Pulmonary phosphatidic acid phosphohydrolase

Pulmonary phosphatidic acid phosphohydrolase

Eiochimica et Biophysics Acta, 665 (1981) 186 Elsevier/North-Holland 186- 194 Biomedical Press BBA 57849 PULMONARY PHOSPHATIDIC DEVELOPMENTAL P...

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Eiochimica et Biophysics Acta, 665 (1981)

186

Elsevier/North-Holland

186- 194

Biomedical Press

BBA 57849

PULMONARY PHOSPHATIDIC DEVELOPMENTAL

PATTERS

ACID PHOSPHOHYDROLASE IN RABBIT LUNG

PAUL G. CASOLA a and FRED POSSMAYER a,b,* a Depart~e~~t of 3io~~~e~is~y and b ~epart~ne~t of Obstetr~es and Gynaecology, London, Ontario N6.4 MS {Canada)

University

of

Western

Ontario,

(Received November ZIst, 1980) (Revised manuscript received March 16th, 1981)

Key wrds:

Pulmonary surfactant: Glycerolipid metabolism; PhosphatidylchoIine;

Phosphatidic acid phosphohydrolase;

Res~~iratory distress sy~ldro~ne; ~Rabb~t kg)

1, The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueoudy dispersed phosphatidic acid (PA,,) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PA~b-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PA,,dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the PA,,- or the PAmbdependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaqdependent activities in the void volume (I’,) (50% total), a peak with an apparent molecular mass (M,) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PA~~dependent peak with 1M,= 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PA,I, dependent activity in the Y0 (15% total), a major peak with an apparent J!Zr = 390 kdaltons (44% total) and minor peaks with 1M, = 240 kdaltons (16% total) and&fr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on the PAmbdependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PA,+, -dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profdes of the PAag-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profdes of the PAaq-dependent activities in adult cytosol were monophasi~.

Introduction

days gestation

which reaches a maximum at day 30 A concomitant increase in the tissue content of PC and disaturated PC occurs during this period [3-51. The levels of PC in fetal lung lavage also increases during this period but a greater efevation occurs after birth 161. These developmental increases in the incorporation of choline into PC and in the tissue content of PC in the rabbit contrast with (term

There is a marked increase in the rate of incorporation of radioactive choline into phosphatidylcholine (PC) in slices of fetal rabbit lung between 27 and 29

* To whom correspondence should be addressed. Abbreviations: PAaq, aqueousiy dispersed phosphatidic acid; PAmb, membrane~ound phosphat~dic acid; PC, phosphatidylcboline. OOOS-2760/81/0000--0000/$02.50

o Elsevier/North-Holland

3 1) [1,2].

the changes observed with the rat where the developmental increases continue after birth 171. As indiBiomedical Press

187

cated in the previous paper [8], these increases will be taken as evidence for an increased production of PC, at least part of which is for surfactant. Although it has been difficult to relate changes in enzyme activity to developmental changes in phospholipid content [4,9] (see also Ref. 10 for an excellent review). A number of workers have observed that the level of phosphatidic acid phosphohydrolase (EC 3 .1.3.4) activity in fetal rabbit lung increases developmentally [2,1 l] and after the induction of pulmonary maturation by glucocorticoids [ 1,2,12,13]. These observations, in which the phosphatidic acid phosphohydrolase activities dependent on aqueously dispersed phosphatidate (PA,,) were assayed, led to the suggestion that this enzyme might be responsible for the control of PC production for surfactant in this species [ 1,2,11]. As indicated in the previous paper [8], the PAaq-dependent phosphohydrolase activities can be distinguished from the activities which are responsible for the degradation of membrane-bound phosphatidic acid (PA,,,). In the present investigation the developmental profiles of the PA,,- and PA,b-dependent activities have been examined in the rabbit in order to investigate more critically the relationship between these two phosphatidic acid phosphohydrolase activities with respect to the increases in PC synthesis which occur in the rabbit fetus near term.

Experimental New Zealand white rabbits, mated under direct vision, were obtained from Rieman’s Fur Ranch, Petersburg, Ontario, Canada. The day of mating was taken as day 0. The does were killed with a lethal dose of sodium pentobarbital administered via the lateral ear vein. The fetuses were removed and killed by severing the spinal cord with a small pair of scissors in such a manner that the trachea was not disturbed. Pooled lower lobes from all living fetuses (6-12/litter) from each doe were homogenized in 19 vol. 0.32 M sucrose/O.1 mM EDTA (pH 7.4) with a serrated Teflon pestle (A.H. Thomas and Co., Philadelphia, PA, U.S.A.). The remaining methods are described in the previous paper or in other publications from this laboratory [8,14,15]. Results Developmental profiles of phosphatidic acid phosphohydrolase in rabbit lung during development Phosphatidic acid phosphohydrolase activities were investigated in the fetal rabbit lung prior to (25 days gestation), during (26 and 27 days gestation) and following (30 days gestation) the marked increases in the total lung PC and disaturated PC con-

TABLE I PHOSPHATIDIC ACID PHOSPHOHYDROLASE ACTIVITIES IN FETAL AND RABBIT LUNG HOMOGENATES Phosphatidic acid phosphohydrolase activities in lung homogenates were determined with PAmb and PAaq as described in Experimental. Results are expressed (mean + S.E.) as specific activity (enzyme activity/mg protein) and total enzyme activity/g lung (enzyme units/g lung) for the number of litters shown (n). Gestational

(n)

age

mg protein/

(days)

25 26 21 30 Adult

PAmb-dependent

activity

PA,,-dependent

activity

g lung

6 5 6 6 6

a pmol/min per mg protein. b nmol/min per protein. c enzyme units per g lung.

83.4 91.1 73.8 88.2 131.0

+ 6.6 + 9.4 + 3.3 f 7.1 + 6.3

Specific activity a

Total activity c

73.0 65.0 90.5 95.6 53.5

6.13 5.91 6.73 8.42 7.07

+ 8.3 f5.0 + 6.4 + 1.2 + 6.4

5 0.7 + 0.5 + 0.4 f 0.9 IO.8

Specific activity b 7.1 5.3 7.1 7.3 4.6

+ k k + k

0.4 0.2 0.4 1.2 0.6

Total activity c ~__ 592.3 f 34.5 480.1 + 15.0 520.0 + 20.5 643.5 + 105.5 596.0 + 75.5

TABLE

II

PII~SFI~.~TIDi~

ACID PIIOSPHOHYDROLAS~

ACTIVITIES

IN FETAL

AND ADULT

RABBIT

Phosphatidic acid phosphohydrolase activities in rabbit lung microsomes were determined I~.xperimental. Results are expressed (mean rt S.E.) as specific activity (enzyme activity/mg lung (enzyme units/g lung) for the number of litters shown (n). _. _.___.____~ ~-____ -___ Gestational

(n)

aL*c (d;,ys)

mg protein/ 6 lung

2s 26 27 30 Adult

6 5 6 6 6

1.2 5.2 6.2 6.6 13.2

+ 0.6 + 0.1 + 0.4 2 0.8 20.6

PA ,,b-dependent ___I-

activity _~_ _--

Specific activity a

Total activity

67.3 77.4 92.1 109.7 41.6

0.48 0.40 0.58 0.72 0.61

+ t i2 +

4.2 0.4 1.9 1.3 7.5

+ k k + i-

LUNG ~ICROSO~~S

with PA,,, protein)

and PA,9 as described in and total enzyme activity/g

--~

-I

PAaq-dependent -___e 0.04 0.00 0.0 1 0.08 0.10

activity _-_-.-

Specific activity b

Total activity

8.5 12.3 14.1 16.6 7.1

30.6 31.3 43.6 54.3 49.5

rf: 0.7 i- 0.4 .i- 0.1 5 1.9 i 0.9

rt + ? t +

e 3.8 1.0 0.3 6.2 5.3

a pmol/min per mg protein. b nlil~)i~ltiir~ per mg protein. c enzyme units/g lung.

tent (3,5,10] and the augmented incorporation of choline into PC [ 1.21. The specific activities of PAmbdependent phosphohydrolase in the microsomes in whole l~onlogenate increase significantly during fetal development, particularly between days 26 to 27 of gestation (Table I, 11) but fell in the adult. In agreement witlt previous observations [2,1 I], the specific activities of the PACT-depel~dent phosphohydrolase in the homogenates and microsomes also increased in

TABLE

fetat lung, principally between 25 or 26 and 27 days gestation. The cytosolic PAmb- and PA,,-dependent activites remained relatively constant during feta1 developinent (Table III). Schultz et al. [I I] have demonstrated an increase in the PA,,-dependent activity in the cytosol between 23 and 25 days gestation but this precedes the increases in PC content by several days. In contrast to the rat [8], only minor changes

III

PHOSPHATIDIC

ACID PHOSPIIOHYDROLASE

ACTIVITIES

IN FETAL

AND ADULT

RABBIT

LUNG CYTOSOLS

Phosphatidic acid phosphohydrolase activities in rabbit lung cytosols were determined with PA,,b and PA,, as described in rimental. Results are expressed (mean t SE.) as specific activity (enzyme activity/mg protein) and totai enzyme activity/a (enzyme units/g lung) for the number of litters shown (II). Gestational age (days)

_ 25 26 27 30 Adult

(n)

mg protein/

PAmbdependent

activity

PAaqdependent

Specific activity a -~ 6 5 6 6 6

a pmoI/min per mg protein. b n~~lol/in~n per nig protein. e enzyme units/g lung.

Total activity

c

k 2.1 _c 1.5 i 1.0 2 0.7 + 0.5

Specific activity b

Totai activity

1.8 1.9 1.8 1.5 1.6

56.05 50.3 42.7 44.8 110.0

c

_~_____

-__-32.0 27.5 24.4 31.2 71.7

activity

-~

p lung

82.1 87.7 87.6 85.8 50.3

rt 6.4 + 6.8 13.7 14.7 _t:7.6

2.62 2.41 2.14 2.66 3.57

f 0.21 k 0.19 & 0.07 i 0.14 + 0.06

-e 0.2 i 0.1 i: 0.1 t 0.3 t 0.3

5.5 k 2.1 t 2.1 i 4.8 i 19.5

Espelung

189

were observed in the protein contents during fetal development in the rabbit. The adult fractions contained approximately twice as much protein. Similar observations have been reported by Tsao and Zachman 191. Consequently, except for being markedly increased in the adult, the total activities largely reflected the specific activities. ~eve~oprne~lt pattems of the phosphat~dic acid phosphohydrolase activities in rabbit lung cytosol

Aliquots of rabbit lung cytosol isolated from 1000 mg fetal and adult lung, were chromatographed on Bio-Gel A5m columns and the apparent molecular weights were estimated [8]. Under the standard assay conditions (PA,b -dependent phosphohydrolase activity, +2.5 mM MgClz; PAag-dependent phosphohydrolase activity, no MgC12), the PA,,- and PA,,dependent phosphohydrolase activities (Fig. 1) demonstrated similar elution profiles to those observed with adult rat lung cytosol 181. The PA,bdependent phosphohydrolase activity was characterized by a major peak at Mr = 390 000 (44% total) followed by a minor activity peak at Mr = 240 000 (16% total) and M, = 110000 (24% total). Approx. 15% of the PA,b-dependent activity was eluted in the void volume (VO) of the column The PA,,,-dependent

Fig. 1. Elution profiles of the PAmbdependent (of and PAa,dependent (0) phosphohydrolase activities from adult rabbit lung cytosol on BioGel ASm. A O-55% ammonium sulfate fraction of rabbit lung cytosol isolated from 1000 mg lung tissue (approx. 54 mg cytosoi protein) was applied to a BioGel A5m column and eluted with 50 mM Tricine, pH 7.4/0.1 M KC1/2.0 mM 2-mercaptoethanol.

phosphohydrolase activity in the rat cytosol is also characterized by the M, = 390000 and IV, = 110000 activity peaks found with rabbit lung cytosol, but not the Mr = 240 000 activity. The PAag-dependent phosphohydrolase activities in rabbit cytosol were characterized by peaks of activity at M, = 150000 (25% total), at M, = 110000 activity (25% total), and a V,, activity (50% total). The distribution of the PA,,dependent phosphohydroiase activities differs somewhat from that described in the rat lung cytosol, where the major activity is in the M, = 130000 peak (50% total). In addition, the PA,,-dependent phosphohydrolase Iw, = 110000 activity in the rabbit represents approx. 25% of the total activity, which is twice that found in the rat. It is possible that the PA,&ependent M, = 150 000 activity in the rabbit is similar to the Iw, = 130000 activity observed in the rat I

Fig. 2. Elution profiles of the PAa~dependent (0) and PAmbdependent (0) phosphohydrolasc activities from fetal rabbit lung cytosols. Ammonium sulphate (0.55%) fractions of cytosol isolated from 1000 mg of (a) 25-day fetal lung (32 mg cytosol protein), (b) 27day fetal lung (24 mg cytosol protein), and (c) 30day fetal lung (31 mg cytosol protein), were chromatographed on Bio-Cel ASm columns.

190

The elution profiles of the PAmr,-dependent phosphohyrolase activities in fetal cytosols were unchanged relative to the adult (Fig. 2). The adult lung cytosol fraction contained more total enzyme activity per g tissue. In each case, the PACT-dependent phosphohydrolase M, = 390000 activity peak was the major component (40-50% total) followed by the Mr = 240 000 activity peak (15-18% total) and the M, = 110000 activity peak (18-24% total). In contrast, the activity profiles of the PACE-dependent phosphohydrolase activity in the fetal cytosols demonstrated some distinct differences from the adult. While the PA,,-dependent phosphohydrolase Mr = 110000 activity peak was present in fetal cytosols, the Mr = 150 000 activity peak observed in the adult cytosol first appeared as a reproducible shoulder of the Mr = 110000 activity peak at 30 days gestation. With both fetal and adult cytosols, approx. 50% of the total PA,,-dependent phosphohydrolase activity eluted in the Ve peak. Chromatography of twice as much fetal cytosol resulted in larger activities of PA~~dependent phosphohydrolase in the V0 and M, = 110000 peaks but still no detectable activity in

the M, = 150 000 region (data not shown). The developmental pattern of the PA,,-dependent phosphohydrolase M, = 150 000 activity component appears to be similar to that described in the previous paper [8] for the rat lung cytosol M, = 130 000 activity peak. In general, the recoveries of both the PAmr,- and PAaq-dependent activities were similar at the various gestational ages examined compared to the adult (Table IV). The PArrrt,-dependent phosphohydrolase Mr = 390000 activity was puri~ed approx. 3-4 fold from fetal and neonatal cytosols (Table V). The PA,,dependent phosphohydrolase activity was enriched to the greatest extent (S-lo-fold) in the V,, of the column. The PA,,-dependent phosphohydrolase M, = 110 000 activity peak demonstrated little enrichment in the fetal cytosols but was purified approx. 4-fold in the adult, as was the M, = 150 000 activity peak. Thermal denaturation of cytosol phosphatidic acid phosphohydrohse activities in the developing rabbit lung Thermal stability plots of the PACT-dependent phosphohydrolase activities at 70°C demonstrated

TABLE IV RECOVERIES OF THE PA,b AND P~4a~-DEPENDENT PHOSPHOHYDROLASE ACTIVITIES FROM FETAL AND ADULT RABBIT LUNG CYTOSOLS ON BIO-GEL ASm Recoveries for the O-55% ammonium sulfate fractions and for column fractions are based on cytosols kept kept at -40°C for the duration of the separation on Rio-Gel A5m (approx. 36-48 h). Numbers in parentheses beside enzyme units refer to recoveries in percent. (a) PAmb-dependent phosphohydrolase (enzyme units)

activity Gestational age (days)

Fraction 25 Cytosol O-55% ammonium sulfate fraction BioGel A5m fractionation

1.39 (100) 1.06 (76) 0.54 (39)

30

21 1.69 (100) 1.08 (64) 0,66 (39)

1.33 (100) 1.55 (117) 0.53 (40) .-__

Adult 3.85 (100) 3.01 (78) 1.47 (38) ~~___.._____~_

(b) [email protected] phosphohydrolaseactivity (enzyme units) Gestational age (days)

Fraction

Cytosol O-55% ammonium sulfate fraction BioGel A5m fractionation

25

27

36.6 (100) 15.5 (42) 10.8 (29)

34.1 14.4 11.9

(100) (42) (34)

30

Adult

24.9 (100) 11.2 (45) 10.8 (44) -

79.1 41.7 36.9

(100) (60) (47)

191 TABLE V PURIFICATION ADULT RABBIT

OF THE PAmb- AND PAa~-DEPENDENT PHOSPHOHYDROLASE LUNG CYTOSOLS ON BIO-GEL A5m

ACTIVITIES FROM FETAL

AND

Specific activity values for cytosols and for the O-55% ammonium sulfate fractions are based on these preparations kept at -40°C for the duration of the separation on Bio-Gel A5m (approx. 36--48 h). Numbers in brackets beside specific activity values refer to relative specific activities. --(a) PA,b-dependent phosphohydrolase (pmol/min per mg protein)

activity Gestational age (days)

Fraction

~~ 25

27

30

Cytosol O-55% ammonium sulfate fraction Bio-Gel A5m fractions: Void volume ( Vo) Mr = 390 000 peak ---(b) PAaq-dependent phosphohydrolase (nmol/min per mg protein) Fraction

Cytosol O-55% ammonium sulfate fraction Bio-Gel A5m fractions:

Void volume f Vo) Mr = 150 000 peak Mr = 110 000 peak

Adult --

-

-

58.0 (1.0) 101.2 (1.7)

66.4 (1.0) 108.6 (1.6)

56.9 (1 .O) 147.7 (2.6)

65.7 (1.0) 140.1 (2.1)

37.9 (0.7) 208.0 (3.6) _.. activity

87.5 (1.3) 271.1 (4.1)

57.9 (1.0) 179.4 (3.2)

164.1 (2.5) 252.0 (3.8)

25

27

30

Adult

1.5 (1.0) 1.5 (1.0)

1.3 (1.0) 1.5 (1.1)

1.1 (1.0) 1.1 (1.0)

1.4 (1.0) 2.2 (1.6)

9.7 (6.3) 0.0 1.4 (0.9)

7.5 (5.6) 0.0 1.6 (1.2)

5.7 (5.3) 0.7 (0.6) 1.9 (1.8)

13.6 (10.0) 4.2 (3.1) 5.6 (4.1)

Gestational age (days)

biphasic inactivation profiles. There was little or no change in the profiles of fetal or adult cytosols. The

rapidly inactivated component has a half-life of 1 min and corresponds to about 70% of the total activity, while the more slowly inactivated component had a half-life of 16 min and accounted for the remaining 30% of the activity. The uniformity of the thermal denaturation profiles observed with the fetal and adult cytosols sug&ested that there was little change in enzyme species. The thermal denaturation profdes of the PA,,dependent phosphohydrolase activities in fetal rabbit cytosols at 7O*Cwere characterized by a rapidly inactivated component with a half-life of approx. 1.0-I .5 min which accounted for 60% of the total activity and a relatively heat-stable component with an approximate half-life of 16-17 min. In contrast, the adult inactivation profiles were virtually monophasic

with a half-life of 4 min, suggesting that one form of the enzyme activity predominated. The PA,,-dependent phosphohydrolase Mr = 150 000 activity present in adult cytosols could have been responsible for these changes. While the heat-lab~e PACT-dependent phosphohydrolase activity in rat lung cytosols had similar half-lives to the fetal rabbit cytosol activities, the heat-stable activities from each species demonstrated some distinct differences. Discussion The present report and the previous paper [8] have examined the developmental patterns of the PA,,- and PA,b-dependent phosphohydrolase activities in rabbit and rat lung in an attempt to relate these activities to the increases in the incorporation of radioactive precursors into PC and the elevations in

192

A

CYTOSOL

100 CYTOSOL

ACTIVITIES

PA&

CYTOSOL

ACTIVITIES

PAnq

ACTIVITIES

PA,,,

25 -

25 -

I

I 2 MINUTES

I 6

AT 704

101

2

6 MINfIES

AT

70”C4

Fig. 3. Thermal denaturation profiles of the PAmb- and PA,qdependent phosphohydrolase activities from fetal and adult rabbit lung cytosols. Rabbit lung cytosols (approx. 2.5 mg cytosol protein/ml) were heated at 7O”C, cooled on ice and subsequently assayed for phosphatidic acid phosphohydrolase activities. The data are plotted as percent activity remaining relative to control incubations which were not heat-treated (A). Semilogarithmic plots of the data are shown in (B). Average values are reported from two separate experiments with cytosols obtained from two separate litters each. Control values were: PAmbdependent phosphohydrolase activities (pmol/min per mg protein), 25day fetal, 76.4; 27day fetal, 85.8; 30day fetal, 80.3; adult, 46.2. PAaqdependent phosphohydrolase activities (nmol/min per mg protein), 25day fetal, 1.6; 27day fetal, 1.4; 30day fetal, 1.7; adult, 1.8. The maximum variation noted between separate experiments for any given point was 3% for the PAmbdependent phosphohydrolase activities and 5% for the PAaq dependent phosphohydrolase activities. 25day fetal cytosol (m), 27day fetal cytosol (o), 30day cytosol (m), adult cytosol (0).

the pulmonary content of this lipid near term. In contrast to rat lung, the specific activity of the PA,,dependent activity in rabbit homogenates increased somewhat between days 25 and 30 of gestation to attain levels which were greater than that observed in the adult. These alterations in the PAaq-dependent activity in the homogenates resembled the more marked changes in the microsomal fraction. In other tissues, PA,,-dependent activities have been detected in mitochondrial, lysosomal and plasma membrane preparations (discussed in Refs. 14 and 15) and in

lung. This activity is also present in lamellar bodies [ 16-191. However, at least in the rat, the contribution of the lamellar bodies to the total PA,,-dependent activity is small (Ref. 20 and Casola, P.G., Macdonald, P., McMurray, W.C. and Possmayer, F., unpublished results). Furthermore, considerable evidence has accumulated which indicates that these organelles do not possess the entire complement of enzymes required for PC synthesis [21-231. Since the de novo synthesis of PC takes place on the cytoplasmic side of the endoplasmic reticulum [24,25],

193 the activities associated with the microsomal and cytosolic fractions could most logically function in the production of this lipid. In both species, the specific and total activities of PAaq-dependent phosphohydrolase in the microsomes demonstrated significant progressive increases during development (rat, 20 days gestation to +3 days, 4.8fold, P< 0.001; rabbit, 25 days to 30 daysgestation, 2-fold, P< 0.01). These increases roughly paralleled the elevations in the incorporation of radioactive precursors and the tissue contents of PC. In contrast, the specific and total activities of PA,,-dependent phosphohydrolase in the cytosol were relatively stable over the developmental periods examined. Schultz et al. [ 111 have observed that the specific activity of PA,,dependent phosphohydrolase increases IO-fold in rabbit fetal cytosol between 23 and 25 days gestation, but this increase precedes the surge of PC synthesis. The absence of PAaq-dependent activity with an apparent MI = 130 kdalton in the cytosols from fetal and +l day neonatal rat lungs and the absence of the M, = 150 kdalton activity in the fetal rabbit provides evidence for a developmental alteration which is not associated with the induction of perinatal surfactant production. In the adult rat, the MI = 130 kdalton PA,,dependent activity represented the major activity (50% total), whereas in the adult rabbit the M, = 150 kdalton activity accounted for approx. 25% of total. In the rat, the PA,,-dependent activity corresponds principally with the Mg-independent phosphatidic acid phosphohydrolase activity and the PA,t,dependent activity mainly corresponds to the Mgdependent activity. Studies in other tissues indicate that the latter activity is associated with glycerolipid metabolism [8]. Certain differences were noted in the developmental patterns of the PA,b-dependent activities in these two species. In the fetal rat, the total and specific activities of the PAmt,-dependent phosphohydrolase increased significantly both in the microsomes (1.5-fold, P< 0.02) and in the cytosol(1.5fold, P< 0.01) between 18 and 20 days gestation and then declined. In the rabbit, the PA,r,-dependent activities progressively increased in the microsomes (1.6-fold, P< 0.001) between 25 and 30 days gestation, but the cytosolic activity remained relatively constant. The changes in the microsomal and cyto-

solic PArnt,-dependent activities were highly correlated in the rat (r = 0.91, P< 0.001) but not with the rabbit fetuses. In the case of the rabbit, the increases in the microsomal activity corresponded to the elevation in PC content, whereas with the rat the increases in the microsomal and cytosolic PA,,,-dependent activity preceded the increased PC production. If, as has been suggested, the PAmi,-dependent activities are important for PC synthesis, there is an apparent difference in the mechanisms by which these activities could function in the control of PC production. In contrast to the situation with the PA,,-dependent activities in the cytosol, in both species there was little change in the distribution of the PA,,dependent activities in fetal and adult cytosols. The fractionation procedures applied in the present investigations will permit a more comprehensive examination of the potential roles of the PA,,- and PA,,dependent activities in the increase in PC synthesis during the perinatal period. In conclusion, these studies have examined the phosphatidic acid phosphohydrolase activities in developing rat and rabbit lung using as the substrate both PA,, and PA,,.,. The most consistent observation with both species was an increase in the PA,,dependent phosphohydrolase activity in the microsomal fraction which paralleled the increase in PC content. However, the relation of the PAaq-dependent activities to PC synthesis remains unknown. It is not possible to draw support from investigations on the control of glycerolipid metabolism in liver and adipose tissue when in general, only the Mg-dependent phosphatidic acid phosphohydrolase activities have been implicated [8]. In addition, although the induction of pulmonary maturation in the rabbit fetus by glucocorticoids is accompanied by an increase in PA,,dependent phosphohydrolase activity [ 1,2,12,13], little effect was noted with the PA,b-dependent activities [ 133. Estradiol treatment of the rabbit fetus results in an increased incorporation of choline into PC and elevated PC levels without a significant increase in either the PAa4- or the PA,t,-dependent activities [26,27]. Consequently, a role for phosphatidic acid phosphohydrolase in the control of surfactant synthesis cannot be concluded until a relationship between one or more of these activities and PC synthesis can be established.

194

Acknowledgements These studies were supported by grants from the Medical Research Council of Canada and the Canadian Lung Association.

13

14

References 15 1 Brehier, A., Benson, B.J., Williams, M.C., Mason, R.J. and Ballard, P.L. (1977) Biochem. Biophys. Res. Commun. 77,883-890 2 Rooney, S.A., Gorban, L.I., Marino, P.A., Maniscalco, W.M. and Gross, I. (1979) Biochim. Biophys. Acta 572, 64-76 3 Gluck, L., Motoyama, E.K., Smits, H.L. and Kulovich, M.V. (1967) Pediatr. Res. 1, 237-246 4 Rooney, S.A., Wai-Lee, T.S., Gobran, L. and Motoyama, E.K. (1976) Biochim. Biophys. Acta 431,447-458 Rooney, S.A. and Gobran, L.I. (1977) Lipids 12,10501054 Rooney, S.A., Gobran, L.I. and Wai-Lee, T.S. (1977) J. CIin. Invest. 60,754-759 Rooney, S.A. (1979) Trends Biochem. Sci. 4,189-191 Casola, P.G. and Possmayer, E.P. (1981) Biochim. Biophys. Acta 665, 177-185 9 Tsao, F.H. and Zachman, R.D. (1977) Pediat. Res. 11, 858-861 10 Ohno, K., Akino, T. and Fujiwara, T. (1978) in Reviews in PerinataI Medicine (Scarpelli, E.M. and Cosmi, E.V., eds.), Vol. 11, pp. 227-3 18, Raven Press, New York 11 Schultz, F.M., Jimenez, J.M., MacDonald, P.C. and Johnston, J.M. (1974) Gynecol. Invest. 5, 222-229 12 Possmayer, F., Duwe, G., Metcalfe, R., Stewart-DeHaan,

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