D-galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.     

Effect of thyroid hormones and diacylglycerols on sphingomyelin metabolism in liver cell nuclei in rats of various ages]

Sphingomyelin metabolism in liver cell nuclei of rats of various age has been studied. It was found that the level of sphingomyelin hydrolysis in cell nuclei is the highest in young animals, showing a decrease in 24-month-old animals. The age-specific fluctuations in the activity of phospholipase C may be one of possible reasons for sphingomyelinase activity changes in liver nuclei during ontogenesis. It has been shown that thyroid hormones and diacylglycerols control the sphingomyelinase activity in rat liver cells.     

Phospholipid dependence of the neutral sphingomyelinase in rat liver plasma membranes

Investigations have been carried out on the influence of the phospholipid composition of rat liver plasma membranes and of their physico-chemical properties on the activity of membrane-bound neutral sphingomyelinase. The membrane phospholipid composition was modified by the incorporation of different phospholipids into the membrane bilayer by means of lipid transfer proteins, n-butanol delipidation or exogenous sphingomyelinase (Staphylococcus aureus) treatment. The results indicate that the activity of neutral sphingomyelinase in liver plasma membranes depends upon phosphatidyl choline presence in the membrane bilayer and not upon membrane fluidity.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.     

Pancreastatin activates pertussis toxin-sensitive guanylate cyclase and pertussis toxin-insensitive phospholipase C in rat liver membranes

We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10^?7 M pancreastatin (EC₅₀ = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30°C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10^?7 M pancreastatin at 10 min (EC₅₀ = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2–5 min) at 30°C by 10^?7 M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10^?7 M pancreastatin, maximal stimulation was obtained (EC₅₀ = 3 nM). GTP (10^?5 M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10^?5 M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with pertussis toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas GMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin. In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, pertussis toxin-sensitive, and -insensitive, respectively. © 1994 Wiley-Liss, Inc.     

Human high density lipoprotein (HDL3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL3 to purified rat liver plasma membranes was studied. 125I-labeled HDL3 bound to the membranes with a dissociation constant of 10.5 micrograms protein/ml and a maximum binding of 3.45 micrograms protein/mg membrane protein. The 125I-labeled HDL3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-labeled HDL3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL3. The 125I-labeled HDL3-binding activity of the membranes was not sensitive to pronase or phospholipase A2; however, prior treatment of the membranes with phospholipase A2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100 degrees C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL3-binding activity.     

Lipocortin I and lipocortin II inhibit phosphoinositide- and polyphosphoinositide-specific phospholipase C. The effect results from interaction with the substrates

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide- and polyphosphoinositide-specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67 microM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol-labeled HL-60 cells, lipocortin II did not affect PI-specific phospholipase C activity in the absence or presence of calcium plus or minus GTP-gamma-S.     

Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes

The influence of the phospholipid composition and the physico-chemical properties of rat liver plasma membranes on the activity of membrane-bound phospholipase A2 has been investigated. The plasma membrane composition was modified by the aid of exogenous phospholipases A2, C and D, and by butanol treatment. The partially delipidated membranes thus obtained were enriched with different phospholipids. The steady-state fluorescent anisotropy of 1,6-diphenyl-1,3,5-hexatriene and the lipid order parameter-SDPH in the modified membranes were calculated. It was established that the activity of the membrane-bound phospholipase A2 was higher in rigid membranes and was decreased when the membrane lipid bilayer was fluidized.     

Different membrane-bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

In the present study we have determinated the acetylcholinesterase molecular forms present in rat liver hepatocytes; we have also studied the association of acetylcholinesterase with the cell surface of the hepatocytes. Subcellular fractionation indicated that rough endoplasmic reticulum and plasma-membrane-enriched fractions contains G4 and G2 acetylcholinesterase forms bound to membranes. Hepatocytes incubated with phosphatidylinositol-specific phospholipase C released about 70% of the surface acetylcholinesterase. Sedimentation analysis showed that all the solubilized acetylcholinesterase activity comes exclusively from a G2 dimer. The G4 hydrophobic form of acetylcholinesterase accounts for the additional cell-surface activity. The existence of these two forms of acetylcholinesterase on the surface of hepatocytes was further established by analyzing the phosphatidylinositol-specific phospholipase C sensitivity of the acetylcholinesterase molecular forms present in isolated rat liver plasma membranes.     

Change in membrane lipid fluidity induced by phospholipase A activation: a mechanism of endotoxic shock

The effects of endotoxin administration on the fluidity of dog liver plasma membranes and their relationship with changes in phospholipase A2 activity were studied. Endotoxin administration decreased the fluidity of liver plasma membranes and this decrease was reversible by phosphatidylcholine. The endotoxin-induced decrease in membrane fluidity could be mimicked by digesting control liver membranes with exogenous phospholipase A2. Endotoxin administration also increased the endogenous phospholipase A2 activity. Endotoxin in vitro had no phospholipase A2-like activity but it activated the hydrolytic activity of exogenous phospholipase A2. Based on these data, it is concluded that endotoxin administration decreased the fluidity of canine liver plasma membranes by acting through activation of phospholipase A2. The decrease in membrane lipid fluidity induced by endotoxin administration may play a significant role in the development of the pathophysiology of endotoxic shock at the cellular level.     

Acceleration by ceramide of calcium-dependent translocation of phospholipase A2 from cytosol to membranes in platelets

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Calpha, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.     

5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol

We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix.     

Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.     

Effect of liver plasma membrane fluidity on endogenous phospholipase A2 activity

Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzyme's microenvironment.     

Phospholipase C activation by ethanol in rat hepatocytes is unaffected by chronic ethanol feeding.

The activation of phosphoinositide-specific phospholipase C by ethanol was compared in hepatocytes isolated from ethanol-fed rats and from pair-fed control animals. Ethanol (100-300 mM) caused a dose-dependent transient increase in cytosolic free Ca2+ levels in indo-1-loaded hepatocytes from both groups of animals. The rate of Ca2+ increase was similar in hepatocytes from control and ethanol-fed rats, but the decay of the Ca2+ increase was somewhat slower in the latter preparation. The ethanol-induced Ca2+ increase caused activation of glycogen phosphorylase, with 50% response at 50 mM-ethanol and a maximal response at 150-200 mM-ethanol, not significantly different in hepatocytes from control and ethanol-fed animals. Ins(1,4,5)P3 formation in response to ethanol (300 mM) or vasopressin (2 nM or 40 nM) was also similar in the two preparations. It is concluded that long-term ethanol feeding does not lead to an adaptive response with respect to the ethanol-induced phospholipase C activation in rat hepatocytes. The ability of ethanol in vitro to decrease membrane molecular order in liver plasma membranes from ethanol-fed and control rats was measured by e.s.r. Membranes from ethanol-fed animals had a significantly lower baseline order parameter compared with control preparations (0.313 and 0.327 respectively), indicative of decreased membrane molecular order. Addition of 100 mM-ethanol significantly decreased the order parameter in control preparations by 2.1%, but had no effect on the order parameter of plasma membranes from ethanol-fed rats, indicating that the plasma membranes had developed tolerance to ethanol, similar to other membranes in the liver. Thus the membrane structural changes associated with this membrane tolerance do not modify the ethanol-induced activation of phospholipase C. The transient activation of phospholipase C by ethanol in hepatocytes may play a role in maintaining an adaptive phenotype in rat liver.     

Nuclear membrane sphingomyelin-cholesterol changes in rat liver after hepatectomy.

Sphingomyelin and cholesterol play an important role in stabilising the plasma membranes architecture and in many physiological process such as cell growth and differentiation. Degradation of sphingomyelin by exogenous sphingomyelinase induces a decrease of cholesterol due either to an increase of esterification or to a reduced biosynthesis. Variations of sphingomyelin due to the presence of a neutral-sphingomyelinase and of sphingomyelin-synthase have been recently shown in rat liver nuclear membranes. The aim of this research is to study the relation between sphingomyelin and cholesterol in the nuclear membranes following sphingomyelinase activation and during cell proliferation. The nuclear membranes, isolated from liver nuclei, were analysed for their content in protein, nucleic acids, and lipids (sphingomyelin and cholesterol) before and after sphingomyelinase activation and during hepatic regeneration. The activities of nuclear membrane SM-syntase and sphingomyelinase were also determined. The results confirmed that also in the nuclear membranes sphingomyelinase, especially exogenous, causes a strong decrease in cholesterol. The increase observed of sphingomyelin during the first 18 h after hepatectomy followed by a decrease at 24 h, due to the different activity of the enzymes, is accompanied by similar behaviour of cholesterol. This confirms the effect of neutral-sphingomyelinase on cholesterol, due to an increase of esterification process. Changes in cholesterol content modify the nuclear membranes fluidity and, as consequence, mRNA transport as previously shown. It can therefore be concluded that the neutral sphingomyelinase, present in the nuclei, may, across this mechanism, regulate the cell function.     

Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet.

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.     

Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5-kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin-hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the phosphatidylcholine-hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster. As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli. The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20-30-fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2.     

Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement.

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.