Effects of vasopressin on the synthesis of phosphatidylethanolamines and phosphatidylcholines by isolated rat hepatocytes

The effect of vasopressin on the biosynthesis of phosphatidylcholines and phosphatidylethanolamines was investigated in freshly isolated rat hepatocytes in suspension. Treatment of hepatocytes with vasopressin inhibits the incorporation of [Me-14C]choline into phosphatidylcholines in a dose-dependent manner. The hormone does not affect the uptake, phosphorylation or oxidation of choline. Pulse-chase studies indicate that CTP:cholinephosphate cytidylyltransferase might be subject to hormonal regulation by vasopressin. In contrast with the inhibitory effect of vasopressin on the synthesis of phosphatidylcholines, this hormone stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines in a dose-dependent manner. Pulse and pulse-chase studies with labelled ethanolamine show that the conversion of ethanolaminephosphate to CDPethanolamine as well as the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerol are enhanced. Determination of the effect of vasopressin on the activity of the enzymes of the synthesis de novo of phosphatidylethanolamines demonstrates an increase of the activity of ethanolaminephosphotransferase, probably as a result of the increased amount of diacylglycerol in vasopressin-treated cells.     

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

Okadaic acid inhibits phosphatidylethanolamine biosynthesis in rat hepatocytes.

Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of CTP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes.     

Dissimilar effects of 1-oleoyl-2-acetylglycerol and phorbol 12-myristate 13-acetate on fatty acid synthesis in isolated rat-liver cells

Exogenous 1-oleoyl-2-acetylglycerol (OAG) is known to mimic the action of tumour-promoting phorbol esters in various cell types. However, in isolated rat hepatocytes OAG depressed the rate of de novo fatty acid synthesis and the activity of the key enzyme acetyl-CoA carboxylase (EC 6.4.1.2), in contrast to the pronounced stimulation of both parameters by phorbol 12-myristate 13-acetate (PMA). The inhibition by OAG appeared to be dose- and time-dependent. On the other hand, medium-chain 1,2-diacylglycerols like 1,2-dioctanoyl-sn-glycerol did mimic the stimulatory action of PMA. The anomalous effect of OAG may well be explained by its metabolic breakdown leading to liberation of oleate and subsequent inhibition of acetyl-CoA carboxylase activity by endogenously formed oleoyl-CoA. The stimulatory effects of both PMA and medium-chain diacylglycerols are likely to be mediated by protein kinase C.     

31P and 1H NMR studies of ethanolamine-linked phosphoglycerides metabolism in human T lymphocytes.

Aqueous and organic extracts of peripheral human T lymphocytes and of T lymphoblastoid cell lines have been examinated by 31P and 1H NMR spectroscopy in order to study the metabolism of ethanolamine (Etn) linked phosphoglycerides. The results show that the Etn concentration in the culture medium determines the composition of Etn-containing metabolites and phospholipids. The effect of phorbol esters, stimulating the synthesis and the breakdown of choline-containing phospholipids has been also studied. A phorbol 12-myristate 13-acetate (PMA) dependent membrane phosphatidylethanolamine hydrolysis, presumably mediated by protein kinase C activity, has been demonstrated.     

Role of protein kinase C in the control of vascular prostacyclin: study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca++ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium.

Exposure of isolated rat hepatocytes to glucagon or chlorophenylthio cyclic AMP led to an inhibition of the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-chase experiments and measurement of the activities of the enzymes involved in the CDP-ethanolamine pathway provided evidence that the inhibitory effect of glucagon on the synthesis de novo of phosphatidylethanolamine was not caused by a diminished conversion of ethanolamine phosphate into CDP-ethanolamine. The observations suggested that the glucagon-induced inhibition of the biosynthesis of phosphatidylethanolamine is probably due to a decreased supply of diacylglycerols, resulting in a decreased formation of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerols.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.     

Increase in chemiluminescence induced by receptor-independent stimulation of polymorphonuclear cells from psoriatic patients

Using a lucigenin-dependent chemiluminescence the authors have measured the "respiratory burst" in polymorphonuclear cells from psoriatic patients and controls. Measurements were performed under stimulation with zymosan, phorbol 12-myristate 13-acetate and latex beads. It has been revealed that the response after stimulation with zymosan increased although there was no significant difference between patients and controls, while the response after stimulation with phorbol 12-myristate 13-acetate and latex beads was significantly increased. Our results suggest an enhanced metabolic activity of polymorphonuclear cells induced by stimuli acting independently from cell-membrane receptors. Therefore an enhanced excitability of polymorphonuclear leukocytes of psoriatic patients is supposed.     

Phorbol12-Myristate13-Acetate Relieves the Inhibitory Effect of A23187 on Erythroid Differentiation of K562 Cells Induced by Dimethylsulfoxide

The differentiating effect of DMSO on K562 cells was studied against the background of pretreatment of the cells by the modulators of activities of protein kinase C and Ca signaling, phorbol 12-myristate 13-acetate, and ionophore A23187. The 2-hour pretreatment of K562 cells with A23187 (1 M), rather than phorbol 12-myristate 13-acetate (0.1 M), led to inhibition of the differentiating effect of DMSO (0.1%) during the subsequent four days of incubation. When the cells were pretreated jointly with A23187 and phorbol 12-myristate 13-acetate, the DMSO-induced erythroid differentiation was restored. Analysis of the DNA-degrading effect of the reagents used on K562 cells by fluorescent dyes under the same conditions suggests that this activity is induced in the presence of DMSO in the cells pretreated with a combination of phorbol 12-myristate 13-acetate and A23187 or without pretreatment.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium

The addition of low concentrations of phorbol 12-myristate 13-acetate to rabbit neutrophils induces cell aggregation, degranulation, increased oxygen consumption and an increase in the amount of actin associated with the cytoskeleton without a rise in the level of intracellular free calcium as measured using the fluorescent probe quin-2. The ability of phorbol 12-myristate 13-acetate to initiate neutrophil responses similar to those produced by the chemotactic factor without causing a rise in the level of intracellular free calcium suggests two possibilities; that there is a second messenger in addition to calcium or that it activates the cells at a point distal to calcium mobilization. The possible role of diacylglycerol in neutrophil activation is discussed.     

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.     

Effect of sphingosine and other amphiphilic amines on the biosynthesis of phosphatidylethanolamine and other glycerolipids in isolated rat hepatocytes.

The importance of ethanolamine and sphingosine as precursors of phosphoethanolamine was investigated by incubating them with [3H]glycerol and isolated rat hepatocytes. Sphingosine (0.1--0.5 mM) stimulated the synthesis of phosphatidylethanolamine from [3H]glycerol, but the stimulation by ethanolamine was more pronounced. Furthermore, more phosphoethanolamine accumulated in the heptatocytes after incubation with ethanolamine than after incubation with sphingosine. It is concluded that ethanolamine is the most important phosphoethanolamine precursor in rat liver. Higher concentrations of sphingosine caused accumulation of [3H]phosphatidate and inhibition of total glycerolipid synthesis in isolated hepatocytes, when incubated in the presence of [3H]glycerol. These effects were very similar to those of fenfluramine and norfenfluramine described previously. Simpler cationic amphiphilic amines, like oleoylamine and octadecyltrimethylammonium bromide, also caused these effects. Variation of alkyl chain length and amphiphile charge showed that both a positive charge and a certain alkyl chain length were necessary for interference with phosphatidate metabolism. A much wider range of compounds inhibited total glycerolipid synthesis from [3H]glycerol.     

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.