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PubMed2001

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C.

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.

Androstadienes↗

PubMed2002

Venom from the ectoparasitic wasp Nasonia vitripennis increases Na+ influx and activates phospholipase C and phospholipase A2 dependent signal transduction pathways in cultured insect cells.

The mode of action of venom from the ectoparasitic wasp Nasonia vitripennis in eliciting cell death was examined using an in vitro approach with BTI-TN-5B1-4 cells, and the cell responses were compared to those evoked by the extensively studied wasp toxin mastoparan. Wasp venom increased plasma membrane permeability to Na+, resulting in cellular swelling and death due to oncosis. When ouabain was used to disable Na+, K+-ATPases, the effects of venom were enhanced. Measurements of intracellular calcium using fluo-4 AM revealed a rearrangement and an increase in cytosolic [Ca+2]i within 30 min after exposure of BTI-TN-5B1-4 cells to venom. This venom-mediated increase in Ca+2 was apparently due to mobilization of intracellular stores since the changes occurred in the absence of extracellular Ca+2. Phospholipase C (PLC) inhibitors, neomycin and U-73122, blocked the venom-induced death temporarily (<3h), but by 24h, all venom-treated cells swelled and lysed. Pre-treatment of cells with caffeine or theophylline but not ryanodine attenuated the induction of oncosis by wasp venom. Anti-inflammatory peptide 1 (antiflammin 1) but not bromophenacyl bromide, agents that block phospholipase A2 (PLA2) activity, abolished the responsiveness of BTI-TN-5B1-4 cells to venom. These results suggest that venom initiates cell death by inducing Ca+2 release from intracellular stores probably via phospholipase C and IP3. A possible mode of action for venom from N. vitripennis requiring dual activation of PLC and PLA2 is discussed and compared to the pathways known to be activated by mastoparan.

Animals↗

PubMed1997

Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages.

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases.

Animals↗

PubMed1997

Induction of cytosolic phospholipase A2 activity by phosphatidic acid and diglycerides in permeabilized human neutrophils: interrelationship between phospholipases D and A2.

Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureus alpha-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor alpha before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In alpha-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs.

Arachidonic Acid↗

PubMed2003

The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling.

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.

Amino Acid Motifs↗

PubMed1984

Studies on phospholipase A inhibitor in blood plasma. I. Purification and characterization of phospholipase A inhibitor in bovine plasma.

Phospholipase A inhibitor was found in bovine, human and porcine plasma. The inhibitor was purified about 300-fold from bovine plasma by ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and anti-BSA antibody-agarose column chromatography to remove albumin. The purified material was homogeneous as judged by immunoelectrophoresis and 0.7% agarose-2% polyacrylamide gel electrophoresis. The inhibitor suppressed not only rat and human plasma phospholipase A2 activity, but also purified Mamushi (Agkistrodon halys blomhoffi) venom phospholipase A2-II. Bovine plasma inhibitor was shown to be a lipoprotein containing phosphatidylcholine, sphingomyelin, cholesterol ester, and triacyl glycerol as major lipid components. The molecular weight of the native inhibitor was nearly the same as that of bovine plasma high density lipoprotein (HDL) when determined by the gel filtration method. The molecular weights of two subunits of the inhibitor observed on polyacrylamide gel electrophoresis in the presence of SDS were about 10K and 42K. The anti-inhibitor antiserum obtained from rabbits immunized with the highly purified inhibitor cross-reacted with bovine plasma alpha-lipoprotein (HDL). The lipid composition of the inhibitor was similar to that of HDL, but apoinhibitor and apoHDL were different in molecular weight and solubility in various buffers.

Animals↗

PubMed1983

Semisynthesis of phospholipase A2. The effect of substitution of amino-acid residues at positions 6 and 7 in bovine and porcine pancreatic phospholipases A2 on catalytic and substrate-binding properties.

The N-terminal alpha-helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg-Ser at positions 6 and 7, whereas the bovine enzyme has Asn-Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of epsilon-amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6-Gly7]AMPA and bovine [Ser7]AMPA and [Arg6-Ser7]AMPA. As we had previously found for the Asn6 leads to Arg bovine substitution, an Asn6-Gly7 leads to Arg5-Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid-water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7 leads to Ser produces almost no effect on these properties. Ser7 leads to Ala and Ser7 leads to Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6 leads to Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6 leads to Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid-water interfaces of Asn6 leads to Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6 leads to Asn substitution in porcine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7 leads to Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.

Amides↗

PubMed1992

Activation of phospholipase A2 and phospholipase C by endothelin-1 in human endometrium.

Human endometrium contains specific binding sites for iodinated endothelin (ET)-1, ET-2 and ET-3, and ET-1 stimulates prostaglandin (PG) F2 alpha synthesis from explants of proliferative endometrium in short-term culture. This study has investigated the cellular responses of normal proliferative endometrium to ET-1. Radioimmunoassay was used to measure PG release and Dowex anion-exchange column chromatography was utilized to assess the accumulation of inositol phosphates. Endothelin-1 induced a significant increase in PGF2 alpha release (basal median: 1465 pg/mg per 60 min (range: 541-3935 pg/mg per 60 min); ET-1-stimulated: 1813 pg/mg per 60 min (1021-5714 pg/mg per 60 min); P < 0.04 using Wilcoxon signed rank test). The effect of ET-1 was attenuated in the presence of the phospholipase A2 inhibitor quinacrine. Endothelin-1 induced a rapid, transient and concentration-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), measured by the accumulation of tritiated inositol phosphates. Following a 1-min stimulation with ET-1 (100 nmol/l), [3H]inositol mono-, bis- and trisphosphate fractions increased from median values of 490.0 d.p.m./mg dry wt (range: 348.0-807.0 d.p.m./mg dry wt), 120.0 d.p.m./mg dry wt (93.6-144.1 d.p.m./mg dry wt) and 67.0 d.p.m./mg dry wt (54.2-85.0 d.p.m./mg dry wt) to 939.0 d.p.m./mg dry wt (635.9-1596.0 d.p.m./mg dry wt; P < 0.03), 145.0 d.p.m./mg dry wt (127.0-293.9 d.p.m./mg dry wt; P < 0.05) and 146.0 d.p.m./mg dry wt (77.5-187.0 d.p.m./mg dry wt; P < 0.03) respectively. These results suggest that ET-1 activates the phospholipase A2 and PtdIns(4,5)P2-specific phospholipase C in human proliferative endometrium, resulting in the generation of PGF2 alpha and second messengers respectively which are pivotal to endometrial function.

Endometrium↗

PubMed2003

Amplification mechanisms of inflammation: paracrine stimulation of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid.

In macrophages and other major immunoinflammatory cells, two phospholipase A(2) (PLA(2)) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A(2) (cPLA(2)) and a secreted phospholipase A(2) (sPLA(2)). In this study, the molecular mechanism underlying cross-talk between the two PLA(2)s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA(2)-dependent manner, and addition of exogenous group V sPLA(2) to the activated cells increases the release. This sPLA(2) effect is abolished if the cells are pretreated with cPLA(2) inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA(2). In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA(2) activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA(2) response to levels found in cells displaying normal cPLA(2) activity. Moreover, 12- and 15-HPETE are able to enhance sPLA(2) activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA(2)-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA(2) with cPLA(2) for a full AA mobilization response.

Arachidonic Acid↗

PubMed1992

[Inhibitory effects of calphobindins on phospholipase A2 and phospholipase C].

Calphobindins (CPBs) I, II and III which we isolated and characterized from placenta belong to a family of calcium and phospholipid binding proteins represented by lipocortins known to inhibit phospholipase A2(PLA2). The amino acid sequence of three CPBs shares much homology with lipocortins I and II, so we investigated the functions of CPBs in affecting the enzymatic activities of PLA2 and also phospholipase C(PLC). In our experiments CPBs I, II and III showed an apparent dose-dependent inhibition of PLA2 and PLC activities, and all three proteins had a more potent inhibitory effect than lipocortins. The inhibition was overcome by high phospholipid substrate concentrations, for example, in the presence of 2.9 x 10(-9)M PLA2 and 2.6 x 10(-7)M CPB I, the inhibition decreased from 85 to 9% as phosphatidylethanolamine was increased from 10 to 100 microM. A similar result was observed in the case of PLC also. The evidence strongly suggests that the inhibitory effect of CPBI results from binding of the proteins to the phospholipid substrate rather than from a direct action on the phospholipase itself.

Annexins↗

PubMed2004

Short report: Lethal malaria in cytosolic phospholipase A2- and phospholipase A2IIA-deficient mice.

Lipid mediators play important roles in the pathogenesis of malaria. Phospholipase A2s are enzymes involved in the production of these mediators, and they function in inflammation. Among them, cytosolic phospholipase A2 (cPLA2) is a key enzyme in the metabolism of arachidonic acid, the first intermediate in the production of lipid mediators. Plasmodium berghei ANKA causes cerebral malaria in CL57B/6 mice, and we recently produced cPLA2-deficient mice with this background. With the expectation of reduced pathogenicity, we performed experimental infection in these mice. Unexpectedly, the infected mice developed cerebral malaria and died at the same time as the control mice, while the parasitemia progressed similarly in both groups. These observations suggest that secretory PLA2s rather than cPLA2 may be involved in the aggravation, although possible compensation by the induction of other enzymes has not been excluded. The present findings are expected to help clarify the involvement of various phospholipase A2s in malaria.

Animals↗

PubMed1985

Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases.

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.

Animals↗

PubMed1994

Arginine vasopressin-stimulated insulin secretion and elevation of intracellular Ca++ concentration in rat insulinoma cells: influences of a phospholipase C inhibitor 1-[6-[[17 beta-methoxyestra-1,3,5(10)-trien- 17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) and a phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid.

The present study investigated the mechanism by which arginine vasopressin (AVP) increases insulin secretion in rat insulinoma (RINm5F) cells by using a specific phospholipase C (PLC) inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), and a phospholipase A2 (PLA2) inhibitor, N-(p-amylcinnamoyl)anthranilic acid (ACA). AVP (0.1-100 nM) increased insulin secretion and cytosolic free Ca++ concentration ([Ca++]i) dose-dependently. AVP-induced increases in the intracellular concentration of inositol 1,4,5-trisphosphate (IP3) and [Ca++]i were dose-dependently inhibited by U-73122 (2-8 microM). At 8 microM, U-73122 abolished AVP's effect on IP3 and [Ca++]i, but AVP-induced increases in insulin secretion were only reduced by 35%. In contrast, 8 microM U-73122 did not reduce the ionomycin (a Ca++ ionophore, 100 nM)-induced increase in [Ca++]i. The discrepancy between the results of [Ca++]i and insulin secretion in U-73122 experiments is indicative of the multiple signal transduction pathways associated with the activation of AVP receptors, specifically the Ca(++)-independent pathway. The phospholipase A2 inhibitor ACA (100 microM) did not antagonize AVP (10 nM)-induced increases in insulin secretion. These results suggested: 1) U-73122 blocks PLC activities but fails to block other signal transduction pathways that trigger insulin secretion in these cells and 2) AVP increases insulin release from RINm5F cells through both the PLC-mediated Ca(++)-dependent and Ca(++)-independent pathways.

Animals↗

PubMed1996

Phospholipase A2 and phospholipase D are involved in macrophage NADPH oxidase-mediated oxidation of low density lipoprotein.

Macrophage-mediated oxidation of low density lipoprotein (LDL) under oxidative stress induces activation of reduced nicotinamide adenine dinuleotide phosphate (NADPH) oxidase. Using J-774 A.1 macrophages, the present study demonstrates that phospholipase A2 (PLase A2) as well as phospholipase D (PLase D) are involved in macrophage NADPH oxidase-mediated oxidation of LDL. Furthermore, the products of these phospholipases, arachidonic acid and phosphatidic acid, can induce NADPH oxidase activation, followed by cell-mediated oxidation of LDL. This LDL oxidation was shown to be dependent on extracellular calcium ions. We conclude that PLase A2 and PLase D can induce macrophage NADPH oxidase-dependent oxidation of LDL and thus can contribute to the formation of atherogenic oxidized lipoprotein.

Arachidonic Acid↗

PubMed2004

Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells.

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.

Arachidonic Acid↗

PubMed1999

Insulin-like growth factor-I-dependent stimulation of nuclear phospholipase C-beta1 activity in Swiss 3T3 cells requires an intact cytoskeleton and is paralleled by increased phosphorylation of the phospholipase.

Swiss 3T3 mouse fibroblasts were exposed to 10 microM colchicine to disrupt microtubules, then stimulated with insulin-like growth factor-I. Immunoprecipitation experiments showed that insulin-like growth factor-I receptor and insulin receptor substrate-1 were tyrosine phosphorylated to the same extent in both cells treated with colchicine and in those not exposed to the drug. Moreover, the activity of phosphatidylinositol 3-kinase was not affected by incubation with colchicine. While in nuclei prepared from cells not exposed to colchicine it was possible to detect an insulin-like growth factor-I-dependent increase in the mass of diacylglycerol, as well as stimulation of phospholipase C activity, no similar changes were observed in nuclei obtained from cells treated with colchicine. Activation of the nuclear phospholipase activity was paralleled by an increase of its phosphorylation. Immunofluorescent studies revealed that mitogen-activated protein kinase did not translocate towards the nucleus when the cytoskeleton was depolymerized. These results show that in Swiss 3T3 cells some as yet unknown events necessary for the insulin-like growth factor-I-dependent activation of nuclear polyphosphoinositide metabolism require the presence of an intact cytoskeleton and are situated down-stream the activation of insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Activation of nuclear phospholipase C-beta1 might be linked to its phosphorylation and translocation of mitogen-activated protein kinase to the nucleus.

3T3 Cells↗

PubMed1999

Guanosine 5'-O-(3-thiotriphosphate)-induced O-2 generation in permeabilized neutrophils requires protein kinase C and phospholipase C but not tyrosine kinase or phospholipase D.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces respiratory burst (O-2 generation) in permeabilized human neutrophils. The signal pathway from GTPgammaS to the enzyme responsible for O-2 generation (NADPH oxidase) is not well defined. To elucidate the signaling pathway activated by GTPgammaS, we used selective inhibitors to test for the involvement of several enzymes, comparing the effects of these inhibitors on fMet-Leu-Phe (fMLP) activation. GTPgammaS-induced respiratory burst was not influenced by genistein, a selective inhibitor of tyrosine kinase, while fMLP-induced response was completely abolished. The respiratory burst by GTPgammaS was efficiently inhibited by the protein kinase C inhibitor GF109203X even more than fMLP activation. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 showed a partial inhibition of both GTPgammaS and fMLP activation. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, completely blocked fMLP activation, but had no effect on the GTPgammaS-induced respiratory burst. Using U73122, phospholipase C is shown to be essential in GTPgammaS signaling as well as fMLP signaling. Butanol blocked fMLP signaling but not GTPgammaS signaling, indicating that only fMLP activation involves phospholipase D. These results suggest that there are several differences between GTPgammaS- and fMLP-induced activation, but both activators share a common pathway including phospholipase C, protein kinase C, and MAPK kinase.

Cell Membrane Permeability↗

PubMed1999

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.

Animals↗