Date of Award

Fall 1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences

Committee Director

Miriam D. Rosenthal

Committee Member

Peter F. Blackmore

Committee Member

William J. Wasilenko

Committee Member

Laura K. Moen

Abstract

The 85 kDa cytosolic phospholipase A2 (cPLA2) is an agonist-responsive effector for intracellular signal transduction through the arachidonate cascade. In vitro 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 in situ mechanisms which govern the activity of cPLA2. The primarily objective of these studies was to develop an in situ system for the study of cPLA2, and investigate mobilization of arachidonate during signal transduction events.

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 vitro. Although undifferentiated U937 cells have exceptionally high quantities of cPLA2, A23187-stimulated arachidonate mobilization was low, and not specific for arachidonate. Thus, differentiation of U937 induced cPLA2 regulatory elements that mediate arachidonate mobilization.

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.

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.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/0zd2-wj86

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