Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells

Background

Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via activation of Ca²⁺-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca²⁺]_i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells.

Results

Maitotoxin produced a concentration-dependent increase in [Ca²⁺]_i followed by a biphasic uptake of ethidium. Comparison of ethidium (M_w 314 Da), YO-PRO-1 (M_w 375 Da), and POPO-3 (M_w 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation.

Conclusions

These results suggest that maitotoxin-induced Ca²⁺ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.     

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.     

Selection and characterization of bovine aortic endothelial cells

This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [3H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotpye throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called "sprouting." This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves.     

Selection and characterization of bovine aortic endothelial cells

Summary This paper reports techniques for isolation, selection and long-term passage of bovine aortic endothelium (BAE). A [³H]thymidine-selection technique was developed to limit overgrowth of cultures by contaminating smooth-muscle cells. The resulting cultures could be passaged for a replicative life span of 35 to 40 doublings and maintained a stable, normal karyotype throughout this period. Despite the fact that these cultures reached a stable monolayer with density-inhibited growth state, postconfluent cells showed focal areas of a second growth pattern called “sprouting.” This was seen only when cultures were maintained at high densities for periods of 1 to 2 weeks. Ultrastructural analysis, as well as immunofluorescence studies with markers for endothelial cells (factor VIII) and smooth-muscle cells (actin), indicates that this phenomenon is not due to overgrowth of a residual population of smooth-muscle cells, but may represent a second growth pattern of the endothelial cells themselves. This research was supported by NIH Grant HL 18645. This work was done during the tenure of an Established Investigationship of the American Heart Association.     

Influence of membrane active substances on cell surface morphology of cultured aortic endothelial cells

1. The changes occurring on the surface of cultured aortic endothelial cells under the influence of exogenous cholesterol, lysolecithin, or cholesterol + lysolecithin were pursued by scanning and transmission electron microscopy. 2. Both compounds were found to cause surface changes under different quantitative relations, as shown by scanning electron microscopy of unfixed preparations. The changes were less significant in preparations fixed prior to the scanning--or transmission electron microscopic examination. 3. Other cell lines maintained in this laboratory and similarly treated with cholesterol, were found to be less responsive than the endothelial cells. 4. Cholesterol content was determined by thin layer chromatography in six different cell lines, before and after the addition of cholesterol. Only the endothelial cells showed a notable rise of cholesterol content after treatment. This fact may confirm our finding that cholesterol induced morphological changes were demonstrable only in the endothelial cells.     

Microcarrier cultivation of bovine aortic endothelial cells in spinner vessels and a membrane stirred bioreactor

Primary bovine aortic endothelial cells were cultivated in serum supplemented medium without any additional growth factors. The anchorage dependent cells were propagated on Dormacell^® microcarriers with covalently bound dimeric DEAE-groups at the surface of the dextrane beads. Cultivations were performed in 200 ml spinner cultures containing 1 g l^–1 to 3 g l^–1 of microcarriers. Out of five types of Dormacell^® microcarriers with different ion exchange capacities ranging from 0.30 up to 0.65 meq g^–1, corresponding to nitrogen contents from 1.2% to 2.9%, respectively, optimal attachment and growth of endothelial cells were obtained with beads of highest nitrogen content (2.9%). Cells were seeded withca. 5 viable cells per microcarrier being sufficient to achieve fully confluent microcarriers after 4 to 5 days. Glucose concentrations decreased from 21 mM to uppermost half of the original concentrations. 4 mM glutamine was rapidly consumed and virtually exhausted after the cells reached confluency. Lactate concentrations raised to a maximum of 7 mM in spinner cultures, but was found to be reutilized in the stationary phase after glutamine limitation occurred. Serine was found to be the second most prominent amino acid being almost exhausted at confluency whereas alanine was produced in noteworthy amounts. Considerable decrease was determined for threonine, lysine and arginine; low consumption rates were observed for leucine, phenylalanine and methionine. All other amino acids did not alter significantly throughout cultivation. These data support that bovine aortic endothelial cells are capable to utilize glucose and glutamine as well as lactic acid (after glutamine exhaustion) as energy and/or carbon source. Finally, batch cultures in a 2 liter membrane stirred bioreactor with bubble-free aeration were performed to produce large quantities of endothelial cells using microcarrier concentrations of 3 g l^–1.Abbreviations BAE cells bovine aortic endothelial cells - NCS newborn calf serum - PBS phosphate buffered saline     

Mechanisms involved in the relaxation of bovine aortic endothelial cells

The importance of endothelial cell contraction in the regulation of vascular biology is being increasingly recognized. Our group has demonstrated that reactive oxygen species, particularly hydrogen peroxide, which are released in pathological conditions such as ischemia-reperfusion, are able to induce contraction in bovine aortic endothelial cells (BAEC). The cGMP-dependent relaxation of contractile cells depends on the ability of the cyclic nucleotide to interfere with intracellular calcium; however, this is not the only mechanism involved. The present experiments were designed to analyse the mechanism by which cGMP induces relaxation in BAEC. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, as well as atrial natriuretic (ANP) and C-type natriuretic (CNP) peptides, activators of particulate guanylate cyclase, blunted the hydrogen peroxide-induced contraction of BAEC and myosin light chain phosphorylation. The inhibitory effect was more marked with SNP and CNP than with ANP, and the action of SNP and CNP were partially reversed by blocking soluble and particulate guanylate cyclases, respectively. Dibutyryl cGMP (db-cGMP), a cGMP analogue, mimicked the effect of SNP and CNP. Cyclic GMP-dependent protein kinase (cGK) protein levels and activity were measured. Hydrogen peroxide induced a significant reduction in cGK activity without any change in protein level. This effect was completely reversed by preincubation with db-cGMP. Calyculin A, a myosin light chain phosphatase inhibitor, prevented the cGMP-induced relaxation of BAEC. SNP, CNP and db-cGMP also partially prevented the hydrogen peroxide-induced increase in intracellular calcium levels. Catalase completely blocked this effect. In summary, the present results support a role for those metabolites which activate guanylate cyclases in the relaxation of BAEC, and suggest that the cGMP-induced BAEC relaxation could be due, at least partially, to the stimulation of cGK and/or myosin light chain phosphatase activity, and to calcium blockade.     

Studies of L-arginine transport in bovine aortic endothelial cells

We have previously demonstrated that p(1),p(4)-diadenosine 5'-tetraphosphate induces the release of NO and modulates the uptake of L-arginine by bovine aortic endothelial cells (BAEC) [Hilderman, R. H., and Christensen, E. F. (1998) FEBS Lett. 407, 320-324; Hilderman, R. H., Casey, T. E., and Pojoga, L. H. (2000) Arch. Biochem. Biophys. 375, 124-130]. In this communication we characterize the uptake of L-Arg by BAEC. L-Arg is transported into BAEC by at least two different transporter systems. One transporter system is protein synthesis dependent, and L-Arg transported by this system is incorporated into proteins. The second transporter system involved in L-Arg uptake is protein synthesis independent, and uptake occurs by facilitated diffusion. The L-Arg transported by facilitated diffusion is metabolized into L-argininosuccinate. Homologous and heterologous competition uptake studies were performed using a fixed concentration of radiolabeled L-Arg, L-lysine, and L-leucine with varying concentrations of competing nonradiolabeled amino acids. The results of these competition uptake studies are consistent with the protein-synthesis-dependent uptake of L-Arg taking place through a transporter system that is highly specific for L-Arg and with the facilitated diffusion uptake taking place through a transporter that is specific for L-Arg and L-Leu.     

Two-dimensional peptide mapping of fibronectins from bovine aortic endothelial cells and bovine plasma

We have made a comparison between plasma and endothelial cell fibronectin, since these cells are in intimate contact with plasma $\text{in vivo}$. Cellular and secreted fibronectins were purified from cloned lines of adult bovine aortic endothelial cells, and compared to purified bovine plasma fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional peptide mapping. When unreduced, all three fibronectins migrated on gels as single bands with M_r 440,000. After reduction, cellular and secreted fibronectins migrated on gels as single bands with M_r 220,000, but plasma fibronectin migrated as two bands with M_r 220,000 and 210,000. All three fibronectins, including the two subunits of plasma fibronectin, had identical structures by peptide mapping analysis.     

Plasma membrane glycoproteins of tumour cells in enzootic bovine leukosis compared with normal bovine lymphoid cells

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.     

DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells

Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium. As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair. Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations. As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators. Treatment with As also increased nitrite production. These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage. The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH. Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2.     

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.     

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.     

Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.     

Cholesterol depletion increases membrane stiffness of aortic endothelial cells

This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.     

Increased expression of urokinase mRNA in bovine aortic endothelial cells treated with propranolol

Propranolol, a beta-adrenergic blocker agent widely used in a number of cardiovascular disorders, increases plasminogen activator (PA) activity in confluent bovine aortic endothelial cells (BAEC). This effect is time and dose dependent (10-100 microM). Hybridization studies with specific cDNA showed that propranolol was able to induce an increase in urokinase mRNA expression in a dose and time dependent manner. The propranolol effect seems to be specific for urokinase mRNA, because it does not affect a-actin mRNA expression. Cycloheximide, similarly to propranolol, also increases urokinase mRNA, indicating that the gene expression may be regulated by some rapidly turning over protein. When compounds were used in combination, a superinduction phenomenon was observed.