1. Endothelial cell activation is correlated with increased cytosolic Ca¥ concentration, often monitored with cytoplasmic Ca¥ dyes, such as fura_2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca¥ elevations occur which are controlled by cell membrane Na¤-Ca¥ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca¥ concentration ([Ca¥]b) was monitored using fura_2 or Calcium Green-1 and subplasmalemmal Ca¥ concentration ([Ca¥]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 1 nmol l¢ Bk or 10 mmol l¢ NaF yielded focal [Ca¥] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 ìmol l¢ nocodazole caused the SER to collapse and unmasked Ca¥ release in response to 1 nmol l¢ Bk and 10 mmol l¢ NaF, similar to low-Na¤ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca¥ elevations in response to small or slow IP× formation occur due to vectorial Ca¥ release from the SER towards the plasmalemma followed by Ca¥ extrusion by Na¤-Ca¥ exchange. While these local Ca¥ elevations are not detectable with Ca¥ dyes for the determination of [Ca¥]b, prevention of Ca¥ extrusion or SER disruption yields increases in [Ca¥]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca¥ release and [Ca¥] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca¥ homeostasis, Ca¥ distribution and Ca¥ entry. The existence of such a subplasmalemmal Ca¥ control unit (SCCU) needs to be considered in discussions of Ca¥ signalling, especially when cytoplasmic Ca¥ dyes, such as fura_2 or Calcium Green-1, are used.
1 We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca 2+ store depletion and Ca 2+ entry was studied further. 2 Microsomal Cyp450 MO was monitored¯uorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca 2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive¯uorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC 4 (5)) and tyrosine kinase was quanti®ed by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine speci®c antibody. 3 Depletion of cellular Ca 2+ pools with inositol 1,4,5-trisphosphate (IP 3 ), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca 2+ store depletion, chelating of intramicrosomal Ca 2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca 2+ failed to in¯uence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP 3 -sensitive stores. 4 Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca 2+ entry pathways, membrane hyperpolarization due to K + channel activation or tyrosine kinase activity. 5 Inhibition of Cyp450 MO by thiopentone sodium diminished Ca 2+ /Mn 2+ entry to Ca 2+ store depletion by 43%, while it did not interfere with intracellular Ca 2+ release by IP 3 or thapsigargin. 6 Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7 Induction of Cyp450 MO with dexamethasone/clo®brate for 72 h yielded increases in thapsigargininduced Cyp450 MO activity (by 35%), Ca 2+ /Mn 2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8 The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9 These data demonstrate that endothelial Cyp450 MO is activated by Ca 2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10 The data presented and our previous ®ndings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca 2+ in¯ux. We propose that Cyp450 MO-derived EETs constitute a signal for Ca 2+ entry activation and increase the driving force for Ca 2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.
Abstract-Although the involvement of free radicals in the development of endothelial dysfunction under pathological conditions, like diabetes and hypercholesterolemia, has been proposed frequently, there is limited knowledge as to how superoxide anions (O 2 Ϫ ) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O 2 Ϫ -generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca 2ϩ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca 2ϩ release and capacitative Ca 2ϩ entry in response to bradykinin and ATP in a time-and concentration-dependent manner. This effect was prevented by superoxide dismutase but not by the tyrosine kinase inhibitor tyrphostin A48. In addition, capacitative Ca 2ϩ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O 2 Ϫ -exposed cells (ϩ38% and ϩ32%, respectively). Increased Ca 2ϩ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (ϩ140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca 2ϩ signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca 2ϩ store depletion with thapsigargin (ϩ103% and ϩ48%, respectively) and tyrosine kinase activity (ϩ97%). A comparison of bradykinin-initiated intracellular Ca 2ϩ release and thapsigargin-induced hyperpolarization with membrane viscosity modulated by XO/HX (decrease in viscosity) or cholesterol (increase in viscosity) reflected a negative correlation between bradykinin-initiated Ca 2ϩ release and membrane viscosity. Because intracellular Ca 2ϩ is a main regulator of endothelial vascular function, our data suggest that O 2 Ϫ anions are involved in regulation of the vascular endothelium.
1. In addition to their contribution to endothelium-derived hyperpolarization, our understanding of the physiological function of epoxyeicosatrienoic acids (EET) within the vascular wall and the actual enzymes involved in the formation of the EET in endothelial cells is very limited. In the present study, the expression of potential cytochrome P450 (CYP) mono/epoxygenases was assessed in endothelial cells isolated from porcine and bovine aortas as well as in the human umbilical vein-derived cell lines EA.hy926 and ECV304. 2. Expression of CYP2B1, CYP2E1 and CYP3A could be found. The latter were inducible by dexamethasone/clofibrate for 72 h, a procedure that also enhanced CYP epoxygenase activity in endothelial cells. 3. Enzyme induction yielded increases in capacitative Ca2+ entry and membrane hyperpolarization in response to autacoids, such as bradykinin and thapsigargin. Thiopentone sodium, an inhibitor of endothelial CYP mono/epoxygenase(s), diminished autacoid-induced capacitative Ca2+ entry and membrane hyperpolarization, while the effect of EET remained unchanged. 4. Epoxyeicosatrienoic acids activated endothelial tyrosine kinase activity in a concentration-dependent manner. Arachidonic acid, at 20-fold higher concentrations, also increased tyrosine kinase activity. Because only the effect of arachidonic acid was inhibited by thiopentone sodium, an inhibitor of CYP mono/epoxygenases, these data suggest that arachidonic acid needs to be converted to the EET in order to stimulate tyrosine kinase. 5. All these data provide clear evidence that the CYP epoxygenase-derived arachidonic acid metabolites (EET) not only serve as potential endothelium-derived hyperpolarizing factors but also constitute highly active intracellular messengers with a physiological role including the control of Ca2+ signalling, membrane potential and tyrosine kinase activity.
1 Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endotheliumdependent relaxation in the presence of L-N0 nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid-activated endothelial Ca2+ signalling, endothelium-dependent relaxation was initiated with the Ca2+ ionophore A23187. 2 The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L-N0 nitroarginine-resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe-234-initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234-induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3
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