Oxidants may play a central role in the pathogenesis of
adult respiratory distress syndrome, and
phospholipase activation is a potential mechanism of
oxidant-induced injury of alveolar epithelial cells. Studies were performed in rat alveolar type II epithelial cells (RAEC) after 3 days in culture. As measured by 51Cr and
lactate dehydrogenase release, H2O2 caused time- and dose-dependent cytotoxicity to RAEC. RAEC
phospholipids labeled with [14C]-
stearic acid ([14C]SA) and [3H]
arachidonic acid ([3H]AA) released
free fatty acids in response to H2O2 in a manner that closely paralleled the cytotoxicity indexes. Analysis of
phospholipid subclasses indicated that
phosphatidylcholine was preferentially affected. Analysis for putative products of
phospholipase activity revealed significant increases in
diacylglycerol and
phosphorylcholine, expected products of
phospholipase C, as well as significant increases in L-alpha-
lysophosphatidylcholine and L-alpha-glycerophosphocholine, expected products of
phospholipase A2. Increases in
phospholipase D activity were not detected. To determine whether H2O2-stimulated
phospholipase activity might be Ca2+ stimulated, RAEC were loaded with
fura-2/AM, and changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored by epifluorescent microscopy. Exposure to H2O2 caused elevations in [Ca2+]i, and the time and dose relationships were consistent with the hypothesis that the release of [14C]SA and [3H]AA is related to changes in cellular Ca2+ concentrations. Additionally, pretreatment with
MAPTAM, an intracellular
chelator of
calcium, partially blocked H2O2-mediated [3H]AA liberation. However, experiments in
saponin-permeabilized RAEC, in which [Ca2+]i was strongly buffered by
ethylene glycol-bis(beta-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, indicate that H2O2-induced
phospholipase activity also has a Ca(2+)-independent component.(ABSTRACT TRUNCATED AT 250 WORDS)