Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits

Biochimica et Biophysics Acta, 962 (1988) 227-233

Elsevier

227

BBA 52896

Reutilization of surfactant phosphatidylglycerol

and lysophosphatidylcholine by adult rabbits

Harris C. Jacobs, David M. Lima, John M. Fiascone and Mark R. Mercurio Department of Pediatrics, Yale University, New Haven, CT (U.S.A.)

(Received 18 January 1988)

Key words: Pulmonary surfactant; Phospholipid metabolism; (Rabbit)

Adult rabbits reutilize the phosphatidylcholine (PC) of surfactant much less efficiently than developing rabbits (22% vs. 95%). Comparisons of reutilization efficiency of other components of surfactant in adult rabbits have not been determined. We injected adult rabbits intratracheally with [3Hjdipalmitoylphosphatid- ylcholine (DPPG) mixed with [ “C]lysophosphatidylcholine (IysoPC) and natural surfactant or [ “C]DPPC mixed with [ 3H]dipalmitoylphosphatidylglycerol (DPPG) and natural surfactant. Recovery in the alveolar wash and lamellar bodies of labelled DPPC, IysoPC and DPPG was determined at different times after injection. By plotting the ratio of 13HjDPPG to [ “C]DPPC in the alveolar wash versus time after injection we found that phosphatidylglycerol was reutilized with an efficiency of only O-7% which was much less than the reutilization of PC in these animals. At early times after injection, adult rabbits injected with [ 14C]lysoPC had a ratio of [ 14C]PC in their alveolar wash to lamellar bodies that was larger than 1.0. By comparison, 3-day old rabbits injected intratracheally with [14C]lysoPC had a ratio of [ “C]PC in alveolar wash to lamellar bodies less than 1.0 at the earliest times measurable. Thus adult rabbits demonstrate a pathway for accumulation of PC in their alveolar space prior to its appearance in lamellar bodies. This was not detected in developing rabbits. As in developing rabbits, adult rabbits reutilize the phosphatidylglycerol of surfactant less efficiently than the PC of surfactant.

Introduction

Chevalier and Collet [l] provided electron mi- croscopic evidence for the pathways involved in the synthesis and secretion of surfactant components by type II cells. Subsequent studies have provided more details on this subject [2-171. The picture that has emerged has been that at least the phospholipid components of surfactant are synthesized in the endoplasmic reticulum of type II cells, stored in lamellar bodies and secreted into the alveolar space. Once in the alveoli, surfactant

Correspondence: H.C. Jacobs, Yale University, Department of Pediatrics, P.O. Box 3333, New Haven, CT 06510, U.S.A.

appears to proceed through several morphologic states.

In developing rabbits, alveolar surfactant is taken up by the lung and efficiently reutilized by the type II cell primarily as intact molecules [9]. Data have been published indicating that in these rabbits and in adult rabbits there is little specific- ity for the form of phosphatidylcholine that can be reutilized by the lung [13,18]. It has also been demonstrated that lysophosphatidylcholine is reutilized as phosphatidylcholine and that phosphatidylglycerol and phosphatidylethanolamine are reutilized but less efficiently than phosphatidylcholine [18,19].

Adult rabbits are also known to reutilize surfactant phosphatidylcholine but with a much

00052760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

228

lower efficiency than in developing rabbits [20]. Most of the phosphatidylcholine appears to be reutilized intact [12,20]. Other investigators have demonstrated uptake of surfactant lipids by cul- tured type II cells isolated from adult lung with at least some breakdown and resynthesis of compo-

nents [17,21]. We asked if, as occurs with phosphatidylcholine, adult rabbit lungs reutilize

lysophosphatidylcholine and phosphatidylglycerol

differently than do developing rabbits.

Materials and Methods

Preparation of phosphatidylglycerol. [ 3 H]Di- palmitoylphosphatidylglycerol was prepared from l-palmitoyl-2-[9,10-3H]palmitoyl-2-sn-glycerol-3- phosphocholine ([ 3H]DPPC) which was purchased from New England Nuclear Corp. The procedure

was that of Dawson [22]. Briefly, [3H]DPPC, glycerol and phospholipase D are incubated over- night at room temperature in a mixture of chlo-

roform and buffer (40 mM CaCl,/O.l M acetate, pH 5.6). Phospholipase D exchanges the choline head-group for glycerol with high efficiency pro-

ducing [ 3H]L-a-dipalmitoylphosphatidyl-D,L-glycerol ([3H]DPPG). The product was isolated by thin-layer chromatography on silica gel TLC plates (Baker) in one dimension (chloroform/methanol/

cont. ammonium hydroxide, 65 : 35 : 5, v/v). [3H]DPPG was eluted from the silica using chloroform/methanol (2 : 1, v/v) and concentrated

under N2 at 50 o C. The purified [ 3 H]DPPG had a specific activity of about 4.6 . 10’ cpm/pmol.

Injection solutions. We prepared five separate injection solutions. The first solution contained 1-palmitoyl-2-[1-‘4C]palmitoyl-sn-glycerol-3-phos-

phocholine ([14C]DPPC) (153 mCi/mmol, New

England Nuclear Corp.) and [ 3H]DPPG. These two phospholipids were mixed in chloroform, dried under N, at 50°C and sonicated into a solution of lactated Ringer’s/water (1 : 1, v/v) at 50°C

[9]. Natural surfactant isolated from adult rabbits was added to this solution so that each rabbit received 1 ml of lactated Ringer’s/ water (1 : 1, v/v) containing 812000 cpm of [3H]DPPG (0.018 pmol), 111200 cpm of [i4C]DPPC and an amount of unlabelled surfactant equivalent to about 5% of the endogenous pool of surfactant.

Four separate injection solutions were prepared using L-1-[1-‘4C]palmitoyl-sn-glycero-3-phosphocholine ([ i4 C]lysoPC) (55 mCi/mmol) purchased from New England Nuclear Corp. This was mixed with [3H]DPPG and sonicated into solution as

indicated above. Three of these solutions were used for adult rabbits. They were mixed with unlabelled natural surfactant prior to injection so

TABLE I

OUTLINE OF EXPERIMENTS

Adult

Injected phospholipids and measured parameters are given for the three experiments described. PC, phosphatidylcholine; PG,

phosphatidylglycerol.

Experiment Animal age

1 Adult

Phospholipids injected

[ l4 C]DPPC and

[‘H]DPPG

Measured parameter

Total alveolar wash [14C]PC

Ratio alveolar wash [ 3H]PG to [ 14C]PC

Lamellar body [ 3H]PC specific activity

Alveolar wash [ ‘H]PC specific activity

[ 3 HJDPPC and [‘4c]1ysoPc

Total alveolar wash [ 3H]PC

Ratio alveolar wash [ ‘H]PC to [‘4C]PC

Lamellar body [ I4 C]PC specific activity

Alveolar wash [ l4 C]PC specific activity

Ratio alveolar wash [t4C]DPPC to [t4C]PC

3 3-day-old [ 3H]DPPC and

[‘4c]lysoPc Ratio alveolar wash [ ‘H]PC to [r4C]PC Lamellar body [14C]PC specific activity

Alveolar wash [‘4C]PC specific activity

229

that each rabbit received 1 ml of solution containing unlabelled surfactant equivalent to about 5% of the endogenous pool. The radioactivity per ml

in the three solutions varied from 134000 to 253000 cpm of [14C]lysoPC and 18000 to 750000 cpm of [ 3H]DPPC.

The fourth injection solution containing [ “C]lysoPC was prepared for injection into 3-day- old rabbits. Each rabbit received 0.1 cc of 1 : 1 lactated Ringer’s/ water containing 340 000 cpm

of [‘4C]lysoPC and 355 000 cpm of [ 3H]DPPC and an amount of unlabelled natural surfactant equiv-

alent to about 5% of the endogenous pool of surfactant.

Samples of injection solutions containing [‘4C]lysoPC were incubated at 37 o C with unlabelled alveolar lavage from adult rabbits for 30 and 90 min. Lipids were then extracted as de-

scribed below and assayed for conversion of [ I4 C]lysoPC to [I4 Clphosphatidylcholine.

Experiments. The details of the experiments are outlined in Table I. 26 female New Zealand White Rabbits weighing 1.20 k 0.03 kg (mean * S.E.) were injected with the solution containing [3H]DPPG (Experiment No. 1). 40 female New Zealand White rabbits weighing 1.14 + 0.02 kg

were injected with one of three solutions prepared with [14C]lysoPC (Experiment No. 2). As was done previously [20], rabbits were lightly anesthe-

tized with ether and a catheter was threaded to about the carina through a needle placed percuta- neously into the trachea. 1 ml of the injection solution was administered and the needle and catheter were withdrawn. These rabbits experienced minimal respiratory distress from the proce-

dure and recovered from anesthesia within minutes. Rabbits given [3H]DPPG were killed in groups from 1 to 20 h following injection. Those given [14C]lysoPC were killed from 0.5 to 20 h following injection.

Twelve 3-day-old rabbits weighing 74.6 k 2.5 g were removed from their litters on the day of injection. These animals were injected as before with a solution containing [14C]lysoPC (Experi- ment No. 3) [9]. Briefly, a small midline incision was made in the neck, under local anesthesia and the trachea was visualized. The solution was injected via a 30 gauge needle into the distal trachea while occluding the trachea proximal to the injec-

tion site manually. Rabbits experienced only brief respiratory distress from the procedure. They were

killed from 30 min to 6 h later. Fraction isolation. Following death, all rabbits

were subjected to a thorough alveolar wash [2,9]. The washed lungs were homogenized in 0.32 M sucrose containing 0.01 M Tris-HCl/O.lS M

NaCl/O.OOl M CaCl JO.0001 M MgSO,/ 0.0001 M EDTA (pH 7.4) [23]. The homogenate was used to isolate lamellar bodies by a series of differential and sucrose density gradient centrifugation steps as previously described [9,23]. The lipids in each

fraction were immediately extracted according to Folch et al. [24] and stored at - 20°C until

analyzed. Lipid analysis. Phospholipids were separated in

duplicate by one-dimensional thin-layer chro-

matography on silica gel plates (Baker) using chlo-

roform/ methanol/ cont. Ammonium hydroxide (65 : 35 : 5, v/v) as the solvent. This resolves lysophosphatidylcholine from phosphatidylcholine from phosphatidylglycerol-phosphatidylethanolamine; these latter two phospholipids comigrate in this system. Depending on the injection solution one set of spots corresponding to phosphatidylcholine and either lysophosphatidylcholine or phosphatidylglycerol-phosphatidylethanolamine were scraped into separate scintillation vials and counted for both 3H and 14C. Duplicate spots for

each sample were analyzed for phosphorus according to Bartlett [25]. These values were used to determine the ratio of the radioactivity in the phospholipid of interest to that in DPPC, total counts/mm recovered, or specific activity in the phospholipid of interest as needed for analysis.

Several alveolar washes from rabbits given [3H]DPPG were extracted in duplicate. The duplicate samples were platted in the system described by Gilfillan et al. [26] which separates phosphatidylglycerol from phosphatidylethanolamine. Lipids separated in this system were analyzed for radioactivity in a liquid scintillation counter to determine if radioactivity accumulated in any lipid other than those administered. No radioactivity was found in any lipid other than phosphatidylcholine or phosphatidylglycerol.

Analysis. The method of analysis was based on previously published information indicating that when radiolabelled DPPC is mixed with surfac-

230

tant and injected intratracheally into adult rabbits, the DPPC behaves like the phosphatidylcholine of surfactant [20]. It was also based on the fact that the decrease in total DPPC radioactivity in the alveolar wash over time is best described by a linear sum of exponent& [20]. The exponential behavior of the components was taken into account in calculating the percent reutilization of phosphatidylglycerol. All values are given as mean f S.E. Statistical comparisons were by Student’s t- test. Error bars not shown on figures fall within the point symbol.

Results

Based on previously published data obtained after giving adult rabbits labelled surfactant by intratracheal injection, we calculated expected percent recovery of radiolabelled DPPC given to rabbits in these experiments. Expected and actual recoveries were compared by determining the slopes of semilog plots of expected and recovered radioactivity [19]. Best-fit lines and slopes were determined by least-squares regression. Slopes of lines for actual recovery were found to be not significantly different than that for expected recovery (data not shown). Thus the labelled DPPC used in these experiments behaved as if it was the phosphatidylcholine of endogenous surfactant.

The ratio of [ 3H]DPPG to [14C]DPPC recovered in the alveolar lavage of each animal injected with this mixture was determined and mean values calculated for rabbits killed at each time. Mean values were plotted against time and the best-fit line was determined by linear-least squares regression (Fig. 1). The slope of this line, -0.06 + 0.02 was statistically different from 0 and was negative indicating that the net clearance of phosphatidylglycerol from the alveolar space was more rapid than the net clearance of phosphatidylcholine. This implies that phosphatidylglycerol was reutilized less efficiently than phosphatidylcholine (see discussion).

The alveolar wash and lamellar body fractions of the lungs were assayed for production of [ 3 Hlphosphatidylcholine from the injected [3H]DPPG which was labelled in palmitate. The specific activity of [ 3H]phosphatidylcholine in the alveolar wash initially increased with time and

O.OOj TIME (hours)

Fig. 1. Ratio of [ ‘H]DPPG to [r4C]DPPC in the alveolar wash. Rabbits were injected intratracheally with a mixture of

[‘H]DPPG and [r4C]DPPC and the ratio of these phospholi-

pids in the alveolar wash was determined over time. Each point

represents the mean f S.E. for all rabbits killed at the indicated

time (n = three or four per group). The line was determined by

linear least-squares regression and has a slope significantly less

than 0 (P < 0.01). This indicates more rapid clearance of

[ 3 H]DPPG compared to [ “C]DPPC.

later decreased while that in lamellar bodies showed a steady decrease (data not shown). This indicated breakdown of the injected DPPC with reutilization of at least some component parts.

As occurred with developing rabbits injected intratracheally with lysophosphatidylcholine, adult rabbits reutilized at least some lysophosphatidylcholine as phosphatidylcholine. This is demonstrated by the ratio of 3H to 14C radioactivity as phosphatidylcholine in alveolar lavage which decreased with time (Fig. 2). We measured the

TIME (hours)

Fig. 2. Ratio of [‘H]PC to [14C]PC in the alveolar wash. After injecting adult rabbits intratracheally with a mixture of

[ 3H]DPPC and [ I4 C]lysoPC, the ratio of counts/mm of [ 3 H]PC

to [r4C]PC was determined in the alveolar wash over time.

Each point represents the meanfS.E. for the 3-7 rabbits

killed at each time. The decrease in the ratio indicates synthesis

of [14C]PC from [r4C]lysoPC which then enters the alveoli.

231

a’4 O.Ooj TIME (hours)

Fig. 3. Ratio of alveolar wash to lamellar body [14C]PC

specific activity in adult rabbits. The ratio of [‘4C]PC specific

activity in alveolar wash to lamellar bodies of rabbits referred

to in Fig. 2 was determined. Values are plotted as mean f S.E.

for animals killed at the indicated times. The ratio is initially

2.0 then drops to less than 1.0 before returning to a value

greater than 1.0 by 6 h. This indicates appearance of [“C]PC in alveoli before its appearance in lamellar bodies.

specific activity of [ l4 Clphosphatidylcholine in the alveolar wash and lamellar bodies. The ratio of the specific activity of [‘4C]phosphatidylcholine in the alveolar wash to that in lamellar bodies was initially greater than 1.0. This ratio then decreased to less than 1.0 and returned to greater than 1.0 after 6 h (Fig. 3). The fact that the ratio was initially greater than 1.0 suggests some early conversion of [ l4 C]lysoPC to alveolar [ l4 Clphosphatidylcholine

0.5

i iA+

0.2.. /

I

b 0.1 .. I

0.04 0 2 4 6

TIME (hours)

Fig. 4. Ratio of alveolar wash to lamellar body [14C]PC

specific activity in 3-day old rabbits. 3-day old rabbits were

injected intratracheally with a mixture containing [14C]lysoPC. The ratio of [14C]PC specific activity in alveolar wash to

lamellar bodies was determined. Values are plotted as mean f

S.E. for rabbits killed at the indicated times (n = three for each

group). This ratio was initially much less than 1.0 and in-

creased with time indicating [14C]PC appearing in lamellar

bodies prior to its appearance in alveoli.

which did not involve lamellar bodies. When alveolar lavages at 30 min and 1 h were analyzed for the fraction of total [ “C]phosphatidylcholine that was saturated the values were found to be 0.42 f 0.07 and 0.57 f 0.02, respectively.

Incubation of injection solution containing [ l4 C]lysoPC with unlabelled alveolar lavage from adult rabbits produced no detectable conversion of [ l4 C]lysoPC to phosphatidylcholine. Thus this early conversion was not an experimental artifact.

We also measured the ratio of alveolar lavage to lamellar body [ l4 Clphosphatidylcholine radioactivity over the first 6 h after injection in 3-day- old rabbits. In contrast to adult rabbits, this ratio in developing rabbits started out at less than 1.0 and increased with time (Fig. 4). (inadequate amounts of [ “C]phosphatidylcholine were found at 30 min to accurately determine a ratio.)

Discussion

The phosphatidylglycerol given to these animals is actually a racemic mixture of L-a-phosphatidyl- D,L-glycerol. We have based our analysis on the assumption that the injected phosphatidylglycerol and lysophosphatidylcholine behaved as their natural counterparts in surfactant. As with previous studies in developing and adult rabbits, the phosphatidylcholine mixed with surfactant and given intratracheally seemed to behave like that of endogenous surfactant [9,18-201. Furthermore, since evidence exists indicating that surfactant is removed from the alveoli by bulk uptake [13,17,18,27,28], we feel that the above assumption is reasonable.

It has been demonstrated previously that surfactant phosphatidylglycerol (as well as other surfactant phospholipids) has the same turnover time as phosphatidylcholine [19]. The reutilization of surfactant phosphatidylcholine has been estimated to be 22% in adult rabbits equivalent to those used in this study [20]. Based on the previously measured turnover time of surfactant phospholipids and the percent reutilization of phosphatidylcholine, it is possible to predict the ratio of alveolar phosphatidylglycerol to phosphatidylcholine for any percent reutilization of phosphatidylglycerol. From the slope of the line in Fig.

232

1 and the standard error of the slope, the reutilization of intact surfactant phosphatidyl~ycerol would be between OS and 7%.

The actual calculation of reutilization is based on the assumption that lamellar bodies and the alveolar space constitute a precursor-product relationship and that reutilization occurs as intact molecules. The first assumption is reasonable based on previous studies demonstrating a close precursor-product relationship between these pools. The second assumption is not strictly valid since some [ 3 H]phosphatidyl~holine was found following injection of [ 3H]DPPG. This is in agreement with recent studies indicating at least some breakdown and resynthesis of surfactant in adult rats [21,29]. However, the quantitative contribution of this pathway is unclear. If this is occur- ring to any significant degree in these rabbits, then we have over estimated the reutilization of intact phosphatidylglycerol. This would not alter the conclusion that phosphatidylglycerol was cleared from the alveoli faster than phosphatidylcholine and hence less efficiently reutilized. The finding that adult rabbits have a lower percent reutilization of phosphatidyl~ycerol compared to phosphatidylcholine is consistent with observations in developing animals [ 191.

Reutilization of lysophosphatidylcholine injected into the alveoli appears in part to be different at early times in adults compared to developing rabbits (Figs. 3 and 4). Developing animals convert lysophosphatidylcholine to phosphatidylcholine which then appears in lamellar bodies prior to its appearance in the alveolar space (i.e. the ratio of ~14C]phosphatidylcho~ne in alveolar wash to lamellar bodies is initially less than one and increases). Adult rabbits manifest an alternate pathway for this conversion which is evident only at early times after injection.

This is demonstrated by the following argu- ment. The net flux of surfactant phosphatidylcholine is from lamellar bodies to alveolar space [2,3,16]. Assume that in these adult rabbits, all conversion of lysophosphatidylcholine to phosphatidylcholine occurred either in the alveolar space or in a #mpartment which emptied into the alveolar space without first entering lamellar bodies. Labelled phosphatidylcholine enters lamellar bodies via reutilization while unlabelled phos-

phatidylcho~ne enters lamellar bodies via new synthesis. Hence the specific activity of phosphatidylcholine entering lamellar bodies at any instant in time would always be less than the simultaneous specific activity in the alveolar space. Therefore the specific activity of phosphatidylcholine in lamellar bodies would never reach that in the alveolar space. The contribution of this early pathway to the conversion cannot be accurately quantitated but does not appear to be the major route since lamellar body specific activity exceeds alveolar wash specific activity by 2 h.

The initial decrease in alveolar wash specific activity of [14C]phosphatidylcholine in adult rabbits suggests a rapid removal of substrate (lysophosphatidylcholine) for the early conversion which must then take place elsewhere. The total radioactivity in the alveolar lavage as lysophosphatidylcholine did drop rapidly (data not shown).

In developing rabbits, the fraction of intratracheally injected lysophosphatidylcholine labelled in palmitate that can be found as phosphatidylcholine exceeds that’ for intratracheally injected palmitate by about IO-fold 1301. This suggests differences in the pathways involved in recy- cling free alveolar pahnitate versus the palmitate of lysophosphatidylcholine. Such a comparison in adult rabbits is not possible since values for incor- poration of alveolar free palmitate into surfactant in adult rabbits are not available.

We have demonstrated that as in developing rabbits, adult rabbits reutilize phosphatidylglycerol less efficiently than phosphatidylcholine. Adult rabbits reutilize lysophosphatidylcholine as phosphatidylcholine but at least in part by an acylation process not detected in developing rabbits. Our data are also in agreement with others indicating that at least some of the component parts of phospholipids are reutilized by breakdown and resynthesis [21,29]. Breakdown and reutilization of component parts of surfactant is also developmentally regulated as developing rabbits show less evidence of this process than adult rabbits.

This work was supported in part by NIH grant No. R23 HL33348 from the National Heart, Lung,

233

and Blood Institute awarded to Harris Jacobs and by NIH grant No. HD07094.

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Documents

Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits