Synthesis and secretion of alpha 2-macroglobulin by human lung fibroblasts.

Synthesis and secretion of alpha 2-macroglobulin were studied with the human lung fibroblast cell line GM 1379. After incubation with [3H]leucine the cells secreted radioactively labeled alpha 2-macroglobulin consisting of subunits with a molecular weight of 180,000. When the cells were treated with tunicamycin the unglycosylated alpha 2-macroglobulin subunit exhibited a molecular weight of 160,000. Poly(A) +RNA was isolated from the cultured cells and translated in a rabbit reticulocyte lysate. From the translation products an alpha 2-macroglobulin species with a molecular weight of 160,000 was immunoprecipitated. The addition of pancreatic microsoms to the translation mixture resulted in the synthesis of an alpha 2-macroglobulin subunit which had molecular weights of 180,000. Thus, a size of approximately 160,000 for the protein moiety and 20,000 for the carbohydrate portion can be estimated for a subunit of alpha 2-macroglobulin from human lung fibroblasts.

Characteristics of angiotensin converting enzyme and its role in the metabolism of angiotensin I by endothelium.

Angiotensin converting enzyme (ACE) was isolated from rabbit lung tissue by affinity chromatography on an N-(1-carboxy-5-aminopentyl)-L-alanylglycine resin. The molecular weight of the enzyme was determined to be 140,000, and upon isoelectric focusing a set of bands between pH 4.3-4.7 was observed. When the enzyme was treated with endoglycosidase F the molecular weight decreased to 100,000, and a virtually identical molecular weight was found for the in vitro translated form of ACE. The ACE-catalyzed hydrolysis of angiotensin I was followed by high-performance liquid chromatography separation and fluorescence monitoring of substrate and products. The kinetic parameters of the angiotensin I hydrolysis were KM = 17 mumol/L and kcat = 420 min-1 at pH 7.5. By the same analytical method the metabolism of angiotensin I by cultured endothelial cells and by the isolated, perfused rabbit aorta was investigated. Only a small fraction of angiotensin I was converted to angiotensin II; the larger part was directly degraded to small peptides. In the presence of ACE inhibitors no angiotensin II was formed but the rate of the angiotensin I hydrolysis was virtually unchanged. Thus, the action of ACE is critical to the generation of angiotensin II but not to the degradation of angiotensin I, and during ACE inhibition no accumulation of angiotensin I occurs.

Role of the endothelium in the vasomotor effects of angiotensin I in isolated arteries with or without inhibition by MK 422.

We investigated in the isolated rabbit aorta the ability of the endothelium to attenuate the vasoconstrictor effects of angiotensin (ANG) I and II. After preincubation with enalaprilat, which inhibited the conversion of ANG I to ANG II by 80%, the contractile response to ANG I (10(-8) to 10(-6) mol/l) was significantly greater in aortae which were endothelium-denuded compared with endothelium-intact segments. There was no such difference for ANG II (10(-10) to 3 x 10(-8) mol/l). We conclude that an endothelium-mediated dilatation is part of the net vasomotor action of ANG I.

Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes.

Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion.

Rat lung tissue is a site of alpha 1-proteinase inhibitor synthesis: evidence by cell-free translation.

Poly(A) +RNA isolated from lungs of normal rats and of rats suffering from experimental inflammation was translated in a cell-free translation mixture from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-proteinase inhibitor and alpha 2-macroglobulin. Comparable levels of mRNA for alpha 1-proteinase inhibitor were found in rat lung tissue from control and experimentally inflamed animals. alpha 2-Macroglobulin mRNA could not be detected in rat lung tissue.