In Situ Regulation of Cytosolic Phospolipase A₂

<p>The 85 kDa cytosolic phospholipase A₂ (cPLA₂) is an agonist-responsive effector for intracellular signal transduction through the arachidonate cascade.<em> In vitro</em> studies have demonstrated that this enzyme is regulated by sub-micromolar calcium and is specific for arachidonate as the sn-2 fatty acyl group of phospholipid substrates. However, very little data is available regarding <em>in situ</em> mechanisms which govern the activity of cPLA₂. The primarily objective of these studies was to develop an<em> in situ</em> system for the study of cPLA₂, and investigate mobilization of arachidonate during signal transduction events.</p> <p>Dimethylsulfoxide differentiation of the human lymphoma cell line, U937, induced an enhanced capacity to mobilize arachidonate in response to the calcium ionophore A23187. The arachidonate mobilizing activity in differentiated cells was consistent with characteristics reported for cPLA₂ in<em> vitro</em>. Although undifferentiated U937 cells have exceptionally high quantities of cPLA_<em>2</em>, A23187-stimulated arachidonate mobilization was low, and not specific for arachidonate. Thus, differentiation of U937 induced cPLA_<em>2</em> regulatory elements that mediate arachidonate mobilization.</p> <p>Differentiation induced significant changes in the capacitative pathway of intracellular calcium elevation. Both the size of intracellular calcium stores, as well as the characteristics of calcium influx channels were altered with differentiation. Agonist-stimulated arachidonate mobilization was coupled to these differentiation-induced alterations. cPLA₂ activity was initiated upon agonist-stimulated depletion of intracellular calcium stores, and continued until maximum elevations of intracellular free calcium were attained. The data suggest that cPLA₂ may be coupled to the generation of a calcium influx factor, which serves as a communication link between intracellular calcium stores and store-operated calcium influx channels. Consistent with this hypothesis, exogenous free arachidonate activated calcium influx in differentiated U937, consistent with activation of store-operated capacitative calcium influx channels.</p> <p>Based on the data obtained in this study, a model for agonist-stimulated cPLA₂ activity is presented. This model suggests a novel role for cPLA₂. Apart from the well known role in initiation of the arachidonate cascade, cPLA₂ may be part of an intracellular effector system which regulates agonist-stimulated influx of extracellular calcium during activation of the capacitative pathway.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biological Sciences

Year dc:date.available

1994

Author dc:creator

• Rzigalinski, Beverly A.

Contributors dc:contributor

• Miriam D. Rosenthal
• Peter F. Blackmore
• William J. Wasilenko
• Laura K. Moen

• Phospholipase A2
• Biochemistry
• Cell Biology
• Pharmacology

Repository record dc:identifier

https://digitalcommons.odu.edu/biomedicalsciences_etds/134

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biomedicalsciences_etds-1130