidylyltransferase [ 61, possibly cholinephosphotransferase [6,14] and lysolecithin acyltransferase [6]. The stimulation of the last enzyme may be particularly significant since it is believed to be involved in the synthesis of the disaturated species of phosphatidylcholine [20]. No significant change in the activity of choline kinase or cholinephosphotransferase was found in the lung explants after exposure to aminophylline. Since simultaneous stimulation of choline incorporation into phosphatidylcholine was observed in explants derived from the same fetuses, it is probable that aminophylline acts by stimulating the activity of other enzymes in the choline incorporation pathway. The effect of corticosteroid on lung phospholipid synthesis in cell culture has been shown to be dependent on gestational age. Smith et al. [19] observed that palmitate incorporation into phosphatidylcholine was stimulated by cortisol after 26 days of gestation in cultures of fetal rabbit lung cells and a marked effect was noted only after 28 days, three days prior to term. The action of aminophylline on fetal rabbit lung also appears to be dependent on gestational age as a significant increase in lung tissue phospholipid content was noted only in 28- and 29day fetuses by Karotkin et al. [ 51. Our data suggest that this is the case in the rat too, as maximal stimulation was observed in explants derived from 19day fetuses. There was then a progressive decline in aminophylline effect in explants derived from fetuses of greater gestational age. Barrett et al. [3] have reported that there is a small increase in the percentage of saturated fatty acids in fetal rabbit lung phosphatidylcholine after aminophylline treatment. The data presented here on choline incorporation into disaturated phosphatidylcholine tend to support this observation. Explants cultured in the presence of dibutyryl cyclic AMP yielded similar data to those cultured in medium containing aminophylline. This suggests that the effect of aminophylline on developing lung is due to its action as a cyclic AMP phosphodiesterase inhibitor. Cyclic AMP is a potent stimulator of glycogenolysis in fetal liver [ 211. Since glycogen is probably an important source of substrate for phospholipid synthesis in fetal lung [ 221, it is possible that the action of cyclic AMP is in part due to the stimulation of glycogen breakdown. The enhancement of fetal lung maturation by corticosteroid may also be at least partially cyclic AMP mediated, as has been suggested by Barrett et al. [3]. However, in view of the differences between the effects of aminophylline and corticosteroid and the fact that aminophylline action appears to be less specific, it is unlikely that cyclic AMP is the sole mediator of corticosteroid action on the developing lung. Acknowledgments The authors are grateful to Mary Rose Czajka, Theresa S. Wai-Lee, and Pat Pasqualini for excellent technical assistance. This work was supported by USPHS Grant No. HL 19752.
269
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431,
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Gross,
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Farrel,
I., Czajka, P.M.
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Funkhouser,
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S.A., N.S.,
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630-637 19
Smith,
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Van
B.T.,
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Kikkawa,
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64,
BBA 57039
AMINOPHYLLIN~ STIMULATES THE INCORPORATION OF CHOLINE INTO PHOSPHOL~~~ IN EXPLANTS OF FETAL RAT LUNG IN ORGAN CULTURE
We have i~~~~~~~~d t&e dire& effect of a~~~~~~l~i~~~ a cyclic AMP phos~hod~esterase inhibitor, and of cyclic AMP on choline ~ncorpo~tion into phosphotipid in exphmts of fetal rat lung in organ culture. Explants from 19 day fetal rat lung (term is 22 days) were cultured for 24 or 48 h in F12 medium containing 1.0 mM aminophylfine or 0.2 mM dibutyryl cyclic AMP. Explants cultured in F12 medium alone served as controls. Exposure to aminophylline resulted in an 88% increase in choline incorporation into phosphatidylcholine (I’ < 0.01) and a 95% increase in choline incorporation into sphingomyelin (P < 0.001~ in the 48-h cultures. Similar changes were noted after 24 h in culture. There was a small increase in the percentage of synthesized ph~spbat~dy~ch~~~ne which was disaturated. The activities of choline kinase and cho~mephosphQ~~sfe~se in the explants were not s~~ific~tly altered by exposure to amin~phy~~e* 71 and 53% st~u~at~on of choline incorporatiun into phos~hat~d~~ch~~jne and sph~~~om~e~~ res~ec~j~e~~ was demonstrated in explants cultured in medium containing cyclic AMP. Am~o~hyll~ne treatment of explants from ‘&I- and 2Xday fetuses resulted in a smaller degree of stimulation of choline incorporation into both phospholipids. These data suggest that aminophylline has a direct stimulatory effect on the incorporation of choline into phospholipid in fetal rat lung and that this effect is probably cyclic AMP mediated.
264
Introduction The alveoli of the lung are lined by surface active material, pulmonary surfactant, which lowers surface tension at the air-alveolar interface, thereby preventing alveolar collapse. The major surface-active components of surfactant are phospholipids; dipalmitoylglycerophosphocholine is thought to be the most important in this regard [ 11. The production of pulmonary surfactant in fetal lung increases toward the end of gestation [Z]. It has been recently suggested by Barrett et al. that the administration of aminophylline to fetal rabbits may enhance pulmonary phosphatidylcholin~ synthesis [3]. Ami~lophylline is the ethyl~ine diamine salt of theophylline, a methyl xanthine compound, which has the property of increasing tissue levels of cyclic AMP through its action as a cyclic AMP phosphodiesterase inhibitor [4]. Karotkin et al. [ 51 have also reported that there is an increase in the phospholipid content of fetal rabbit lung tissue and lung lavage (source of surfactant) after aminophylline administration to the mother. Corticosteroid is another agent that is believed to stimulate fetal lung surfactant production [ 2,6]. Since Barrett et al. [ 31 found that exposure of fetal rabbits to either corticosteroid or aminophylline resulted in increased lung tissue cyclic AMP levels, they have suggested that cyclic AMP may be a mediator of fetal lung phosphatidylcholine synthesis. We have examined the direct effect of ~inophylline and cyclic AMP on phospholipid synthesis in explants of fetal rat lung grown in organ culture, in order to further evaluate the role of these agents in fetal lung maturation. Materials and Methods Culture qinterial. Timed 1% to 21-day pregnant Sprague-Dawley rats (term is 22 days) were used in all experiments. Pregnant rats were killed by a blow to the head and the fetuses removed immediately under sterile conditions. All further procedures were performed in a laminar flow hood and sterility was carefully maintained. The lungs from two to three litters of fetuses were immediately excised, pooled, and chopped into cubes with 0.8 mm sides by means of a McIlwain tissue chopper (Brinkm~n Instruments, Inc., Westbury, N.Y.). A stainless steel grid with a 25 X 25 mm surface (Frankel Company, Philadelphia, Pa.) was placed in a plastic tissue culture dish to which 5.5 ml F12 medium (Grand Island Biological Company, Grand Island, N.Y.) was added so that it just reached the undersurface of the grid. A sterile 25-mm diameter Metricel filter (Gelman Instrument Company, Ann Arbor, Mich.) was placed on the grid and 30 lung explants placed on the filter. The undersurface of the explants was thus exposed to the medium and the upper surface to the air. The explants were then incubated in a humidified atmosphere of 95% O2 and 5% CO:! at 37°C. Control cultures were incubated in F12 medium only. ~xperiment~ cultures were grown in F12 medium containing either aminophylliile, 1.0 mM, or dibutyryl cyclic AMP, 0.2 mM. F12 is an enriched synthetic medium which contains choline chloride, 0.1 mM.
Phospholipid synthesis. After 24 or 48 h in culture the medium was removed and replaced with 5 ml of F12 medium containing 1.25 @Z!i [Me3H]choline chloride, 2.5 Ci/mol. The explants were cultured for 6 h in the presence of the isotope after which time the medium was removed and the explants washed with ice-cold 0.9% NaCl. The explants from each flask were then sonicated in 2 ml of 0.9% NaCl prior to lipid analysis and protein determination. Lipid was extracted from the sonicate with chloroform and methanol by the method of Bligh and Dyer [7]. Phospholipids were identified by thin layer chromatography on silica gel (LQD plates, Quantum Industries, Fairfield, N.J.) in chloroform/methanol/7 M NH40H (60 : 35 : 5, v/v). Lipids were visualized on the chromatograms by exposure to iodine vapor. The phosphatidylcholine and sphingomyelin spots were scraped off the plates into scintillation vials and the radioactivity in these fractions was determined in 0.5 ml H,O and 14.5 ml Aquasol (New England Nuclear, Boston, Mass.). Protein content was determined by the method of Lowry et al. [S] and DNA content by a modification of the method of Burton [9]. Disaturated phosphatidylcholine was determined by the method of Mason et al. [lo]. Enzyme actiuities. After 48 h in culture the explants were sonicated in chilled 0.33 M Tris HCl, pH 7.4, containing 0.001 M EDTA. Choline kinase (EC 2.7.1.32) was assayed in the homogenate by measuring the rate of incorporation of radioactivity from [Me-‘4C]choline into phosphocholine as described previously [ 111. Cholinephosphotransferase (EC 2.7.8.2) was assayed in the homogenate by measuring the rate of incorporation of radioactivity from CDP [Me-14C]choline into lipid as described previously [12], except that the reaction was carried out at pH 8.5 and 19 mM dioleoylglycerol and 0.08 mM CDP choline were used as substrates. Chemicals. All radiochemicals were purchased from New England Nuclear, Boston, Mass. Phospholipids were obtained from Serdary, London, Ont. Aminophylline U.S.P. was obtained from G.D. Searle and Company, Chicago, Ill. and dibutyryl cyclic AMP and all other biochemicals from Sigma Chemical Company, St. Louis, MO. All other chemicals were reagent grade. Results Fetal rat lung explants remain viable under the cnditions described in Materials and Methods. This has been determined previously [13] by examining the histologic appearance by light and electron microscopy and by measuring the rate of glucose oxidation and comparing it to that obtained with fresh fetal lung tissue. As is shown in Table I, there was an 88% increase in choline incorporation into phosphatidylcholine in explants of 19-day fetal lung after exposure to 1.0 mM aminophylline for 48 h. A similar degree of stimulation of choline incorporation into sphingomyelin was also observed. These changes are statistically significant. Varying the dose between 0.5 and 5.0 mM had little effect on the rate of choline incorporation. The rate of choline incorporation into phosphatidylcholine was thirty times greater than that into sphingomyelin. Exposure of the explants to aminophylline for 24 h resulted in a nearly two-fold increase in choline incorporation into phospholipid. Similar results were obtained when
266
TABLE
I
THE
INFLUENCE
AND
ON
RAT
THE
LUNG
OF
AMINOPHYLLINE
ACTIVITY
IN
OF
ORGAN
Explants
were
nificance
was evaluated
for by
48
h.
Values
reported
t test for
Student’s
Choline
INCORPORATION
WITH
INTO
PHOSPHOLIPID
are the means
dependent
protein
of 3-5
variables.
PHOSPHOLIPID
SYNTHESIS
experiments
n.s..
not
Enzyme
incorporation
(pmol/mg ---
_-
CHOLINE
INVOLVED
IN
FETAL
CULTURE
cultured
_
ON
ENZYMES
Statistical
siy-
activity
(pmol/mg
per h) into .._____-.
? S.E.
significant.
protein
per min)
Phosphatidyl-
Sphingo-
Choline
Cholinephosopho-
choline _____________.__~__
my&n
kinase
transferase
_“~_____~______
Control
1813
i 325
58.2
i
11.6
1125
1 142
1319
1 261
Aminophylline
3257
?- 413
106.1
i
10.5
1262
t
1118
t 315
52
Aminophylline: control
1.88
P
1
0.17
1.95
_+
0.17
1.17
_+
0.83
0.22
t
0.14
n.s.
n.s.
the explants were cultured for 48 h in medium containing 0.2 mM dibutyryl cyclic AMP. Under these conditions choline incorporation into phosphatidylcholine was increased by 77% and incorporation into sphingomyelin by 53%. There was a small increase in the percentage of radioactive phosphatidylcholine which was disaturated. When the distribution of radioactivity in phosphatidylcholine was examined, it was found that 27% of the radioactivity in the phosphatidylcholine fraction of the control cultures was in the disaturated species, whereas 32% of that obtained from the aminophylline treated cultures was disaturated (P < 0.01). The DNA:protein ratio in the control cultures was 0.107 pg DNA per pg protein, while that in the aminophylline treated cultures was 0.118 1.18DNA per lug protein. Thus when the data was expressed per pg DNA, the degree of stimulation of choline incorporation into phosphatidylcholine or sphingomyelin was about 10% less than when expressed per protein. Despite the stimulation of choline incorporation into phosphatidylcholine, no significant differences were found in the activities of choline kinase or cholinephosphotransferase after exposure of the explants to aminophyll~e for 48 h. TABLE THE
II INFLUENCE
EXPLANTS The
OF
results
Explants Gestational
are
were
OF
AMINOPHYLLINE
FETAL
RAT
expressed
cultured
for
age (days)
LUNG
as the 48
ON OF
mean
CHOLINE
DIFFERENT of
the
INCORPORATION GESTATIONAL
INTO
aminophylline:control
ratios
Aminophylline:control
ratio
of choline
3-5
incorporation
experiments
18
1.44
t 0.12
*
1.30
+ 0.20
**
1.88
* 0.17
**
1.95
i 0.17
*
20
1.25
* 0.14
1.38
i 0.21
***
21
1.13
r 0.17
1.19
+ 0.23
difference
from
control
into
Sphingomyelin
19
significant
of
h.
Phosphatidylcholine
* Statistically
PHOSPHOLIPID
value
(P <: 0.001).
IN
AGES
** P <
0.01.
***
P < 0.05.
f
S.E.
267
The influence of am~ophylline on choline incorporation into phospholipid was also examined in 18-, 20- and 21day gestation fetal rats to determine whether aminophylline action was dependent on the degree of lung maturity. As is shown in Table II, the degree of stimulation of choline incorporation into both phosphatidylcholine and sphingomyelin was maximal in explants from 19day fetal rats and then declined progressively. Discussion Aminophylline has multiple effects on the organs of the body through its action as an inhibitor of cyclic AMP phosphodies~r~e, the enzyme responsible for the degradation of cyclic AMP [4 1. Barrett et al. 13) have recently reported that the administration of aminophylline to pregnant rabbits results in decreased phosphodiesterase activity, increased cyclic AMP concentrations, and stimulation of in vivo choline incorporation into phosphatidylcholine in the lungs of the fetuses. They also reported that administration of corticosteroid had a similar effect and suggested that the well-described corticosteroidinduced enhancement of lung phosphatidylcholine synthesis [2,6,14] may be cyclic AMP mediated. Karotkin et al. [ 5] have shown that when pregnant rabbits are injected with aminophylline there is an increase in the lung tissue and lung lavage phospholipid content in the fetuses. They did not further analyze the phospholipid composition of the lung tissue or lavage. Associated with the stimulation of phospholipid synthesis there was an increase in lung compliance, as determined by pressure-volume studies. There was, however, no increase in the number of lamellar bodies in the alveolar type II cells of the aminophylline-treated animals. Lamellar bodies are believed to be the site of storage and possibly of synthesis of surfactant [ 151. In contrast to this, corticosteroid administration has been reported to result in increased numbers of lamellar bodies in fetal rabbit lung [ 161. In addition administration of corticosteroids to fetal rabbits results in the specific stimulation of pulmonary phosphatidylcholine production so that the phosphatidylcholine:sphingomyelin ratio of lung lavage fluid is markedly increased [ 61. We have attempted to determine whether ~~ophylline has a specific effect on lung phosphatidylchol~e synthesis by examining its effect on fetal lung in organ culture. Organ culture is a useful model for the study of fetal lung maturation and has been used extensively in the past for studies of morphogenesis 1171 and more recently for studies of biochemical development [18]. Under the conditions described in this paper, fetal rat lung continues morphologic development in short term organ culture with the formation of larger and more numerous alveoli and flatter alveolar lining cells. On electron microscopy there is a progressive decrease in the glycogen content of the alveolar type II cells and an increase in the number of lamellar bodies [ 131. The data reported here indicate that aminophylline may stimulate fetal lung phosphohpid synthesis, but that this stimulation may be non-specific as the incorporation of choline into sphingomyelin was enhanced to the same degree as that into phosphatidylcholine. This aminophylline effect appears to differ from that observed with corticosteroid. Smith et al. (191 have reported that exposure of monolayer
268
cultures of mixed fetal rabbit lung cells to cortisol results in an increase in palmitate incorporation into phosphatidylcholine, but not into sphingomyelin. The administration of corticosteroid to pregnant rabbits has been shown to result in increased activity of cholinephosphate cytidylyltransferase [ 61, possibly cholinephosphotransferase [6,14] and lysolecithin acyltransferase [6]. The stimulation of the last enzyme may be particularly significant since it is believed to be involved in the synthesis of the disaturated species of phosphatidylcholine [20]. No significant change in the activity of choline kinase or cholinephosphotransferase was found in the lung explants after exposure to aminophylline. Since simultaneous stimulation of choline incorporation into phosphatidylcholine was observed in explants derived from the same fetuses, it is probable that aminophylline acts by stimulating the activity of other enzymes in the choline incorporation pathway. The effect of corticosteroid on lung phospholipid synthesis in cell culture has been shown to be dependent on gestational age. Smith et al. [19] observed that palmitate incorporation into phosphatidylcholine was stimulated by cortisol after 26 days of gestation in cultures of fetal rabbit lung cells and a marked effect was noted only after 28 days, three days prior to term. The action of aminophylline on fetal rabbit lung also appears to be dependent on gestational age as a significant increase in lung tissue phospholipid content was noted only in 28- and 29day fetuses by Karotkin et al. [ 51. Our data suggest that this is the case in the rat too, as maximal stimulation was observed in explants derived from 19day fetuses. There was then a progressive decline in aminophylline effect in explants derived from fetuses of greater gestational age. Barrett et al. [3] have reported that there is a small increase in the percentage of saturated fatty acids in fetal rabbit lung phosphatidylcholine after aminophylline treatment. The data presented here on choline incorporation into disaturated phosphatidylcholine tend to support this observation. Explants cultured in the presence of dibutyryl cyclic AMP yielded similar data to those cultured in medium containing aminophylline. This suggests that the effect of aminophylline on developing lung is due to its action as a cyclic AMP phosphodiesterase inhibitor. Cyclic AMP is a potent stimulator of glycogenolysis in fetal liver [ 211. Since glycogen is probably an important source of substrate for phospholipid synthesis in fetal lung [ 221, it is possible that the action of cyclic AMP is in part due to the stimulation of glycogen breakdown. The enhancement of fetal lung maturation by corticosteroid may also be at least partially cyclic AMP mediated, as has been suggested by Barrett et al. [3]. However, in view of the differences between the effects of aminophylline and corticosteroid and the fact that aminophylline action appears to be less specific, it is unlikely that cyclic AMP is the sole mediator of corticosteroid action on the developing lung. Acknowledgments The authors are grateful to Mary Rose Czajka, Theresa S. Wai-Lee, and Pat Pasqualini for excellent technical assistance. This work was supported by USPHS Grant No. HL 19752.
269
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