Platelet-activating factor mobilizes intracellular calcium in vascular smooth muscle cells

The effect of platelet-activating factor (PAF) on polyphosphoinositide metabolism and 45Ca2+ efflux was examined in a vascular smooth muscle cell line (A7r5). PAF stimulated a rapid but transient production of inositol trisphosphate and inositol bisphosphate which, in the presence of lithium, resulted in an accumulation of inositol monophosphate. In addition, PAF induced a rapid efflux of 45Ca2+ from preloaded cells, an effect which was concentration-dependent. These data suggest that PAF mobilizes intracellular Ca2+ via the production of inositol trisphosphate.     

Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines

Platelet-activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date, little is known of the effect of PAF on B lymphocytes. Using two Ig-secreting B lymphoblastoid cell lines, we have demonstrated that PAF can enhance Ig production by these cells in a dose-dependent fashion. PAF also causes significant alteration of the kinetics of Ig secretion in these lymphoblastoid cell lines. The effect of PAF is rapid, with detection of 6- to 12-fold increases in Ig production in the first 24 h of cell culture, followed by a plateau during the next 24 to 48 h. The specificity of the PAF effect on Ig secretion is emphasized by lyso-PAF having no Ig-enhancing properties and by the inhibition of Ig enhancement in the presence of the structural analogue PAF antagonist CV3988 and the soluble nonstructural analogue PAF receptor antagonist Web 2086. PAF does not cause an increase in the kinetics of cell proliferation or an increase in cell numbers at any time during a 72-h culture period. In an attempt to explain the increase in Ig secretion in the absence of changes in growth parameters, an ELISA spot assay for enumeration of Ig-secreting cells was developed. This assay demonstrated that the increase in Ig production is likely due to enhancement of single cell Ig secretion rather than an increase in cell number. These data indicate that PAF may have an important immunomodulatory role in the production of Ig by B lymphocytes.     

Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca(2+)-dependent microtubule disassembly and Ca(2+)-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.     

Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration

Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228-7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca(2+)](i)) in Caco-2 cells, since Ca(2+) is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca(2+)](i) from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca(2+)](i) was related only to an increase in Ca(2+) permeability of plasmalemma. At a late stage of infection, [Ca(2+)](i) elevation was due to both extracellular Ca(2+) influx and Ca(2+) release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca(2+)](i) increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca(2+) release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca(2+)](i) of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca(2+) from the ER and by extracellular Ca(2+) influx. These supernatants induced a Ca(2+)-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.     

Platelet-activating factor induces tyrosine phosphorylation in human neutrophils

The addition of platelet-activating factor (PAF) to human neutrophils increases the levels of the tyrosine phosphorylation in several proteins. These proteins have molecular weights of 41 (pp41), 54 (pp54), 66 (pp66), 104 (pp104), and 116 (pp116) kDa. The effect of PAF was dose-dependent and could be seen at concentrations as low as 1 nM. The nonmetabolizable bioactive PAF analog, C-PAF, caused an increase in the level of phosphorylation of the same proteins in a time- and dose-dependent manner. On the contrary, lyso-PAF, enantio-PAF, and L-beta,gamma-dihexadecyl-alpha-lecithin failed to stimulate the phosphorylation of any of the aforementioned proteins. The response to PAF was prevented by the PAF antagonist BN-52021. The PAF-induced increases in tyrosine phosphorylation in pp66, pp116, and pp104 were selectively inhibited by pertussis toxin. In contrast, the level of pp41 phosphorylation remained unchanged after the pertussis toxin treatment. The calcium chelator EGTA significantly inhibited the PAF-produced phosphorylation of the pp41 protein. The intracellular calcium chelator 1,2-bis-(O-aminophenoxil)ethane-N,N,N',N'-tetraacetic acid (BAPTA) potentiated the PAF-enhanced levels of tyrosine phosphorylation on the pp41 protein. On the other hand, the PAF-induced phosphorylations of pp66, pp104, and pp116 were inhibited in BAPTA-treated cells. The calcium ionophore A23187 selectively potentiated the phosphorylation of the pp41 protein and reduced the phosphorylation in the pp54 protein. This phosphorylation was dependent on the extracellular calcium and was inhibited in toxin-treated cells. The results suggest that PAF is able to affect either directly or indirectly tyrosine kinase and/or phosphotyrosine phosphatase activities. The phosphorylation of the high and low molecular weight proteins are mediated by two different sets of kinases and/or phosphatases.     

Glioma cell-derived placental growth factor induces regulatory B cells

Tumor specific immune regulatory cells play an important role in the pathogenesis of glioma. The mechanisms have not been fully understood yet. It is suggested that placenta growth factor (PlGF) is involved in the generation of immune regulatory cells. This study aims to investigate the role of glioma cell-derived PlGF in the generation of regulatory B cells (Breg). Glioma cells were isolated from surgically removed glioma tissue. Cytokines were measured by enzyme-linked immunosorbent assay, quantitative real time RT-PCR and Western blotting. Immune suppressor functions of Bregs were assessed by T cell proliferation assay. The results showed that glioma cells expressed PlGF, which was increased after a non-specific activation. Naïve B cells captured the PlGF to differentiate into transforming growth factor-β positive Bregs. The Bregs were activated upon exposure to protein extracts of glioma tissue to suppress the CD8⁺ T cell proliferation and the release of perforin and granzyme B. We conclude that glioma cell-released PlGF can induce Bregs to suppress CD8⁺ T cell activities.     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.     

A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors

Inositol-trisphosphate was pressure-injected into Limulus ventral photoreceptors; these injections induced electrical responses that mimic several aspects of the electrical responses induced by light. Single cells were also injected with aequorin. Injections of inositol-trisphosphate into such cells induced an increase in luminescence from the intracellular aequorin, even in the absence of extracellular calcium ions. These aequorin responses show directly that inositol-trisphosphate induces an increase in ionized calcium concentration within intact and functioning cells that arises from release of calcium ions from intracellular stores.     

Cytochalasin induces an increase in cytosolic free calcium in murine B lymphocytes

Cytochalasin promotes the progression of anti-immunoglobulin-treated B lymphocytes to S phase. However, the intracellular events induced by cytochalasin which may mediate signaling for progression have not been elucidated. In this study, the effect of cytochalasin on the level of intracellular free calcium in murine splenic B lymphocytes was assessed by using the fluorescent calcium indicator Indo-1. Cytochalasins A, B, D, and E induced a rapid and sustained elevation of intracellular free calcium. The calcium response to cytochalasin derived largely from the influx of extracellular calcium, although a small, transient elevation in intracellular calcium persisted when the suspension medium was made calcium-free with EGTA, implicating an intracellular source for a portion of the calcium response. Single cell fluorescence studies revealed that cytochalasin elicited a calcium response in most splenic B cells in suspension, indicating that this phenomenon is not restricted to a subpopulation of responding B cells. Phorbol esters inhibited the B cell calcium response to cytochalasin, and an established response to cytochalasin was rapidly and completely reversed by subsequently administered phorbol ester. T cells that lack the cytochalasin pathway showed a markedly diminished calcium response that was only apparent at higher cytochalasin concentration. However, B cells from xid-defective [CBA/N X DBA/2]F1 males, which fail to respond to anti-immunoglobulin plus cytochalasin, showed a calcium response to cytochalasin similar to that of phenotypically normal F1 females. These data, along with the finding that the rise in intracellular calcium occurred in naive B cells as well as B cells previously treated with anti-immunoglobulin, suggest that there is no clear association between the calcium response induced by cytochalasin and the ability of cytochalasin to stimulate progression to S phase. However, this effect of cytochalasin may suggest a connection between actin filaments and calcium influx in B cells.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.     

Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells

Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of O2. and H2O2 and that exogenously added H2O2 (12 microm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dose-dependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.     

Platelet-activating factor induces the production of leukotrienes by human monocytes

The aim of this study was to evaluate the role of platelet-activating factor (PAF) as a stimulator of leukotriene production by human monocytes. The production of leukotrienes was time- and concentration-dependent. Release of leukotrienes was half-maximal after 2 min and reached a maximum after 10 min. At a concentration of 10(-8) M, PAF induced the production of 0.14 +/- 0.01 ng LTB4/10(6) cells (mean +/- S.E., n = 8). At concentrations of 10(-6) M, PAF induced the production of 1.0 +/- 0.04 ng LTB4 and 0.22 +/- 0.03 ng peptidoleukotrienes (mean +/- S.E., n = 16). There was no metabolism of LTB4 as judged from stability of [3H]LTB4 added to the incubations. LTC4 was slowly metabolized by human monocytes to LTD4 and LTE4. The two specific PAF-receptor antagonists BN 52021 and WEB 2086 in concentrations of 10(-4) and 10(-6) M, respectively, inhibited the PAF (10(-6) M) stimulated LTB4 production completely. In this study, we demonstrate that nanomolar concentrations of PAF can stimulate the production of LTB4 and peptidoleukotrienes in human monocytes by a receptor-mediated mechanism.     

Mitochondrial calcium response in human transformed lymphoblastoid cells

Human lymphoblastoid cell line (LCL) transformed by Epstein-Barr Virus (EBV) is a unique cellular model for the study of human diseases. Although pathophysiological significance of mitochondrial calcium regulation is drawing attention, it is not known whether or not mitochondria in LCLs play a role in intracellular calcium signaling. In this study, role of mitochondria of the lymphoblastoid cell line in calcium signaling was examined. Intra-mitochondrial calcium concentration ([Ca2+]m) was successfully measured using dihydro-Rhod-2, revealed by the decrease of fluorescence after application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and intracellular localization patterns imaged by fluorescent microscope. Platelet activating factor (PAF) concentration-dependently increased cytosolic calcium concentration ([Ca2+]i), while no increase of [Ca2+]m was observed. In contrast, 10 microM thapsigargin increased [Ca2+]i as well as [Ca2+]m. LCLs may be used for the study of possible pathophysiological role of mitochondrial calcium regulation in human diseases.     

High-level expression of functional platelet-activating factor receptors on a human B lymphoblastoid cell line.

Binding of platelet-activating factor (PAF) was characterized in a human b lymphoblastoid cell line, ASK.0. [3H]PAF binding to these cells was time-dependent, reaching equilibrium at 60 minutes, and saturable. Scatchard analyses of saturation binding experiments revealed a single class of PAF binding sites (108,000 +/- 17,000 per cell) with a KD of 2.16 +/- 0.41 nM. That the binding sites were specific for PAF was demonstrated by competition studies. PAF was shown to increase the intracellular calcium concentrations of ASK.0 cells in a dose-dependent manner with an EC50 of 7 nM. We have, therefore, identified a B cell line expressing large numbers of functional PAF receptors.     

Gonadotropin-releasing hormone stimulation of pituitary gonadotrope cells produces an increase in intracellular calcium

Gonadotropin-releasing hormone (GnRH) stimulates pituitary gonadotrope cells to release luteinizing hormone (LH). Previous studies have indicated a role for Ca+2 in this process; however, the present study provides the first measurements of an increased intracellular Ca+2 concentration. Pituitary cell cultures enriched for gonadotropes were loaded with quin 2, a fluorescent Ca+2-sensitive molecule. Subsequent addition of GnRH to these cells produced a rapid (within 10 sec) increase in fluorescence (indicating an increase in intracellular free Ca+2). In contrast, two GnRH analogs, des1 GnRH (a very low-affinity binder to the GnRH receptor) and Ac[D-pCl-Phe1,2] DTrp3 DLys6 DAla10-GnRH (a pure GnRH antagonist) produced no such Ca+2 change, thus showing a correlation between increased intracellular Ca+2 and LH release. A functional relationship between increased Ca+2 and LH release was suggested by experiments in which LH release was inhibited from cells loaded with high levels of intracellular quin 2 (in order to chelate intracellular Ca+2). Since this inhibition was completely reversed by addition of the Ca+2 ionophore A23187, quin 2 was not toxic at the concentrations used and apparently inhibited LH release by buffering intracellular Ca+2. The results presented here are consistent with the hypothesis that GnRH-stimulated LH release is mediated by increased intracellular Ca+2 and support the notion that the rate-limiting step in GnRH-stimulated LH release is distal to Ca+2 mobilization.     

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (?log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.     

Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskeleton structures

Platelet-activating factor (PAF), but not its deacetylated and biologically inactive metabolite lyso-PAF, in a dose dependent-manner (0.1 to 10 nM), induces human endothelial cells (EC) in culture to change their shape. EC retract and tend to lose reciprocal contact, whereas the distribution of stress fibers is changed and the cells tend to assume a migratory phenotype. The normal localization of vinculin at streaks corresponding to adhesion plaques and located at stress fiber endings is mostly lost and replaced by diffuse distribution of this protein related to the cell-substratum adhesion complex. The effects of PAF are appreciable after 10 min, become maximal after 30 min, and are fully reversible. The disappearance of F-actin in stimulated EC was analyzed by measuring the fluorescence of the cells after staining with fluorosceinated phalloidin, PAF, but not lyso-PAF and the enantiomer of PAF ([S] form), decreases in a time- and concentration-dependent fashion the fluorescence of the cells stained with fluoresceinated phalloidin. EC grown on fibronectin-coated polycarbonate filters restrict the diffusion of 125I-albumin; PAF promotes 125I-albumin diffusion with a concentration dependency similar to that for the PAF-induced cytoskeleton changes. Four different PAF-receptor antagonists belonging to four different series, CV-3988 (PAF-related framework), BN52021 (a natural terpenoid), BN53013 (a natural lignan) and 48740 RP (a synthetic antagonist) prevent the alteration induced by PAF. These findings, coupled to the lack of effect of the enantiomer of PAF ([S] form), support the existence of a specific mechanism of PAF-mediated activation of EC, most probably mediated by a putative receptor. These results explain part of the PAF mechanism of action in inducing the increase of vascular permeability in vivo.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.