Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts.

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.

Altered rates of collagen synthesis in in vitro aged human lung fibroblasts.

Absolute rates of protein and collagen synthesis based on prolyl-tRNA as the precursor were determined in two age groups of IMR-90 human lung fibroblasts. Compared with midrange fibroblasts [population doubling level (PDL) = 20 to 30] aged fibroblasts (PDL greater than 40] were larger in size in terms of protein and RNA per cell, generally proliferated more slowly, exhibited different steady state [3H] proline transfer RNA (tRNA) precursor pool specific radioactivities, synthesized collagen at a substantially lower rate, and exhibited a reduction in the percent commitment to collagen synthesis. Total protein synthetic rates were reduced slightly in aged versus midrange fibroblasts but the difference was not statistically significant. Proliferative capacity (PDL/wk) correlated better with these changes than cumulative PDL. Cell size (protein/cell) was the variable that had the highest correlation with the reduction in collagen synthesis observed in human lung fibroblasts. Thus, an important differentiated function of human lung fibroblasts, collagen synthesis, is greatly diminished in vitro in large, slowly dividing fibroblasts.

Surfactant release in excised rat lung is stimulated by air inflation.

Because increased ventilation has been associated with an acceleration of lung surfactant turnover, we investigated the effect of fluid and air inflations on the release of surfactant into the air spaces. We found that excised rat lungs, initially lavaged three times at 23 degrees C, release approximately 40-90 micrograms of phospholipid/g wet lung wt into the air spaces in response to a further infusion of fluid into the airway equal to total lung capacity. A single air inflation to the same volume, followed by degassing and lavage, contributes approximately 230 micrograms to the yield of phospholipid. We estimated basal release of phospholipid as 112 micrograms wet lung wt-1 . h-1, which is far less than the 2,050 micrograms -1 . h-1 retrieved during a series of air and fluid inflations. The above findings are consistent with the hypothesis that air inflation to total lung capacity is a major physiological stimulus to release of lung surfactant into the alveolar space. The lung lavage process itself also causes the release of surfactant.

Prolyl-tRNA-based rates of protein and collagen synthesis in human lung fibroblasts.

Knowledge of the dynamics of collagen turnover requires information regarding rates of synthesis of this group of connective-tissue proteins. The relationship of various amino acid pools to the tRNA precursor pool used for protein synthesis is known to vary between different cell types and tissues, even for essential amino acids. We studied extracellular, intracellular and tRNA-proline pools in cultured human lung IMR-90 fibroblasts to determine the relationship between them as candidate proline precursor pools for total protein and collagen synthesis. Time-course experiments showed that the three proline pools attained distinctly different steady-state specific radioactivities (extracellular greater than intracellular greater than tRNA) at the extracellular proline concentration of 0.2 mM. The kinetics of radioisotope incorporation into cell protein and collagenase-digestible protein indicated that the intracellular free proline pool could not be used reliably as a precursor for calculating synthetic rates. However, tRNA-proline behaved isotopically as if it were the precursor and provided synthesis rates 2-3-fold higher than those calculated by using either free proline pool. The incorporation of labelled lysine and leucine was constant over a wide range of extracellular proline concentrations. Fractional rates of protein synthesis based on tRNA-amino acid were the same with [3H]phenylalanine as with [3H]proline. The specific radioactivity of cell-associated hydroxyproline reached a steady-state value 8-10h after radioisotope administration which matched the mean tRNA-proline specific radioactivity, suggesting that tRNA-proline is not isotopically compartmentalized. A model of cellular proline-pool relationship is presented and discussed.

Pulmonary surface film stability and composition.

Stability of pulmonary alveoli at end expiration requires a very low air-water surface tension (e.g., less than 10 mN.m-1). Another important requirement is that the surface film maintain this low surface tension for a sufficiently long time at fixed lung volume. We measured monolayer collapse rates at 37 degrees C of lung surface-active material (SAM) and certain lipids found in this material and compared them with alveolar monolayer collapse rates calculated from published lung compliance changes. We found collapse rates for purified SAM or a mixture of dipalmitoyl lecithin (DPPC):monoenoic lecithin (PC):cholesterol (CHOL) (3.03:1.65:1 molar ratios) to be much greater than collapse rates of alveolar films estimated from indirect measurements. Monolayers of pure DPPC or DPPC with 10 mol% monoenoic PC and/or CHOL had collapse rates equal to or less than those estimated from lungs. We conclude that the alveolar monolayer is enriched in DPPC to the extent of 90 mol% or greater. Enrichment may exclude more mobile components from the monolayer during expiration when surface tension reaches verry low values.