Calcium, calmodulin, and the production of prostacyclin by cultured vascular endothelial cells

1983 ◽  
Vol 3 (11) ◽  
pp. 1007-1015 ◽  
Author(s):  
J. M. Seid ◽  
S. Macneil ◽  
S. Tomlinson
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Vascular Endothelial ◽  
Calmodulin Antagonist ◽  
Aortic Endothelial Cells ◽  
Intracellular Calcium Mobilization ◽  
Porcine Aortic Endothelial Cells

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell Jysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.

Swelling-activated K fluxes in vascular endothelial cells: volume regulation via K-Cl cotransport and K channels

1993 ◽  
Vol 265 (3) ◽  
pp. C763-C769 ◽  
Author(s):  
P. B. Perry ◽  
W. C. O'Neill
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Ionophore A23187 ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
K Channels ◽  
Volume Decrease ◽  
Aortic Endothelial

K efflux pathways responsible for regulatory volume decrease (RVD) were examined in bovine aortic endothelial cells. Hypotonic swelling produced a rapid and reversible threefold increase in bumetanide-insensitive 86Rb efflux. Swelling-activated 86Rb efflux was inhibited 43% when Cl was replaced with NO3, and this Cl-dependent efflux was inhibited by 1 mM furosemide. Neither Cl replacement nor furosemide inhibited the efflux stimulated by a Ca ionophore (A23187) in isotonic medium. Swelling-activated 86Rb efflux was also inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate but not by dinitrostilbenedisulfonate. Cell swelling induced a volume-regulatory K loss that was incomplete in hypotonic medium but complete and more rapid when bumetanide was added or when cells were swollen isosmotically. K loss in the presence of bumetanide was partially blocked by furosemide. We conclude that two separate swelling-activated K fluxes mediate RVD in aortic endothelial cells: a Cl-dependent, furosemide-sensitive, but bumetanide-insensitive flux that is consistent with K-Cl cotransport, and a Cl-independent efflux that presumably is mediated by K channels.

Oxidative-stress response in vascular endothelial cells exposed to acellular hemoglobin solutions

1995 ◽  
Vol 269 (2) ◽  
pp. H648-H655 ◽  
Author(s):  
R. Motterlini ◽  
R. Foresti ◽  
K. Vandegriff ◽  
M. Intaglietta ◽  
R. M. Winslow
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Cell Injury ◽  
Vascular Endothelial Cells ◽  
Stress Protein ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Methemoglobin Formation ◽  
Porcine Aortic Endothelial Cells

We investigated the effect of different hemoglobins on the activation of endothelial heme oxygenase (HO), an inducible "stress" protein, which is responsible for heme catabolism, and we determined whether the propensity of hemoglobins to autoxidize correlates with endothelial heme uptake and cell injury. Porcine aortic endothelial cells were incubated for 6 h in the presence of 60 microM unmodified hemoglobin A0 (HbA0), hemoglobin cross-linked between the alpha-chains with bis-(3,5-dibromosalicyl)fumarate (alpha alpha Hb), or cyanomet-alpha alpha-hemoglobin (CNmet alpha alpha Hb). Endothelial HO activity augmented 4.1-fold in the presence of alpha alpha Hb, 2.7-fold with HbA0, and 1.8-fold with CNmet alpha alpha Hb over the control value. Deferoxamine, but not catalase or dimethylthiourea, partially attenuated the HO induction produced by alpha alpha Hb. The rates of methemoglobin formation exhibited a linear relationship over the time of incubation (r = 0.94), and the apparent rate constant was 1.8-fold higher for alpha alpha Hb (0.023 h-1) than for HbA0 (0.013 h-1). Endothelial heme content and lactate dehydrogenase (LDH) release, an index of cell injury, were also higher in alpha alpha Hb compared with HbA0 and CNmet alpha alpha Hb groups (P < 0.05). Deferoxamine but not catalase markedly reduced the release of LDH induced by alpha alpha Hb, whereas dimethylthiourea provided only a partial cytoprotection. These studies suggest that 1) the higher rate of oxidation of alpha alpha Hb contributes to the augmented endothelial HO activity, and 2) both heme release and iron-mediated oxygen radical formation are major contributors to endothelial oxidative stress and cytotoxicity generated by the cross-linked hemoglobin.

Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC

1998 ◽  
Vol 275 (1) ◽  
pp. C113-C119 ◽  
Author(s):  
A. F. Castro ◽  
C. Amorena ◽  
A. Müller ◽  
G. Ottaviano ◽  
M. T. Tellez-Iñon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Extracellular Atp ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Relaxing Factor ◽  
Calphostin C ◽  
Kinase C ◽  
Porcine Aortic Endothelial Cells

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732–735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359–1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.

Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification

1993 ◽  
Vol 265 (4) ◽  
pp. H1424-H1433 ◽  
Author(s):  
R. C. Ziegelstein ◽  
L. Cheng ◽  
P. S. Blank ◽  
H. A. Spurgeon ◽  
E. G. Lakatta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Cytosolic Calcium ◽  
Ph Sensitive ◽  
Vascular Endothelial ◽  
Intracellular Acidification ◽  
Cytosolic Ph ◽  
Aortic Endothelial Cells ◽  
Endothelial Monolayers ◽  
Aortic Endothelial

Acidosis produces vasodilation in a process that may involve the vascular endothelium. Because synthesis and release of endothelium-derived vasodilatory substances are linked to an increase in cytosolic calcium concentration ([Ca2+]i), we examined the effect of intracellular acidification on cultured rat aortic endothelial cells loaded either with the pH-sensitive probe carboxy-seminaphthorhodafluor-1 or the Ca(2+)-sensitive fluorescent probe indo 1. The basal cytosolic pH (pHi) of endothelial monolayers in a 5% CO2-HCO3- buffer was 7.27 +/- 0.02 and that in a bicarbonate-free solution was 7.22 +/- 0.03. Acidification was induced either by removal of NH4Cl (delta pHi = -0.10 +/- 0.02), changing from a bicarbonate-free to a 5% CO2-HCO3(-)-buffered solution at constant buffer pH (delta pHi = -0.18 +/- 0.03), or changing from a 5% to a 20% CO2-HCO3- solution (delta pHi = -0.27 +/- 0.07). Regardless of the method used, intracellular acidification increased [Ca2+]i as indexed by indo 1 fluorescence. The increase in [Ca2+]i induced by changing from a 5 to a 20% CO2-HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin- and thapsigargin-sensitive intracellular Ca2+ stores. Thus intracellular acidification of vascular endothelial cells releases Ca2+ into the cytosol either from pH-sensitive intracellular buffer sites, mitochondria, or from bradykinin- and thapsigargin-insensitive intracellular stores. This Ca2+ mobilization may be linked to endothelial synthesis and release of vasodilatory substances during acidosis.

Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells

1993 ◽  
Vol 264 (5) ◽  
pp. H1599-H1608 ◽  
Author(s):  
W. Kuhne ◽  
M. Besselmann ◽  
T. Noll ◽  
A. Muhs ◽  
H. Watanabe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Actin Filaments ◽  
Fold Increase ◽  
Metabolic Inhibition ◽  
Ionophore A23187 ◽  
Aortic Endothelial Cells ◽  
Monolayer Cultures ◽  
Cell Retraction ◽  
Porcine Aortic Endothelial Cells ◽  
Reversible Phase

In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.

Intracellular pathways of insulin transport across vascular endothelial cells

1988 ◽  
Vol 255 (4) ◽  
pp. C459-C464 ◽  
Author(s):  
H. L. Hachiya ◽  
P. A. Halban ◽  
G. L. King
Keyword(s):  
Endothelial Cells ◽  
Insulin Receptor ◽  
Vascular Endothelial Cells ◽  
Insulin Degradation ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Lysosomal Protease ◽  
Insulin Transport ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stereoselectivity of ectonucleotidases on vascular endothelial cells

1983 ◽  
Vol 214 (3) ◽  
pp. 975-981 ◽  
Author(s):  
N J Cusack ◽  
J D Pearson ◽  
J L Gordon
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Thiol Group ◽  
Side Chain ◽  
Vascular Endothelial ◽  
Nucleotide Analogues ◽  
Aortic Endothelial Cells ◽  
Nucleotide Analogue ◽  
High Degree ◽  
Aortic Endothelial

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5′-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5′-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5′-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.

Sign in / Sign up

Export Citation Format

Share Document