Effect of CO, concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions

An investigation of the effect of change of total COP concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-I4C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-14C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO, concentration increased [U-14C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-14C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-14C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO, concentration. The increase in the incorporation of [U-14C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO, concentration. The results suggest that alteration in extracellular CO, concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung. Supplementary key words [U-'4C]glucose * palmitic acid . diplamitoyl phosphatidylcholine Variation of medium C02 concentration within physiological limits of pH has been previously reported to alter the incorporation of [U-'4C]glucose into phospholipids of the isolated perfused rat lung (1). In those studies, glucose incorporation into the phosphatidylcholine and phosphatidylethanolamine fractions of whole lung was measured. Increasing the medium C02 concentration from 6 to 45 mM was found to increase the incorporation of [U-14C]glucose into phosphatidylcholine fatty acids over 100%. Recent studies have shown that phosphatidylglycerol, in addition to phosphatidylcholine, is an important lung phospholipid. It has been reported to be a major component of rat and dog lung surfactant (2, 3) and to be a metabolically active phospholipid in rat lung (3, 4). Continued interest in factors that may regulate or otherwise alter the synthesis of the specific phospholipids of the surfactant fraction has prompted the present study. In this study, the effect of medium CO, concentration on [U-14C]glucose incorporation into phosphatidylcholine and phosphatidylglycerol of the rat lung surfactant complex and into the same phospholipids present in the remaining lung tissue are compared. The surface-active lipoprotein complex of lung is separated from whole lung after ex vivo perfusion using a discontinuous sucrose density gradient centrifugation procedure (4). The surfactant fraction isolated by this procedure differs from that obtained by lavage of lung in that it contains both the intracellular and extracellular components. These two pools of surfactant material have been found to have a similar chemical composition and surface activity (5). EXPERIMENTAL PROCEDURE Male Wistar strain rats (Hilltop Lab Animals, Inc., Scottdale, PA) were used. They were fed commercial laboratory chow ad libitum. Preparation of the lungs for perfusion, perfusate preparation, and procedures of the perfusion have been previously reported (6). The perfusion medium was adjusted to contain approximately 7 or 43 mM total Cot by the addition of the appropriate ratio of NaHC03NaCl to the medium so that equilibration with COP0, gas mixtures in the range of 2-13% C02 (and 98-87% 0,) gave initial pH values of 7.327.40. Following removal of lung tissue during or at the termination of perfusion, the surfactant and residual fractions were separated by the method of Frosolono et al. (7) as modified by Sanders and Longmore (4). Lipids were extracted from these fractions Journal of Lipid Research Volume 18, 1977 309 by gest, on N ovem er 3, 2017 w w w .j.org D ow nladed fom with 45 ml of chloroform-methanol 2:l (v/v). Preliminary separation of lipids was carried out by silicic acid column chromatography using the previously reported procedure designated method 2 (3). The fraction containing primarily phosphatidylglycerol and phosphatidylethanolamine was then separated by thin-layer chromatography on silica gel H with tetrahydrofuranmethylalmethanol 2 M aqueous ammonia 10:5:5: 1 (v/v). The fraction containing phosphatidylcholine was likewise isolated by thin-layer chromatography using a solvent system containing chloroform-methanol-acetic acid-water 12.5:7.5:2.1 (v/v). Procedures concerning pH and COS measurements, analysis of medium glucose concentration, tissue phosphorus and protein content, phospholipid hydrolysis, and the preparation, separation, quantitation, and trapping of fatty acid methyl esters by gas-liquid chromatography were as previously reported (1, 6). The amount of glucose incorporated into the phospholipid fraction was calculated from the specific activity of glucose in the medium and the pool size and specific activity of the phospholipid fraction at the time of sampling. [ U ' 4 C l G l ~ c ~ ~ e was purchased from New England Nuclear Corp., Boston, MA. Radioactivity was determined using scintillation counting techniques with efficiency calculated using the channels ratio method.