MOCINNAMYL(AMINO)ETHYL]-5-ISOQUINOLINESULPHONAMIDE (H-89) INHIBITS INCORPORATION OF CHOLINE INTO PHOSPHATIDYLCHOLINE VIA INHIBITION OF CHOLINE KINASE AND HAS NO EFFECT ON THE PHOSPHORYLATION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE

We have shown previously that mocinnamyl(amino)ethyl]-5-isoquinolinesu lphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent prote in kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cel ls. In the present study, we elucidated the mechanism underlying the d escribed inhibition. Treatment of cells with 10 mu M H-89 had no effec t on the phosphorylation of CTP:phosphocholine cytidylyltransferase. H owever, H-89 slightly affected the distribution of cytidylyltransferas e between cytosol and membranes, but the cellular 1,2-diacylglycerol c ontent was not influenced. Furthermore, pulse-chase experiments reveal ed that H-89 did not affect cytidylyltransferase activity. Instead, H- 89 inhibited choline kinase, the enzyme catalysing the first step in t he CDP-choline pathway. In the presence of 10 mu M H-89, choline kinas e activity was inhibited by 36+/-7.6 % in vitro. Additionally, the pho sphorylation of choline to phosphocholine was inhibited by 30+/-3% in cell-culture experiments. This inhibitory effect could be partly preve nted by simultaneous addition of 10 mu M forskolin, indicating that ch oline kinase is regulated in part by PKA activity.