Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49(AR). ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49(AR) cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49(AR) cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-beta-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.     

Proapoptotic effects of P. aeruginosa involve inhibition of surfactant phosphatidylcholine synthesis

Pseudomonas aeruginosa causes sepsis-induced acute lung injury, a disorder associated with deficiency of surfactant phosphatidylcholine (PtdCho). P. aeruginosa (PA103) utilizes a type III secretion system (TTSS) to induce programmed cell death. Herein, we observed that PA103 reduced alveolar PtdCho levels, resulting in impaired lung biophysical activity, an effect partly attributed to caspase-dependent cleavage of the key PtdCho biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Expression of recombinant CCTalpha variants harboring point mutations at putative caspase cleavage sites in murine lung epithelia resulted in partial proteolytic resistance of CCTalpha to PA103. Further, caspase-directed CCTalpha degradation, decreased PtdCho levels, and cell death in murine lung epithelia were lessened after exposure of cells to bacterial strains lacking the TTSS gene product, exotoxin U (ExoU), but not ExoT. These observations suggest that during the proapoptotic program driven by P. aeruginosa, deleterious effects on phospholipid metabolism are mediated by a TTSS in concert with caspase activation, resulting in proteolysis of a key surfactant biosynthetic enzyme.     

Inhibition of phosphatidylcholine synthesis is not the primary pathway in hexadecylphosphocholine-induced apoptosis

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.     

Uncoupling protein 2 protects testicular germ cells from hyperthermia-induced apoptosis

The mRNA of the mitochondrial uncoupling protein 2 (UCP2) was up-regulated by cryptorchidism, a testicular hyperthermic condition under which germ cells undergo severe apoptosis. We investigated whether UCP2 was able to protect germ cells from hyperthermia-induced apoptosis. UCP2 was predominantly present in elongate spermatids under normal conditions, and was detected in all germ cells with its level significantly increased if the testes were exposed to 43 degrees C for 5 min. Such a short heat exposure was non-lethal and enabled the preconditioned cells to be resistant to apoptosis induced by a longer hyperthermic treatment (15 min). While hyperthermia resulted in oxidative stress in mouse testes, it did not change the total anti-oxidative capacity. Indeed, overexpression of UCP2 in the GC-2 germ cell line protected the cells from radical oxygen species (ROS)-induced apoptosis. Taken together, we propose that UCP2 may represent an effective weaponry used by germ cells to combat ROS-induced apoptosis.     

Caspase processing and nuclear export of CTP:phosphocholine cytidylyltransferase alpha during farnesol-induced apoptosis

CTP:phosphocholine cytidylyltransferase alpha (CCT alpha) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway, the primary route for synthesis of phosphatidylcholine (PtdCho) in eukaryotic cells. Induction of apoptosis by farnesol (FOH) and other cytotoxic drugs has been shown to alter PtdCho synthesis via the CDP-choline pathway. Here we report that FOH-induced apoptosis in CHO cells caused a dose-dependent activation of CCT alpha and inhibition of the final step in the pathway, resulting in a biphasic effect on PtdCho synthesis. Activation of CCT alpha was accompanied by enzyme translocation to the nuclear envelope within 30 min of FOH addition to cells. Following translocation to membranes, CCT alpha was exported from the nucleus and underwent caspase-mediated proteolysis that coincided with poly(ADP-ribose) polymerase cleavage. Site-directed mutagenesis and in vivo and in vitro expression studies mapped a caspase 6 and/or 8 cleavage site to TEED(28 downward arrow)G, the final residue in the CCT alpha nuclear localization signal. Nuclear export of CCT alpha appeared to be an active process in FOH-treated CHO cells that was independent of caspase removal of the nuclear localization signal. Caspase cleavage of CCT alpha occurred during UV or chelerythrine-induced apoptosis; however, nuclear membrane translocation and nuclear export were not evident under these conditions. Thus, caspase cleavage of CCT alpha was a late feature of several apoptotic programs that occurred in the nucleus or at the nuclear envelope. Activation and nuclear export of CCT alpha were early events in FOH-induced apoptosis that contributed to altered PtdCho synthesis and, in conjunction with caspase cleavage, excluded CCT alpha from the nucleus.     

Detection and quantification of farnesol-induced apoptosis in difficult primary cell cultures by TaqMan protein assay

Apoptosis can be detected reliably by assaying for cleaved caspase-3, for which active caspase-3 antibodies are used in several methods, such as immunocytochemistry, enzyme-linked immunosorbent assay, and western blot. In this study, we used TaqMan protein assay (TPA), a novel method for protein detection and quantification that detects proteins by amplification of substitute DNA templates. TPA uses antibodies and proximity ligation for quantitative real-time PCR. Meningiomas are primarily benign intracranial tumors. Primary cell cultures of meningiomas are often unsuitable for sensitive protein detection methods. We optimized a TPA to detect active caspase-3 and evaluated its ability to detect farnesol-induced apoptosis in primary meningioma cells. The specificity and sensitivity of the inactive and active caspase-3 assay were determined using recombinant caspase-3. Apoptosis was induced in meningiomas in the presence of 0.2 μM farnesol as shown by immunocytochemistry of single-stranded DNA. Also, viability decreased by over 90 % after treatment with 1.2 μM farnesol for 24 h. The TPA detected a significant increase in active caspase-3 after treatment with 2 and 4 μM farnesol for 2 h, which could not be detected using standard methods such as western blot and immunofluorescence. In addition, TPA determined that meningiomas show disparate sensitivities to low concentrations of farnesol. Caspase-3 expression fell significantly in cells that were treated with 0.25 μM farnesol for 2 h. Further, by TPA, active caspase-3 peaked after 2 h and declined with longer incubation times. This study demonstrates that cleaved caspase-3 is detected and quantified reliably in meningiomas by TPA.     

De novo CDP-choline-dependent glycerophosphorylcholine synthesis and its involvement as an intermediate in phosphatidylcholine synthesis

The activity of CDP-choline-dependent glycerophosphorylcholine synthetase (CDP-choline:sn-3-glycerophosphate cholinetransferase), a newly discovered enzyme involved in the recently proposed pathways of acyl-specific phosphatidylcholine synthesis, is reported in rat liver. Endogenous CDP-choline, synthesized via the CMP-driven back reaction of phosphorylcholine transferase, is also shown to be a choline donor for this glycerophosphorylcholine synthetase. The function of glycerophosphorylcholine as an intermediate in phosphatidylcholine synthesis is demonstrated by specific isotope trapping whereby unlabelled glycerophosphorylcholine inhibited label incorporation from sn-[14C]glycerol-3-phosphate into phosphatidylcholine in mouse gastrocnemius, a tissue that is essentially devoid of the cytidine pathway for phosphatidylcholine synthesis and uses a non-allelic glycerophosphorylcholine synthetase (exogenous PC:sn-3-glycerophosphate cholinetransferase) in the synthesis of glycerophosphorylcholine.     

1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells.

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.     

The coordination of sterol and phospholipid synthesis in cultured myogenic cells: Effect of cholesterol synthesis inhibition on the synthesis of phosphatidylcholine

The coordination of biosynthesis of cholesterol and phosphatidylcholine has been investigated in a myoblast cell line, L₆, grown in lipid-depleted medium. The addition of 25-hydroxycholesterol or compactin to this medium inhibits cholesterol synthesis by over 95%. The rate of [³H]choline incorporation into phosphatidylcholine begins to decline after 6 h and eventually falls to 45% of control. Measurements of choline flux through the CDPcholine pathway and of the pool sizes of choline-containing intermediates indicate that the formation of CDPcholine is the rate-limiting step in phosphatidylcholine synthesis in L₆. The rate of CDPcholine synthesis was measured in vivo by pulse-chase experiments. Culturing cells with 25-hydroxycholesterol or compactin results in an inhibition of this step, which parallels the inhibition of incorporation of [³H]choline into phosphatidylcholine. The specific activities of the enzymes of phosphatidylcholine synthesis were assayed under optimal substrate conditions. Growth in the presence of sterol-synthesis inhibitors for 24 h has a significant, but variable, effect on the activity of microsomal and cytosolic cholinephosphate cytidylyltransferase. Inhibition is seen in approximately one-half of the preparations and ranges up to 60%. The degree of inhibition of the enzyme in vitro correlates with an elevation of cytosolic triacylglycerol and phospholipid levels, and is not eliminated by the inclusion of excess stimulatory phospholipids in the assay. The pool sizes of the substrates, cholinephosphate and CTP, are unaffected by cholesterol synthesis inhibition. In contrast to the effects on cholinephosphate cytidylyltransferase, the microsomal enzymes glycerol-3-phosphate acyltransferase and choline phosphotransferase are stimulated 2-fold or more. Choline kinase specific activity was inhibited 2-fold after 24 h of treatment with 25-hydroxycholesterol; however, no effect on this step was observed in vivo. These results indicate that the coordination of cholesterol and phosphatidylcholine synthesis involves regulation at the cytidylyltransferase-catalyzed step.     

Cytokine secretion requires phosphatidylcholine synthesis

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT alpha-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT alpha is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT alpha-deficient cells. Pro-tumor necrosis factor alpha and interleukin-6 remain lodged in the secretory compartment of CCT alpha-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1 beta secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT alpha-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor alpha or interleukin-6. These results identify CCT alpha as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.     

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.     

p53-induced inhibition of protein synthesis is independent of apoptosis.

Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-independent events that can be dissociated from the induction of apoptosis per se.     

Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli

Summary The synthesis of the bacteriocin cloacin DF13 and its release into the culture medium were genetically uncoupled by subcloning the gene encoding the bacteriocin release protein (BRP) from pCloDF13. The gene was cloned under the control of the IPTG-inducible lpp-lac promoter-operator system on the expression vector pINIIIA1, giving pJL1. A 4 kb DNA fragment of pJL1, containing the tandem lpp-lac promoter, the BRP gene and lacI (BRP cassette), was cloned into the pCloDF13 derivative plasmid pJN67, which encodes cloacin DF13 but not the release protein. Furthermore, the pCloDF13 immunity protein gene was subcloned downstream of the temperature-inducible P _L promoter of the expression vector pPLc236, together with the BRP cassette. Growth, induction and excretion experiments with Escherichia coli cells harbouring the constructed plasmids revealed that: i) the BRP is the only pCloDF13-derived gene product responsible for the observed growth inhibition and apparent lysis of strongly induced cells. This growth inhibition and lysis can be prevented by Mg²⁺ ions added to the culture medium, and involves induction of phospholipase A activity. (ii) The expression of the BRP gene can be regulated by varying the IPTG concentration. (iii) A separately controlled and moderate induced BRP synthesis can be used to bring about the release of large amounts of cloacin DF13 under conditions that allow a strong induction of the bacteriocin and which do not result in lysis of cells. (iiii) Preliminary results indicated that the BRP can stimulate the release of immunity protein in the absence of cloacin or cloacin fragments.     

Yeast mutant defective in phosphatidylcholine synthesis

The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine. The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline. Inositol, but not choline, stimulated growth of the mutant. The opi3-3 mutation was recessive and was genetically linked to the ino4 locus. When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains. In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The incorporation of label from [methyl-14C]methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains. However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine. The first methylation reaction appeared to be occurring at normal or even elevated levels. Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline. Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline.     

Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis

The mitochondrial uncoupling protein-3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein-1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein-3 was expressed in 293 cells using the tetracycline-inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein-3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP-inhibition of state 4 respiration and a GDP-induced re-polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein-3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein-3 expression had been induced showed a higher mitochondrial Bax/Bcl-2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein-3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein-3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways.     

Apaf1 inhibition promotes cell recovery from apoptosis

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.     

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.