Dual signaling by the alpha(v)beta(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells

Vitronectin, which ligates the alpha(v)beta(3)-integrin, increases both lung capillary permeability and lung endothelial Ca(2+). In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located alpha(v)beta(3)-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha(v)beta(3) monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha(v)beta(5). Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA(2) inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca(2+) or cPLA(2) translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca(2+) chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical alpha(v)beta(3) in BPAECs caused ERK2 activation and an increase of intracellular Ca(2+), both conjointly required for cPLA(2) activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated "two-hit" signaling pathway that regulates an integrin-induced proinflammatory response.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

4-Bromophenacyl bromide induces Ca2+ influx in human gingival fibroblasts

4-Bromophenacyl bromide (BPB) is generally used as a phospholipase A(2) (PLA2) inhibitor. In the present study, we demonstrate that BPB induces Ca2+ influx in human gingival fibroblasts. In fura-2-loaded human gingival fibroblasts, BPB evoked a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The BPB-induced Ca2+ mobilization was also shown in a single fluo-3-loaded-fibroblast. The BPB-induced increase in [Ca2+]i was completely abolished by the elimination of the external Ca2+. Ca2+ influx induced by the Ca2+-mobilizing agonist histamine was markedly enhanced in the presence of BPB. These suggest that the BPB-induced Ca2+ mobilization is due to the influx of extracellular Ca2+. However, it is unlikely that the effect of BPB is dependent on the inhibition of PLA2 activity, because other PLA2 inhibitors, such as AACOCF3, quinacrine dihydrochloride and manoalide, failed to induce Ca2+ mobilization. Chemical compounds similar to BPB, but which have no -CH2-Br at position 1 in the benzene ring failed to evoke Ca2+ mobilization, indicating that the position of -CH2--Br in BPB is important for causing the Ca2+ influx.     

Macrophage arachidonate release via both the cytosolic Ca2+-dependent and -independent phospholipases is necessary for cell spreading

We have observed that phospholipase A₂ (PLA₂) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA₂s in these processes. The PLA₂-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca²⁺-independent PLA₂ (iPLA₂)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca²⁺-dependent phospholipase (cPLA₂)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA₂-inhibited cells. AA release during spreading was also inhibited by Ca²⁺ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA₂. Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca²⁺ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA₂ (which appears to be constitutively active) and cPLA₂ (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA₂.     

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2)

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1 beta by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1 beta and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1 beta processing was not due to perturbation of the export machinery for pro-IL-1 beta and IL-1 beta or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1 beta processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1 beta maturation.     

Essential fatty acid deficiency impairs macrophage spreading and adherence. Role of arachidonate in cell adhesion

Dietary polyunsaturated fatty acid manipulation exerts a strikingly protective effect in models of tissue inflammation and injury. A critical element of this effect appears to revolve around leukocyte trafficking but underlying mechanisms are ill understood. In the current study it was observed that essential fatty acid (EFA) deficiency markedly impaired the capacity of resident macrophages to spread and adhere. This effect was not a simple function of the alteration of membrane fatty acid composition. Elicited EFA-deficient macrophages were equally adherent to elicited control cells, despite the fact that they were equally EFA-deficient relative to resident EFA-deficient cells. With respect to the mechanism underlying defective macrophage adherence in EFA deficiency, no change in the expression of cell surface adherence molecules (Fc receptor, Mac-1, or LFA-1) was noted with the deficiency state. Also, an adherence defect could not be induced in normal cells pharmacologically with cyclooxygenase blockade, lipoxygenase blockade, or a platelet-activating factor receptor antagonist. In contrast, phospholipase inhibition was able to induce a spreading and adherence defect in resident macrophages similar to that seen with EFA deficiency. Using several phospholipase inhibitors, a correlation between phospholipase inhibition and impairment of adherence was observed. Adding back exogenous fatty acids to cells after phospholipase inhibition demonstrated that normal adherence was reconstituted with arachidonate. This alteration in macrophage spreading and adherence with EFA deficiency may be an important component of the anti-inflammatory effect of dietary polyunsaturated fatty acid manipulation. Additionally, these results suggest that arachidonate may be an intracellular mediator of leukocyte adherence.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Neomycin is a potent agent for arachidonic acid release in human platelets

Neomycin (10 microM - 1 mM) was found to induce considerable release of [3H]arachidonic acid from phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine in saponin-permeabilized human platelets prelabeled with [3H]arachidonic acid. The magnitude of arachidonate liberation was almost equal to that induced by A23187 (400 nM) or even greater than that caused by thrombin (1 U/ml). Moreover, neomycin enhanced arachidonic acid release induced by thrombin. Since no significant formation of diacylglycerol and phosphatidic acid via phospholipase C was observed, the arachidonate liberation was considered to be mainly catalyzed by phospholipase A2 action. Addition of neomycin (100 microM) to 45Ca2+-preloaded platelets elicited 45Ca2+ mobilization from intracellular stores. These results indicate evidence that neomycin evokes Ca2+ mobilization from internal stores, which leads to activation of phospholipase A2 to release arachidonic acid in human platelets.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts

RANK ligand (RANKL) induces activation of NFkappaB, enhancing the formation, resorptive activity, and survival of osteoclasts. Ca(2+) transduces many signaling events, however, it is not known whether the actions of RANKL involve Ca(2+) signaling. We investigated the effects of RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp. RANKL induced transient elevation of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to maxima 220 nm above basal, resulting in activation of Ca(2+)-dependent K(+) current. RANKL elevated [Ca(2+)](i) in Ca(2+)-containing and Ca(2+)-free media, and responses were prevented by the phospholipase C inhibitor. Suppression of [Ca(2+)](i) elevation using the intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NFkappaB was found predominantly in the cytosol of untreated osteoclasts. RANKL induced transient translocation of NFkappaB to the nuclei, which was maximal at 15 min. or BAPTA delayed nuclear translocation of NFkappaB. Delays were also observed upon inhibition of calcineurin or protein kinase C. We conclude that RANKL acts through phospholipase C to release Ca(2+) from intracellular stores, accelerating nuclear translocation of NFkappaB and promoting osteoclast survival. Such cross-talk between NFkappaB and Ca(2+) signaling provides a novel mechanism for the temporal regulation of gene expression in osteoclasts and other cell types.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.