Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties

Journal of Inflammation, Oct 2008

Background Lysophosphatidylcholines (lysoPCs) are products of phospholipase A2 (PLA2) enzyme activity, and like the enzyme, have a direct role in toxic inflammatory responses in variety of organ systems. Paradoxically, reduced plasma lysoPC levels have been noted in sepsis patients and systemic treatment with lysoPCs is therapeutic in rodent models of sepsis and ischemia. These observations suggest that elevation of plasma levels of these lipids can actually help to relieve serious inflammatory conditions. We demonstrate that specific lysoPCs act as uncompetitive product inhibitors of plasma secreted PLA2 enzymes (sPLA2s), especially under conditions of elevated enzyme activity, thus providing a feedback mechanism for the observed anti-inflammatory effects of these compounds. Methods Thin layer chromatography and mass spectroscopy were used to estimate total lysoPC concentration and the relative contributions of different lysoPC species in rat plasma samples. Kinetic studies of sPLA2 enzyme activity were conducted on these samples ex vivo and on purified group IA sPLA2 in vitro after addition of specific lysoPC species to the reaction mixture. Enzyme activity was also measured in plasma samples of rats injected with these same lysoPCs. Results Palmitoyl (16:0), stearoyl (18:0) are the most abundant lysoPCs in rat plasma consistent with other reports. Kinetic studies demonstrated that both were uncompetitive inhibitors of plasma sPLA2 enzyme activity. In vitro experiments with group IA sPLA2 confirmed the inhibition and the kinetic properties of these lysoPC species. Decanoyl lysoPC (10:0), which was not detected in plasma, did not inhibit enzyme activity in vitro. LysoPC injections into normal rats resulted in "buffering" of plasma sPLA2 activity in a narrow low range, consistent with the activity-dependent inhibition suggested by the ex vivo and in vitro experiments. Conclusion The results may explain the efficacy of lysoPC therapy during periods of elevated inflammatory activity and further highlight the utility uncompetitive enzyme inhibitors. In this case, the inhibitor is a product of the enzyme reaction, and therefore represents an example of activity-driven feedback inhibition.

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Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties

Journal of Inflammation Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties Timothy J Cunningham 0 Lihua Yao 0 Angel Lucena 0 0 Address: Neurobiology and Anatomy, Drexel University College of Medicine , Philadelphia, PA 19129 , USA Background: Lysophosphatidylcholines (lysoPCs) are products of phospholipase A2 (PLA2) enzyme activity, and like the enzyme, have a direct role in toxic inflammatory responses in variety of organ systems. Paradoxically, reduced plasma lysoPC levels have been noted in sepsis patients and systemic treatment with lysoPCs is therapeutic in rodent models of sepsis and ischemia. These observations suggest that elevation of plasma levels of these lipids can actually help to relieve serious inflammatory conditions. We demonstrate that specific lysoPCs act as uncompetitive product inhibitors of plasma secreted PLA2 enzymes (sPLA2s), especially under conditions of elevated enzyme activity, thus providing a feedback mechanism for the observed anti-inflammatory effects of these compounds. Methods: Thin layer chromatography and mass spectroscopy were used to estimate total lysoPC concentration and the relative contributions of different lysoPC species in rat plasma samples. Kinetic studies of sPLA2 enzyme activity were conducted on these samples ex vivo and on purified group IA sPLA2 in vitro after addition of specific lysoPC species to the reaction mixture. Enzyme activity was also measured in plasma samples of rats injected with these same lysoPCs. Results: Palmitoyl (16:0), stearoyl (18:0) are the most abundant lysoPCs in rat plasma consistent with other reports. Kinetic studies demonstrated that both were uncompetitive inhibitors of plasma sPLA2 enzyme activity. In vitro experiments with group IA sPLA2 confirmed the inhibition and the kinetic properties of these lysoPC species. Decanoyl lysoPC (10:0), which was not detected in plasma, did not inhibit enzyme activity in vitro. LysoPC injections into normal rats resulted in "buffering" of plasma sPLA2 activity in a narrow low range, consistent with the activity-dependent inhibition suggested by the ex vivo and in vitro experiments. Conclusion: The results may explain the efficacy of lysoPC therapy during periods of elevated inflammatory activity and further highlight the utility uncompetitive enzyme inhibitors. In this case, the inhibitor is a product of the enzyme reaction, and therefore represents an example of activitydriven feedback inhibition. - Background Upregulation of circulating phospholipase A2 enzymes, principally the secreted isoforms (sPLA2s), is associated with the activity of the innate immune system and a number of inflammatory disorders [1-4]. Experimental and correlative studies suggest increased levels of sPLA2s in the blood contribute to or are predictive of the tissue destruction that occurs following trauma, heart and lung disease, local and systemic infections, brain damage, and autoimmune disorders [4-9]. PLA2 enzyme activity and the lipid mediators regulated by that activity are directly linked to a variety of cell death effector mechanisms including the production of reactive oxygen species (both directly and via inflammatory cells), excitotoxicity, and expression of the death receptor family members in a variety of cells [10-12]. Many of these activities have been studied relative to the fatty acid branch of PLA2 pathways, which includes arachidonic acid and its metabolites (prostanoids, leukotrienes)[13]. The other products of PLA2 hydrolysis, the lysophosphatides, are also potent biological mediators but their mechanisms are more enigmatic and their actions are often contradictory[14]. There is current interest in lysophosphatidylcholines (lysoPCs) in particular, as some of these are proposed for treatment of systemic inflammatory disorders. This suggestion is based on reports that plasma lysoPC levels are diminished with the onset of sepsis in human patients [15,16], and in rodent models of sepsis and ischemia, lysoPC treatment is an effective therapy [17-21]. In previous studies, we investigated sPLA2 enzyme activity in plasma of rats and humans because inhibition of this group of enzymes, and subsequent tissue repair and protection, is a long-standing goal of pharmacotherapeutics [22]. Our studies were expedited by the fact that plasma sPLA2 enzymes will follow Michaelis-Menten kinetics when incubated with a broad-spectrum substrate, even though the specific classes of sPLA2s active in plasma are diverse. This property made it possible to characterize a peptide inhibitor of sPLA2 activity (called CHEC-9), also with broad-spectrum activity, as well as demonstrate the therapeutic potential of sPLA2 inhibition in vivo. CHEC-9 treatment of both traumatic and autoimmune models of neurodegeneration resulted in significant cytoprotection and reduction of cell-mediated inflammation [5,6,23]. These results have been supported by other experime (...truncated)


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Timothy J Cunningham, Lihua Yao, Angel Lucena. Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties, Journal of Inflammation, 2008, pp. 17, 5, DOI: 10.1186/1476-9255-5-17