Anoxia induces phospholipase A2 activation in rabbit renal proximal tubules.
Phospholipase A2 (PLA2) activation during anoxic cell injury was determined by use of a variety of approaches in rabbit proximal renal tubules. Arachidonic acid (AA) mass release increased from 4 +/- 1 (normoxia control) to 40 +/- 6 ng/mg protein after 20 min and 106 +/- 16 ng/mg protein after 40 min of anoxia. PLA2 activity was measured by estimating the amount of sn-2 fatty acid released from either 14C-labeled Escherichia coli membranes or [14C]phosphatidylethanolamine (PE) micelles incubated with membrane and cytosolic fractions obtained from normoxic or anoxic tubules. At pH 7.4 and 1 mM Ca, PLA2 activity increased in the 20-min anoxic membrane fractions from 8.1 +/- 2.3 (normoxic) to 15.2 +/- 2.1 pmol.min-1.mg protein-1 (anoxic). When the proximal tubules were homogenized in the absence of Ca, the anoxia-induced PLA2 activity was found to be soluble. Preincubation with pancreatic PLA2 antibody inhibited 50% of both basal and anoxia-stimulated PLA2 activity. Two protein bands (40- and 21-kDa species) immunoreactive to PLA2 antibody were detected in the membrane fraction. A sixfold increase in the immunoreactivity of the 40-kDa band was detected after 40 min of anoxia of proximal tubules. These results suggest that anoxia induces an intracellular PLA2 activity in kidney cells that could be immunologically related to pancreatic PLA2.
Portilla
D
D
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Mandel
L J
LJ
Bar-Sagi
D
D
Millington
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D015687:Cell Hypoxia; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology
D001095:Arachidonic Acids; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D010741:Phospholipases A; D054467:Phospholipases A2