Cell Signal. 2004 Jun;16(6):751-62
Role of an aprotinin-sensitive
protease in protein kinase Calpha-mediated activation of
cytosolic phospholipase A2 by calcium ionophore (A23187) in
pulmonary endothelium.
Chakraborti S, Michael JR, Chakraborti T.
Department of Biochemistry and Biophysics, University of
Kalyani, Kalyani 741235, West Bengal, India.
saj_chakra@rediffmail.com
Treatment of bovine pulmonary artery endothelial cells with
the calcium ionophore, A23187, stimulates the cell membrane
associated protease activity, phospholipase A2 (PLA2) activity,
and arachidonic acid (AA) release from the cells. Pretreatment
of the cells with arachidonyl-trifluomethylketone (AACOCF3), a
cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2
inhibitor, prevents A23187 stimulated PLA2 activity and AA
release without producing an appreciable alteration of the
protease activity. Pretreatment of the cells with aprotinin, an
ambient protease inhibitor, prevents the increase in the
protease activity and cPLA2 activity in the membrane and AA
release from the cells caused by both low and high doses of
A23187, and also inhibits protein kinase C (PKC) activity caused
by high doses of A23187. Immunoblot study of the endothelial
cell membrane isolated from A23187 (10 microM)-treated cells
with polyclonal PKCalpha antibody elicited an increase in the 80
kDa immunoreactive protein band along with an additional 47 kDa
immunoreactive fragment. Pretreatment of the cells with
aprotinin abolished the 47 kDa immunoreactive fragment in the
immunoblot. Immunoblot study of the endothelial membrane with
polyclonal cPLA2 antibody revealed that treatment of the cells
with A23187 dose-dependently increases cPLA2 immunoreactive
protein profile in the membrane. It therefore appears from the
present study that treatment of the cells with a low dose of
A23187 (1 microM) causes a small increase in an
aprotinin-sensitive protease activity and that stimulates cPLA2
activity in the cell membrane without an involvement of PKC. By
contrast, treatment of the cells with a high dose of 10 microM
of A23187 causes optimum increase in the protease activity and
that plays an important role in activating PKCalpha, which
subsequently stimulates cPLA2 activity in the cell membrane.
Although pretreatment of the cells with pertussis toxin caused
ADP ribosylation of a 41 kDa protein in the cell membrane, it
did not inhibit the cPLA2 activity and AA release caused by both
low and high doses of A23187.
PMID: 15093616 |
