Inhibition of Phosphatidylinositol 4-phosphate 5-kinase by Cyclic AMP in Human Platelets.
The effects of cyclic AMP (cAMP) on phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) synthesis were examined in human platelets. In (32)P-prelabeled intact platelets, although the level of [(32)P]phosphatidylinositol 4-phosphate (PI 4-P) was increased by forskolin and prostaglandin-I(2) (PGI(2)), the formation of [(32)P]PI 4,5-P(2) time-and concentration-dependently decreased, suggesting inhibition of phosphatidylinositol 4-phosphate 5-kinase (PI 4-P 5-kinase). In saponin-permeabilized platelets, formation of PI 4-P and PI 4,5-P(2) can be measured by utilizing [γ-(32)P] ATP. In this system, PGI(2) and cAMP inhibited the generation of [(32)P)PI 4,5-P(2). The PI 4-P 5-kinase activity was mostly located in the platelet membrane fraction and was inhibited by cAMP; H-8 and H-89, inhibitors of cAMP-dependent protein kinase (PKA), abolished this inhibitory effect, suggesting that cAMP exerted its action on PI 4-P 5-kinase via PKA. Adenosine, which is reported to directly inhibit phosphatidylinositol 4-kinase (PI 4-kinase) in some types of cells, had no effect on platelet membrane PI 4-P 5-kinase activity. In dbc AMP-pretreated membranes, PI 4-P 5-kinase activity was lower than that of control membranes. The involvement of PKA with the inhibitory action of cAMP in PI 4-P 5-kinase activity was further confirmed using the catalytic subunit of PKA. The synthesis of [(32)P] PI 4,5-P(2) in permeabilized platelets and the specific activity of partially purified PI 4-P 5-kinase were decreased by incubation with the PKA catalytic subunit. The present results indicate that the cAMP-PKA system inhibits PI 4-P 5-kinase activity, leading to decreased formation of PI 4,5-P(2) in human platelets.
Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, 500, Japan.
T
T
Suzuki
S
S
Nakashima
Y
Y
Nozawa