D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca(2+) influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na(+) entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na(+) concentration (approx. 1-2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na(+) influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na(+) influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na(+) concentration. Both thapsigargin- and DOG-induced Na(+) increases were not affected in the presence of Na(+)/H(+) antiport inhibitor, HOE609, or Na(+)/Ca(2+) antiport inhibitor, dimethylthiourea, as well as in the presence of Co(2+) and Ni(2+), which block store-operated Ca(2+) entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na(+) influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I(CRANC)). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na(+) entry.     

Phospholipase A(2) is involved in thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that emptying of intracellular Ca(2+) pools with endoplasmatic Ca(2+)-ATP-ase inhibitor thapsigargin leads to the Na(+) influx in human lymphocytes (M. Tepel et al., 1994, J. Biol. Chem. 269, 26239-26242). In the present study we examined the mechanism underlying the thapsigargin-induced Na(+) entry. We found that the thapsigargin-induced increase in Na(+) concentration was effectively inhibited by three structurally unrelated phospholipase A(2) (PLA(2)) inhibitors, p-bromophenacyl bromide, 3-(4-octadecyl)-benzoylacrylic acid (OBAA), and bromoenol lactone (BEL). The thapsigargin-induced Na(+) influx could be mimicked by PLA(2) exogenously added to the lymphocyte suspension. In addition, thapsigargin stimulated formation of arachidonic acid (AA), the physiological PLA(2) product. AA induced Na(+) entry in a time- and concentration-dependent fashion. Both, thapsigargin-induced Na(+) influx and AA liberation were completely inhibited in the presence of tyrosine kinase inhibitor genistein but not in the absence of extracellular Ca(2+). Collectively, these data show that thapsigargin-induced Na(+) entry is associated with tyrosine kinase-dependent stimulation of PLA(2).     

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:     

Extracellular ATP triggers superoxide production in human neutrophils

The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.     

Extracellular ATP causes loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors

Lymphocytes from normal subjects or patients with chronic lymphocytic leukemia are known to possess receptors for extracellular ATP termed P₂Z purinoceptors whose physiological role is undefined. Addition of extracellular ATP (50–500 μM) to both normal and leukemic lymphocytes caused loss of binding of monoclonal antibodies to L-selectin (CD62L) on the cell surface. UTP, ADP, and adenosine (all at 500 μM) had no effect on L-selectin expression. Several features of the ATP-induced loss of L selectin indicate that this effect is mediated by lymphocyte P₂Z purinoceptors. First the loss was attenuated in isotonic NaCl medium compared to 150 mM KCl medium. Second the loss of L-selectin was immediately halted by addition of Mg²⁺ ions in molar excess of ATP. The most potent nucleotide causing L-selectin loss was benzoylbenzoic ATP (>10 μM) which is also the most potent agonist for the P₂Z purinoceptor. Finally preincubation of lymphocytes with oxidized ATP, an irreversible inhibitor of P₂Z purinoceptors, also inhibited ATP induced loss of L-selectin. Extracellular ATP is known to open an ion channel associated with the P₂Z purinoceptor on B-lymphocytes which allows influx of Ca²⁺. However, ATP-induced loss of L-selectin did not require extracellular Ca²⁺. Moreover addition of the calcium ionophore, ionomycin, had minimal effect on L-selectin expression. Staurosporine (500 nM), an inhibitor of protein kinase C, inhibited only 10% of ATP induced loss of L-selectin but completely inhibited the loss of L-selectin caused by 50 nM PMA. Thus extracellular ATP interacts with lymphocyte P₂Z purinoceptors which leads to shedding of L-selectin via a pathway which requires neither Ca²⁺ influx nor activation of protein kinase C. ATP may have a physiological role in the loss of L-selectin which occurs during the interactions of lymphocytes with other cells. © 1996 Wiley-Liss, Inc.     

Extracellular ATP induces cell death in human intestinal epithelial cells

Background

Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP.

Methods

We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique.

Results

ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells.

General significance

The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.     

Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts

The effect of Lys-bradykinin on net Na+ influx in serum-deprived cultured human fibroblasts (HSWP cells) was measured. It was found that Lys-bradykinin stimulates net Na+ influx with a K1/2 of 1 nM. When Lys-bradykinin was combined with epidermal growth factor, vasopressin and insulin, the net Na+ influx stimulated was comparable with that measured in response to 10% serum. The above combination of growth factors also was found to stimulate DNA synthesis to 92% of the serum-stimulated levels in HSWP cells and to support cell growth over a period of 6 days. In addition, it was observed that the Na+ influx stimulated by Lys-bradykinin or by the combination of four growth factors was completely inhibited by the amiloride analog benzamil. Thus Lys-bradykinin presumably stimulates the same Na+ transport system as is stimulated by serum. Finally, the present results suggest that an increase in Na+ influx always accompanies DNA synthesis in HSWP cells. On the basis of these observations, it can be hypothesized that Na+ influx is a necessary event to stimulate DNA synthesis.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

Self-exchange of sodium in human lymphocytes.

Self-exchanges of Na and K in human lymphocytes were measured by isotopic efflux techniques. In washed cells, K exchanged in a single slow exponential fraction, but the Na exchange had a marked curvature. It was shown that the curvature was not caused by simple bulk-phase diffusion, and it was resolved into three major fractions: fast (F) (half-time, t1/2 = 2-4 min), intermediate (I) (t1/2 = 12 min), and slow (S) (t1/2 = 125 min). Each of these appeared to follow an exponential function. The I fraction contained approximately 10 mmol Na/kg cells (25-30% of normal cellular Na), was not affected by manipulations that cause lymphocytes to gain Na, and had little or no temperature dependence. The S fraction of Na in normal cells (S1) contained approximately 10 mmol Na/kg cells, had only a slight temperature dependence, and the amount and rate of S1 were independent of external K concentration (Kex). Another slow fraction (S2) appeared when the cells underwent a net gain of Na in exchange for K, and was characterized by a steep temperature dependence and a peak rate around the transition point (the point at which half of cellular K is replaced by Na) at 0.4 mM Kex. The results are discussed within context of a theory that assigns the exchange of the major part of K in its slow exponential fraction and the Na exchange in S2 to interactions of these ions with fixed anionic sites, on intracellular macromolecules, which have been shown previously to interact cooperatively in their association with K and Na.     

Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells

BackgroundApoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5′-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC.Methods and resultsTo simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po₂ < 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/?0.4; P < 0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/?0.5; P < 0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αβ-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP.ConclusionThe data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.     

Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride-sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.     

Extracellular ATP activates Ca2(+)-dependent K+ conductance via Ca2+ influx in mouse macrophages

1. Using the perforated patch recording, the effects of ATP on membrane current were investigated in mouse peritoneal macrophages. 2. Extracellularly applied ATP induced a biphasic current consisting of a initial inward current [Ii(ATP)] followed by an outward current [Io(ATP)]. These currents were associated with a marked increase in conductance at their peaks. 3. Ii(ATP) reversed close to 0 mV and was attenuated by removal of external Na+. 4. Io(ATP) reversed near -80 mV and was increased by decreasing the external concentration of K+. 5. Io(ATP) was completely abolished by removal of external Ca2+, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (BAPTA-AM) and bath applied quinidine but not tetraethylammonium (TEA) or apamin. 6. These results suggest that Ii(ATP) and Io(ATP) are due to an activation of nonspecific cationic and Ca2(+)-dependent K+ conductances, respectively, and raise the possibility that the putative ATP receptor may be important in regulating macrophage functions, motility, phagocytosis and cytokines secretion.     

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.     

Changes in the content of potassium, sodium and potassium influx in human peripheral blood lymphocytes during long-term cultivation with phytohemagglutinin

Intracellular potassium, sodium and potassium influx were examined in PHA-activated human lymphocytes within 6 days of cultivation. DNA flow cytometry was used to estimate the percentage of cells in G1, S and G2 + M phases. Potassium influx and content per g protein were found to be increased, whereas sodium content decreased with the progression of cells from G1 to S phases, being maximum on the 3rd day. Later on the percentage of cells in S phase was seen diminished, and the potassium content decreased just as sodium content increased. It is concluded that ionic changes may correlate with the entering of cells into S phase.