Uptake of [14C] Choline and Incorporation into Lung Phospholipid by the Isolated Perfused Rat Lung RODGER G. JOHNSON, MOIRA A. LUGG, and TERENCE E. NICHOLAS, Department of Human Physiology, School of Medicine, The Flinders University of South Australia, Bedford Park, S.A. 5042 and Department of Physiology, School of Medicine, University of Hawaii, Honolulu, Hawaii, USA 96822. ABSTRACT
We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 raM. The rote of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extraceUular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Kin. INTRODUCTION
[n the mammalian lung, more than 70% by weight of surfactant is phosphatidylcholine (PC) (1). The major route for the de novo synthesis of PC in the lung is via the incorporation of choline, which in turn must either be derived from plasma or be reutilized after degradation of PC within the lung. Evidence for the first source is provided by Chevalier and Collet (2), who demonstrated that an intraperitoneal injection of [3HI choline in mice was rapidly followed by the appearance of radioactive label in the alveolar type lI cells. Whereas the uptake of choline from the vascular compartment of the lung has not been investigated, choline uptake in other non-neural tissue is carrier-mediated (3-5). Moreover, inhibition of carrier-mediated transport of choline into rat brain synaptosomes has effectively inhibited the synthesis of acetylcholine (6-8). If choline uptake in tung is also carrier-mediated, we postulate that the process may be rate-limiting in the biosynthesis of surfactant PC. In the present study, we have investigated the characteristics of choline uptake by the isolated perfused rat lung (IPL). METHODS
Lungs from male Porton rats (200-250 g) were perfused at l0 ml/min with Krebs bicarbonate solution containing 4.5% albumin and ventilated with 5% CO 2 in oxygen (9). In all cases, the lungs were perfused for 10 rain before [t4 C-choline was introducted. Sodiumfree medium was prepared as previously reported (10). [3H-methyl] choline chloride (10.1 Ci/mmol) and [14C-methyl] choline chloride (52 mCi]mmoI) were obtained from the Radiochemical Centre (Amersham, England). 1-Pal-
mitoyl-2-palmitoyl [ 9,10-3 H ] phosphatidylcho line (20Ci/mmol)([3H]DPPC) was obtained from Applied Sciences Laboratories (State College, PA). The purity of labeled compounds was checked regularly by thin layer chromatography (TLC). TLC plates were scanned for radioactivity with a Berthold TLC scanner II (model LB273, Wildbad, Germany). Hemicholinium-3(HC-3) was obtained from Aldrich (Milwaukee, WI). Unlabeled choline chloride was added to the standard medium containing [14C] choline when concentrations of 0.1 mM and greater were required. Choline chloride, dipalmit oyl-DL-a-phosphatidylcholine, mixed PCs and sphingomyelin were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals and solvents were obtained from normal commercial sources. Lipids were extracted from the homogenized lung tissue by the method of Folch et al. (11), and samples for counting were taken from both aqueous and organic phases. Samples of the total lipid extract were applied to silicic acid columns (500-750 mg silicic acid in Pasteur pipets), and neutral lipids were eluted with 10 ml chloroform. The phospholipid fraction was eluted with 10 ml methanol, dried at 35 C under reduced pressure, redissolved in methanol (200/~1), and duplicate samples (25/11) were taken for counting. Samples (I0/11) were spotted on precoated Silica Gel G TLC plates (Macherey Nagel, Germany). A standard carrier mixture of PCs was spotted as a marker with each sample, and [3Hi DPPC with unlabeled carrier was run separately as a reference standard. The plates were developed according to Gluck et al. (12). The identity of PC was checked by two dimensional TLC with a second solvent system (13). Lipid spots were visualized by exposure to iodine vapors or to a
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R.G. JOHNSON, M.A. LUGG, AND T.E. NICHOLAS
556 120
~'~ -6 Y
.
c 40
-~ Uptake i=0 992 13
We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 raM. The rote of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extraceUular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Kin. INTRODUCTION
[n the mammalian lung, more than 70% by weight of surfactant is phosphatidylcholine (PC) (1). The major route for the de novo synthesis of PC in the lung is via the incorporation of choline, which in turn must either be derived from plasma or be reutilized after degradation of PC within the lung. Evidence for the first source is provided by Chevalier and Collet (2), who demonstrated that an intraperitoneal injection of [3HI choline in mice was rapidly followed by the appearance of radioactive label in the alveolar type lI cells. Whereas the uptake of choline from the vascular compartment of the lung has not been investigated, choline uptake in other non-neural tissue is carrier-mediated (3-5). Moreover, inhibition of carrier-mediated transport of choline into rat brain synaptosomes has effectively inhibited the synthesis of acetylcholine (6-8). If choline uptake in tung is also carrier-mediated, we postulate that the process may be rate-limiting in the biosynthesis of surfactant PC. In the present study, we have investigated the characteristics of choline uptake by the isolated perfused rat lung (IPL). METHODS
Lungs from male Porton rats (200-250 g) were perfused at l0 ml/min with Krebs bicarbonate solution containing 4.5% albumin and ventilated with 5% CO 2 in oxygen (9). In all cases, the lungs were perfused for 10 rain before [t4 C-choline was introducted. Sodiumfree medium was prepared as previously reported (10). [3H-methyl] choline chloride (10.1 Ci/mmol) and [14C-methyl] choline chloride (52 mCi]mmoI) were obtained from the Radiochemical Centre (Amersham, England). 1-Pal-
mitoyl-2-palmitoyl [ 9,10-3 H ] phosphatidylcho line (20Ci/mmol)([3H]DPPC) was obtained from Applied Sciences Laboratories (State College, PA). The purity of labeled compounds was checked regularly by thin layer chromatography (TLC). TLC plates were scanned for radioactivity with a Berthold TLC scanner II (model LB273, Wildbad, Germany). Hemicholinium-3(HC-3) was obtained from Aldrich (Milwaukee, WI). Unlabeled choline chloride was added to the standard medium containing [14C] choline when concentrations of 0.1 mM and greater were required. Choline chloride, dipalmit oyl-DL-a-phosphatidylcholine, mixed PCs and sphingomyelin were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals and solvents were obtained from normal commercial sources. Lipids were extracted from the homogenized lung tissue by the method of Folch et al. (11), and samples for counting were taken from both aqueous and organic phases. Samples of the total lipid extract were applied to silicic acid columns (500-750 mg silicic acid in Pasteur pipets), and neutral lipids were eluted with 10 ml chloroform. The phospholipid fraction was eluted with 10 ml methanol, dried at 35 C under reduced pressure, redissolved in methanol (200/~1), and duplicate samples (25/11) were taken for counting. Samples (I0/11) were spotted on precoated Silica Gel G TLC plates (Macherey Nagel, Germany). A standard carrier mixture of PCs was spotted as a marker with each sample, and [3Hi DPPC with unlabeled carrier was run separately as a reference standard. The plates were developed according to Gluck et al. (12). The identity of PC was checked by two dimensional TLC with a second solvent system (13). Lipid spots were visualized by exposure to iodine vapors or to a
555
R.G. JOHNSON, M.A. LUGG, AND T.E. NICHOLAS
556 120
~'~ -6 Y
.
c 40
-~ Uptake i=0 992 13
