Bovine Coronary Endothelial Cells Research Articles

Role of the endothelial caveolae microdomain in shear stress–mediated coronary vasorelaxation

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress-mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI2) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca2+ ([Ca2+] i ) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae-TRPV4-SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.

Endothelial Caveolae-TRPV4-SK Channel Interactions in Shear Stress-Mediated Coronary Arteriole Dilation

We have previously reported that shear stress-mediated dilation (SSD) of coronary arterioles is caveolae-dependent. We found that both TRPV4 and SK channels are targeted to the low-buoyant density caveolin-rich membrane fraction in freshly isolated bovine coronary endothelial cells (BCEC) and TRPV4 and SK3 channels are co-immunoprecipitated by anti-caveolin-1 antibody. Isoproterenol (1 µM) enhanced the whole-cell K+ currents in BCEC by 5-fold that are inhibited by apamin (200 nM), an SK channel-specific inhibitor, but not by iberiotoxin (100 nM) or Tram34 (200 nM). Exposure of the BCEC to the TRPV4 activator, GSK1016790A (150 nM) resulted in a 3.5-fold activation of SK currents. Acute exposure of BCEC seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in a 4-fold increase in TRPV4 currents that are inhibited by HC067047 (200 nM), a TRPV4-specific inhibitor, and a 3.5-fold increase in SK currents that are inhibited by apamin (200 nM). However, after incubation with HC067047, a TRPV4 specific inhibitor, SK currents could no longer be activated by SS, suggesting SK activation by SS was mediated through TRPV4. In addition, pre-incubation of isolated coronary arterioles with apamin (200 nM) resulted in a significant diminution of SSD while pre-incubation with HC067047 (150 nM) results in vasoconstriction by SS. Exposure of BECE to SS (10 dynes/cm2 12 h) enhanced the production of endothelium-derived vasodilators, NO and PGI2, and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated [Ca2+]i increase in BECE. These novel results indicate that there is a dynamic interaction in the vascular endothelium between caveolae, TRPV4 and SK channels in the modulation of SSD.

Diabetes-induced Coronary Vascular Dysfunction Involves Increased Arginase Activity

Increases in arginase activity have been reported in a variety of disease conditions characterized by vascular dysfunction. Arginase competes with NO synthase for their common substrate arginine, suggesting a cause and effect relationship. We tested this concept by experiments with streptozotocin diabetic rats and high glucose (HG)-treated bovine coronary endothelial cells (BCECs). Our studies showed that diabetes-induced impairment of vasorelaxation to acetylcholine was correlated with increases in reactive oxygen species and arginase activity and arginase I expression in aorta and liver. Treatment of diabetic rats with simvastatin (5 mg/kg per day, subcutaneously) or L-citrulline (50 mg/kg per day, orally) blunted these effects. Acute treatment of diabetic coronary arteries with arginase inhibitors also reversed the impaired vasodilation to acetylcholine. Treatment of BCECs with HG (25 mmol/L, 24 hours) also increased arginase activity. This effect was blocked by treatment with simvastatin (0.1 micromol/L), the Rho kinase inhibitor Y-27632 (10 micromol/L), or L-citrulline (1 mmol/L). Superoxide and active RhoA levels also were elevated in HG-treated BCECs. Furthermore, HG significantly diminished NO production in BCECs. Transfection of BCECs with arginase I small interfering RNA prevented the rise in arginase activity in HG-treated cells and normalized NO production, suggesting a role for arginase I in reduced NO production with HG. These results indicate that increased arginase activity in diabetes contributes to vascular endothelial dysfunction by decreasing L-arginine availability to NO synthase.

Endothelium-derived 2-arachidonylglycerol: an intermediate in vasodilatory eicosanoid release in bovine coronary arteries

Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC) AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K(+) channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone.

AP-4F, antennapedia peptide linked to an amphipathic α helical peptide, increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells

Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic α helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)–DNA typically yields transfection efficiencies of 35.4 ± 3.3% with low levels of cell death (8.1 ± 1.0%). Pre-treatment of the Lipofectamine 2000–GFP–DNA complexes with AP-4F for 5 min increased transfection to 58.2 ± 2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10–20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.

Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (.NO) and superoxide anion (O(2)) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates.NO. As eNOS is capable of generating both.NO and O(2), we hypothesized that Hsp90 might also mediate eNOS-dependent O(2) production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 microg/ml; 17.8 microm) and then stimulated with the calcium ionophore, (5 microm). GA significantly decreased -stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased -stimulated eNOS-dependent O(2) production (p < 0.001, n = 8). increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent.NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O(2) production.

Mechanism of damnacanthal-induced [Ca 2+] i elevation in human dermal fibroblasts

Damnacanthal is a potent and selective inhibitor of p56 lck tyrosine kinase in a variety of tissues. We have found, however, using the Ca 2+ microfluorimetry technique, that damnacanthal releases intracellular Ca 2+ stores and promotes Ca 2+ entry in human dermal fibroblasts. The effect of damnacanthal on the peak [Ca 2+] i values and the latent time to the peak was concentration-dependent. Damnacanthal releases Ca 2+ from thapsigargin-sensitive Ca 2+ stores, and the Ca 2+ stores responding to damnacanthal were overlapped with those of bradykinin. Damnacanthal-induced Ca 2+ entry was mediated by voltage-dependent and voltage-independent Ca 2+ channels. This effect of damnacanthal on intracellular Ca 2+ mobilization was also observed in cultured bovine coronary endothelial cells but not demonstrated in freshly isolated rat basilar smooth muscle cells. Our study suggests that damnacanthal increases intracellular Ca 2+ by releasing Ca 2+ from internal stores and promoting Ca 2+ entry. The relationship between the actions of damnacanthal on tyrosine kinase and intracellular Ca 2+ requires further investigation.

N-arachidonylethanolamide relaxation of bovine coronary artery is not mediated by CB1 cannabinoid receptor.

It has been reported that the endogenous cannabinoid N-arachidonylethanolamide (AEA), commonly referred to as anandamide, has the characteristics of an endothelium-derived hyperpolarizing factor in rat mesenteric artery. We have carried out studies to determine whether AEA affects coronary vascular tone. The vasorelaxant effects of AEA were determined in isolated bovine coronary artery rings precontracted with U-46619 (3 x 10(-9) M). AEA decreased isometric tension, producing a maximal relaxation of 51 +/- 9% at a concentration of 10(-5) M. Endothelium-denuded coronary arteries were not significantly affected by AEA. The CB1 receptor antagonist SR-141716A (10(-6)M) failed to reduce the vasodilatory effects of AEA, suggesting that the CB1 receptor is not involved in this action of AEA. Because AEA is rapidly converted to arachidonic acid and ethanolamine in brain and liver by a fatty acid amide hydrolase (FAAH), we hypothesized that the vasodilatory effect of AEA results from its hydrolysis to arachidonic acid followed by enzymatic conversion to vasodilatory eicosanoids. In support of this hypothesis, bovine coronary arteries incubated with [3H]AEA for 30 min hydrolyzed 15% of added substrate; approximately 9% of the radiolabeled product was free arachidonic acid, and 6% comigrated with the prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs). A similar result was obtained in cultured bovine coronary endothelial cells. Inhibition of the FAAH with diazomethylarachidonyl ketone blocked both the metabolism of [3H]AEA and the relaxations to AEA. Whole vessel and cultured endothelial cells prelabeled with [3H]arachidonic acid synthesized [3H]PGs and [3H]EETs, but not [3H]AEA, in response to A-23187. Furthermore, SR-141716A attenuated A-23187-stimulated release of [3H]arachidonic acid, suggesting that it may have actions other than inhibition of CB1 receptor. These experiments suggest that AEA produces endothelium-dependent vasorelaxation as a result of its catabolism to arachidonic acid followed by conversion to vasodilatory eicosanoids such as prostacyclin or the EETs.

Vesnarinone inhibits adenosine uptake in endothelial cells, smooth muscle cells and myocytes, and mediates cytoprotection.

Vesnarinone is a novel synthetic inotropic agent. Recently, it has been reported that vesnarinone inhibits adenosine uptake in the B-lymphocytoid cell line. Since extracellular adenosine is cardioprotective, we examined whether vesnarinone inhibits adenosine uptake in cells constituting the cardiovascular system. 1 microCi of -3H-adenosine was added to cells of the myocyte cell line (C2C12), human coronary smooth muscle cell line (HCASMC), human and bovine coronary endothelial cell lines (HCAEC and BCAEC), bovine arterial endothelial cell line (BAEC), and human umbilical venous endothelial cell line (HUVEC). After 10 s-5 min, cells were separated from free [3H]adenosine, and the radioactivity was measured. When 0.1-100 microM of vesnarinone was added to each cell line, the uptake of adenosine was inhibited dose-dependently {% inhibition of -3H-adenosine uptake at 10 and 30 microM of vesnarinone: 14 and 33% (C2C12), 47 and 72% (HCASMC), 37 and 58% (HCAEC), 42 and 68% (BCAEC), 19 and 68% (BAEC), 29 and 59% (HUVEC)}. The cellular viability of HCAEC exposed to 60 min of hypoxia and 60 min of reoxygenation increased from 34+/-5 to 67+/-6% (Trypan blue exclusion test) and 23+/-5 to 78+/-6% (LDH release), which was completely blunted by 8-sulfophenyltheophylline, an adenosine receptor antagonist, and was partially blunted by alpha,beta-methyleneadenosine 5'-diphosphate, an inhibitor of ecto-5'-nucleotidase. We also found that vesnarinone is cytoprotective against hypoxia and reoxygenation in C2C12 and HCASMC. We conclude that vesnarinone inhibits the uptake of adenosine in cardiovascular cells, which contributes to cytoprotection.

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ 14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F 1α, PGE 2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12( S)-, 15( S)-, and 11( R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11( R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF 1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF 1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.

Nitro-L-arginine inteferes with the cadmium reduction of nitrate/griess reaction method of measuring nitric oxide production.

Nitro-L-arginine is used as an inhibitor of nitric oxide synthase in many biological Systems. Nitric oxide (NO) is unstable and degrades to nitrite NO(2)- and nitrate NO(3)-. The colorimetric reaction of N0(2)- with Griess reagent is commonly used to measure NO(2)-. NO(3)- may be measured as NO(2)- following reduction by cadmium or cadmium/copper. We found that bradykinin increased the formation of NO(2)- by bovine coronary endothelial cells. Nitro-L-arginine further increased the formation of NO(2)-. This increase is due to the interference of nitro-L-arginine in determination of NO(3)- by the cadmium reduction to NO(2)- and Griess reagent reaction. Incubation of nitro-L-arginine with cadmium or cadmium/copper produced a product that reacts with Griess reagent to form a compound that has an absorption spectrum identical to the product formed by NO(2)- and Griess reagent. Caution must be exercised when using the NO(2)-/NO(3)- measurement by the Griess reaction to assess inhibition of nitric oxide synthase by nitro-L-arginine.

Cultured bovine coronary arterial endothelial cells synthesize HETEs and prostacyclin.

Arachidonic acid metabolism was examined in endothelial cells cultured from bovine coronary arteries. In culture, these cells exhibit specific characteristics of endothelial cells. They form a contact-inhibited monolayer with a cobblestone appearance, contain immunoreactive von Willebrand's factor antigen, and have angiotensin I converting enzyme activity. Prostacyclin was the major prostaglandin synthesized from exogenous and endogenous arachidonic acid in these cells. In addition, exogenous arachidonic acid was metabolized to small amounts of prostaglandin E2 (PGE2) and several relatively nonpolar metabolites including 12-, 15-, and 11-hydroxyeicosatetraenoic acids (12-, 15-, and 11-HETE). Histamine, bradykinin, and thrombin increased PGI2 synthesis in these bovine coronary endothelial cells. Of these agonists, bradykinin was the most potent, increasing basal PGI2 release by fourfold. More vigorous stimulation of the cells with mechanical disruption of the cell monolayer, melittin, or A23187 resulted in release of both PGI2 and PGE2. Pretreatment of cells with exogenous arachidonic acid (10(-5) M) abolished their responsiveness to subsequent stimulation by arachidonic acid or vasoactive agents, but not PGH2. Furthermore, treatment of cells with 15-HPETE (10(-7)-10(-4) M), but not 15-HETE, specifically inhibited basal as well as A23187-stimulated PGI2 release. PGE2 release was increased slightly after 15-HPETE treatment. These studies indicate that bovine coronary endothelial cells can metabolize arachidonic acid to several biologically active products and that PGI2 synthesis by these cells is specifically related to the type of vasoactive agent employed. Both the qualitative pattern and quantity of eicosanoids synthesized by bovine coronary endothelial cells differ substantially from endothelial cells isolated from noncardiac vascular beds.

Prostaglandin synthesis in bovine coronary endothelial cells: comparison with other commonly studied endothelial cells

Comparison of arachidonic acid metabolism by bovine coronary artery endothelial cells, bovine aortic endothelial cells and human umbilical endothelial cells indicated potentially important differences in relative amounts of the different prostaglandins produced. Bovine coronary endothelial cells converted 14C-arachidonic acid to radioactive 6-keto PGF 1α (the stable metabolite of PGI 2) and to a lesser extent PGE 2. Bovine aortic cells synthesized 6-keto PGF 1α and 6,15-diketo PGF 1α as the major products. PGE 2, 6-keto PGE 1, PGE 2α, and PGD 2 were minor metabolites. By comparison, endothelia cells isolated from human umbilical artery or vein formed mainly 6-keto PGF 1α and substantial amounts of PGF 2α, PGE 2 and PGD 2. Basal concentrations of 6-keto PGF 1α were two-fold higher in bovine coronary cells than in bovine aortic endothelial cells, but seven-fold less than in endothelial cells cultured from human umbilical vessels. Histamine, bradykinin and thrombin stimulated PGI 2 synthesis in both coronary endothelial cells and human umbilical cells, but only bradykinin stimulated PGI 2 synthesis in bovine aortic cells. This comparative study indicates that endothelial cells vary in the metabolites of arachidonic acid that they produce depending upon the vascular origin of the cells. Also, endothelial cells from different vascular beds respond differently to specific vasoactive agents.