Oleic acid promotes changes in the subcellular distribution of protein kinase C in isolated hepatocytes.

The effect of oleate on the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes and in perfused rat liver in the presence of physiological concentrations of serum albumin. A time- and dose-dependent translocation of PKC from the cytosol towards the membranes was observed at oleate concentrations that fell within the range of concentrations reached under several physiological conditions. Analysis of the membrane-bound isoenzymes of PKC by hydroxylapatite chromatography revealed that the beta isoenzyme was preferentially translocated to this compartment in hepatocytes incubated with oleate. Activation of PKC after incubation of hepatocytes with oleate involved at least three different effectors of the enzyme: the fatty acid itself, the diacylglycerol synthesized from oleate, and the rise in the cytosolic calcium concentration elicited by oleate. As a result of PKC activation, protein phosphorylation of intact hepatocytes in response to oleate exhibited an enhancement in the phosphate content of a protein of 82 kDa, similar to that phosphorylated in the presence of phorbol dibutyrate.     

Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.

Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.     

Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition

The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.     

Mechanism of the stimulating effect of estradiol on protein kinase C in plasma membranes of target cells

Using inhibitors and activators of protein kinase C, it was demonstrated that in isolated plasma membranes of target cells estradiol-17 beta selectively stimulates protein phosphorylation by endogenous protein kinase C. In estradiol-dependent tissues, estradiol effectuates the translocation of protein kinase C from the cytosol to the membrane fraction within 10-12 minutes. Estradiol activates protein kinase C in cellular membranes of target tissues via a mechanism which is different from that of phorbol ester (TPA): 3H-estradiol, in contrast with 3H-TPA, it is not bound by protein kinase C and, in contrast with TPA, estradiol-17 beta does not activate purified protein kinase C in vitro. In this case, the specific stimulation of protein kinase C translocation to membranes and the estradiol-induced increase in the phosphorylation of plasma membrane proteins seem to be due to the estradiol-induced activation of the transmembrane system of polyphosphoinositide degradation, eventually resulting in the formation of diacylglycerol, a protein kinase C activator.     

Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.     

Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on protein kinase C was studied by metabolically labeling GH3 cells with [35S]methionine and using a polyclonal antibody raised against rat brain protein kinase C to immunoprecipitate the enzyme. PMA accelerates the loss of immunologically reactive protein kinase C from the cells in a time- and dose-dependent manner. The half-life of the enzyme in cells treated with 400 nM PMA was 2 h whereas in control cells 60-70% of the enzyme was still detectable after 24 h. The concentration of PMA required to reduce cellular protein kinase C 50% after 24 h was 130 nM. PMA also induced the translocation of [35S]Met-labeled protein kinase C from the cytosol to the membranes in a concentration-dependent manner. Less protein kinase C was translocated to membranes when cells were treated with 20 nM PMA than when they were exposed to 400 nM PMA. In the latter case, most of the labeled protein kinase C became membrane-associated. Maximal translocation was evident after 15 min of incubation with either concentration of PMA and was followed by degradation of the membrane-associated enzyme. The rate of degradation of membrane-associated protein kinase C was the same with both concentrations of PMA. In cells treated with 20 nM PMA, disappearance of [35S]Met-labeled protein kinase C from the cytosolic fraction occurred in two phases, a rapid decrease characteristic of the membrane-associated enzyme, followed by a slower loss similar to that seen in control cells. The results indicate that turnover of protein kinase C is enhanced by membrane association.     

Translocation of Protein Kinase C in Anterior Pituitary Tumor Cells

Previous studies have shown that phorbol esters and lithium each stimulate the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary tumor cell line AtT20/D16-16. Pretreatment with either lithium or phorbol ester desensitizes the cells to subsequent stimulation by phorbol ester. An early consequence of phorbol ester action in other systems is the translocation of protein kinase C from cytosol to membranes. We have assayed protein kinase C activity in cytosol and membranes of AtT20 cells after treatment with phorbol dibutyrate, lithium, or other agents that stimulate secretion of ACTH in these cells. Phorbol dibutyrate clearly induced translocation of protein kinase C, but lithium treatment did not cause translocation itself, nor did pretreatment with lithium affect the translocation induced by phorbol dibutyrate. These results are consistent with a role for translocation of protein kinase C in the stimulatory and desensitizing effects of phorbol esters but fail to implicate translocation in the actions of lithium on AtT20 cells.     

Adrenergic and calcium-mediated subcellular redistribution of protein kinase C in primary neuronal cultures

Incubation of primary neuronal cultures prepared from the brains of neonatal rats with 50 microM epinephrine resulted in the transient redistribution of protein kinase C from the cytosol to the particulate fraction. This effect occurred after 1 and 5 min of incubation and resulted in a decrease in cytosolic protein kinase C activity with a corresponding increase in particulate protein kinase C of approximately 30% and 15%, respectively. The epinephrine-stimulated translocation of protein kinase C was blocked by 1 microM prazosin indicating the involvement of alpha 1-adrenergic receptors. Further, inclusion of 0.1 microM Ca2+ in the homogenization buffer was found to significantly enhance the binding of protein kinase C to cellular membranes prepared from neuronal cultures. These results indicate that alpha 1-adrenergic receptors in neuronal brain cell cultures are linked to the activation of protein kinase C and that the mobilization of Ca2+ may enhance this effect.     

Activation of protein kinase C from B lymphocytes by lipid A

The effect of lipid A, a residue of the lipopolysaccharide molecule, on protein kinase C from B lymphocytes has been studied. Lipid A activates and promotes the translocation of protein kinase C from the soluble to the particulate membrane fraction in a cell-free system reconstituted with purified enzyme and membranes isolated from B lymphocytes. These results demonstrate that the activating effect of lipopolysaccharide on protein kinase C from B cells is due to the lipid moieties of this molecule.     

Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor-Mediated Calmodulin Redistribution in SK-N-SH Neuroblastoma Cells

Abstract: Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 nM TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 µM. In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the α-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the ε-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.     

Translocation of CTP: phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol

Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.     

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Hepatic protein kinase C: translocation stimulated by prolactin and partial hepatectomy

Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.     

Translocation of diacylglycerol kinase in response to chemotactic peptide and phorbol ester in neutrophils

When neutrophils were stimulated by the chemotactic peptide, fMLP, a rapid, transient increase in the activity of diacylglycerol(DG) kinase in the membrane fraction was detected. DG kinase in cytosol, on the contrary, showed a transient decrease. The total activity in homogenates was not affected. Tetradecanoylphorbol acetate(TPA) and 1-oleoyl-2-acetylglycerol(OAG) also caused an increase in DG kinase activity in the membrane fraction. Km value of DG kinase in membranes was not changed by the treatment of fMLP or TPA, though Vmax was increased. Considering these results, DG kinase may translocate from cytosol to membranes on stimulation by fMLP, TPA or OAG in neutrophils. The translocation may play important roles in regulation of protein kinase C activity, since DG kinase competes with protein kinase C for DG, which is formed by receptor-activation.     

Platelet aggregation is inhibited by long chain acyl-CoA

Oleoyl coenzyme A and other acyl-CoA derivatives inhibited ADP or thrombin-induced aggregation of platelets. Arachidonic acid-induced aggregation was also inhibited, but not the slower aggregation caused by 1-oleoyl-2-acetylglycerol or tetradecanoyl-phorbol-13-acetate. Coenzyme A and free fatty acids had little or no effect, and transfer of labeled oleate from oleoyl Co-A to other lipid classes was not detected. Because acyl Co-A compounds have recently been shown to modulate protein kinase C activity, acyl Co-A may provide a useful tool for investigating activation sequences in platelets and other membranes.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.