Route of incorporation of alveolar palmitate and choline into surfactant phosphatidylcholine in rabbits

Biochimica et Biophysics Acta. 752 (1983) 178- 18 1

178

Elsevier

BBA Report BBA 50042

ROUTE OF INCORPORATION OF ALVEOLAR PALMITATE SURFACTANT PHOSPHATIDYLCHOLINE IN RABBITS HARRIS

JACOBS,

ALAN

JOBE, MACHIKO

Fetal- Maternal Research Laboratories, (Received

December

IKEGAMI,

Department

SALLY

JONES

AND CHOLINE

and DEBORAH

of Pediatrics, Harbor -UCLA

INTO

MILLER

Medical Center, Torrance, CA 90509 (U.S.A.)

15th, 1982)

Key worak Lung surjactani;

Phosphatidylcholine;

Precursor incorporation;

(Rabbit)

Intratracheal injection of 3-day-old rabbits with radioactively labeled palmitic acid and choline results in an S-lo-fold increase in the efficiency of their incorporation into surfactant phosphatidylcholine when compared to the intravenous injection of these precursors. Based on labeling patterns in microsomal, lamellar body and alveolar wash fractions, the incorporation appears to be via normal surfactant synthetic pathways. Intratracheal injection of phospholipid precursors is useful for producing relatively high specific activity natural surfactant.

Surfactant is a complex mixture of lipids and protein which is synthesized by pulmonary type 11 pneumocytes and packaged into lamellar bodies for secretion to the alveolar surface [ 1,2]. Most of the phospholipid of surfactant is phosphatidylcholine (PC) [3]. One approach to the study of surfactant metabolism has been to inject animals intravenously with radioactively labeled precursors of PC. The radioactively labeled PC can then be followed through lung subcellular fractions to the alveolar space by autoradiography [4,5] or by isolating subcellular fractions and measuring PC specific activities [1,2,6-g]. Another approach has been to administer a solution of radioactively labeled natural or synthetic surfactant directly into the airways and again use autoradiography, specific activities or total count recovery to study metabolism [9-131. The present experiment was designed to compare the pattern of incorporation of radioactively labeled palmitate and choline into surfactant PC by intratracheal injection versus that after administration by intravenous injection.

Abbreviation:

PC, phosphatidylcholine.

0005-2760/83/$03.00

0 1983 Elsevier Science Publishers

B.V.

Three injection solutions were prepared with [ “C]choline (50 Ci/mol) and 9,10-[3H]palmitic acid 918 Ci/mmol) that was complexed to albumin [ 1,6]. Solutions 1 and 2 contained 8.23 PCi of [‘4C]choline/ml and 137 PCi of [3H]palmitic acid/ml in a 1 : 1 mixture of Lactated Ringers Injection (Travanol Lab., Inc.) and distilled water. Unlabeled natural rabbit surfactant was isolated as previously described [ 131 and was added to solution 1 to a final concentration of 0.75 pm01 of surfactant PC/ml of solution. No surfactant was added to solution 2. Solution 3 was made with 0.9% saline and contained 7.5 PCi of [14C]-. choline/ml and 125 PCi of [ 3H]palmitic acid/ml. 44 3-day-old New Zealand White rabbits were taken from their litters on the day of injection. 12 rabbits were injected intratracheally with solution 1 and 12 other rabbits were injected intratracheally with solution 2. The trachea of each rabbit was isolated and 0.35 ml/ 100 g body weight was injected with a 30 gauge needle [ 131. This provided 2.88 pCi of [‘4C]choline/100 g body weight and 48 PCi of [ 3H]palmitic acid/ 100 g body weight. Three rabbits from each group were killed at 1, 5, 15 and 25 h after injection by an intraperitoneal

injection of pentobarbital followed by exanguination. 20 rabbits were injected via an external jugular vein with 0.4 ml/ 100 g body weight of solution 3, providing 3.0 PC1 of [‘4C]choline/ 100 g body weight and 50 pCi of [3H]palmitate/100 g body weight. These doses were approximately the same as those received by rabbits given solution 1 or 2. These rabbits were killed in groups of four at 1, 5, 13, 17 and 25 h after injection. From each of the 44 rabbits we isolated the alveolar wash, a microsomal and a lamellar body fraction. Immediately after killing, the chest of each animal was opened, the trachea cannulated and the lungs were thoroughly washed with saline to yield an alveolar wash fraction [ 1,131. The lungs of each rabbit then were homogenized in 8 ml of 0.32 m sucrose, 0.01 M Tris-HCl, 0.15 M NaCl, 0.001 M CaCl,, 0.001 M MgSO, and 0.0001 M EDTA at pH 7.4 [ 1,131, and 0.5 ml of the homogenate was saved. From the rest of the homogenate we isolated lamellar bodies and a microsomal fraction by differential and sucrose density gradient centrifugation [ 1,6,7,13]. The lamellar body fraction was that fraction recovered between 0.45 and 0.55 M sucrose buffers while the microsomal fraction was recovered as the pellet which sedimented through 1 M sucrose buffer at 100000 x g. For each rabbit, lipids in each fraction were extracted [14] and PC was isolated in duplicate from each lipid extract by one-dimensional thinlayer chromatography [1,6,13]. One spot was assayed for phosphate according to the method of Bartlett [ 151 and its duplicate for radioactivity in Aquasolscintillation fluid (New England Nuclear Corporation). All 3H and I4 C counts/ min were corrected for cross-channel contamination, and the specific activity of PC was determined on each fraction and expressed as counts/min per pm01 PC. The specific activity of PC in the lung homogenate and microsomal fractions were maximal at 1 h, which was the first time of killing. The specific activity of PC in the lamellar body fraction and in the alveolar wash increased with time after injection. The time course of this change in PC specific activity from the incorporation of [3H]palmitic acid following the intravenous and the intratracheal injections is shown in Fig. 1. The peak

0 0

10

20

0

10

20

HOURS

Fig. 1. Specific activity of phosphatidylcholine versus time after injection of [ 3H]palmitic acid in the lung homogenate, microsomes (Micro), lamellar bodies and alveolar wash. Rabbits were injected intravenously (A) or intratracheally (A) with [ ‘Hlpalmitic acid. They were killed in groups at the indicated times and phosphatidylcholine specific activity was measured in each fraction. Each symbol is the mean f SE. for the rabbits killed at that time. If the S.E. falls within the symbol only the mean value is shown.

activities of PC in each fraction resulting from the intratracheally injected precursor were about 8 times the peak specific activities in the same fractions labeled by the same precursor injected intravenously. The specific activities of PC versus time after injection of [‘4C]choline for the rabbits injected intravenously and intratracheally with the same amount of [ “C]choline/g body weight are again shown together for comparison (Fig. 2). The peak specific activities reached in all fractions following an intratracheal injection of [ “C]choline exceeded that reached following an intravenous injection by about IO-fold. The specific activity of the palmitateand choline-labeled PC increased first in the micro-

180

Ojd 0

I& IO

20

0

s 10

20

HOURS

Fig. 2. Specific activity of phosphatidylcholine versus time after injection of [ “C]choline in the lung homogenates, microsomes (Micro), lamellar bodies. and the alveolar wash. A, Intravenous injection; A, intratracheal injection.

somes, then the lamellar bodies, and finally in the alveolar wash. For each route of injection, when the specific activities in each fraction are plotted as the % maximum specific activity reached in that fraction, the shapes of these curves were similar for the intravenous and intratracheal injections (data not shown). The [3H]palmitic acid and [‘4C]choline, when injected intrathacheally without natural surfactant, gave similar results (data not shown). That is, microsomal specific activity increased before that in the lamellar body fraction, which increased before that in the alveolar wash for both isotopes. The shapes of the curves of specific activity versus time for the microsomes, the lamellar bodies and the alveolar wash were similar to those shown in Fig. 1. Also the peak specific activities of PC in the alveolar wash, in the lamellar body fraction and in the microsomes were about IO-fold greater than those achieved in the corresponding fractions after

injection intravenously with the same amount of labeled palmitic acid or choline. The route of incorporation of the intratracheally injected palmitate and choline into PC appears to be via microsomes. When injected intravenously, the radioactively labeled palmitate and choline are incorporated into the PC in microsomes and transport into lamellar bodies before appearing in the alveolar wash [ 1,2,4-61. If an acyl exchange at the alveolar surface occurred (or if choline exchange occurred) as the major route of incorporation after intratracheal precursor injection, then the specific activity of PC from the alveolar wash would tend to increase before that of the lamellar body fraction. In fact, the increase in PC specific activity of lamellar bodies would then be via reutilization of intact PC molecules [13]. Furthermore, very little, if any, radioactivity would be found in microsomes under these circumstances. Similarly, if acyl or head-group exchange occurred within lamellar bodies as the major route of incorporation, then again we would expect to find very little radioactivity in microsomes. Our results indicate that this type of exchange cannot be more than a minor route of incorporation for either precursor. One reservation is that our microsomal fractions were recovered from the entire lung homogenate and the percent of that fraction derived from type II cells is unknown. When radioactively labeled palmitic acid and choline are injected intravenously into rabbits the isotopes are rapidly cleared from serum [ 161. However, the fractions of these isotopes which are actually incorporated into surfactant phospholipids are very small [ 1,6]. In 3-day-old rabbits, only about 0.4% of the [ “C]choline can be recovered as radioactively labeled PC in the alveolar wash at peak alveolar wash specific activity [l]. For the [‘Hlpalmitic acid label, this value is only about 0.02% [ 11. When injected intratracheally, these percents increase to about 4.0% for [ “C]choline and 0.2% for [jH]palmitate. Since there is no evidence that acyl or head-group exchange takes place, the IO-fold higher specific activities reached in the different fractions after an intratracheal injection of precursors must be due to the type II cell taking up an increased fraction of the radioactively labeled precursors. While type II cells are known

181

to take up surfactant phospholipids from the alveolar surface [9,11- 131, phagocytosis is not generally considered to be a major property of these cells. The surface of the cell adjacent to the basement membrane is nearest to the capillary bed and is the surface which has been assumed to transport precursors used for surfactant synthesis. The type II cells in this experiment were exposed to the precursors on their alveolar surface. Nevertheless, they were capable of internalizing both precursors and utilizing them quite effectively for surfactant synthesis. This occurred independent of whether the precursors were mixed with surfactant prior to injection. This lo-fold increase in alveolar wash PC specific activity after an intratracheal injection versus an intravenous injection is useful for production of a naturally labeled surfactant with a high specific activity. A radioactively labeled surfactant which has a high specific activity allows for the use of only a small quantity in intratracheal labeling experiments. The advantage of this is that one can be certain of minimal perturbation of endogenous pools while still administering sufficient radioactivity for the required measurements.

Acknowledgements This work was supported by NIH Grant 11932 from Child Health and Development,

HDDe-

partment of Health and Human Services, by Research Career Development Award HD-HL-00252 to A.J. and NIH Research Service Award HL06544 to H.J. References 1 Jacobs, H.C., Jobe, A.H., Ikegami, M. and Jones, S. (1981) J. Biol. Chem. 257, 1805-1810 2 Young, S.L., Kremers, S.A., Apple, J. Crapo, J.D. and Brumley, G.W. (1981) J. Appl. Physiol. 51, 248-253 3 King, R.J. (1974) Fed. Proc. 33, 2238 4 Chevalier, G. and Collet, A.J. (1972) Anat. Rec. 174, 289-310 5 Askin, F.B. and Kuhn, C. (1971) Lab. Invest. 25, 260-268 6 Jobe, A.H. (1977) Biochim. Biophys. Acta 489, 440-453 7 Jobe, A.H., Kirkpatrick, Chem. 253, 3810-3816

E. and Gluck,

L. (1978) J. Biol.

8 Baritussio, A.G., Magoon, M.W., Goerke, J. and Clements, J.A. (1981) B&him. Biophys. Acta 666, 382-393 9 Geiger, K., Gallagher, M.L. and Hedley-Whyte, J. (1975) J. Appl. Physiol. 39 759-766 10 Scarpelh, E.M., Condorelli, S., Colacicco, G. and Cosmi, E.V. (1975) Pediat. Res. 9, 195-201 11 Hallman, M., Epstein, B.L. and Gluck, L. (1981) J. Clin. Invest. 68, 742-75 I 12 Glatz, T.H., Ikegami, M. and Jobe, A.H. (1982) Pediat. Res. 16, 711-715 13 Jacobs, H.C., Jobe, A.H., Ikegami, M. and Conaway, D.J. and Jones, S.J. (1983) J. Biol. Chem. 258, 4159-4165 14 Bligh, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37,911-917 1s Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468 16 Jobe, A.H. (1979) Biochim. Biophys. Acta 574, 268-279