Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis.
Fibronectin (Fn) is an adhesive protein found in the plasma and extracellular tissue matrix. Locally synthesized tissue or cellular Fn (cFn) has extra domains (ED1 and ED2) not present in liver synthesized plasma Fn (pFn). In the lung, Fn is found in the endothelial and epithelial basement membranes, as well as in the interstitial matrix. Utilizing murine monoclonal antibodies to ED1 of cFn, we studied the release of total Fn as well as ED1-Fn into the plasma-free perfusate of the isolated perfused rabbit lung in relation to changes in lung weight due to fluid accumulation after oxidant (H2O2) challenge. Both parameters were also studied after addition of cycloheximide (20 micrograms/ml perfusate) to the perfusion medium to inhibit lung protein synthesis. After continuous H2O2 challenge (11 nmol.ml buffer-1.min-1), there was a 2.25 +/- 0.62 g increase in lung weight over 60 min. Measurement of 125I-labeled albumin clearance at 20 min after the start of H2O2 infusion confirmed an increase in lung endothelial protein permeability after H2O2 treatment. Fn antigen was released into the perfusate as early as 15 min after oxidant challenge. By 60 min, total perfusate Fn increased in H2O2-treated lungs (n = 6) to 2.10 +/- 0.48 micrograms/ml compared with only 0.35 +/- 0.09 micrograms/ml in normal control lungs (n = 5). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of nonreduced samples revealed that the Fn released consisted of primarily intact (440 kDa) Fn as well as Fn fragments. A rapid release of ED1-Fn paralleled the increased release of total Fn.(ABSTRACT TRUNCATED AT 250 WORDS)
Vincent
P A
PA
Department of Physiology and Cell Biology, Albany Medical College of Union University, New York 12208.
Rebres
R A
RA
Lewis
E P
EP
Hurst
V
V
Saba
T M
TM
D000818:Animals; D000911:Antibodies, Monoclonal; D003513:Cycloheximide / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity*; D015151:Immunoblotting; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008168:Lung / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000293:injuries / Q000473:pathology; D010477:Perfusion; D011651:Pulmonary Artery / Q000187:drug effects* / Q000293:injuries / Q000473:pathology; D011654:Pulmonary Edema / Q000378:metabolism; D011817:Rabbits
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase