Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

Effect of calmodulin antagonists on lysosomal enzyme secretion and phospholipid metabolism in guinea-pig macrophages

The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-β-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca²⁺ ionophore A23187. The effect of calmodulin antagonists on the incorporation of [³²P]P_i or [³H]glycerol into glycerolipids as well as on the redistribution of [¹⁴C]glycerol or [³H]arachidonic acid in [¹⁴C]glycerol- or [³H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [³²P]P_i incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [³²P]P_i into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [³²P]P_i incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [³H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [³H]glycerol into PtdA and PtdIns was greatly enhanced. But [³H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [³H]glycerol was maximally activated by 10μm-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [¹⁴C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [¹⁴C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [³H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [³H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-³H]diacylglycerol and non-esterified [³H]-arachidonic acid was also enhanced, but the increase in [³H]arachidonic acid was only observed at concentrations between 1 and 50μm. [Arachidonate-³H]PtdIns was not significantly affected. The activated formation of [arachidonate-³H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.     

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E₂ and F_2α which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of ³H-arachidonic acid labelled phospholipids, releasing free ³H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases ³H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-³H]inositol labelled phospholipids. Phospholipase A₂ (PLA₂), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, ³H-arachidonic acid labelled phospholipid breakdown, and ³H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo-[2-³H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA₂ and PLD, but not by activation of phosphatidylinositol-specific PLC. © 1993 Wiley-Liss, Inc.     

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-¹⁴C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-¹⁴C]glycerol and by determining incorporation of ¹⁸O from H₂ ¹⁸O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-¹⁴C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca²⁺-dependent phospholipase A₂ activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

Synergistic Effects of Acetylcholine and Glutamate on the Release of Arachidonic Acid from Cultured Striatal Neurons

Abstract: The activation of muscarinic and NMDA receptors by carbachol and NMDA, respectively, stimulated the release of [³H]arachidonic acid ([³H]AA) from cultured striatal neurons. Striking synergistic effects were observed when both agonists were coapplied. This synergistic response was suppressed by atropine or (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate and inhibited by magnesium. It was markedly reduced in the absence of external calcium and suppressed by mepacrine. NMDA strongly elevated the intracellular calcium concentration ([Ca²⁺]_i), but carbachol was ineffective. Ionomycin, α-amino-3-hydroxy-5-methylisoxazole-4-propionate, or potassium depolarization, which increased [Ca²⁺]_i but was ineffective on [³H]AA release, also potentiated the carbachol response. Sphingosine and Ro 31-8220 suppressed the responses evoked by carbachol, NMDA, or both agonists. However, no synergistic responses could be observed when phorbol 12-myristate 13-acetate was associated with either carbachol or NMDA. Together, these results suggest that both the massive influx of calcium induced by NMDA and the coupling of muscarinic receptors with a putative phospholipase A₂ are required for the strong synergistic effects of carbachol and NMDA on [³H]AA release. Synergistic effects were also observed with acetylcholine and glutamate in the presence of magnesium, further revealing the physiological relevance of this process.     

Phospholipase A2 Stimulation by Methyl Mercury in Neuron Culture

Abstract: In primary prelabeled cultures of cerebellar granule cells, methyl mercury (MeHg) induced a concentration- and time-dependent release of [³H]arachidonic acid. MeHg-induced [³H]arachidonate release was partially dependent on the extracellular Ca²⁺ concentration. MeHg at 10–20 µM also stimulated basal ⁴⁵Ca²⁺ uptake after 20 min of incubation at 37°C, and at 10 µM inhibited K⁺ depolarization-stimulated uptake. MeHg stimulated [³H]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A₂ (PLA₂) activation preceded cytotoxicity, but at higher concentrations of MeHg such dissociation was not evident. Inhibition of MeHg-induced PLA₂ activation by 100 µM mepacrine failed to modify cytotoxicity. MeHg-induced lipoperoxidation, measured as the production of thiobarbituric acid-reacting products, was inhibited by α-tocopherol without inhibition of [³H]arachidonate release. The absence of α-tocopherol inhibition of MeHg-induced arachidonate release precludes a causal role for lipoperoxide-induced PLA₂ activation in this system. Moreover, MeHg induced an increased susceptibility of unilamellar vesicles to exogenous PLA₂ in the presence of low Ca²⁺ concentrations without evidence of lipid peroxidation. [³H]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relatively high proportional incorporation was found in the combined phosphatidylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the phosphatidylinositol fraction, indicating a preferential turnover in this phospholipid species in the presence of MeHg.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.     

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [³H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A₂ and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.     

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phospahtidylinositol turnover measured as modification of incorporation of [³²P]P_i and [³H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca²⁺, without a decrease in the total quantity of Ca²⁺ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca²⁺ concentration [Ca²⁺]_i, without a decrease in the magnitude of the final increase of [Ca²⁺]_i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca²⁺-phospholipid-dependent protein kinase C.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

Effect of Retinoic Acid on the Ca2+-Independent Phospholipase A2 in Nuclei of LA-N-1 Neuroblastoma Cells

LA-N-1 neuroblastoma cell cultures contain Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.     

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca²⁺sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺([Ca²⁺]_i) due to plasmalemmal Ca²⁺influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca²⁺stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺]_iper se causes slowing of peptide chain elongation is lacking. L-type Ca²⁺channel activity of GH₃pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺]_ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺]_ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca²⁺channel antagonist, nisoldipine, lowered [Ca²⁺]_iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺]_ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca²⁺contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺]_ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca²⁺]_ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.     

U-73122 inhibits carbachol-induced increases in [Ca2+]i,IP3, and insulin release in β-TC3 cells

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca²⁺]_i, and IP₃ in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca²⁺]_i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP₃) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca²⁺]_i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca²⁺]_i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP₃, [Ca²⁺]_i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.     

Phosphatidylinositol turnover in mitogen-activated lymphocytes. Suppression by low-density lipoproteins

Low-density (LD) lipoproteins inhibit phytohaemagglutinin-enhanced turnover of phosphatidylinositol in human peripheral lymphocytes. Turnover was assessed by ³²P incorporation into phospholipids and by loss of ³²P from [³²P]phosphatidylinositol. Inhibition of lipid turnover by LD lipoproteins is not the result of a change in the amount of phytohaemagglutinin required for maximum cellular response. Neither phytohaemagglutinin nor LD lipoproteins influence ³²P incorporation into phosphatidylethanolamine and phosphatidylcholine during the first 60min after mitogenic challenge. The extent of inhibition of phosphatidylinositol turnover by LD lipoproteins depends on the concentration of LD lipoproteins present in the incubation medium: 50% of maximum inhibition occurs at a low-density-lipoprotein protein concentration of 33μg/ml and maximum inhibition occurs at low-density-lipoprotein protein concentrations above 100μg/ml. Phytohaemagglutinin stimulates ³²P incorporation into phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. However, LD lipoproteins abolish ³²P incorporation into phosphatidylinositol without affecting incorporation into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The ability of LD lipoproteins to inhibit phytohaemagglutinin-induced phosphatidylinositol turnover is mimicked by EGTA. Furthermore, inhibition of LD lipoproteins by phytohaemagglutinin-induced ³²P incorporation into phosphatidylinositol correlates directly with inhibition by LD lipoproteins of Ca²⁺ accumulation. These results suggest that Ca²⁺ accumulation and turnover of phosphatidylinositol are coupled responses in lymphocytes challenged by mitogens. The step in phosphatidylinositol metabolism that is sensitive to LD lipoproteins and, by inference, that is coupled to Ca²⁺ accumulation is release of [³²P]phosphoinositol from phosphatidylinositol.     

Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, α-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes

Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [³H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A₂ and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A₂, cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C. This revised version was published online in August 2006 with corrections to the Cover Date.