The hydrolysis of glycerophosphocholine by rat brain microsomes: Activation and inhibition

Experiments with glycerophosphocholine phosphodiesterase (GPC diesterase, EC 3.1.4.2.) in rat brain microsomes suggest that, although its activity is inhibited by low concentrations of calmidazolium, its dependence on Ca²⁺ ions is not modulated by calmoulin. The activity of glycerophosphocholine choline phosphodiesterase (choline phosphohydrolase, EC 3.1.4.38) was much lower than that of the GPC diesterase. A relatively inexpensive method for the preparation ofsn-glycero-3-phospho [Me-¹⁴C]choline is described.Special Issue Dedicated to Dr. Abel Lajtha.     

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline.

The uptake and metabolism of [14C]choline in dissociated rat brain embryo cell cultures was examined as a function of the extracellular choline concentration. Choline uptake did not follow normal Michaelis-Menten kinetics, but rather exhibited two components with apparent Km of 0.016 mM and 0.96 mM. At low choline concentrations (high affinity uptake) most of the [14C]choline label was present in the phosphocholine fraction prior to the appearance of label in phospholipids. At high choline concentrations (low affinity uptake) a large proportion of the radioactivity was converted into acetylcholine. The dissimilarities between the formation of phosphocholine and acetylcholine as a function of choline concentration might be explained by the existence of two mutually independent enzymatic activities with different Km affinities for choline. Kinetic data augmented by double label studies, suggested that formation of choline phosphoglyceride proceeds entirely via a phosphocholine intermediate. Nearly all radioactivity in the lipid fraction is incorporated into choline phosphoglycerides. A higher turnover rate of choline incorporation into choline phosphoglycerides, accompanied by an increase in the levels of glycerophosphocholine, was observed in older cultures as compared to younger cultures. The metabolic implications of these findings in cultured brain cells in comparison with other in vitro systems are discussed.     

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.     

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.     

The transfer of choline from the host to the bacterial cell surface requires glpQ in Haemophilus influenzae

Haemophilus influenzae incorporates choline obtained from environmental sources onto its lipopolysaccharide as phosphorylcholine (ChoP). The decoration of the bacterial surface with ChoP contributes to pathogenesis by allowing for mimicry of the host. As the main reservoir for choline in the host is phosphatidylcholine, we tested whether other choline-containing molecules associated with eukaryotic membranes could provide an alternative source of choline. H. influenzae was able to use glycerophosphorylcholine (GPC), an abundant degradation product of phospholipids, as efficiently as free choline. Utilization of GPC required glpQ, which expresses an enzyme with glycerophosphodiester phosphodiesterase activity. In the absence of free choline, this gene was required for adherent H. influenzae to obtain choline directly from epithelial cells in culture. GlpQ therefore allows choline to be transferred from the host to the bacterial cell surface.     

Choline deficiency induces apoptosis in primary cultures of fetal neurons.

Treatment of rats with choline during brain development results in long-lasting enhancement of spatial memory whereas choline deficiency has the opposite effect. Changes in rates of apoptosis may be responsible. We previously demonstrated that choline deficiency induced apoptosis in PC12 cells and suggested that interruption of cell cycling due to a decrease in membrane phosphatidylcholine concentration was the critical mechanism. We now examine whether choline deprivation induces apoptosis in nondividing primary neuronal cultures of fetal rat cortex and hippocampus. Choline deficiency induced widespread apoptosis in primary neuronal cells, indicating that cells do not have to be dividing to be sensitive to choline deficiency. When switched to a choline-deficient medium, both types of cells became depleted of choline, phosphocholine and phosphatidylcholine, and in primary neurons neurite outgrowth was dramatically attenuated. Primary cells could be rescued from apoptosis by treatment with phosphocholine or lysophosphatidylcholine. As described previously for PC12 cells, an increase in ceramide (Cer) was associated with choline deficiency-induced apoptosis in primary neurons. The primary neuronal culture appears to be an excellent model to explore the mechanism whereby maternal dietary choline intake modulates apoptosis in the fetal brain.     

Choline kinase and ethanolamine kinase activity in the cytosol of nerve endings from rat forebrain.

Both choline kinase and ethanolamine kinase are present in the cytosol of nerve endings prepared from rat brain are the products of their action, phosphocholine (84 nmol/g fresh wt. of brain) and phosphoethanolamine (190 nmol/g fresh wt. of brain). In contrast with the enzymes from the cytosol of whole brain, both are as equally active at pH 7.5 as 9.0. Determination of kinase activity in membrane-containing tissue samples at pH9 gives low values because of the activity of alkaline phosphatase. Choline kinase, but not ethanolamine kinase, requires Mg2+ in excess of that required for the formation of the MgATP complex and is inhibited by an excess of free ATP. The Km for choline is 2.6mM and for ethanolamine is 2.2mM. The differing requirements for ATP and Mg2+ and the inhibition of choline kinase, but not ethanolamine kinase, by hemicholinium-3 suggest either the presence of two separate enzymes or two different active sites on the same enzyme.     

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.     

Cellular responses to excess phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.     

A spectrophotometric assay of Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase using p-nitrophenylphosphorylcholine.

A direct spectrophotometric assay for the glycerophosphorylcholine phosphocholine phosphodiesterase requiring zinc ions for activity is described. This assay is based on the continuous measurement of p-nitrophenol produced from the enzymatic hydrolysis of p-nitrophenylphosphorylcholine. The assay method, which showed a good linearity with time and amount of protein, was found to be rapid, simple, and, at the same time, accurate and sensitive enough to allow the quantitation of nanomolar amounts of product. With an alkaline buffer containing Triton X-100, the Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase activity in the tissue homogenate can be directly and selectively measured by this technique. The specific activity of the phosphodiesterase in brain and kidney was determined to be 80 and 6.5 nmol/h mg protein, respectively, and much lower activity was present in other tissues.     

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.     

Phospholipid-Metabolizing Enzymes in Alzheimer's Disease: Increased Lysophospholipid Acyltransferase Activity and Decreased Phospholipase A2 Activity

Abstract: Damage to brain membrane phospholipids may play an important role in the pathogenesis of Alzheimer's disease (AD); however, the critical metabolic processes responsible for the generation and repair of membrane phospholipids affected by the disease are unknown. We measured the activity of key phospholipid catabolic and anabolic enzymes in morphologically affected and spared areas of autopsied brain of patients with AD and in matched control subjects. The activity of the major catabolic enzyme phospholipase A₂ (PLA₂), measured in both the presence and absence of Ca²⁺, was significantly decreased (−35 to −53%) in parietal and temporal cortices of patients with AD. In contrast, the activities of lysophospholipid acyltransferase, which recycles lysophospholipids into intact phospholipids, and glycerophosphocholine phosphodiesterase, which returns phospholipid catabolites to be used in phospholipid resynthesis, were increased by ∼50–70% in the same brain areas. Brain activities of enzymes involved in de novo phospholipid synthesis (ethanolamine kinase, choline kinase, choline phosphotransferase, phosphoethanolamine cytidylyltransferase, and phosphocholine cytidylyltransferase) were either normal or only slightly altered. The activities of PLA₂ and acyltransferase were normal in the degenerating cerebellum of patients with spinocerebellar atrophy type 1, whereas the activity of glycerophosphocholine phosphodiesterase was reduced, suggesting that the alterations in AD brain were not nonspecific consequences of neurodegeneration. Our data suggest that compensatory phospholipid metabolic changes are present in AD brain that reduce the rate of phospholipid loss via both decreased catabolism (PLA₂) and increased phospholipid resynthesis (acyltransferase and glycerophosphocholine phosphodiesterase).     

Effect of phospholipase C treatment on the activity of the particulate cyclic nucleotide phosphodiesterase of rat brain

1. The activity of cAMP phosphodiesterase (PDE) was studied in a 10,000 g particulate fraction prepared from rat brain. 2. Phospholipase C such as sphingomyelin choline phosphodiesterase (SMase), phosphatidylinositol phosphodiesterase (PIase) and phosphatidylcholine phosphohydrolase (PCase) were used to deplete phospholipid(s) from the particulate fraction and their effects on PDE activity were investigated. 3. Treatment with SMase or PIase did not affect PDE activity whereas treatment with PCase resulted in inhibition. 4. It was also found that the PCase used not only hydrolyzed phosphatidylcholine but also other phospholipids such as phosphatidylethanolamine, phosphatidylserine and sphingomyelin.     

Developmental changes in rat blood choline concentration.

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Metabolites in the developing rat liver--a proton nuclear magnetic resonance spectroscopic study

We have used 1H-NMR spectroscopy in vitro to investigate metabolite changes in the rat liver in the first 21 days of life. The principle findings are firstly that betaine, a metabolite of choline, was relatively low (1-2 mumol/g) on days 1-7, then rose sharply to 5-6 mumol/g by day 19, whereas approximately reciprocal changes occurred in taurine levels. Secondly the lactate levels were remarkably low (0.1-0.8 mumol/g) on days 1-7. Changes in two other choline derivatives, phosphocholine (PC) and glycerophosphorylcholine (GPC) are also reported. The results are discussed in the context of the origin of these metabolites in the neonatal period, their levels in the adult (180 day-old) rat and the significance of the measured changes in metabolite levels during liver development.     

Active Site of Brain Zn2+-Glycerophosphocholine Cholinephosphodiesterase and Regulation of Enzyme Activity

Properties of active site of Zn²⁺-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quaternary ammonium compounds such as phosphocholine, choline or carnitine inhibited the enzyme, divalent metal ions such as Co²⁺, Mn²⁺ or Zn²⁺ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6–9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or diethylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tyrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tyrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen peroxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.     

Lipid metabolism in T47D human breast cancer cells: 31P and 13C-NMR studies of choline and ethanolamine uptake.

31P and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or 1,2-13C-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphoryl-choline (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with 13C and the reduction of label was monitored. 31P spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 +/- 1 fmol/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 50% over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The 31P and 13C data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine kinase were both on the order of 1.0 fmol/cell per h, and the rate constants for CTP:phosphocholine cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-1 for both enzymes. The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.     

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805-2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both D- and L-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [3H]glycerol from [3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.