- Email: [email protected]
The rate-limiting step in the biosynthesis of phosphatidylcholine by alveolar type ii cells from adult rat lung
Biochrmica et Blophysica Acta. 7 12 (I 982) 390- 394
390
Elsevier Biomedical
Pre.\s
BBA 51180
THE RATE-LIMITING STEP IN THE BIOSYNTHESIS OF PHOSPHATIDYLCHOLINE ALVEOLAR TYPE II CELLS FROM ADULT RAT LUNG MARTIN
POST, JOSEPH
J. BATENBURG,
Laboratory of Veterinary Biochemistry, (Received
February
ELISABETH
A.J.M.
SCHUURMANS
State University of Utrecht, Biltstraat
and LAMBERT
BY
M.G. VAN GOLDE
172, 3572 BP Utrecht (The Netherlandsi
16th, 1982)
Key words: Phosphatidylcholine lung type II cell)
synthesis;
Phospholipid
regulation;
Cholrnephosphate
cytidylyltransferase;
Pulmonary
surfactant;
(Rat
1. The rate-limiting reaction in the biosynthesis of surfactant phosphatidylcholine by type II cells isolated from adult rat lung was examined. 2. Studies on the uptake of [Me - 14C]choline and its incorporation into its metabolites over a 5 h period indicated that in these cells the cholinephosphate pool was much larger than both the choline and CDPcholine pool. This is consistent with the idea that the rate-limiting reaction is that catalyzed by cholinephosphate cytidylyltransferase. 3. Evidence that cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine was also obtained from pulse-chase experiments. [Me -‘4C]Choline taken up by the cells was rapidly converted into cholinephosphate during the pulse period. As the radioactivity disappeared from cholinephosphate during the chase period, the label was incorporated immediately into phosphatidylcholine, without much change in the labelling of CDPcholine. This indicates that the cholinephosphotransferase is at least as fast as the cholinephosphate cytidylyltransferase. 4. Inclusion of palmitate in the chase medium accelerated the conversion of labelled cholinephosphate into phosphatidylcholine and decreased the radioactivity associated with CDPcholine. This indicates that under these conditions the cholinephosphate cytidylyltransferase reaction cannot keep up with increased utilization of the CDPcholine in the terminal step of the CDPcholine pathway.
dylcholine in a variety of mammalian tissues [5-91. It has been suggested that cholinephosphate cytidylyltransferase also catalyses the rate-limiting reaction in the biosynthesis of pulmonary phosphatidylcholine (lo- 131. The best evidence so far for this suggestion was obtained in the studies on the pool sizes of choline and its derivatives in the developing rat and rabbit lung [ 14- 161. The cholinephosphate pool was much larger than the CDPcholine pool, suggesting that the formation of the nucleotide may be limited. These studies, however, were carried out with preparations of whole lung. Because of the cellular heterogeneity of the lung, these results are not necessarily pertinent to type II cells. The present study was undertaken to investigate
Introduction Phosphatidylcholine is the major lipid component of pulmonary surfactant which lowers the surface tension in the alveoli and prevents their collapse during expiration [I]. The alveolar type II cells of the lung are the most important, if not sole, producers of surfactant lipids [2]. The de novo synthesis of surfactant phosphatidylcholine proceeds in the type II cell mainly by the CDPcholine pathway [3,4]. How the biosynthesis of phosphatidylcholine in the type II cell is controlled has not yet been elucidated. There is experimental evidence which suggests that cholinephosphate cytidylyltransferase catalyses the rate-limiting step in the production of phosphatiOOOS-2760/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
391
which of the enzymes is rate-limiting in type rat lung. To the best first of its kind carried
of the CDPcholine pathway II cells isolated from adult of our knowledge, it is the out with this cell type.
Materials and Methods
Materials. Unlabelled biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. [Me-i4C]Choline (spec. act., 58 Ci/mol) was bought from the Radiochemical Centre Amersham, U.K. Male Wistar rats, weighing 180-200 g, were obtained from the Central Institute for Breeding of Laboratory Animals in Zeist, The Netherlands. The rats had free access to laboratory chow and water. Isolation of type II cells. Type II cells were isolated from the rat lungs by trypsinization, density gradient centrifugation and differential adherence in primary monolayer culture [ 17-191. After 20 h in culture, the type II cells were used for the biochemical experiments. Purity and viability of the cells were in the same order as reported previously [20]. Incubation of type II cells. The cells, attached to the 35mm culture dishes were rinsed with a standard medium containing 5.6 mM glucose, 125 mM NaCl, 5 mM KCl, 2.5 mM Na,HPO,, 2.5 mM CaCl,, 1.2 mM MgSO,, 17 mM N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid and 50 pg/ml gentamycin at a pH of 7.4. Subsequently, the cells were either used for experiments in which the uptake and utilization of [Me-‘4C]choline was studied over a 5 h period or for pulse-chase experiments with [Me-‘4C]choline. In the first type of experiments the cells were incubated in standard incubation medium (1.5 ml/dish) supplemented with 0.1 mM [Me-‘4C]choline (spec. act., 6.59. lo4 dpm/nmol). During the incubation the dishes were each held in a 50 ml beaker covered with parafilm which was placed in a reciprocating waterbath (25 cycles/mm) kept at 37°C. After the desired incubation times the medium was removed and the cells were washed with ice-cold (choline-free) standard medium to remove excess labelled choline. Immediately afterwards, 1.5 ml of ice-cold methanol/H,0 (5 : 4, v/v) was added to the dishes. In the pulse-chase experiments the washed cells
were first incubated as described above for 1 h in standard medium supplemented with 0.025 mM choline. After this preincubation period the medium was removed and replaced with standard medium supplemented with 10 PCi [Me“C]choline/dish. At the end of a 15 min pulse period, the radioactive medium was removed and the dishes were rinsed with (choline-free) standard medium to remove excess labelled choline, after which chase medium, with or without 0.2 mM palmitate (complexed to bovine serum albumin in a molar ratio 5.3 : l), was added to the cells. This chase medium consisted of standard medium supplemented with 0.025 mM choline. At various times after the onset of the chase period, the incubation was terminated by removal of the medium, washing of the cells with ice-cold standard medium and addition of 1.5 ml methanol/H,0 (5 : 4, v/v). Analysis of choline and its derivatives. In both types of experiment, the cells were scraped from the dishes with a rubber policeman after the addition of methanol/H,0 and transferred with the methanol/H,0 into centrifuge tubes for extraction. To these tubes, lung lipid (0.2 pmol phosphorus), choline (2 pmol), cholinephosphate (8 pmol) and CDPcholine (2 pmol) were added as carriers. After addition of chloroform, lipids were extracted by the method of Bligh and Dyer [21]. Phosphatidylcholine was isolated from the lipid extract by thin-layer chromatography on silica gel G plates with chloroform/methanol/H ,O (65: 35 :4, v/v) as eluent. After detection by exposure to iodine, the phosphatidylcholine fraction was scraped into scintillation vials and assayed for radioactivity. The aqueous phase remaining after lipid extraction was evaporated to dryness in a vacuum rotary evaporator. The residue was dissolved in water. Choline, cholinephosphate and CDPcholine were separated by thin-layer chromatography on silica gel H plates with 0.15 M NaCl/methanol/conc. NH, (50 : 50 : 5, v/v) as developing solvent. Choline and its metabolites were visualized with iodine vapour, scraped into scintillation vials and assayed for radioactivity. Radioactivity was measured in the liquid scintillation mixture described by Pande [22]. Counting efficiency was determined by the channels ratio method.
392
Results and Discussion Fig. 1 shows the uptake of [Me- “C]choline by type II cells and its incorporation into cholinephosphate, CDPcholine and phosphatidylcholine. It can be seen that the cells rapidly phosphorylated choline to cholinephosphate. At any time more than 90% of the water-soluble radioactivity extracted from the cells was associated with cholinephosphate. The radioactivity in intracellular choline was maximal and constant after 2 h of incubation, whereas the incorporation of label into cholinephosphate, CDPcholine and phosphatidylcholine increased up to at least 5 h of incubation. Assuming no change in pool size, the
012365
Fig. 1. The uptake of [Me-i4C[choline into type II cells and its incorporation into cholinephosphate, CDPcholine and phosphatidylcholine. Isolated rat lung type 11 cells were incubated for the indicated periods with 0.1 mM [ Me-t4C]choline in the standard medium. The radioactivity in choline, cholinephosphate, CDPcholine and phosphatidylcholine was determined as described in Materials and Methods. The data are representative of two experiments, each carried out in duplicate.
much higher label incorporation into cholinephosphate as compared to that into intracellular choline indicates that the cholinephosphate pool is much larger than the intracellular choline pool. The observation that the incorporation of the label into phosphatidylcholine is only slightly lower than the incorporation into cholinephosphate while the incorporation into CDPcholine is much lower, indicates that the CDPcholine pool is much smaller than the cholinephosphate pool. A cholinephosphate pool that is much larger than both the intracellular choline and CDPcholine pool is consistent with the idea that the cholinephosphate cytidylyltransferase is rate-limiting in the formation of phosphatidylcholine. Further support for this idea comes from pulse-chase studies on the metabolism of choline, shown in Fig. 2. It can be seen that at the end of a 15 min pulse period most of the radioactivity was associated with the cholinephosphate pool. The high ratio between the labelling of cholinephosphate and that of intracellular choline again argues against a rate-limiting role of choline kinase. The observation that the radioactivity of free choline did not decrease during the chase period is in agreement with earlier studies with perfused hamster heart [8]. The radioactivity in the cholineposphate decreased rapidly during the chase period. The radioactivity lost from cholinephosphate appeared in phosphatidylcholine. A similar transfer of radioactivity from cholinephosphate to phosphatidylcholine was observed in studies with isolated hepatocytes [23]. The radioactivity in CDPcholine remained almost constant in the type II cells during the chase period and comprised less than 3.5% of the total radioactivity extractable from the cells (Fig. 2). This indicates that the cholinephosphotransferase reaction proceeds relatively fast and that the rate of phosphatidylcholine synthesis is limited by the activity of cholinephosphate cytidylyltransferase. Inclusion of palmitate in the chase medium caused a 2-fold increase in the rate of disappearance of the label from cholinephosphate and in the rate of its appearance in phosphatidylcholine (Fig. 2). In the presence of palmitate the radioactivity of the CDPcholine pool was decreased during the chase period, while the labelling of the
393
0.3!ii>o ’
0.20.
l ,/’ ,’
0.2 -
“qyo
0.’ 5\
9 2 %
z $j
0.1 .
,’ \
\
: \
\
0.1 0 \
\
? 1 /w-----0 :Q
\ t
‘0 c
005-
:/
s f
0
go
120
0
60
120
H
0.12-
0.0
h
G==-==~
II
O.Oh
0
12
60 Chase
time
fmin
)
Fig. 2. Pulse-chase study on the metabolism of choline in alveolar type II cells. Isolated rat lung type II cells were preincubated for 1 h in the standard incubation medium supplemented with 0.025 mM choline. Subsequently, the type II cells were pulsed with [ Me-‘4Cjcholine for 15 min and chased in the presence or absence of palmitate in standard medium containing 0.025 mM choline. Radioactivity in choline (V, V), cholinephosphate (0, l), CDPcholine (a, A) and phosphatidylcholine (0, n ) was determined. Open symbols, solid line, without palmitate; closed symbols, dashed line,with palmitate. The start of the chase is indicated as time zero. The data are representative of two experiments, each carried out in dupli-
cytidylyltransferase reaction cannot keep up with the increased utilization of the CDPcholine by the cholinephosphotransferase. In conclusion, the results of the present studies indicate that although the rate of phosphatidylcholine formation by the type II cells can be modulated by the supply of diacylglycerols cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine. These results may indicate that cholinephosphate cytidylyltransferase becomes regulatory for phosphatidylcholine synthesis when the cells are under ample supply of plasma free fatty acids in viva. In order to obtain more evidence for the ratelimiting role of cholinephosphate cytidylyltransferase future studies should be directed towards measurement of the pool sizes of the intermediates and cofactors involved in the CDPcholine pathway in type II cells. Moreover, the activities and kinetic properties of the enzymes of the type II cell involved in this pathway should be determined. Acknowledgements
The investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and by the Dutch Asthma Foundation (Nederlands Astma Fonds).
cate.
References
intracellular choline was unaffected (Fig. 2). A stimulatory effect of palmitate on the incorporation of choline into phosphatidylcholine by isolated type XI cells had been observed previously [20]. The most likely explanation for this effect in the previous [20] and present studies is that by addition of palmitate the supply of diacylglycerols for the cholinephosphotransferase reaction was increased, resulting in a faster conversion of CDPcholine into phosphatidylcholine. The observation that palmitate decreased the amount of radioactivity in CDPcholine indicates that the supply of CDPcholine by the choiinephosphate
King, R.J.and Clements, J.A. (1972) Am. J. Physiol. 223, 715-726 Goerke, J. (1974) B&him. Biophys. Acta 344, 241-261 Kikkawa, Y., Yoneda, K., Smith, F., Packard, B. and Suzuki, K. (1975) Lab. Invest. 32, 295-302 Batenburg, J.J. and Van Golde, L.M.G. (1979) in Reviews in Perinatal Medicine (Scarpelli, E.M. and Cosmi, E.V., eds.), Vol. 3, pp. 73- 114, Raven Press, New York Vance, D.E. and Choy, P.C. (1979) Trends B&hem. Sci. 4, 145-148 Vance, D.E., Trip, E.M. and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069 Sleight, R. and Kent, C. (1980) J. Biol. Chem. 255, 1064410650 ZRlinski, T.A., Savard, J.D., Man, R.Y.K. and Choy, P.C. (1980) 3. Bid. Chem. 255, 11423- 11428
394
9 Pritchard, P.H. and Vance, D.E. (1981) Biochem. J. 196, 26 l-267 10 Rooney, S.A. (1979) Trends Biochem. Sci. 4, 189-191 11 Stern, W., Kovac, C. and Weinhold, P.A. (1976) Biochim. Biophys. Acta 441, 280-293 12 Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco, W.M. and Gross, I. (1979) Biochim. Biophys. Acta 572, 64-76 13 Weinhold, P.A., Feldman, D.A., Quade, M.M., Miller, J.C. and Brooks, R.L. (1981) B&him. Biophys. Acta 665, 134144 14 Tokmakjian, S. and Possmayer, F. (1981) Biochim. Biophys. Acta 666, 176-180 15 Tokmakjian, S.. Haines, D.S.M. and Possmayer, F. (1981) Biochim. Biophys. Acta 663, 557-568 16 Possmayer, F., Casola, P.G., Chun, F., MacDonald, P., Ormseth, M.A., Wong, T., Harding, P.G.R. and Tokmakjian, S. (1981) Biochim. Biophys. Acta 664, lo-21 17 Mason, R.J., Williams, M.C., Greenleaf, R.D. and Clements, J.A. (1977) Am. Rev. Respir. Dis. 115, 1015-1026
18 Mason, R.J., Williams, M.C. and Dobbs, L.G. (1977) in Pulmonary Macrophage and Epithelial Cells, Proc. Annu. Hanford Biol. Symp. 16th, Energy Res. Dev. Admin. Symp. Ser. 43 (Sanders, CL., Schneider, R.P.. Dagle, G.E. and Ragan, H.A. eds), pp, 280-297, Technical Information Center, Energy Research and Development Administration, Springfield, VA. 19 Mason, R.J.and Williams, M.C. (1977) Am. Rev. Respir. Dis. 115 Suppl., 81-91 20 Batenburg, J.J., Longmore, W.J. and Van Golde, L.M.G. (1978) Biochim. Biophys. Acta 529, 160-170 21 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 22 Pande, S.V. (1976) Anal. Biochem. 74, 25-34 23 Pelech, S.I., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286
390
Elsevier Biomedical
Pre.\s
BBA 51180
THE RATE-LIMITING STEP IN THE BIOSYNTHESIS OF PHOSPHATIDYLCHOLINE ALVEOLAR TYPE II CELLS FROM ADULT RAT LUNG MARTIN
POST, JOSEPH
J. BATENBURG,
Laboratory of Veterinary Biochemistry, (Received
February
ELISABETH
A.J.M.
SCHUURMANS
State University of Utrecht, Biltstraat
and LAMBERT
BY
M.G. VAN GOLDE
172, 3572 BP Utrecht (The Netherlandsi
16th, 1982)
Key words: Phosphatidylcholine lung type II cell)
synthesis;
Phospholipid
regulation;
Cholrnephosphate
cytidylyltransferase;
Pulmonary
surfactant;
(Rat
1. The rate-limiting reaction in the biosynthesis of surfactant phosphatidylcholine by type II cells isolated from adult rat lung was examined. 2. Studies on the uptake of [Me - 14C]choline and its incorporation into its metabolites over a 5 h period indicated that in these cells the cholinephosphate pool was much larger than both the choline and CDPcholine pool. This is consistent with the idea that the rate-limiting reaction is that catalyzed by cholinephosphate cytidylyltransferase. 3. Evidence that cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine was also obtained from pulse-chase experiments. [Me -‘4C]Choline taken up by the cells was rapidly converted into cholinephosphate during the pulse period. As the radioactivity disappeared from cholinephosphate during the chase period, the label was incorporated immediately into phosphatidylcholine, without much change in the labelling of CDPcholine. This indicates that the cholinephosphotransferase is at least as fast as the cholinephosphate cytidylyltransferase. 4. Inclusion of palmitate in the chase medium accelerated the conversion of labelled cholinephosphate into phosphatidylcholine and decreased the radioactivity associated with CDPcholine. This indicates that under these conditions the cholinephosphate cytidylyltransferase reaction cannot keep up with increased utilization of the CDPcholine in the terminal step of the CDPcholine pathway.
dylcholine in a variety of mammalian tissues [5-91. It has been suggested that cholinephosphate cytidylyltransferase also catalyses the rate-limiting reaction in the biosynthesis of pulmonary phosphatidylcholine (lo- 131. The best evidence so far for this suggestion was obtained in the studies on the pool sizes of choline and its derivatives in the developing rat and rabbit lung [ 14- 161. The cholinephosphate pool was much larger than the CDPcholine pool, suggesting that the formation of the nucleotide may be limited. These studies, however, were carried out with preparations of whole lung. Because of the cellular heterogeneity of the lung, these results are not necessarily pertinent to type II cells. The present study was undertaken to investigate
Introduction Phosphatidylcholine is the major lipid component of pulmonary surfactant which lowers the surface tension in the alveoli and prevents their collapse during expiration [I]. The alveolar type II cells of the lung are the most important, if not sole, producers of surfactant lipids [2]. The de novo synthesis of surfactant phosphatidylcholine proceeds in the type II cell mainly by the CDPcholine pathway [3,4]. How the biosynthesis of phosphatidylcholine in the type II cell is controlled has not yet been elucidated. There is experimental evidence which suggests that cholinephosphate cytidylyltransferase catalyses the rate-limiting step in the production of phosphatiOOOS-2760/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
391
which of the enzymes is rate-limiting in type rat lung. To the best first of its kind carried
of the CDPcholine pathway II cells isolated from adult of our knowledge, it is the out with this cell type.
Materials and Methods
Materials. Unlabelled biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. [Me-i4C]Choline (spec. act., 58 Ci/mol) was bought from the Radiochemical Centre Amersham, U.K. Male Wistar rats, weighing 180-200 g, were obtained from the Central Institute for Breeding of Laboratory Animals in Zeist, The Netherlands. The rats had free access to laboratory chow and water. Isolation of type II cells. Type II cells were isolated from the rat lungs by trypsinization, density gradient centrifugation and differential adherence in primary monolayer culture [ 17-191. After 20 h in culture, the type II cells were used for the biochemical experiments. Purity and viability of the cells were in the same order as reported previously [20]. Incubation of type II cells. The cells, attached to the 35mm culture dishes were rinsed with a standard medium containing 5.6 mM glucose, 125 mM NaCl, 5 mM KCl, 2.5 mM Na,HPO,, 2.5 mM CaCl,, 1.2 mM MgSO,, 17 mM N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid and 50 pg/ml gentamycin at a pH of 7.4. Subsequently, the cells were either used for experiments in which the uptake and utilization of [Me-‘4C]choline was studied over a 5 h period or for pulse-chase experiments with [Me-‘4C]choline. In the first type of experiments the cells were incubated in standard incubation medium (1.5 ml/dish) supplemented with 0.1 mM [Me-‘4C]choline (spec. act., 6.59. lo4 dpm/nmol). During the incubation the dishes were each held in a 50 ml beaker covered with parafilm which was placed in a reciprocating waterbath (25 cycles/mm) kept at 37°C. After the desired incubation times the medium was removed and the cells were washed with ice-cold (choline-free) standard medium to remove excess labelled choline. Immediately afterwards, 1.5 ml of ice-cold methanol/H,0 (5 : 4, v/v) was added to the dishes. In the pulse-chase experiments the washed cells
were first incubated as described above for 1 h in standard medium supplemented with 0.025 mM choline. After this preincubation period the medium was removed and replaced with standard medium supplemented with 10 PCi [Me“C]choline/dish. At the end of a 15 min pulse period, the radioactive medium was removed and the dishes were rinsed with (choline-free) standard medium to remove excess labelled choline, after which chase medium, with or without 0.2 mM palmitate (complexed to bovine serum albumin in a molar ratio 5.3 : l), was added to the cells. This chase medium consisted of standard medium supplemented with 0.025 mM choline. At various times after the onset of the chase period, the incubation was terminated by removal of the medium, washing of the cells with ice-cold standard medium and addition of 1.5 ml methanol/H,0 (5 : 4, v/v). Analysis of choline and its derivatives. In both types of experiment, the cells were scraped from the dishes with a rubber policeman after the addition of methanol/H,0 and transferred with the methanol/H,0 into centrifuge tubes for extraction. To these tubes, lung lipid (0.2 pmol phosphorus), choline (2 pmol), cholinephosphate (8 pmol) and CDPcholine (2 pmol) were added as carriers. After addition of chloroform, lipids were extracted by the method of Bligh and Dyer [21]. Phosphatidylcholine was isolated from the lipid extract by thin-layer chromatography on silica gel G plates with chloroform/methanol/H ,O (65: 35 :4, v/v) as eluent. After detection by exposure to iodine, the phosphatidylcholine fraction was scraped into scintillation vials and assayed for radioactivity. The aqueous phase remaining after lipid extraction was evaporated to dryness in a vacuum rotary evaporator. The residue was dissolved in water. Choline, cholinephosphate and CDPcholine were separated by thin-layer chromatography on silica gel H plates with 0.15 M NaCl/methanol/conc. NH, (50 : 50 : 5, v/v) as developing solvent. Choline and its metabolites were visualized with iodine vapour, scraped into scintillation vials and assayed for radioactivity. Radioactivity was measured in the liquid scintillation mixture described by Pande [22]. Counting efficiency was determined by the channels ratio method.
392
Results and Discussion Fig. 1 shows the uptake of [Me- “C]choline by type II cells and its incorporation into cholinephosphate, CDPcholine and phosphatidylcholine. It can be seen that the cells rapidly phosphorylated choline to cholinephosphate. At any time more than 90% of the water-soluble radioactivity extracted from the cells was associated with cholinephosphate. The radioactivity in intracellular choline was maximal and constant after 2 h of incubation, whereas the incorporation of label into cholinephosphate, CDPcholine and phosphatidylcholine increased up to at least 5 h of incubation. Assuming no change in pool size, the
012365
Fig. 1. The uptake of [Me-i4C[choline into type II cells and its incorporation into cholinephosphate, CDPcholine and phosphatidylcholine. Isolated rat lung type 11 cells were incubated for the indicated periods with 0.1 mM [ Me-t4C]choline in the standard medium. The radioactivity in choline, cholinephosphate, CDPcholine and phosphatidylcholine was determined as described in Materials and Methods. The data are representative of two experiments, each carried out in duplicate.
much higher label incorporation into cholinephosphate as compared to that into intracellular choline indicates that the cholinephosphate pool is much larger than the intracellular choline pool. The observation that the incorporation of the label into phosphatidylcholine is only slightly lower than the incorporation into cholinephosphate while the incorporation into CDPcholine is much lower, indicates that the CDPcholine pool is much smaller than the cholinephosphate pool. A cholinephosphate pool that is much larger than both the intracellular choline and CDPcholine pool is consistent with the idea that the cholinephosphate cytidylyltransferase is rate-limiting in the formation of phosphatidylcholine. Further support for this idea comes from pulse-chase studies on the metabolism of choline, shown in Fig. 2. It can be seen that at the end of a 15 min pulse period most of the radioactivity was associated with the cholinephosphate pool. The high ratio between the labelling of cholinephosphate and that of intracellular choline again argues against a rate-limiting role of choline kinase. The observation that the radioactivity of free choline did not decrease during the chase period is in agreement with earlier studies with perfused hamster heart [8]. The radioactivity in the cholineposphate decreased rapidly during the chase period. The radioactivity lost from cholinephosphate appeared in phosphatidylcholine. A similar transfer of radioactivity from cholinephosphate to phosphatidylcholine was observed in studies with isolated hepatocytes [23]. The radioactivity in CDPcholine remained almost constant in the type II cells during the chase period and comprised less than 3.5% of the total radioactivity extractable from the cells (Fig. 2). This indicates that the cholinephosphotransferase reaction proceeds relatively fast and that the rate of phosphatidylcholine synthesis is limited by the activity of cholinephosphate cytidylyltransferase. Inclusion of palmitate in the chase medium caused a 2-fold increase in the rate of disappearance of the label from cholinephosphate and in the rate of its appearance in phosphatidylcholine (Fig. 2). In the presence of palmitate the radioactivity of the CDPcholine pool was decreased during the chase period, while the labelling of the
393
0.3!ii>o ’
0.20.
l ,/’ ,’
0.2 -
“qyo
0.’ 5\
9 2 %
z $j
0.1 .
,’ \
\
: \
\
0.1 0 \
\
? 1 /w-----0 :Q
\ t
‘0 c
005-
:/
s f
0
go
120
0
60
120
H
0.12-
0.0
h
G==-==~
II
O.Oh
0
12
60 Chase
time
fmin
)
Fig. 2. Pulse-chase study on the metabolism of choline in alveolar type II cells. Isolated rat lung type II cells were preincubated for 1 h in the standard incubation medium supplemented with 0.025 mM choline. Subsequently, the type II cells were pulsed with [ Me-‘4Cjcholine for 15 min and chased in the presence or absence of palmitate in standard medium containing 0.025 mM choline. Radioactivity in choline (V, V), cholinephosphate (0, l), CDPcholine (a, A) and phosphatidylcholine (0, n ) was determined. Open symbols, solid line, without palmitate; closed symbols, dashed line,with palmitate. The start of the chase is indicated as time zero. The data are representative of two experiments, each carried out in dupli-
cytidylyltransferase reaction cannot keep up with the increased utilization of the CDPcholine by the cholinephosphotransferase. In conclusion, the results of the present studies indicate that although the rate of phosphatidylcholine formation by the type II cells can be modulated by the supply of diacylglycerols cholinephosphate cytidylyltransferase is the slowest of the three enzymes incorporating choline into phosphatidylcholine. These results may indicate that cholinephosphate cytidylyltransferase becomes regulatory for phosphatidylcholine synthesis when the cells are under ample supply of plasma free fatty acids in viva. In order to obtain more evidence for the ratelimiting role of cholinephosphate cytidylyltransferase future studies should be directed towards measurement of the pool sizes of the intermediates and cofactors involved in the CDPcholine pathway in type II cells. Moreover, the activities and kinetic properties of the enzymes of the type II cell involved in this pathway should be determined. Acknowledgements
The investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and by the Dutch Asthma Foundation (Nederlands Astma Fonds).
cate.
References
intracellular choline was unaffected (Fig. 2). A stimulatory effect of palmitate on the incorporation of choline into phosphatidylcholine by isolated type XI cells had been observed previously [20]. The most likely explanation for this effect in the previous [20] and present studies is that by addition of palmitate the supply of diacylglycerols for the cholinephosphotransferase reaction was increased, resulting in a faster conversion of CDPcholine into phosphatidylcholine. The observation that palmitate decreased the amount of radioactivity in CDPcholine indicates that the supply of CDPcholine by the choiinephosphate
King, R.J.and Clements, J.A. (1972) Am. J. Physiol. 223, 715-726 Goerke, J. (1974) B&him. Biophys. Acta 344, 241-261 Kikkawa, Y., Yoneda, K., Smith, F., Packard, B. and Suzuki, K. (1975) Lab. Invest. 32, 295-302 Batenburg, J.J. and Van Golde, L.M.G. (1979) in Reviews in Perinatal Medicine (Scarpelli, E.M. and Cosmi, E.V., eds.), Vol. 3, pp. 73- 114, Raven Press, New York Vance, D.E. and Choy, P.C. (1979) Trends B&hem. Sci. 4, 145-148 Vance, D.E., Trip, E.M. and Paddon, H.B. (1980) J. Biol. Chem. 255, 1064-1069 Sleight, R. and Kent, C. (1980) J. Biol. Chem. 255, 1064410650 ZRlinski, T.A., Savard, J.D., Man, R.Y.K. and Choy, P.C. (1980) 3. Bid. Chem. 255, 11423- 11428
394
9 Pritchard, P.H. and Vance, D.E. (1981) Biochem. J. 196, 26 l-267 10 Rooney, S.A. (1979) Trends Biochem. Sci. 4, 189-191 11 Stern, W., Kovac, C. and Weinhold, P.A. (1976) Biochim. Biophys. Acta 441, 280-293 12 Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco, W.M. and Gross, I. (1979) Biochim. Biophys. Acta 572, 64-76 13 Weinhold, P.A., Feldman, D.A., Quade, M.M., Miller, J.C. and Brooks, R.L. (1981) B&him. Biophys. Acta 665, 134144 14 Tokmakjian, S. and Possmayer, F. (1981) Biochim. Biophys. Acta 666, 176-180 15 Tokmakjian, S.. Haines, D.S.M. and Possmayer, F. (1981) Biochim. Biophys. Acta 663, 557-568 16 Possmayer, F., Casola, P.G., Chun, F., MacDonald, P., Ormseth, M.A., Wong, T., Harding, P.G.R. and Tokmakjian, S. (1981) Biochim. Biophys. Acta 664, lo-21 17 Mason, R.J., Williams, M.C., Greenleaf, R.D. and Clements, J.A. (1977) Am. Rev. Respir. Dis. 115, 1015-1026
18 Mason, R.J., Williams, M.C. and Dobbs, L.G. (1977) in Pulmonary Macrophage and Epithelial Cells, Proc. Annu. Hanford Biol. Symp. 16th, Energy Res. Dev. Admin. Symp. Ser. 43 (Sanders, CL., Schneider, R.P.. Dagle, G.E. and Ragan, H.A. eds), pp, 280-297, Technical Information Center, Energy Research and Development Administration, Springfield, VA. 19 Mason, R.J.and Williams, M.C. (1977) Am. Rev. Respir. Dis. 115 Suppl., 81-91 20 Batenburg, J.J., Longmore, W.J. and Van Golde, L.M.G. (1978) Biochim. Biophys. Acta 529, 160-170 21 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 22 Pande, S.V. (1976) Anal. Biochem. 74, 25-34 23 Pelech, S.I., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286