Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester.

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.

Phosphatidylserine decarboxylase is located on the external side of the inner mitochondrial membrane.

It is shown that the trypsin-treatment of rat liver mitochondria, depleted of the outer membrane, causes a strong inactivation of phosphatidylserine decarboxylase. This inactivation is dependent on trypsin concentration and the time of digestion in a similar manner as the inactivation of cytochrome oxidase. Under these conditions only a moderate inactivation of succinate dehydrogenase is observed. Phosphatidylserine decarboxylase is thus localized in the outer leaflet of the inner mitochondria membrane or, at least, is accessible from the outer surface of the inner membrane.

Serine base-exchange in rat liver nuclei.

It has been shown that the incorporation of [(14)C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base-exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells.

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase.

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.

Lipids and signal transduction in the nucleus.

During the last few years a growing amount of data has accumulated showing phospholipid participation in nuclear signal transduction. Very recent data strongly support the hypothesis that signal transduction in the nucleus is autonomic. Local production of inositol polyphosphates, beginning with the activation of phospholipase C is required for their specific function in the nucleus. Enzymes which modify polyphosphoinositols may control gene expression. Much less information is available about the role of other lipids in nuclear signal transduction. The aim of this minireview is to stress what is currently known about nuclear lipids with respect to nuclear signal transduction.

Is the glutathione conjugate of trans-4-hydroxy-2-nonenal transported by the multispecific organic anion transporting-ATPase of human erythrocytes?

Trans-4-hydroxy-2-nonenal (4-HNE), a cytotoxic end product of lipid peroxidation, is present in normal human blood plasma at concentrations of 0.1-1.0 microM. It can be, however, further metabolized within a cell, and one of the main products is 4-HNE glutathione conjugate (HNE-SG). In human erythrocyte membrane the system for active extrusion of glutathione (GSH) conjugates of various endo- and xenobiotics has been described; it exhibits either a low (Km at submillimolar concentration range) or a high (Km at low micromolar range) affinity for the transported substrates, such as for example S-(2,4-dinitrophenyl)glutathione (Dnp-SG). In the present study it has been shown that the high affinity transport system for Dnp-SG is competitively inhibited by HNE-SG with Ki of 0.2 microM, while 4-HNE inhibits non-competitively the activity of the transport system for Dnp-SG with Ki of 220microM. These observations point to the possibility that HNE-SG shares the same transport system with GSH conjugates of other endo- and xenobiotics in erythrocytes. This may be of importance for overall detoxification of the organism under oxidative stress.

Decarboxylation of phosphatidylserine by rat liver mitochondria.

1. The decarboxylation of phosphatidylserine was studied using particles obtained by sonication of rat liver mitochondria as the source of the enzyme, and liposomes prepared from total microsomal phospholipids labelled in phosphatidylserine. The reaction was followed by measuring formation of either CO2 or phosphatidylethanolamine. 2. The reaction was inhibited when isotonic sucrose was substituted by equiosmotic solutions of electrolytes (acetate or phosphate). 3. Optimum pH for the reaction was 5.0 - 5.2. 4. At pH 7.4 the reaction was stimulated by the cytoplasmic fraction from rat liver, most likely due to the action of phospholipid transfer protein(s). 5. The reaction was stimulated by Mg2+ and Mn2+. Maximum stimulation occurred at 2 - 3 mM-concentration of the divalent cation.

Exogenous sphingosine 1-phosphate and sphingosylphosphorylcholine do not stimulate phospholipase D in C6 glioma cells.

In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phospholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-14C]palmitic acid and PLD-mediated synthesis of [14C]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [14C]phosphatidylethanol formation either at low (0.1-10 microM) or high (25-100 microM) concentrations. On the other hand, sphingosine at concentrations of 100-250 microM strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.

Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal.

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.

Serine base-exchange in rat liver microsomes: effect of phospholipids.

We enriched liver microsomes in lipid classes and molecular species disrupting membranes with octyl glucoside and reassembling them by detergent removal. Phosphatidylethanolamine incorporated into membranes better than phosphatidylserine or phosphatidylcholine. In addition, the degree of incorporation depended on the unsaturation of fatty acyl-chains. The enrichment of the membranes with phosphatidylserine or phosphatidylcholine inhibited serine base-exchange, whereas the addition of phosphatidylethanolamine usually stimulated it. The effect of exogenous lipids also depended on molecular species; egg yolk phosphatidylcholine and dipalmitoyl phosphatidylcholine inhibited base-exchange whereas the effect of palmitoyl-oleoyl phosphatidylcholine depended on the incorporated amount. The degree of unsaturation also modulated the effect of phosphatidylethanolamine.

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomes.

Properties of Ca(2+)-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent K(m) values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their K(m) values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCl. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCl and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were also found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca(2+)-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.