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R D Greenleaf

Publications and source records attributed to R D Greenleaf.

5 recordsLinked to original sources
PubMed1984

Characteristics of amino acid metabolism by isolated alveolar type II cells.

Alveolar type II cells of the lung are important in producing the lipoprotein surfactant. Most studies about metabolism in type II cells have focussed on lipid precursors for phospholipid metabolism. Surfactant contains a unique apoprotein; yet relatively little is known about the metabolism of amino acids by type II cells. Type II cells were isolated using density gradient centrifugation followed by centrifugal elutriation. Alanine (Ala), leucine (Leu), valine (Val), and phenylalanine (Phe) incorporation into protein and lipid and oxidation to CO2 was measured after the cells were incubated for 2 h. For alanine metabolism, 22% of total radioactivity from alanine was incorporated into protein, 20% into lipid, and 58% oxidized to CO2. For leucine, 51% was incorporated into protein, 23% into lipid, and 22% oxidized to CO2. Fifty percent of radioactivity from valine metabolism was incorporated into protein, 5% into lipid, and 47% oxidized to CO2. Virtually all (95%) of phenylalanine, however, was utilized for protein synthesis only. Puromycin and cycloheximide decreased protein synthesis from Ala, Leu, and Phe but had little affect on Ala and Leu metabolism to lipid or CO2. The hypolipidemic drug clofibrate inhibited all aspects of amino acid metabolism. In summary, type II cell amino acid metabolism is regulated similar to that of cells from skeletal muscle and adipose tissue, but in contrast to hepatocytes, type II cells readily oxidize valine and utilize leucine for lipid as well as protein synthesis.

Amino Acids↗
PubMed1980

Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase.

We used porcine pancreatic elastase to isolate type II cells from the lungs of rats; the yield and purity of the type II cells was better than that obtained by methods using trypsin. In 102 experiments we obtained 82 +/- 23 . 10(6) cells/rat, 68 +/- 11% (mean +/- S.D.) of which type II cells. This preparation of cells, when centrifuged over a discontinuous density gradient, yielded 25 +/- 10 . 10(6) cells/rat, 80 +/- 13% of which were type II cells (n = 102). The cells, after density gradient centrifugation, could be futher purified by centrifugal elutriation (94 +/- 3% type II cells, n = 22) or adherence in primary culture (94 +/- 2% type II cells, n = 34). Type II cells isolated with elastase are similar morphologically and biochemically to type II cells isolated from rats with trypsin. The preparations of cells appeared healthy by several different criteria: ultrastructure, exclusion of vital dye, lack of stimulation of oxygen consumption by exogenous sodium succinate, and linear rates of oxidation of [1-14C]palmitic acid and of incorporation of [1-14C]acetate into fatty acids. Type II cells consumed 75 +/- 20 nmol O2/10(6) cell per h, oxidized [1-14C]palmitic acid at a rate of 0.4 nmol/10(6) cells per h, and incorporated [1-14C]acetate into fatty acids at a rate of 7.5 nmol/10(6) cells per h.

Acetates↗
PubMed1979

Isolation of alveolar type II cells by centrifugal elutriation.

Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9 +/- 1.0 x 10(6) cells per rat lung (mean +/- S.D., n=30) were recovered of which 86 +/- 6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101 +/- 21 nmol per hr . 10(6) cells (mean +/- S.D., n=4), and their oxygen consumption increased only 10% after 10 mM sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugral elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells.

Animals↗
PubMed1977

Isolation and properties of type II alveolar cells from rat lung.

Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.

Animals↗