ABSTRACT
By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.
Subject(s)
Angiotensin II/pharmacology , Annexin A1/metabolism , Glomerular Mesangium/metabolism , Phosphoproteins/metabolism , Tyrosine , Animals , Annexin A1/isolation & purification , Calcium/metabolism , Cells, Cultured , Chromatography, Affinity , Egtazic Acid , Electrophoresis, Polyacrylamide Gel , Male , Molecular Weight , Phosphates/metabolism , Phospholipids/metabolism , Phosphoproteins/isolation & purification , Phosphorus Radioisotopes , Phosphorylation , Phosphotyrosine , Rats , Rats, Wistar , Tyrosine/analogs & derivatives , Tyrosine/analysisABSTRACT
Several sets of data suggest that specific classes of anti-DNA antibodies could be implicated in the genesis of glomerular lesions in SLE. The goal of this work is to investigate if this pathogenic role could be related to the antibodies' genetic origin--from BALB/c or NZBxNZW/F1 mice--or to their physiological origin--induced either by DNA or by polyclonal B cell activation in normal mice. For this purpose, anti-DNA antibody hybridoma clones produced from different origins were subcutaneously injected in BALB/c or NZBxNZW/F1 female mice, followed by studies of immunological parameters and kidney lesions. Results concur that the induced anti-DNA antibodies can play a role in fatal disease development, related to clonal specificity but not to the way of stimulation which was either polyclonal B cell activation or DNA immunization. Also, they emphasize the possible very lethal role of serum circulating DNA.
Subject(s)
Antibodies, Antinuclear/administration & dosage , DNA/immunology , Hybridomas/transplantation , Lupus Erythematosus, Systemic/immunology , Acute Disease , Animals , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/analysis , B-Lymphocytes/immunology , Clone Cells , Female , Fluorescent Antibody Technique , Immunoglobulins/analysis , Injections, Subcutaneous , Kidney/pathology , Lupus Erythematosus, Systemic/genetics , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NZBABSTRACT
Levels of circulating DNA increase under treatment by an artificial kidney. Using a new assay, levels of plasma DNA are studied in 45 patients during 99 sessions of hemodialysis or hemofiltration. Before the session, plasma DNA levels are increased in 41/99 samples and, among them, in 18/24 samples collected from hepatitis B surface antigen carriers. During the first 3 h of the session, plasma DNA levels increase whatever the method of treatment. At the 30th and 60th minute of hemodialysis, a positive gradient of plasma DNA exists between the output and the input of the artificial kidney. It is concluded that: (1) the increase in plasma DNA is related to the overall procedure of artificial kidney therapy; (2) death of leukocytes in the artificial kidney is responsible for the release and the increase in circulation of extracellular DNA.
