Induction of surfactant protein (SP-A) biosynthesis and SP-A mRNA in activated type II cells during acute silicosis in rats.

B. E. Miller, W. E. Bakewell, S. L. Katyal, G. Singh, G. E. Hook

Research output: Contribution to journalArticle

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Abstract

The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation. Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments. Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis. Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes. Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB. The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells. Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells. SP-A synthesis was measured by incubating freshly isolated cells with [35S]Translabel (70% [35S]methionine, 15% [35S]cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein. The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells. Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000. In type II cells from control rats, the 34-kD protein accounted for approximately 93% of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species. The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA. These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.

Original languageEnglish (US)
Pages (from-to)217-226
Number of pages10
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume3
Issue number3
DOIs
StatePublished - Jan 1 1990
Externally publishedYes

Fingerprint

Pulmonary Surfactant-Associated Protein A
Silicosis
Biosynthesis
Surface-Active Agents
Rats
Messenger RNA
Silicon Dioxide
Methionine
Molecular weight
Rat control
Proteins
Pancreatic Elastase
Radioactivity
Cysteine
Immunoglobulin G
Molecular Weight
Alveolar Epithelial Cells
Somatotypes
Lung
Immunoprecipitation

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Induction of surfactant protein (SP-A) biosynthesis and SP-A mRNA in activated type II cells during acute silicosis in rats. / Miller, B. E.; Bakewell, W. E.; Katyal, S. L.; Singh, G.; Hook, G. E.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 3, No. 3, 01.01.1990, p. 217-226.

Research output: Contribution to journalArticle

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abstract = "The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation. Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments. Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis. Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes. Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB. The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells. Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells. SP-A synthesis was measured by incubating freshly isolated cells with [35S]Translabel (70{\%} [35S]methionine, 15{\%} [35S]cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein. The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells. Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000. In type II cells from control rats, the 34-kD protein accounted for approximately 93{\%} of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species. The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA. These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.",
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