Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of β1 Integrin

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKCϵ, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plama membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of β1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. J. Cell. Physiol. 173:361–370, 1997. © 1997 Wiley-Liss, Inc.     

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.     

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca²⁺ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca²⁺. Addition of exogenous AA to cells spreading in the absence of extracellular Ca²⁺ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A₂ (cPLA₂) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA₂ phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca²⁺, all cPLA₂ became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca²⁺, the time for complete cPLA₂ phosphorylation was lengthened to <4 min. Maximal translocation of cPLA₂ from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca²⁺ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA₂ translocated to the membrane in the presence of extracellular Ca²⁺ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca²⁺ only 55–65% of the total cPLA₂ was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca²⁺. Although translocation of cPLA₂ from cytosol to membrane was Ca²⁺ dependent, phosphorylation of cPLA₂ was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA₂, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca²⁺. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA₂ phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA₂ and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA₂ translocation and phosphorylation remained unaffected. In conclusion, although cPLA₂-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA₂ phosphorylation and the release of AA.     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

Mg2+ mediates the cell-substratum interaction of Arg-Gly-Asp-dependent HeLa cell collagen receptors

Three HeLa cell surface collagen receptors of apparent molecular mass 102/58, 87, and 38/33 kDa were eluted from gelatin-Sepharose with salt gradients or Arg-Gly-Asp-containing peptides. To understand how the collagen receptors are involved in HeLa cell spreading on collagen we investigated the effects of divalent cations and Arg-Gly-Asp-containing peptides on adhesion to gelatin, since HeLa cells behave similarly on both native type I collagen and gelatin substrata and also whether Arg-Gly-Asp-containing substrata would substitute for gelatin in facilitating cell spreading. Gly-Arg-Gly-Asp-Ser-containing peptides in solution inhibited HeLa cell spreading onto gelatin and promoted only partial HeLa cell spreading when bound to tissue culture plastic. Both partial spreading of HeLa cells on the Gly-Arg-Gly-Asp-Ser substratum and full spreading on gelatin was dependent on Mg2+, but not on Ca2+. Binding of the 102/58-, 87-, and 38/33-kDa collagen receptors to gelatin-Sepharose was increased fourfold in the presence of Mg2+, and subsequent elution of the collagen receptors and a 45-kDa collagen-binding protein not thought to be involved in HeLa cell spreading was achieved with EDTA. In contrast, affinity chromatography on Gly-Arg-Gly-Asp-Ser-Sepharose eluted predominantly the 45-kDa collagen-binding protein and the 38/33-kDa collagen receptor. In summary, the Mg2(+)-dependent interaction of the collagen receptors with the Arg-Gly-Asp sequence in collagen appears to be essential for the initial events in HeLa cell spreading but is not sufficient for full cell spreading.     

Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion

Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.     

Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.     

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B₄ (LTB₄) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E₂ (PGE₂) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB₄ signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB₄. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE₂ reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro. leukotriene B₄; prostaglandin E₂; spreading; migration; bridges     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

The identification and characterization of collagen receptors involved in HeLa cell-substratum adhesion

Four proteins of molecular mass 102, 87, 45, and 38 kDa were isolated from plasma membrane preparations by affinity chromatography. The 102-, 87-, and 38-kDa proteins were shown to be collagen receptors involved in the adhesion of HeLa cells to a gelatin substratum. All four proteins were eluted by high salt from affinity columns made of either types I or IV collagen or type I gelatin. Generally, a total of six major proteins were found in the high salt eluates, although the relative amounts of each varied among experiments. Immunoprecipitation, immunoblotting, and limited peptide mapping indicated that the 102-kDa protein was most sensitive to proteolysis leading to the formation of proteins of molecular mass 58 and 54 kDa. Even in the presence of a mixture of protease inhibitors the 58-kDa fragment was usually the more abundant species. Lectin binding indicated that the 102-, 87-, and 38-kDa proteins contain carbohydrate. Phase-partitioning with Triton X-114 and the need to solubilize the proteins in Triton X-100 indicated that the 102-, 87-, 45-, and 38-kDa proteins have a hydrophobic domain. The 87-kDa protein partitioned exclusively with the detergent-rich phase, suggesting that it is the most hydrophobic. Cell surface labeling with 125I indicated that the four proteins have an extracellular domain. Four criteria were used to determine which of the four proteins are collagen receptors mediating cell-substrate adhesion: 1) during HeLa cell adhesion, proteins with Mr values similar to all four proteins or their peptide fragments were cross-linked to a gelatin substratum derivatized with a photoactivatable probe; 2) a pentapeptide containing the Arg-Gly-Asp cell recognition sequence eluted the same four proteins as those found by high salt elution of collagen affinity columns; 3) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, inhibited the spreading of HeLa cells on a gelatin substratum; 4) monospecific antibodies to the 102-, 87-, and 38-kDa proteins, but not the 45-kDa protein, bound to culture dishes substituted for gelatin in mediating the spreading of HeLa cells. Taken together, the data suggest that the 102-, 87-, and 38-kDa proteins are collagen receptors involved in HeLa cell adhesion. Although the 45-kDa protein has two of the characteristics of a collagen receptor defined here, it does not fit the criteria for one involved in cell-substratum adhesion.     

Involvement of arachidonic acid metabolism and EGF receptor in neurotensin-induced prostate cancer PC3 cell growth

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.     

Protein kinase C dependent and independent activation of phospholipase A2 under calcium ionophore (A23187) exposure in rabbit pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to the calcium ionophore A23187, dose-dependently stimulates arachidonic acid (AA) release and phospholipase A2 (PLA2) activity. The protein kinase C (PKC) inhibitor, sphingosine does not prevents AA release and PLA2 activity caused by low doses of A23187. In contrast, sphingosine markedly prevents AA release and PLA2 activity caused by higher doses of A23187. PKC activity profile indicates that treatment of the cells with low doses of A23187 does not cause significant alteration of PKC translocation from cytosol to membrane whereas higher concentrations of the ionophore dose-dependently enhance PKC translocation from cytosol to membrane in the smooth muscle cells.     

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.     

Production of factors required for cell attachment and spreading is a constitutive property in mouse A9 cells.

It is demonstrated here that cells in a suspension culture of an established mammalian cell line release non-dialyzable factors into their growth medium. These factors are capable of promoting the adhesion and spreading of these cells on a generally non-attachable substratum and also promote spreading on an adhering substrate. Evidence is presented which demonstrates that the spreading promotion activity of the condition medium is dependent on the cell density of the culture from which it was derived. Dilution of the conditioned medium results in a proportionate dilution of the spreading promotion activity. The results clearly demonstrate that the production of this spreading promotion factor is continued even in the absence of cell to substrate attachment.     

Monochloramine potently inhibits arachidonic acid metabolism in rat platelets

In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH(2)Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB(2), and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4x10(8)/ml) were preincubated with drugs for 5min at 37 degrees C prior to the incubation with AA (40microM) for 2min at 37 degrees C. HOCl (50-250microM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5-67% inhibition) and COX metabolites (TXB(2), 33-73% inhibition; 12-HHT, 27-74% inhibition). Although Tau-Cl and Glu-Cl up to 100microM were without effect on the formation of 12-HETE, TXB(2) and 12-HTT, NH(2)Cl showed a strong inhibition on the formation of all three metabolites (10-100microM NH(2)Cl, 12-HETE, 21-92% inhibition; TXB(2), 58-94% inhibition; 12-HHT, 36-92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH(2)Cl, and taurine restoring that induced by both NH(2)Cl and HOCl. These results suggest that NH(2)Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH(2)Cl-induced alterations in the COX and LOX pathways in vivo.     

Dual effect of the phorbol ester TPA on arachidonic acid release from HeLa cells

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces release of arachidonic acid (AA) from HeLa cells with a maximum at 2-3 h. Subsequently the extracellular level of AA decreases. Cycloheximide (CH, 10(-5) M does not influence the release of AA, however, it causes the AA level to remain elevated. In the presence of TPA and CH (i) re-uptake of AA is not altered, (ii) re-incorporation of AA into phosphatidylinositol (and phosphatidylethanolamine) is largely increased, and (iii) the level of lysophosphatidylinositol is elevated. The latter two phenomena can be prevented by fluocinolone acetonide (10(-8) M), i.e. by inhibition of phospholipase A2 (PLA2). These data point to a continuously elevated PLA2 activity in the presence of TPA and CH. The phorbol ester appears to induce a proteinaceous principle which diminishes PLA2 activity.     

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.     

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.     

Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D

Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.     

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.