Contribution of lipid mediators to the regulation of phosphatidylcholine synthesis by angiotensin.

Angiotensin stimulates a cellular mitogenic response via the AT1 receptor. We have examined the effect of angiotensin on the rate of phosphatidylcholine (PC) synthesis and have begun to dissect the pathway linking the AT1 receptor to the rate-limiting enzyme in PC synthesis, CTP: phosphocholine cytidylyltransferase (CCT), using CHO cells engineered to express the AT1a receptor. Since CCT can be directly activated by lipid mediators, we probed for their involvement in the PC synthesis response to angiotensin. Angiotensin stimulated CCT activity and PC synthesis two- to threefold after a 30-min delay. The kinetics of this stimulation most closely paralleled an increase in diacylglycerol (DAG) derived from myristic acid-enriched phospholipids. The production of arachidonic acid, phosphatidic acid, or reactive oxygen species either peaked much earlier or not at all. Moreover, manipulation of the intracellular supply of oxygen free radicals, arachidonic acid, HETEs, or phosphatidic acid (using inhibitors and/or exogenous addition) did not generate parallel effects on the rate of PC synthesis. Restricting the production of DAG by inhibition of PLCbeta with U73122 reduced both basal and angiotensin-stimulated PC synthesis. The U73122 inhibition of PC synthesis was accompanied by a similar inhibition of ERK1/2 phosphorylation. Addition of exogenous DAG stimulated basal and angiotensin-dependent PC synthesis, and partially reversed the effect of the PLC inhibitor on PC synthesis. These results do not provide support for lipid mediators as direct stimulators of CCT and PC synthesis downstream of angiotensin, but give rise to the idea that angiotensin effects might be mediated via ERK1/2.

Angiotensin stimulates phosphatidylcholine synthesis via a pathway involving diacylglycerol, protein kinase C, ERK1/2, and CTP:phosphocholine cytidylyltransferase.

We are probing the regulation of phosphatidylcholine (PC) synthesis by angiotensin II. In the accompanying paper, we showed that manipulation of the lipid second messengers, arachidonic acid or hydroxyeicosatetraenoic acid, produced downstream of the angiotensin AT1a receptor did not affect the PC synthesis rates in a manner consistent with direct activation of the rate limiting enzyme in the pathway, CTP:phosphocholine cytidylyltransferase (CCT). However, suppression of diacylglycerol (DAG) production with an inhibitor of phospholipase C-beta reduced angiotensin-dependent PC synthesis as well as ERK1/2 phosphorylation. Here, we show that the stimulation of PC synthesis and activation of CCT by angiotensin requires a signaling pathway that involves protein kinase C and ERK1/2. The inhibitors bis-indolylmaleimide I and PD98059 blocked ERK1/2 phosphorylation and completely eliminated angiotensin stimulation of the CCT-catalyzed reaction and PC synthesis. Exogenous addition of DAG using a lipid vesicle delivery system exactly mimicked the kinetics of angiotensin-promoted PC synthesis, suggesting that this mode of DAG delivery can effectively substitute for the DAG generated downstream of the activated AT1a receptor. Moreover, exogenous DAG activated ERK1/2, and the activation of PC synthesis by DAG was blocked by inhibition of protein kinase C and MEK. These data suggest that angiotensin-dependent DAG and the exogenously supplied DAG stimulate PC synthesis, not solely by direct action on CCT, but via a signaling pathway involving protein kinase C and ERK1/2. Angiotensin did not alter the net phosphorylation state of CCT as probed by immunoprecipitation of 32P-labeled CCT. Angiotensin stimulation of ERK1/2 likely mediates effects on CCT via a process other than CCT dephosphorylation.

Contribution of lipid second messengers to the regulation of phosphatidylcholine synthesis during cell cycle re-entry.

During entry into the cell cycle a phosphatidylcholine (PC) metabolic cycle is activated. We have examined the hypothesis that PC synthesis during the G(0) to G(1) transition is controlled by one or more lipid products of PC turnover acting directly on the rate-limiting enzyme in the synthesis pathway, CTP: phosphocholine cytidylyltransferase (CCT). The acceleration of PC synthesis was two- to threefold during the first hour after addition of serum to quiescent IIC9 fibroblasts. The rate increased to approximately 15-fold above the basal rate during the second hour. The production of arachidonic acid, diacylglycerol (DAG), and phosphatidic acid (PA) preceded the second, rapid phase of PC synthesis. However, an increase in the cellular content of these lipid mediators was detected only for DAG. CCT activation and translocation to membranes accompanied the second phase of the PC synthesis acceleration. Bromoenol lactone (BEL), an inhibitor of calcium-independent phospholipase A(2) and PA phosphatase, blocked production of fatty acids and DAG, inhibited both phases of the PC synthesis response to serum, and reduced CCT activity and membrane affinity. The effect of BEL on PC synthesis was partially reversed by in situ generation of DAG via exogenous PC-specific phospholipase C to generate approximately 2-fold elevation in PC-derived DAG. Exogenous arachidonic acid also partially reversed the inhibition by BEL, but only at a concentration that generated a supra-physiological cellular content of free fatty acid. 1-Butanol, which blocks PA production, had no effect on DAG generation, or on PC synthesis. We conclude that fatty acids and DAG could contribute to the initial slow phase of the PC synthesis response. DAG is the most likely lipid regulator of CCT activity and the rapid phase of PC synthesis. However, processes other than direct activation of CCT by lipid mediators likely contribute to the highly accelerated phase during entry into the cell cycle.