Mitogenic activity and inositide metabolism in thrombin-stimulated pig aorta endothelial cells

The mitogenic activity of thrombin in endothelial cells is not well understood. The inositide metabolism is an ubiquitous transducing mechanism that seems to be involved in the control of cell growth. Thrombin is a potent stimulant of the release of inositol phosphates in platelets. The data presented here suggest that thrombin is able to induce competence in pig aorta endothelial cells to proliferate in response to insulin. Also thrombin is a potent stimulant of the inositide metabolism what suggests that the activation of this pathway might be at least one of the mechanisms through which thrombin induces competence in this cells.     

Arachidonic acid incorporation in cardiomyocytes,endothelial cells and fibroblast-like cells isolated from adult rat heart

The incorporation of radiolabeled arachidonic acid (3[H]-AA) in normoxic cardiomyocytes (MC), cardiac endothelial cells (EC) and fibroblast-like cells (FL) isolated from adult rat heart was studied. Deposition of 3[H]-AA in the cellular lipid pool was assessed with biochemical and autoradiographic techniques. Extraction and subsequent analysis of lipids from the three different cell types revealed that MC contained significantly more triacylglycerols than EC and FL. The proportion of (unlabeled) AA was also higher in MC triacylglycerols than in EC and FL. The quantity of phospholipids did not differ among the three cell types studied. However, the content of (unlabeled) AA in the MC phospholipid pool was twice as high as in EC and FL. The amount of 3[H]-AA incorporated in the cellular lipid pool of MC, EC and FL depended on the concentration of AA in the incubation medium and the incubation time. In EC and FL incorporation of 3[H]-AA was highest in the cellular phospholipid pool (0.01 microM AA, 30 min incubation). With increased concentration of AA and longer incubation times, the cellular triacylglycerol pool became more important as site of 3[H]-AA incorporation. In MC, comparable amounts of 3[H]-AA were incorporated in the cellular triacylglycerol and phospholipid pools (0.01 and 1 microM AA). At higher AA concentrations (10 microM) the triacylglycerol pool was the preferred site of 3[H]-AA deposition. Autoradiographic analysis at the light microscopic level revealed that the extra-nuclear space was readily stained when the three cell types were incubated with 3[H]-AA. These findings indicate that all cellular lipid pools and membranes are most likely site of deposition of radiolabeled arachidonic acid.     

Arachidonic acid metabolism in guinea pig Langerhans cells: studies on cyclooxygenase and lipoxygenase pathways

Epidermal Langerhans cells are macrophage-like la+ leukocytes that are critically involved in cutaneous immune reactions. Because macrophages exert their immunoregulatory activity in part by generation of oxygenated arachidonic acid metabolites, we systematically studied arachidonic acid transformations by purified guinea pig Langerhans cells and compared them with mixed epidermal cells and Langerhans cell-depleted keratinocytes. Products formed from arachidonic acid by cell homogenates were measured after thin-layer or reverse-phase high-pressure liquid chromatographic separation. In addition, leukotriene B4 and C4 formation was assessed in supernatants of Ca ionophore A23187-challenged intact cells by radioimmunoassay. Mixed epidermal cells converted arachidonic acid predominantly via cyclooxygenase and 12-lipoxygenase pathways. The main products were prostaglandin D2 (PGD2) and 12-hydroxyeicosatetraenoic acid (12-Hete), although significant amounts of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were formed as well. PGD2 synthesis was dependent on the presence of reduced glutathione. The product spectrum formed by Langerhans cell-depleted keratinocytes was virtually indistinguishable from mixed epidermal cells. In contrast, Langerhans cells showed a markedly different metabolism of arachidonic acid. They exhibited an exceedingly high PGD2-generating capacity, whereas only minor amounts of 12-HETE and very low amounts of other prostaglandins were synthesized. The PGD2/12-HETE ratio was 1.22 for mixed epidermal cells and 4.37 for Langerhans cells. Leukotriene production from exogenous or endogenous arachidonic acid could not be demonstrated by either radioenzymatic or radioimmunologic detection methods. We conclude that guinea pig Langerhans cells transform arachidonic acid predominantly to PGD2, which might mediate significant immunoregulatory, inflammatory, and antitumoral activity in the skin.     

Arachidonic acid and calcium signals in human breast tumor-derived endothelial cells: a proteomic study

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released upon proangiogenic stimulation, arachidonic acid (AA) and its metabolites play a key role, and their effects are strictly related to calcium homeostasis. In human breast tumor-derived endothelial cells (B-TECs) AA stimulates proliferation and tubulogenesis in a calcium-dependent way. Here, to characterize the proteins whose expression is regulated by AA-induced calcium entry, we used a proteomic approach (two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, 2-DE and MALDI-MS) and we compared the proteomes of B-TECs stimulated with AA in presence or in absence of calcium entry (with addition to the culture medium of the calcium chelator EGTA, which completely prevents calcium fluxes throughout the plasma membrane). We found that six proteins increased their levels of expression, all higher when AA-induced calcium entry was abolished. These proteins have been identified by mass spectrometry and database search, and their potential roles in AA-stimulated pathway and in angiogenesis are discussed.     

Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Arachidonic acid inhibits 5-lipoxygenase in human T cells

The data on whether T cells produce leukotrienes or other 5-lipoxygenase metabolites of arachidonic acid is conflicting. We report that exogenous arachidonic acid added to phytohemagglutin-stimulated human T cells profoundly inhibits leukotriene B4 production, with 90% inhibition caused by 10(-6) M arachidonic acid. The 12- and 15-lipoxygenase pathways were also inhibited by arachidonic acid. Recent reports that human T cells produce no 5-lipoxygenase metabolites of arachidonic acid might be explained by the fact that the studies used greater than or equal to 10(-5)M arachidonic acid in the incubation media.     

Arachidonic acid metabolism in purified human lung mast cells

Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na⁺−H⁺ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH₄Cl) to the superfusate, or by changing the bath solution saturated with 10% CO₂+90% O₂ to a solution saturated with 100% O₂. Both approaches evoked hyperpolarization of EC. After intracellular Ca²⁺ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH₄Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH₄Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na⁺-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca²⁺-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca²⁺ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na⁺−H⁺-antiporter, inhibits influx of extracellular Ca²⁺ into EC under ATP stimulation.     

Arachidonic acid randomizes endothelial cell motion and regulates adhesion and migration

Cell adhesion and migration are essential for the evolution, organization, and repair of living organisms. An example of a combination of these processes is the formation of new blood vessels (angiogenesis), which is mediated by a directed migration and adhesion of endothelial cells (ECs). Angiogenesis is an essential part of wound healing and a prerequisite of cancerous tumor growth. We investigated the effect of the amphiphilic compound arachidonic acid (AA) on EC adhesion and migration by combining live cell imaging with biophysical analysis methods. AA significantly influenced both EC adhesion and migration, in either a stimulating or inhibiting fashion depending on AA concentration. The temporal evolution of cell adhesion area was well described by a two-phase model. In the first phase, the spreading dynamics were independent of AA concentration. In the latter phase, the spreading dynamics increased at low AA concentrations and decreased at high AA concentrations. AA also affected EC migration; though the instantaneous speed of individual cells remained independent of AA concentration, the individual cells lost their sense of direction upon addition of AA, thus giving rise to an overall decrease in the collective motion of a confluent EC monolayer into vacant space. Addition of AA also caused ECs to become more elongated, this possibly being related to incorporation of AA in the EC membrane thus mediating a change in the viscosity of the membrane. Hence, AA is a promising non-receptor specific regulator of wound healing and angiogenesis.     

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium.

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Ascorbate uptake in pig coronary artery endothelial cells

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When ¹⁴C-ascorbate uptake in endothelial cells was examined as ¹⁴C or by HPLC, the two values did not differ from each other. ¹⁴C-ascorbate uptake was Na⁺-dependent, stereoselective for l-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: K_m = 27± 3 M (Hill coefficient = 1) for ascorbate and K_m = 73± 14 mM (Hill coefficient = 2) for Na⁺. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect. (Mol Cell Biochem 271: 43–49, 2005)     

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells

Cyclic AMP (cAMP)is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP3), Ca2+ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of extracellular Ca2+. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8-9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca2+ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P3 -receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca2+ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.     

Arachidonic acid activation of guinea pig lung guanylate cyclase by two independent mechanisms

The purpose of this study was to elucidate the mechanisms by which arachidonic acid activates guanylate cyclase from guinea pig lung. Guanylate cyclase activities in both homogenate and soluble fractions of lung were examined. Guanylate cyclase activity was determined by measuring formation of [32-P] cyclic GMP from α-[32-P] GTP in the presence of Mn²⁺, a phosphodiesterase inhibitor and a suitable GTP regenerating system. Arachidonic acid, and to a slight extent dihomo-γ-linolenic acid, activated guanylate cyclase in homogenate but not soluble fractions. Similarly, phospholipase A₂ activated homogenate but not soluble guanylate cyclase. Methyl arachidonate, linolenic, linoleic and oleic acids did not activate guanylate cyclase in either fraction. High concentrations of indomethacin, meclofenamate and aspirin inhibited activation of homogenate guanylate cyclase by arachidonic acid and phospholipase A₂, without altering basal enzyme activity. These data suggested that a product of cyclooxygenase activity, present in the microsomal fraction, may have accounted for the capacity of arachidonic acid to activate homogenate guanylate cyclase. This view was supported by the findings that addition of the microsomal fraction to the soluble fraction enabled arachidonic acid to activate soluble guanylate cyclase, an effect which was reduced with cyclooxygenase inhibitors. Lipoxygenase activated guanylate cyclase in homogenate and soluble fractions. Arachidonic acid potentiated the activation of soluble guanylate cyclase by lipoxygenase, and this effect was inhibited with nordihydroguaiaretic acid, 1-phenyl-3-pyrazolidone and hydroquinone, but not with high concentrations of indomethacin, meclofenamate or aspirin. These data suggest that arachidonic acid activates guinea pig lung guanylate cyclase indirectly, via two independent mechanisms, one involving the microsomal fraction and the other involving lipoxygenase.     

Arachidonic acid inhibits phosphate transport by cultured renal cells

Because arachidonic acid and its metabolites are reported to be intracellular messengers of various exogenous stimuli, we studied whether arachidonic acid influences phosphate transport by cultured mouse renal epithelial cells. Arachidonic acid, at 10(-7)-10(-4)M, inhibited phosphate transport without influencing cyclic adenosine 3':5'-monophosphate production. Nordihydroguaiaretic acid and indomethacin, inhibitors of arachidonic acid metabolism, did not cancel the arachidonic acid-induced inhibition of phosphate transport. Furthermore, unsaturated fatty acids other than arachidonic acid also inhibited phosphate transport and their inhibitory effect increased as the number of double bond increased. These data demonstrate that arachidonic acid inhibits the phosphate transport by the cultured renal epithelial cells, probably not via conversion to its metabolites.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.