Constitutive shedding of the amyloid precursor protein ectodomain is up-regulated by tumour necrosis factor-alpha converting enzyme.

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells.

Acceleration of phosphatidylcholine synthesis and breakdown by inhibitors of mitochondrial function in neuronal cells: a model of the membrane defect of Alzheimer's disease.

Brain cells in Alzheimer's disease (AD) exhibit a membrane defect characterized by accelerated phospholipid turnover. The mechanism responsible for this defect remains unknown. Recent studies indicate that impairment of mitochondrial function is frequently observed in AD and may be responsible for certain aspects of its pathophysiology. We show that when PC12 cells are exposed to inhibitors of mitochondrial bioenergetics, the turnover of their major membrane phospholipid, phosphatidylcholine, is accelerated, producing a pattern of metabolic changes that mimics that observed in brains of AD patients. Abnormalities of mitochondrial function may therefore underlie the membrane defect in AD.

The m3 muscarinic acetylcholine receptor is coupled to mitogen-activated protein kinase via protein kinase C and epidermal growth factor receptor kinase.

The acetylcholine analogue carbachol rapidly activated mitogen-activated protein kinase (MAPK), and caused tyrosine phosphorylation of the adapter protein p52 Shc and the epidermalgrowth factor (EGF) receptor, in human embryonic kidney cells stably expressing m3 muscarinic receptors. The protein kinase C (PKC) inhibitor GF109203X caused a significant partial inhibition of m3 receptor-mediated activation of MAPK. The PKC-independent MAPK activity elicited by carbachol in the presence of GF109203X was reproducibly abolished by AG1478, an inhibitor of EGF-receptor tyrosine kinase activity, and by the Src tyrosine kinase inhibitor PP1. In a subset of these experiments, GF109203X concomitantly increased carbachol-induced tyrosine phosphorylation of p52 Shc and the EGF receptor. In co-stimulation experiments, carbachol and EGF activated MAPK in a non-additive fashion; moreover, EGF-induced association of Shc with the phosphorylated EGF receptor was inhibited by carbachol. This effect of carbachol was blocked by GF109203X. The results indicate that MAPK activation by m3 receptor stimulation is regulated by two pathways; one dependent on PKC, and the other mediated via the EGF receptor and Src. Moreover, the EGF-receptor-dependent pathway may be subject to negative-feedback regulation via m3 receptor-coupled activation of PKC.

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix.

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine-glycine-aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine-glycine-glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell's interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.

Rapid stimulation of amyloid precursor protein release by epidermal growth factor: role of protein kinase C.

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved by an uncharacterized enzyme known as alpha-secretase within its extracellular/intraluminal domain after the activation of guanine nucleotide-binding protein-coupled receptors linked to phosphoinositide hydrolysis. The secretory process results in the release of large soluble derivatives of APP (APPs), and, when elicited by muscarinic receptor activation, exhibits both protein kinase C (PKC)-dependent and tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. In this report we examine the regulation of the release of APPs by epidermal growth factor (EGF) receptors, which possess intrinsic tyrosine kinase activity, and are coupled to a variety of effectors including phosphoinositide-specific phospholipase Cgamma. In A431 cells, EGF caused time-dependent and dose-dependent increases in the formation of inositol phosphates in cultures prelabelled with myo--3H-inositol, and in the release of APPs into the culture medium; the two responses exhibited similar time courses and EC50 values for EGF. Concomitant with these effects, there were concentration-dependent (3-300 ng/ml) increases in the phosphorylation of tyrosine residues in several proteins, including the EGF receptor itself. The specific PKC antagonist GF 109203X decreased the effect of EGF by approx. 35% at a concentration that abolished the stimulation of the release of APPs by the PKC activator PMA. Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, abolished the EGF-induced release of APPs. These results demonstrate that in A431 cells, activation of the EGF receptor stimulates alpha-secretase activity by a mechanism that is partly dependent on PKC activity.

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization.

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd ( approximately 0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 x 10(5) and 0.3 x 10(5) for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.

Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism.

Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism.

Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons.

The mechanism by which populations of brain cells regulate the flux of choline (Ch) into membrane or neurotransmitter biosynthesis was investigated using electrically stimulated superfused slices of rat corpus striatum. [Me-14C]Ch placed in the superfusion medium for 30 min during a 1-h stimulation period was incorporated into tissue [14C] phosphorylcholine (PCh) and [14C]phosphatidylcholine (PtdCh). Stimulation also caused a profound inhibition of PCh synthesis and a 10-fold increase in [14C]ACh release into the medium; it failed to affect tissue [14C]ACh levels. This effect was not explained by changes in ATP levels nor in the kinetic properties of Ch kinase (E.C. 2.7.1.32) or Ch acetyltransferase (ChAT) (E.C.2.3.1.7). To investigate the mechanism of these effects, Ch uptake studies were performed with and without hemicholinium-3 (HC3), a selective inhibitor of high affinity Ch uptake. A two-compartment model accurately fit the observed data and yielded a K(m) for Ch uptake of 5 microM into cholinergic structures and 72 microM into all other cells. Using this model it was estimated that cholinergic neurons account for 60% of observed uptake of Ch at physiologic Ch concentrations, even though they represent fewer than 1% of the total cells in the slice. The model also predicts that an increase in Ch uptake within cholinergic neurons, reported to be associated with depolarization [4,27,32], would significantly inhibit Ch uptake into all other cells, and would account for the observed decrease in PCh synthesis.

Tyrosine phosphorylation-dependent stimulation of amyloid precursor protein secretion by the m3 muscarinic acetylcholine receptor.

Stimulation of m1 and m3 muscarinic acetylcholine receptors, which are coupled to phosphoinositide hydrolysis and protein kinase C activation, has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs). The effect is mimicked by phorbol esters, which directly activate protein kinase C. Using human embryonic kidney cells expressing individual muscarinic receptor subtypes, we found that stimulation of APPs release by the muscarinic agonist carbachol was only partially reduced by a specific inhibitor of protein kinase C (the bisindolylmaleimide GF 109203X), while the response to phorbol 12-myristate 13-acetate (PMA) was abolished. The increase in APPs release elicited by carbachol and PMA was accompanied by elevated tyrosine phosphorylation of several proteins and reduced by tyrosine kinase inhibitors; GF 109203X significantly reduced the stimulation of tyrosine phosphorylation by carbachol and PMA. Inhibition of protein tyrosine phosphatases by vanadyl hydroperoxide markedly increased cellular tyrosine phosphorylation and enhanced APPs release as effectively as PMA and carbachol. Direct phosphorylation of amyloid precursor protein on tyrosine residues following treatment with carbachol, PMA, or vanadyl hydroperoxide was not observed. The results implicate both tyrosine phosphorylation and protein kinase C-dependent mechanisms in the regulation of APPs release by G protein-coupled receptors, and suggest that carbachol and PMA increase APPs release from human embryonic kidney cells expressing m3 muscarinic receptors via partially divergent pathways that converge at a tyrosine phosphorylation-dependent step.

Phorbol ester stimulates choline uptake in Swiss 3T3 fibroblasts following introduction of the gene encoding protein kinase C alpha.

Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKC-subtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKC alpha. A retroviral expression system was used to introduce the genes for PKC alpha and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKC alpha at levels that were 20-fold higher than those found in the control (neomycin-resistant) line, or in the wild-type cells. In control Swiss 3T3 fibroblasts, 1 microM PMA elevated choline levels by only 30%, whereas, in Swiss 3T3 cell lines that stably over-expressed PKC alpha, PMA caused a 5-fold enhancement in [14C]choline accumulation. This concentration of PMA significantly increased [14C]PtdCho levels in both control and PKC alpha-over-expressing lines, although the effect in the latter was significantly greater. The effects of PMA were inhibited by the PKC antagonist sphingosine. These results implicate PKC alpha in the regulation of choline accumulation and phospholipid synthesis in fibroblasts. Although additional PKC subtypes appear to participate in the control of PtdCho synthesis in these cells, PMA-stimulated choline uptake in Swiss 3T3 fibroblasts is almost entirely dependent on the presence of PKC alpha.

Regulation of proteolytic processing of the amyloid beta-protein precursor of Alzheimer's disease in transfected cell lines and in brain slices.

beta A4 is the principal component of Alzheimer's disease brain amyloid. It is derived from proteolytic processing of amyloid beta-protein precursors (APP), a family of transmembrane glycoproteins. Secretion of APPs, a secreted proteolytic derivative that is cleaved within the beta A4 domain of APP, is increased many-fold by the activation of cell-surface receptors, like the muscarinic m1 and m3 receptor subtypes, which are coupled to protein kinase C. Concomitantly, their activation decreases the formation of both secreted soluble beta A4 and of endosomal-lysosomal C-terminal APP derivatives. These data suggest that muscarinic m1 and m3 receptors accelerate non-amyloidogenic APP processing and depress the formation of potentially amyloidogenic derivatives. Other receptors that stimulate APPs secretion include those for bradykinin, vasopressin, and interleukin-1 receptors. A similar control mechanism is present in rat brain tissue slices, in which the release of both APPs and endogenous neurotransmitters is increased by electrical depolarization. This increase is tetrodotoxin-sensitive and frequency-dependent, suggesting that APPs release may normally depend on neuronal activity. Taken together, our findings suggest that specific receptor agonists might be effective in reducing the formation of potentially amyloidogenic APP derivatives in vivo.

Regulation by phorbol esters of amyloid precursor protein release from Swiss 3T3 fibroblasts overexpressing protein kinase C alpha.

Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheimer's disease was measured in two Swiss 3T3 fibroblast cell lines (designated SF1.4 and SF3.2), overexpressing the alpha subtype of protein kinase C, and in two control cell lines (SC1 and SC2) (Eldar, H., Zisman, Y., Ullrich, A., and Livneh, E. (1990) J. Biol. Chem. 265, 13290-13296). Basal release of APP was significantly increased in SF1.4 cells, but not in SF3.2 cells, relative to controls. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, elicited a concentration-dependent increase in APP release in all four cell lines. However, the estimated EC50 for this effect was lower in the two cell lines overexpressing protein kinase C-alpha (7 and 6 nM, in SF1.4 and SF3.2 cells, respectively) than in control SC1 and SC2 cells (56 and 22 nM, respectively). The absolute amount of APP released by maximal concentrations of phorbol ester was not altered by overexpression of protein kinase C alpha. The protein kinase C inhibitor H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) significantly reduced the response to phorbol esters in control (SC1) cells but not in cells (SF1.4) that overexpress protein kinase C alpha. Levels of cell-associated APP were slightly elevated, and rates of APP turnover were unchanged, in SF1.4 cells relative to controls. However, cell-associated APP levels were lower in SF3.2 cells than in controls. The results demonstrate that protein kinase C alpha regulates APP release in Swiss 3T3 fibroblasts, and perhaps in other tissues, including brain, and may be the isozyme that mediates receptor-evoked release of APP.

Receptor-coupled amyloid precursor protein processing.

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.

Regulation of amyloid precursor protein release by protein kinase C in Swiss 3T3 fibroblasts.

Release of the amyloid precursor protein (APP) of Alzheimer's disease from Swiss 3T3 fibroblasts was stimulated in a concentration-dependent manner by phorbol 12-myristate 13-acetate. In fibroblasts overexpressing protein kinase C alpha (PKC alpha), the EC50 for this response was 7 nM, while in control cells the EC50 was 63 nM. The effect of PMA was inhibited by the PKC antagonist H-7 in control cells, but not in cells that overexpressed PKC alpha. Basal release of APP was higher in cells that overexpressed PKC alpha, and was not affected by the phosphatase inhibitor okadaic acid, although this compound doubled APP release from control cells. The results suggest that PKC alpha regulates APP processing in mammalian cells. Alterations in the activity of PKC have been reported to occur in Alzheimer's disease and might potentially contribute to abnormalities of APP metabolism characteristic of this disorder.

Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors.

Altered processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease. To investigate whether cellular APP processing is controlled by cell-surface neurotransmitter receptors, human embryonic kidney (293) cell lines were transfected with the genes for human brain muscarinic acetylcholine receptors. Stimulation of m1 and m3 receptor subtypes with carbachol increased the basal release of APP derivatives within minutes of treatment, indicating that preexisting APP is released in response to receptor activation. Receptor-activated APP release was blocked by staurosporine, suggesting that protein kinases mediate neurotransmitter receptor-controlled APP processing.

Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line.

Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a membrane defect in Alzheimer disease brain.

To determine whether neurodegeneration in Alzheimer disease brain is associated with degradation of structural cell membrane molecules, we measured tissue levels of the major membrane phospholipids and their metabolites in three cortical areas from postmortem brains of Alzheimer disease patients and matched controls. Among phospholipids, there was a significant (P less than 0.05) decrease in phosphatidylcholine and phosphatidylethanolamine. There were significant (P less than 0.05) decreases in the initial phospholipid precursors choline and ethanolamine and increases in the phospholipid deacylation product glycerophosphocholine. The ratios of glycerophosphocholine to choline and glycerophosphoethanolamine to ethanolamine were significantly increased in all examined Alzheimer disease brain regions. The activity of the glycerophosphocholine-degrading enzyme glycerophosphocholine choline-phosphodiesterase was normal in Alzheimer disease brain. There was a near stoichiometric relationship between the decrease in phospholipids and the increase of phospholipid catabolites. These data are consistent with increased membrane phospholipid degradation in Alzheimer disease brain. Similar phospholipid abnormalities were not detected in brains of patients with Huntington disease, Parkinson disease, or Down syndrome. We conclude that the phospholipid abnormalities described here are not an epiphenomenon of neurodegeneration and that they may be specific for the pathomechanism of Alzheimer disease.

Production of diacylglycerol by exogenous phospholipase C stimulates CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in human neuroblastoma cells.

The involvement of endogenous diacylglycerol production in the stimulation of phosphatidylcholine synthesis by exogenous phospholipase C was examined using a neuroblastoma (LA-N-2) cell line. Phospholipase C treatment (0.1 unit/ml) of intact cells stimulated CTP:phosphocholine cytidylyltransferase activity significantly more effectively than did maximally effective concentrations of the synthetic diacylglycerol sn-1,2-dioctanoylglycerol (1 mM). When added to cells together with phospholipase C, oleic acid, but not dioctanoylglycerol, further increased cytidylyltransferase activity with respect to phospholipase C treatment alone, indicating that the enzyme was not maximally activated by the lipase. This suggests that the lack of additivity of diacylglycerol and phospholipase C reflects a common mechanism of action. The time course of activation of cytidylyltransferase by phospholipase C paralleled that of [3H]diacylglycerol production in cells prelabeled for 24 h with [3H]oleic acid. Diacylglycerol mass was similarly increased. Significant elevations of [3H]oleic acid and total fatty acids occurred later than did the increases in cytidylyltransferase activity and diacylglycerol levels. No significant reduction in total or [3H]phosphatidylcholine was elicited by this concentration of phospholipase C, but higher concentrations (0.5 unit/ml) significantly reduced phosphatidylcholine content. The stimulation of cytidylyltransferase activity by phospholipase C or dioctanoylglycerol was also associated with enhanced incorporation of [methyl-14C]choline into phosphatidylcholine. Dioctanoylglycerol was more effective than phospholipase C at stimulating the formation of [14C]phosphatidylcholine, and the effects of the two treatments were additive. However, further analysis revealed that dioctanoylglycerol served as a precursor for [14C]dioctanoylphosphatidylcholine as well as an activator of cytidylyltransferase; and when corrections were made for this effect, the apparent additivity disappeared. The results indicate that the generation of diacylglycerol by exogenous phospholipase C (and possibly the subsequent production of fatty acids via diacylglycerol metabolism) activates cytidylyltransferase activity in neuronal cells under conditions in which membrane phosphatidylcholine content is not measurably reduced.

Alterations of phospholipid metabolites in postmortem brain from patients with Alzheimer's disease.

To test the hypothesis that brain cell membranes degenerate in Alzheimer's disease (AD), we measured the levels of phospholipids, their water-soluble metabolites, and glycerophosphocholine (GPC) cholinephosphodiesterase activity in postmortem brain tissue from patients with AD and age-matched controls. We found significantly higher levels of the phospholipid catabolite GPC in AD brain. In contrast, choline and ethanolamine levels were significantly lower in AD, and phospholipid levels were slightly decreased. Furthermore, in AD the activity of the GPC-degrading enzyme GPC cholinephosphodiesterase was unaltered. Our results indicate that membrane phospholipid catabolism is increased in AD brain. Inasmuch as the tissue levels of initial phospholipid precursors were decreased, we suggest that phospholipid turnover is elevated in this neurodegenerative disease.