My hypothesis is that rapid activation of a novel intracellular Ca2+- independent PLA2 results in accelerated hydrolysis of not only diacylglycerophospholipids but also of plasmalogens, and is the major event responsible for the accumulation of unesterified arachidonic acid and lysophospholipids during hypoxic cell injury. This hypothesis is based on our preliminary studies which show for the first time that 55% of the AA present in proximal tubule ethanolamine phospholipids is esterified to plasmalogen molecular species as well as the demonstration that hypoxia in isolated proximal tubules results in the rapid activation of a calcium- independent, plasmalogen-active PLA2. This novel enzyme has at least 10 fold higher specific activity than the previously described diacylphospholipid calcium-dependent PLA2 enzyme.
The specific aims are: 1) To investigate the nature of membrane phospholipid catabolism during hypoxia/reoxygenation in rabbit proximal tubules. We will utilize rabbit proximal tubules exposed to conditions of hypoxia/reoxygenation in vitro to determine the relative contribution of diacylglycerophospholipid and plasmalogen phospholipolysis to the total production of unesterified arachidonic acid and to establish which phospholipase catalyzed enzymic pathways are involved. These studies will also identify the specific amphiphiles (i.e. lysophospholipids/lysoplasmalogen, alkenylacylglycerol) generated during hypoxia/reoxygenation. 2) To examine the factors which activate and/or regulate the calcium- independent PLA2 activity in rabbit proximal tubules. These studies will be performed in intact cells and isolated subcellular fractions in order to study regulation under conditions where the enzyme is present in its native environment. Although the phospholipase A2 activity can be measured in the absence of calcium, the role of calcium in membrane translocation and/or activation of calcium-independent plasmalogen PLA2 activity will be examined. Similarly the role of PKC activation and the effect of reactive oxygen metabolites on calcium-independent plasmalogen active PLA2 activity will be examined. 3) To complete the purification of the diacyl and plasmalogen active PLA2 enzyme(s). I have obtained a high degree of purity of a soluble calcium independent 44 kDa PLA2 protein with activity against both plasmalogen and diacylglycerophospholipid substrate. I plan to obtain sufficient amounts of this protein to perform the following studies: i) amino acid sequence composition; ii) kinetics of substrate specificity (to determine whether diacylglycerophospholipids or plasmalogens are the preferred substrates for the enzyme) and iii) comparison between the characteristics of our isolated protein(s) with the features of the enzyme activity defined in the studies outlined in specific aims 1 and 2.
| Portilla, D; Crew, M D; Grant, D et al. (1998) cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities. J Am Soc Nephrol 9:1178-86 |
| Portilla, D (1997) Carnitine palmitoyl-transferase enzyme inhibition protects proximal tubules during hypoxia. Kidney Int 52:429-37 |
| Schonefeld, M; Noble, S; Bertorello, A M et al. (1996) Hypoxia-induced amphiphiles inhibit renal Na+, K(+)-ATPase. Kidney Int 49:1289-96 |
| Portilla, D; Dai, G (1996) Purification of a novel calcium-independent phospholipase A2 from rabbit kidney. J Biol Chem 271:15451-7 |
| Portilla, D; Creer, M H (1995) Plasmalogen phospholipid hydrolysis during hypoxic injury of rabbit proximal tubules. Kidney Int 47:1087-94 |
