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Published online 20 February 2001. doi:10.1083/jcb.152.4.717
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© The Rockefeller University Press, 0021-9525/2001/2/717/ $5.00
The Journal of Cell Biology, Volume 152, Number 4, February 19, 2001 717-728


Original Article

A Specific Role of Phosphatidylinositol 3–Kinase {gamma}: A Regulation of Autonomic Ca2+ Oscillations in Cardiac Cells

Claire Bonya, Serge Rocheb, Ueno Shuichic, Takehiko Sasakid, Michael A. Crackowerd, Josef Penningerd, Hiroyuki Manoc, and Michel Pucéatb
a The French Institute of Health and Medical Research, CNRS UPR1086 Montpellier 34293, France
b the Center for Research of Macromolecular Biochemistry, CNRS UPR1086 Montpellier 34293, France
c Division of Functional Genomics, Jichi Medical School, Tochigi, 329-04 Japan
d Amgen Institute, Ontario Cancer Institute, Department of Medical Biophysics and Immunology, Toronto, Ontario, MSG 2C1 Canada

Correspondence to: Michel Pucéat, Centre de Recherches de Biochimie Macromoléculaire, CNRS UPR1086, 1919 route de Mende, 34293 Montpellier, France. Tel:33-4-6761-3350 Fax:33-4-6752-1559 E-mail:puceat{at}crbm.cnrs-mop.fr.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLC{gamma} and generates IP3, a breakdown product of phosphatidylinositol 4,5–bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca2+ spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3–kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5–trisphosphate, regulates activity of PLC{gamma}. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLC{gamma}, Ca2+ homeostasis. However, although PI3K{alpha} and -ß have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3K{gamma}.

We report that neonatal rat cardiac cells in culture express PI3K{alpha}, -ß, and -{gamma}. The purinergic agonist predominantly activates PI3K{gamma}. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLC{gamma} as well as IP3 generation in ATP-stimulated cells. Furthermore, an anti-PI3K{gamma}, but not an anti-PI3Kß, injected in the cells prevents the effect of ATP on cell Ca2+ spiking. A dominant negative mutant of PI3K{gamma} transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca2+ spiking of wild-type but not of PI3K{gamma}2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLC{gamma}. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca2+ spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3K{gamma} antibody and a dominant negative mutant of PI3K{gamma} injected or transfected into cells prevent the latter event.

We conclude that PI3K{gamma} activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca2+ spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3K{gamma} to fully activate PLC{gamma} in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP3 generation and thus cardiac autonomic activity.

Key Words: phosphoinositide kinase, calcium, tyrosine kinase, heart, automaticity


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