Evaluation of phospholipase C and D activity in stimulated human neutrophils using a phosphono analog of choline phosphoglyceride.

A phosphono analog of choline phosphoglyceride was used to examine the relative contributions of phospholipase C and D in the generation of diglycerides in fMLP- and A23187-stimulated human neutrophils. The phosphono analog, 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphonocholine, contains a carbon-phosphorus bond adjacent to the base moiety and is resistant to phospholipase D hydrolysis, while remaining susceptible to phospholipase C hydrolysis. fMLP stimulated the production of [3H]phosphatidic acid and subsequently [3H]diglyceride from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine, but not from cells prelabeled with the phosphono analog. Treatment with A23187 also resulted in the formation of these products from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine. Additionally, A23187 stimulated the conversion of the phosphono analog to phosphodiester-containing choline phosphoglyceride which then resulted in the generation of [3H]phosphatidic acid and subsequently [3H]diglyceride. This study demonstrates the use of a phosphono analog in assessing phospholipase C and D activity in cells and provides evidence that in fMLP- and A23187-stimulated human neutrophils, diglyceride is generated indirectly from choline phosphoglycerides by the combined activities of phospholipase D and phosphatidate phosphohydrolase.

Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine.

A resistant cell line (HL-60R) was selected by incubating HL-60 cells with increasing concentrations of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) and used to examine the mechanism of resistance to the antineoplastic ether-linked lipid. The HL-60R cells exhibited a > 10-fold increase in resistance when measured by [3H]-thymidine incorporation in comparison to the HL-60 cell line. ET-18-OCH3 binding occurred at 4 degrees C and was not saturable at the concentrations tested (1-100 microM), indicating that the binding was receptor-independent. At 4 degrees C, association of ET-18-OCH3 was low for each cell line. AT 37 degrees C, uptake in the HL-60 cells was approximately 5-fold greater in comparison to HL-60R cells at each concentration tested. However, when the cellular content of ET-18-OCH3 was equal, both cell lines experienced similar declines in cell growth. Cellular incorporation of ether lipid was determined using serum-free media and in the presence of serum albumin or lipoproteins. Reduced uptake by the resistant cell line was observed only in the presence of albumin. A greater proportion of ether lipid could be removed from prelabeled HL-60R cells than from HL-60 cells, by an albumin wash procedure, indicating an increased rate of internalization and retention by the sensitive cell line. ET-18-OCH3 uptake in the HL-60 cell line was also more sensitive to treatment with endocytic (chloroquine, monensin) or metabolic (NaF, KCN) inhibitors. These results suggest that uptake is the principal determinant influencing sensitivity of the resistant cell line and consists of receptor-independent binding followed by internalization. Differential uptake requires the presence of serum albumin and is dependent on the energy-dependent endocytosis of the ether lipid.

Identification of a lysophospholipase C that may be responsible for the biosynthesis of choline plasmalogens by Madin-Darby canine kidney cells.

The biosynthesis of choline plasmalogens was investigated in Madin-Darby canine kidney cells to determine the source of the vinyl ether linkage. 1-O-[3H] Alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine was a better precursor than 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine for the synthesis of 1-O-[3H]alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine; this suggests that the vinyl ether linkage in choline phosphoglycerides originates from ethanolamine plasmalogens. The contribution of N-methylation and base exchange enzymes to choline plasmalogen biosynthesis was assessed using 1-O-[3H]alkenyl-2-lyso-sn-glycero-3-[32P]phosphoethanolamine. While 1-O-[3H]alkenyl-2-acyl-sn-glycero-3-phosphocholine was formed from this precursor, the 32P was lost indicating that N-methylation and base exchange enzymes do not contribute significantly to the synthesis of choline plasmalogens. The conversion of a phosphono analog of 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphoethanolamine, which is resistant to phospholipase D hydrolysis, to 1-O-[3H]alkenyl-2-acyl-sn-glycero-2-phosphocholine was observed demonstrating that phospholipase D is not required for choline plasmalogen biosynthesis. A Mg(2+)-dependent lysophospholipase C activity was detected in microsomes that actively hydrolyzed ether-linked lysophosphoglycerides as well as the lysophosphono analog. To assess the role of lysophospholipase C in shuttling 1-O-alk-1'-enyl-sn-glycerol (alkenylglycerol) from ethanolamine plasmalogens to choline plasmalogens, cells prelabeled with 1-O-[3H]alkenyl-2-lyso-sn-glycero-3-phosphoethanolamine were treated with 12-O-tetradecanoylphorbol-13-acetate. This resulted in the rapid deacylation of 1-O-[3H]alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine to 1-O-[3H]alkenyl-2-lyso-sn-glycero-3-phosphoethanolamine and the subsequent generation of 1-O-[3H]alkenylglycerol. A concomitant 2-3-fold increase in 1-O-[3H]alkenyl-2-acyl-sn-glycero-3-phosphocholine was observed. These studies suggest that the alkenyl linkage in choline phosphoglycerides may originate from 1-O-alkenyl-2-lyso-sn-glycero-3-phosphoethanolamine through an enzymatic pathway involving lysophospholipase C to generate alkenylglycerol that is subsequently converted to choline plasmalogens.

Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling.

Products of glycerolipid and sphingolipid metabolism are now known to fulfill second messenger functions in a variety of cellular signaling pathways. Evidence for glycerolipid-derived second messengers was first obtained from the "phosphatidylinositol cycle," which involves a signal-dependent hydrolysis of phosphatidylinositol bisphosphate yielding diacylglycerol and inositol trisphosphate. The role of diacylglycerol in the regulation of protein kinase C activity and its site of interaction with PKC are now well known. Recently, another glycerolipid second messenger, phosphatidic acid, was found to interact with the protooncogenic Raf-1 kinase. In cultured cells, a signal-induced generation of phosphatidic acid was critical for Raf-1 translocation to the cell membrane. Thus, different glycerolipid second messengers appear to regulate distinct targets with exquisite specificity. Analogous to the PI cycle, a "sphingomyelin cycle" was also found to exist, generating sphingolipid second messengers. Ceramide, derived from the agonist-induced hydrolysis of sphingomyelin, is a potent biomolecule with effects in multiple cell signaling pathways. The steroid hormone progesterone stimulated sphingomyelin hydrolysis in Xenopus oocytes. Ceramide, derived from the "sphingomyelin cycle," was sufficient for meiotic cell cycle progression in the oocytes. These results demonstrate the various effects of lipid-derived second messengers and promise exciting discoveries into the roles of lipids in cell signaling.

Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. A novel pathway for the metabolism of ether-linked phosphoglycerides.

Madin Darby canine kidney (MDCK) cells convert 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine [( 3H]alkylacylGPC) to a product tentatively identified as an ethanolamine-containing phosphoglyceride (PE) (Daniel, L. W., Waite, B. M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132). In the present study, analysis of the radiolabeled phosphoglycerides as diradylglycerobenzoate derivatives indicated that [3H] alkylacylGPC was initially converted to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkylacylGPE) which was subsequently desaturated to 1-O-[3H]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkenylacylGPE). The conversion of [3H]/[32P]alkyl-lysoGPC to [3H]alkenylacylGPE indicated that base exchange enzymes were not involved in this pathway. A phosphono analog of alkyl-lysoGPC, resistant to phospholipase D hydrolysis and radiolabeled in the 1-O-alkyl chain was readily incorporated, acylated, and subsequently metabolized to [3H]alkylacylGPC and [3H]alkenylacylGPE. Therefore, the involvement of phospholipase D in the conversion pathway was ruled out. The conversion of [3H]alkylacylGPC or its phosphono analog to [3H]alkenylacylGPE was significantly enhanced by the addition of 100 microM ethanolamine to the culture media, suggesting that [3H]alkylacylglycerol is an intermediate in the cytidine-dependent pathway of PE synthesis. MDCK cell cytosol and microsomes contained no detectable phospholipase C activity. However, incubation of microsomes with CMP resulted in the degradation of [3H]alkylacylGPC and accumulation of [3H]alkylacylglycerol. Furthermore, the addition of CDP-ethanolamine to microsomes following preincubation with CMP, resulted in a decrease in [3H]alkylacylglycerol with a concomitant increase in [3H]alkenylacylGPE. Overall, these results suggest that the reverse reaction of choline phosphotransferase may be responsible for the conversion of alkylacylGPC to alkylacylGPE.

1-beta-D-Arabinofuranosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells.

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on phospholipid metabolism was investigated in HL-60 cells. Treatment of cells with ara-C resulted in a time- and dose-dependent increase in the mass of ceramide and diglyceride. When cells were treated with 10 microM ara-C, an increase in the mass of ceramide and diglyceride was detectable at 5 min and reached a plateau at 30 min with a 2-fold increase compared to control cells. Treatment of cells with 10 microM ara-C resulted in the activation of neutral sphingomyelinase with a peak at 30 min that represented a 2-fold increase in specific activity. ara-C also increased expression of the transcription factor NF-kappa B which is related to the control of monocyte differentiation. These findings suggest that ara-C activates phospholipid hydrolytic pathways to generate second messengers that control gene expression and differentiation in the HL-60 cell line.

Ceramide triggers meiotic cell cycle progression in Xenopus oocytes. A potential mediator of progesterone-induced maturation.

The role of sphingomyelin-derived second messengers in progesterone-induced reinitiation of the meiotic cell cycle of Xenopus laevis oocytes was investigated. A brief treatment of defolliculated oocytes with sphingomyelinase (Staphylococcus aureus) was sufficient to induce maturation as measured by H1 kinase activity and germinal vesicle breakdown (GVBD). Pretreatment with cycloheximide inhibited sphingomyelinase-induced GVBD demonstrating a requirement for protein synthesis. Microinjection of ceramide or sphingosine, potential products of sphingomyelin hydrolysis, were capable of inducing GVBD in the absence of hormone. Metabolic labeling studies suggested the conversion of sphingosine to ceramide was necessary for sphingosine-induced GVBD. Additionally, fumonisin b1, an inhibitor of sphingosine N-acyltransferase, blocked sphingosine-induced GVBD demonstrating that ceramide is the more proximal biologically active metabolite. Treatment of oocytes with progesterone, the physiological inducer of oocyte maturation, resulted in a time- and concentration-dependent increase in the mass of ceramide and decrease in the mass of sphingomyelin through activation of a Mg(2+)-dependent neutral sphingomyelinase. These observations suggest that the generation of ceramide from sphingomyelin is part of the signal transduction pathway activated in response to progesterone and that the increase in ceramide is likely to be functionally important in resumption of the meiotic cell cycle.

Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

Recovery and expression of messenger RNA from postmortem human brain tissue.

The Bryan Alzheimer's Disease Research Center Rapid Autopsy Program at Duke University Medical Center obtains postmortem human brain tissue for experimental investigations. We evaluated 19 brains for RNA integrity and mRNA gene expression. Nine were from patients diagnosed with Alzheimer's disease, and ten were from nondemented controls. In all cases, the following variables were recorded: postmortem procurement delay (range, 1 hour and 10 minutes to 14 hours), pH of cerebrospinal fluid, premortem fever or sepsis, provision of supplemental oxygen in the agonal period, and temporal relation to time of death (either sudden death or protracted illness). Total RNA was extracted, quantified, and evaluated by agarose gel electrophoresis and quantitative gene expression analysis of 18S rRNA and edg-1 using TaqMan technology. All samples appeared to yield intact RNA without significant degradation, and expression of the edg-1 gene was detected by the real time reverse transcriptase polymerase chain reaction in all cases. We conclude that intact RNA can be obtained from postmortem human brain tissue, even in patients with severe premortem illnesses and delayed postmortem tissue procurement intervals. However, we caution that the successful expression of certain genes from postmortem brain tissue may require enhanced procurement efforts to maximize RNA integrity.

The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras.

Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.

Phospholipase D hydrolysis of choline phosphoglycerides is selective for the alkyl-linked subclass of Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells were used to study the synthesis of diglycerides from choline phospholipids (PC) in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). In this system, diglyceride formation was blocked in the presence of ethanol (0.5%), and a corresponding amount of phosphatidylethanol (PEt) was formed, indicating that phospholipase D is responsible for the diglyceride production. Analysis of the subclasses of phosphatidylethanol revealed 1-O-alkyl-(alkyl), 1-O-alk-1'-enyl-(alkenyl), and 1-acyl species of PEt (38.0, 8.3, and 53.7%, respectively). The molecular species of the alkyl-PEt most closely matched the alkyl-PC. No change in the relative amounts of alkyl- versus acyl-PEt was observed with time after stimulation. Comparison of the alkyl content of PEt (38.0%) and the parent PC (15.2%) indicated a marked selectivity for the alkyl subclass of PC. A cell-free assay (Huang, C., Wykle, R. L., Daniel, L. W., and Cabot, M. C. (1992) J. Biol. Chem. 267, 16859-16865) for phospholipase D was also used to confirm the selectivity of the enzyme for alkyl-PC versus acyl-PC. The predominant molecular species of PEt contained saturated acyl or alkyl chains in position-1 and monounsaturated residues in position-2 accounting for approximately 50% of the total PEt. 1-O-Octadecyl-2-oleoyl-sn-glycerol, a representative alkyl molecular species, was synthesized and tested for its effect upon protein kinase C derived from MDCK cells. This alkyl-diglyceride (DG) neither stimulated protein kinase C nor inhibited its activation by diacylglycerol. In summary, TPA-stimulated phospholipase D is selective for the alkyl-PC subclass in MDCK cells. The alkyl-DG subsequently formed does not appear to function as a second-messenger in activating protein kinase C.

Increases in neutral, Mg2+-dependent and acidic, Mg2+-independent sphingomyelinase activities precede commitment to apoptosis and are not a consequence of caspase 3-like activity in Molt-4 cells in response to thymidylate synthase inhibition by GW1843.

Thymidylate synthase (TS) inhibition causes cell death, and this enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis. The role of the ceramide in apoptotic signaling was studied in Molt-4 human T-cell leukemia cells undergoing cell death after treatment with GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3-like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blebbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.