Phosphatidate-dependent protein phosphorylation was observed in soluble extracts from rat liver, brain, lung, and testis. The phosphorylation was stimulated by free Ca2+ in the range of 360-0 nM. Incubation mixtures contining phosphatidate provided markedly different profiles of protein phosphorylation from thise with phosphatidylserine plus 1,2-diolein. Phosphatidate-dipendent phosphorylation of a 30-kDa protein in the soluble fraction from heart was also observed. This phosphorylation did not require Ca2+. Soluble fractions from liver, testis, brain, and lung phosphorylated the 30-kDa heart protein in a phosphatidate-dependent Ca2+-independent manner. We propose that part of the action of phosphatidate in cells may be mediated by a protein kinase(s).Phosphatidic acid (PA) has been suggested to be a second messenger (1, 2) and recently has been the object of much study. In a wide variety of cell types, a phospholipase D acting on phosphatidylcholine has been shown to be activated rapidly by diverse agonists (for review, see ref.3). Agonists can also elicit PA through the phosphorylation of diacylglycerol and through a phospholipase D acting on phosphatidylinositol (4-6). In studies designed to investigate the cellular actions of PA, workers have incubated cells with exogenous PA or with microbial phospholipase D. These studies have implicated PA in the regulation of DNA synthesis (7-10); platelet aggregation (11); superoxide formation by neutrophils (12); the induction of c-myc (7), c-fos (7), and plateletderived growth factor (10); the release of insulin (13) (Harlan-Sprague-Dawley) were killed by cervical dislocation. Their tissues were removed immediately and placed in ice-cold 250 mM sucrose/1 mM EGTA/5 mM NaHepes, pH 7.5, containing leupeptin (40 ,ug/ml) and antipain (40 ,ug/ml). The tissues were minced in this buffer, washed several times by decantation, and homogenized in this buffer (28 ml/g of tissue) using a Dounce homogenizer. The homogenates were centrifuged at 37,000 x g for 20 min and the supernatant fraction was removed for assay.

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTPyS stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone-dependent phospholipase D activity that can also catalyze the formation of phosphatidylethanol.

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Background:The role of extracellular loop regions during substrate translocation in the serotonin transporter (SERT) is not well understood. Results: A mutation in the extracellular loop 4 of SERT disrupts the conformational equilibrium by favoring an outward-facing conformation. Conclusion: Extracellular loop 4 is important for conformational transitions in SERT.Significance: New insights are gained into the coordinated conformational changes associated with substrate translocation in SERT.