Roger Sundler

Pathways for the incorporation of choline into rat liver phosphatidylcholines in vivo

1. 1. [1,2-14C2]Choline was injected intraportally into male rats. The incorporation of radioactivity into liver phosphorylcholine, betaine, CDP-choline and phosphatidyl-cholines over the first 300 s was determined. 2. 2. Labeled choline taken up by the liver did not equilibrate with the total tissue pool of free choline prior to incorporation into phosphorylcholine. The oxidation of [14C]choline to betaine was at least as rapid as the incorporation into phosphoryl-choline. 3. 3. The data were entirely consistent with phosphorylcholine being the precursor of CDP-choline which is the precursor of phosphatidylcholines. Any formation of CDP-choline from phosphatidylcholines must be very slow compared to that from phosphorylcholine. The rate of phosphatidylcholine synthesis via CDP-choline was 0.20–0.25 μmole/min per liver. 4. 4. Part of the [14C]choline was incorporated into phosphatidylcholines by a pathway not involving CDP-choline, probably an exchange reaction. The rate of such base exchange was only 120 compared to the rate of phosphatidylcholine synthesis via CDP-choline. 5. 5. A high proportion of the radioactivity incorporated through base exchange was found in arachidonoyl-phosphatidylcholines whereas the CDP-choline pathway preferentially gave rise to oleoyl- and linoleoyl-phosphatidylcholines.

A phospholipase A2 hydrolyzing arachidonoyl-phospholipids in mouse peritonea macrophages

A calcium‐dependent phospholipase A2 with half‐maximal activity at approx. 0.7 μM free Ca2+ has been identified in the cytosolic fraction from macrophages. The enzyme eluted as a 70 kDA protein upon gel chromatography and showed increased activity after 10 min pretreatment of the cells with 10 nM phorbol myristate acetate. No significant activity could be detected in the membrane fraction. The enzyme hydrolyzed arachidonic acid‐containing phosphatidylcholine and ‐ethanolamine as well as phosphatidylinositol. The release of arachidonic acid in the vitro assay was inhibited in a dose‐dependent manner by nordihydroguaiaretic acid and quercetin that are also potent inhibitors of the mobilization of arachidonic acid in intact macrophages.

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.

Biosynthesis of rat liver phosphatidylethanolamines from intraportally injected ethanolamine.

Abstract 1. 1. Following intraportal injection into male rats of radioactively labeled ethanolamine, the incorporation of radioactivity into liver phosphorylethanolamine, CDP-ethanolamine and different phosphatidylethanolamines was followed over the first 5 min. 2. 2. The time course for the phosphorylation of injected ethanolamine indicated that ethanolamine taken up by the liver does not equilibrate with the endogenous liver pool of free ethanolamine. The fact that the specific radioactivity of CDP-ethanolamine rapidly became twice as high as that of phosphorylethanolamine further suggests that phosphorylethanolamine formed from exogenous ethanolamine is not completely mixed with other liver pool(s) of phosphorylethanolamine. 3. 3. The distribution of radioactive ethanolamine among phosphatidylethanol-amines of different degrees of unsaturation indicated that it had been incorporated mainly via CDP-ethanolamine and not to any significant extent by base exchange. The calculated rate of phosphatidylethanolamine synthesis from CDP-ethanolamine was of the order 0.06–0.08 μmole/mm per liver. From analysis of the individual molecular species of phosphatidylethanol-amine it is suggested that the specificity of CDP - ethanolamine :1,2-diacyl- sn -glycerol ethanolaminephosphotransferase (EC 2.7.8.1) is less sensitive to the chain length of the saturated fatty acid in the diacylglycerol molecules than to their degree of unsaturation.

Macrophage arachidonate-mobilizing phospholipase A2: Role of Ca2+ for membrane binding but not for catalytic activity

A recently purified Ca(2+)-dependent intracellular phospholipase A2 from spleen, kidney and macrophage cell lines is activated by Ca2+ at concentrations achieved intracellularly. Using enzyme from the murine cell line J774 we here demonstrate the formation of a ternary complex of phospholipase, 45Ca2+ and phospholipid vesicle, and provide evidence for a single Ca(2+)-binding site on the enzyme involved in its vesicle binding. Although Ca2+ binds to and functions as an activator of the enzyme, this ion does not appear to be involved in its catalytic mechanism, since enzyme brought to the phospholipid vesicle by molar concentrations of NaCl or NH4+ salts exhibited Ca(2+)-independent catalytic activity.

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.

Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan.

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.

Antimalarial Drugs Inhibit Phospholipase A2 Activation and Induction of Interleukin lβ and Tumor Necrosis Factor α in Macrophages: Implications for Their Mode of Action in Rheumatoid Arthritis

1. The effects of antimalarial drugs on the intracellular signaling leading to activation of the phospholipase C and phospholipase A2 pathways and the induction of proinflammatory cytokines have been studied in mouse macrophages. 2. Both chloroquine and quinacrine, and to a lesser extent hydroxychloroquine, inhibited arachidonate release and eicosanoid formation induced by phorbol diester. This inhibition was due to that of the activation of the arachidonate-mobilizing phospholipase A2. 3. All three antimalarials potently inhibited arachidonate release induced by zymosan. They also inhibited the zymosan-induced formation of inositol phosphates, which hints that an inhibitory effect at the phospholipase C level might explain the inhibition of the response to zymosan. 4. Quinacrine, and to a lesser extent chloroquine, has an inhibitory effect on the lipopolysaccharide- or zymosan-induced expression of interleukin 1beta and tumor necrosis factor alpha, both at the mRNA and protein levels. This, in particular, has important implications for the mode of action of these compounds in rheumatoid arthritis.

Auranofin inhibits the induction of interleukin 1β and tumor necrosis factor α mRNA in macrophages

Gold compounds are widely used in the treatment of rheumatoid arthritis, but their mechanisms of action remain unclear. We demonstrate here that auranofin (AF) (0.1-3 microM), but neither the hydrophilic gold compounds aurothiomalate (ATM) and aurothioglucose nor methotrexate or D-penicillamine, inhibits the induction of interleukin 1 beta and tumor necrosis factor (TNF) alpha mRNA and protein by either zymosan, lipopolysaccharide (LPS), or various bacteria in mouse macrophages. The auranofin-mediated inhibition of the induction of TNF-alpha mRNA was stronger than that of interleukin (IL) 1 beta mRNA. AF, but not the other drugs, also inhibited zymosan-induced mobilization of arachidonate. The fact that AF inhibited the induction of mRNA for both these proinflammatory cytokines, irrespective of which stimulus was used, may indicate that it affects some common signal transduction step vital to their induction.