phosphatidylethanol and 1-2-dioctanoylglycerol

The effects of 4 beta-phorbol-12-myristate-13-acetate (PMA) and 1,2-sn-dioctanoylglycerol (DOCG) on the rate of labeling of phosphatidylcholine (PC) with (32P)phosphate and the rate of formation of (3H)phosphatidylethanol (PET) from PC labeled with (3H)myristic acid were investigated in vitro in minced placentae obtained from first trimester human pregnancies. Maximally effective concentrations of PMA (1 microM) or DOCG (125-250 microM) stimulate PC-labeling with (32P)phosphate along different time courses: responses to DOCG and PMA require 30 and 60 min, respectively. The early response to DOCG is attended by a rapid accumulation of 32P)PCDOCG followed by a decline from the peak value in the second 30 min. The PMA effect is accompanied by increased rate of formation of (32P)phosphatidic acid (PA). Importantly, the effects of PMA and DOCG on PC-labeling are additive and PMA does not have any effect on the labeling of PCDOCG. These findings indicate that PMA stimulates degradation and the attendant turnover of PC, whereas a greater part of the DOCG-effect comes from the stimulation of PC synthesis de novo. Consistent with this notion is the finding that PMA enhances the PC-selective phospholipase D activity (measured by the formation of PET) 2.4-fold, whereas the effect of DOCG is smaller (1.4-1.8-fold) and not additive with that of PMA. The results provide evidence for the presence of functional PC-cycle in the primordial human placenta. The cycle can be triggered by a single addition of PMA and to a lesser extent by DOCG. The smaller effect of DOCG may be related to its short lifetime in the tissue, which is sufficient, however, to stimulate the activity of the regulatory enzyme (CTP: choline cytidylyl transferase) of PC synthesis. Since the effect of PMA on PC-labeling is diminished by protein kinase C inhibitors, this enzyme appears to be involved in the stimulation of PC-cycle by DAG and its analogs.

Topics: Diglycerides; Female; Glycerophospholipids; Humans; In Vitro Techniques; Kinetics; Myristic Acid; Myristic Acids; Phosphates; Phosphatidic Acids; Phosphatidylcholines; Phospholipids; Phosphorus Radioisotopes; Placenta; Pregnancy; Signal Transduction; Tetradecanoylphorbol Acetate; Tritium

Surfactant phospholipid secretion can be stimulated by a variety of agonists acting via a number of signal-transduction mechanisms. To determine whether phospholipase D has a role in surfactant secretion, we examined phosphatidylethanol formation in response to surfactant secretagogues in primary cultures of rat type II cells. Phosphatidylethanol formation was stimulated by ATP, 12-O-tetradecanoylphorbol-13 acetate (TPA), and dioctanoylglycerol, surfactant secretagogues that also activate protein kinase C. Surfactant secretagogues that act via other signaling mechanisms had no effect on phosphatidylethanol formation. The effect of ATP on phosphatidylethanol formation was dependent on time, with the maximum stimulation being achieved in approximately 10 min. It was also dependent on ATP concentration. The ATP concentration eliciting 50% of the maximum effect (EC50) was 2.45 x 10(-6) M. This was similar to the EC50 reported for ATP stimulation of surfactant secretion. ATP analogues also stimulated phosphatidylethanol formation with a potency order generally similar to that reported for surfactant secretion. The effects of ATP, TPA, and dioctanoylglycerol were antagonized by protein kinase C inhibitors. We speculate that activation of protein kinase C either directly by TPA and dioctanoylglycerol or indirectly subsequent to phosphoinositide-specific phospholipase C activation by ATP leads to initial stimulation of surfactant secretion as well as activation of phospholipase D. The action of phospholipase D on cellular phospholipids then leads to further generation of diacylglycerols, continued activation of protein kinase C, and sustained surfactant secretion.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Glycerophospholipids; Lung; Male; Osmolar Concentration; Phosphatidic Acids; Phospholipase D; Phospholipids; Protein Kinase C; Pulmonary Surfactants; Rats; Tetradecanoylphorbol Acetate