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Thrombin stimulation of human endothelial cell phospholipase D activity.
Regulation by phospholipase C, protein kinase C, and cyclic adenosine
3'5'-monophosphate
JG Garcia, JW Fenton and V Natarajan
Department of Medicine, Indiana University School of Medicine,
Indianapolis.
The activation of membrane-bound phospholipase D (PLD) resulting in the
generation of phosphatidic acid (PA) is increasingly recognized as an
integral event in the initiation of a variety of cellular responses. We
explored whether alpha-thrombin is a physiologic agonist for PLD activation
in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were
labeled with [32Pi] and PLD activity determined by formation of the PLD
metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol
by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased
PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with
maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a
threefold to fourfold increase in PA and a sixfold to eightfold increase in
PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10
nmol/L) and gamma- thrombin (1 mumol/L), but not inactive
DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of
Ca2+ flux in human endothelial cell PLD activation was investigated and PEt
formation was significantly enhanced by Ca2+ ionophores A23187 and
ionomycin (1 mumol/L, three-fold to fourfold increase in PEt).
Alpha-Thrombin- stimulated PEt formation was abolished (greater than 90%
inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment
with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30%
inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5
mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced
alpha-thrombin-induced PEt formation in a dose-dependent manner (10
mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment
(18 hours) also resulted in marked inhibition of alpha- thrombin-induced
PEt formation. Neither pertussis nor botulinum C bacterial toxins
significantly altered alpha-thrombin-induced PLD responses. In contrast,
similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes)
consistently augmented alpha-thrombin- stimulated PLD activity by 50% to
90%. Comparable results were observed with agents which increased cAMP such
as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin
augmentation was abolished by 2- dideoxyadenosine, a competitive inhibitor
of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin
is a potent stimulus for human PLD-mediated PA formation and that cyclic
adenosine nucleotides modulate agonist-induced cellular PLD activity. In
this model of PLD activation, alpha-thrombin receptor occupancy leads to
the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by
phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT
TRUNCATED AT 400 WORDS)
Volume 79,
Issue 8,
pp. 2056-2067,
04/15/1992
Copyright © 1992 by The American Society of Hematology
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