1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells

It has recently been proposed that degradation products of sphingolipids may serve as physiologic inhibitors of protein kinase C. The present study was performed to determine the effect of 1,2-diacylglycerols and phorbol esters, known activators of protein kinase C, on sphingomyelin metabolism. 1,2-Dioctanoylglycerol (diC8) caused time- and concentration-dependent reduction in the level of sphingomyelin labeled to equilibrium with [3H]choline. diC8 (200 micrograms/ml) reduced [3H]sphingomyelin to 81 +/- 3% of control (p less than 0.005) by 15 min, and the level was 58 +/- 5% of control after 1 h; an EC50 for this event was 56 micrograms/ml. To evaluate the mechanisms of stimulated hydrolysis, the sphingoid base backbone of sphingomyelin was labeled with [14C] serine, and the effects of diC8 were quantitated. diC8 (100 micrograms/ml) reduced the level of sphingomyelin to 66 +/- 7% of control by 1 h from 375 +/- 12 pmol/10(6) cells to 245 +/- 26 pmol/10(6) cells. There was a concomitant increase in ceramide from 89 +/- 4 pmol/10(6) cells to 252 +/- 27 pmol/10(6) cells consistent with activation of the enzyme, sphingomyelinase (EC 3.1.4.12). In support of this contention, 1,2-diacylglycerols appeared to enhance the activity of an acid, but not a neutral, sphingomyelinase in homogenates of GH3 cells. The 1,2-diacylglycerol, 1-oleyl-2-acetylglycerol, produced similar effects. In contrast, the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate, failed to stimulate sphingomyelin hydrolysis. Further, these effects of the 1,2-diacylglycerols occurred in cells down-modulated for protein kinase C. These studies demonstrate that 1,2-diacylglycerols stimulate sphingomyelin hydrolysis by a mechanism independent of the protein kinase C which mediates phorbol ester action. This is the first report of stimulated sphingomyelin hydrolysis by a physiologic effector molecule.     

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.     

Mechanism of Arachidonic Acid Liberation During Ischemia in Gerbil Cerebral Cortex

Once brain ischemia was induced in the gerbil cerebral fronto-parietal cortex, serial changes occurred in energy metabolites and various lipids. The amounts of inositol-containing phospholipids began to decrease immediately after energy failure, followed by an increase in the amount of 1,2-diacylglycerol with a subsequent liberation of arachidonic acid and other free fatty acids. The fatty acid compositions of inositol-containing phospholipids, of 1,2-diacylglycerols produced by ischemia, and of free fatty acids liberated during ischemia were quite similar. The amount of stearic acid liberated was much larger than that of arachidonic acid between 30 s and 1 min of ischemia. On the other hand, there was no significant decrease in the amount of the other phospholipids except for phosphatidic acid. Furthermore, there was also no change in the fatty acid composition of phosphatidylcholine or phosphatidylethanolamine throughout 15 min of ischemia. The amount of cytidine-monophosphate reached a peak (36.7 nmol/g wet wt) at 2 min of ischemia. These results indicated that arachidonic acid was predominantly liberated from inositol-containing phospholipids by phospholipase C, and by the diglyceride lipase and monoglyceride lipase system rather than from phosphatidylcholine or phosphatidylethanolamine by phospholipase A2 or plasmalogenase or choline phosphotransferase during the early period of ischemia.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets. A determination using purified phospholipases and a rapid method for membrane isolation.

1. Non-lytic degradation of human platelet phospholipids have been performed using a combination of bee venom phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) and Staphylococcus aureus sphingomyelinase C (sphingomyelin choline phosphohydrolase). Under these conditions, 25.4% of total phospholipds are degraded and 6.4% of total platelet arachidonic acid is released. 2. A new method for rapid isolation of platelet plasma membrane is described, based on the use of [3H]concanavalin A as a membrane marker and of self-generating gradients of Percoll. Plasma membranes are enriched 5.2 fold in lectin marker and 0.43 in N-acetyl-beta-D-glucosaminidase, the main contaminant. This method allows to estimate that 57% of the total cell phospholipids and 61% of the total arachidonic acid content are located in the plasma membrane. 3. The distribution of phospholipids and arachidonic acid between the two leaflets of the plasma membrane has been deduced by using these values and those obtained from non-lytic treatment of intact platelets by phospholipases. It is concluded that 45% of plasma membrane phospholipids, comprising 93% of sphingomyelin, 45% of phosphatidylcholine, 9% of phosphatidylserine, 16% of phosphatidylinositol and 20% of phosphatidylethanolamine form the outer half of the human platelet plasma membrane. The phospholipids appear to bear only 10% of the total membrane arachidonic acid.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes.

About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.     

Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase.

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.     

Physiologic 1,2-diacylglycerol levels induce protein kinase C-independent translocation of a regulatory enzyme

Phorbol esters and 1,2-diacylglycerols have been used interchangeably to study protein kinase C action. This laboratory first suggested that 1,2-diacylglycerols may also act independent of protein kinase C using protein kinase C-"down-modulated" cells (Kolesnick, R. N., and Paley, A. E. (1987) J. Biol. Chem. 262, 9204-9210). Unfortunately, down-modulation was never complete. The present studies establish an in vitro system of enzyme translocation to resolve this issue. Choline phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme for phosphatidylcholine biosynthesis, was utilized. Cytidylyltransferase translocation from cytosol to membranes mediates phorbol ester-induced phosphatidylcholine synthesis in GH3 pituitary cells. In the present studies, 1,2-diacylglycerols similarly induced phosphatidylcholine synthesis and cytidylyltransferase translocation. 1,2-Diacylglycerol-induced phosphatidylcholine synthesis, however, was not concentration-dependent but proportional to the moles of 1,2-diacylglycerol added per cell, i.e. subject to surface dilution. For instance, at constant cell number (1.67 x 10(6)/sample) and 1,2-dioctanoylglycerol concentration (diC8; 20 micrograms/ml), 32Pi incorporation into phosphatidylcholine varied from 150 to 350% above control as the incubation volume increased from 0.3 to 1.2 ml. Hence, the effective diC8 concentrations 0.5-30 micrograms/ml are preferably referred to as doses and reported as 0.25-15 nmol/10(6) cells. These doses increased cellular 1,2-diacylglycerol levels within a few fold of basal (374 pmol/10(6) cells). In vitro, diC8 also induced translocation of purified cytidylyltransferase devoid of protein kinase C to microsomes. Translocation was again subject to surface dilution. Translocation occurred with the same ratio of diC8 to microsomal membrane as phosphatidylcholine synthesis in intact cells (1-10 nmol of diC8/10(6) cell membranes). Despite stimulating cytidylyltransferase translocation in intact cells, phorbol esters failed to stimulate translocation in vitro. Hence, 1,2-diacylglycerols are not always interchangeable with phorbol esters and at physiologic levels may stimulate enzyme translocation by an alternative mechanism to protein kinase C.     

The phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation

Agonist-induced degradation of phosphatidylcholine (PC) is of interest as this pathway of diacylglycerol (DG) generation may provide added opportunities for the regulation of protein kinase C (PKC). In REF52 cells [3H]myristic acid is preferentially incorporated into PC; this, coupled with the use of [3H]choline, allows for quantitation of both the water-soluble and the lipid products generated when PC is degraded. In cells prelabeled with [3H]choline, TPA stimulated a time-dependent release, into the medium, of choline and not phosphocholine or glycerophosphocholine. Treatment of [3H]myristic acid-labeled cells with either phorbol diesters, sn-1,2-dioctanoylglycerol, or vasopressin elicited the formation of labeled phosphatidate (PA) and DG. The temporal pattern of PC hydrolysis in cells treated with TPA is indicative of a precursor (PA)-product (DG) relationship for an enzymatic sequence initiated by phospholipase D. Adding propranolol, a phosphatidate phosphohydrolase inhibitor, eliminated TPA-induced DG formation, whereas PA generation was unaffected. From these data we conclude that TPA elicits DG formation from PC by the sequential actions of phospholipase D and phosphatidate phosphohydrolase.     

Age features of glycerolipid metabolism in rat liver under short term exposure to thyroxine

The age specificity of the regulation by thyroid hormones of 1,2-diacylglycerol production in rat liver has been studied. It was found that L-thyroxine-stimulation of the 3-month old rats liver cells resulted in a rapid rise in 1,2-diacylglycerol concentration in hepatocytes and simultaneous degradation of phospholipids. The endogenous phosphatidylcholine and phosphatidylethanolamine are the sources of 1,2-diacylglycerol in a liver. Under the action of hormone liver cells of young rats may product 1,2-diacylglycerol from exogenous 1-acyl, 2-[14C]arachidonyl-phosphatidylethanolamine. Thyroxine had no effect on de novo 1,2-diacylglycerols formation and their release from triacylglycerol. In liver cells of elder rats, 1,2-diacylglycerol and individual phospholipids content are unaffected by hormones.     

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes.

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.     

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack.Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.     

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A1 and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).