Hypoxia factors suppression effect on the energy metabolism of a malignant melanoma cell SK-MEL-30.

OBJECTIVE: Malignant melanoma (MM), as well as other cancers, is a disorder in the cell life cycle at many levels. In terms of energy, the sync of cytosolic and mitochondrial metabolism is required for each cell. Mismatches also caused by hypoxic factors accumulate defects leading to the formation, development and invasiveness of malignant melanoma. Our aim was to compare the effect of HIF-1α and miR-210 on the metabolism of malignant melanoma cells in normoxia and pseudohypoxia. Further, we also investigated how gene silencing affects the viability in order to evaluate the potential of gene therapy in the treatment of MM. MATERIALS AND METHODS: We targeted oxidative phosphorylation by genetically suppressing HIF-1α and miR-210. We have examined mitochondrial activity, cytosolic glycolysis and cell viability. RESULTS: The ratio of NADH/NAD+ in the cytoplasm under normal conditions is stable and can thus serve as a specific cellular metabolic marker. Therefore, the study was aimed at finding the cause of the reduction in NADH levels in increasing hypoxia under ideal in vitro conditions on the SK-MEL-30 malignant melanoma cells. The relationship between HIF-1α and miR-210, their effect on transcriptional level, and the subsequent effect on metabolic process attenuation in cells was investigated. Obtained results indicate that the NADH which is accumulated by cells in hypoxia was significantly decreased upon gene silencing. CONCLUSIONS: Our studies have shown that small regulatory molecules with organelle-specific effect (such as miRs) need to be targeted, and that the resultant effect is comparable to silencing of "general" hypoxic transcription factors.

Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples.

Vital cells maintain a steep potassium ion (K+ ) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+ ]) and especially the [K+ ] within the extracellular matrix are strictly regulated, the latter within a narrow range of ∼3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+ ] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+ ]. Generally, [K+ ] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+ ]. In addition to the determination of [K+ ] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells. © 2019 The Authors.

C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca(2+) from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl-/- and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl-/- macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl-/- macrophages, FB1 treatment rescued Atgl-/- macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl-/- macrophages by activation of the mitochondrial apoptosis pathway.

Intercellular signalling within vascular cells under high D-glucose involves free radical-triggered tyrosine kinase activation.

AIMS/HYPOTHESIS: Diabetes mellitus is associated with endothelial dysfunction in human arteries due to the release of superoxide anions (*O(2)(-)) that was found to occur predominantly in smooth muscle cells (SMC). This study was designed to elucidate the impact of high glucose concentration mediated radical production in SMC on EC. Pre-treatment of vascular SMC with increased D-glucose enhanced release of *O(2)(-). METHODS: Microscope-based analyses of intracellular free Ca(2+) concentration (fura-2), immunohistochemistry (f-actin) and tyrosine kinase activity were performed. Furthermore, RT-PCR and Western blots were carried out. RESULTS: Interaction of EC with SMC pre-exposed to high glucose concentration yielded changes in endothelial Ca(2+) signalling and polymerization of f-actin in a concentration-dependent and superoxide dismutase (SOD) sensitive manner. This interaction activated endothelial tyrosine kinase(s) but not NFkappaB and AP-1, while SOD prevented tyrosine kinase stimulation but facilitated NFkappaB and AP-1 activation. Erbstatin, herbimycin A and the src family specific kinase inhibitor PP-1 but not the protein kinase C inhibitor GF109203X prevented changes in endothelial Ca(2+) signalling and cytoskeleton organization induced by pre-exposure of SMC to high glucose concentration. Adenovirus-mediated expression of kinase-inactive c-src blunted the effect of pre-exposure of SMC to high glucose concentration on EC. CONCLUSIONS/INTERPRETATION: These data suggest that SMC-derived *O(2)(-) alter endothelial cytoskeleton organization and Ca(2+) signalling via activation of c-src. The activation of c-src by SMC-derived radicals is a new concept of the mechanisms underlying vascular dysfunction in diabetes.

Impact of apolipoprotein(a) on in vitro angiogenesis.

Angiostatin, which consists of the kringle I-IV domains of plasminogen and which is secreted into urine, is an efficient inhibitor of angiogenesis and tumor growth. Because N-terminal apolipoprotein(a) [apo(a)] fragments, which also contain several types of kringle IV domains, are found in urine as well, we evaluated the potential angiostatic properties of these urinary apo(a) fragments and of a recombinant form of apo(a) [r-apo(a)]. We used human microvascular endothelial cell (hMVEC)-based in vitro assays of tube formation in 3-dimensional fibrin matrixes. Purified urinary apo(a) fragments or r-apo(a) inhibited the basic fibroblast growth factor/tumor necrosis factor-alpha-induced formation of capillary-like structures. At concentrations varying from 0.2 to 10 microgram/mL, urinary apo(a) fragments inhibited tube formation by as much as 70%, whereas there was complete inhibition by r-apo(a). The highest concentrations of both inhibitors also reduced urokinase plasminogen activator production of basic fibroblast growth factor-induced hMVEC proliferation. The inhibitors had no effect on plasminogen activator inhibitor-1 expression. If our in vitro model for angiogenesis is valid for the in vivo situation as well, our data point toward the possibility that apo(a) may also be physiologically operative in modulating angiogenesis, as the concentration of free apo(a) found in humans exceeds that tested herein.

Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase.

Using single cell microfluorometry to monitor changes in bulk Ca2+ concentration ([Ca2+]bulk) and the whole-cell configuration of the patch clamp technique to measure K+ currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamine-induced Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. In single cells, histamine (10 microM) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+]o = 10-30 microM). In contrast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 microM EGTA) or in the presence of 2.5 mM extracellular Ca2+. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activated K+ (KCa) channels and membrane hyperpolarization of 18.1 +/- 3.9 mV. Hence, cell depolarization with 70 mM extracellular K+ or the inhibition of non-selective cation channels (NSCCs) and KCa channels by 10 microM Loe 908 and 10 mM tetrabutylammonium prevented histamine-evoked Ca2+ oscillations. Preventing Na+-Ca2+ exchange (NCX) by 10 microM 2', 4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillations. Lowering the extracellular Na+ concentration and thus promoting the reversed mode of NCX (3Na+ out and 1Ca2+ in) increased the amplitude and frequency of histamine-induced Ca2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca2+, 10 microM histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal domains. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2,5-di-tert-butyl-1, 4-benzo-hydroquinone (15 microM) prevented histamine-induced Ca2+ oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release (RsCR) by 25 microM ryanodine blunted Ca2+ oscillations. In endothelial cells that were treated for 16 h with 10 microM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca2+ oscillations were found. We conclude that in low [Ca2+]o conditions histamine-induced Ca2+ oscillations depend on transmembrane Na+ loading through NSCCs that leads to Ca2+ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca2+ spiking. These data further support our previous work on localized Ca2+ signalling as a key phenomenon in endothelial Ca2+ homeostasis.

Subplasmalemmal ryanodine-sensitive Ca2+ release contributes to Ca2+-dependent K+ channel activation in a human umbilical vein endothelial cell line.

The whole-cell configuration of the patch clamp technique was used to assess the involvement of ryanodine-sensitive Ca2+ release (RsCR) in histamine-activated Ca2+-dependent K+ (KCa) channels in the human umbilical vein endothelial cell line EA.hy926. Histamine (10 microM) induced a transient outward current that reached 18.9 +/- 5.5 pA pF-1 at +20 mV. This current was diminished by 1 mM tetraethylammonium or 50 nM iberiotoxin, by 90 % and 80 %, respectively, suggesting that this current results from the stimulation of large-conductance KCa (BKCa) channels. In about 50 % of the cells tested, stimulation of RsCR with 200 nM ryanodine initiated a small outward current that was also sensitive to iberiotoxin. Following the ryanodine-mediated RsCR, the potency of 10 microM histamine to activate KCa channels was reduced by about 60 %. In agreement, an inhibition of RsCR with 25 microM ryanodine diminished KCacurrent in response to histamine by about 70 %. The effect of 100 microM histamine on KCa channel activity was not reduced by previous RsCR with 200 nM ryanodine, or by an inhibition of RsCR by 25 microM ryanodine. Histamine (10 microM)-induced Ca2+ elevation was reduced by 30 % following ryanodine-mediated RsCR, whereas no inhibition occurred in the case of 100 microM histamine stimulation. In cells treated with 10 microM nocodazole for 16 h to collapse the superficial endoplasmic reticulum, 200 nM ryanodine failed to initiate any KCa current. Furthermore, the inhibitory effect of previous RsCR on 10 microM histamine-induced KCa current was not obtained in nocodazole-treated cells. Our data suggest that during moderate cell stimulation (10 microM histamine), subplasmalemmal RsCR greatly contributes to the activation of KCa channels in endothelial cells. Thus, the function of the subplasmalemmal Ca2+ control unit (SCCU) described previously must be extended as a regulator for KCa channels.

In human hypercholesterolemia increased reactivity of vascular smooth muscle cells is due to altered subcellular Ca(2+) distribution.

There is evidence that, besides an attenuated endothelium-dependent relaxation, functional changes in smooth muscle contractility occur in experimental hypercholesterolemic animals. Unfortunately, little is known of the situation in human arteries, and the intracellular mechanisms involved in the modulation of vascular smooth muscle function in human hypercholesterolemia are still unclear. Thus, besides acetylcholine-induced endothelium-dependent relaxation, smooth muscle reactivity to KCl, norepinephrine (NE) and phenylephrine (PE) was evaluated in uterine arteries from 34 control individuals (CI) and 22 hypercholesterolemic patients (HC). Contractions to KCl, norepinephrine and phenylephrine were enhanced by 1.3-, 2.1- and 3.5-fold in vessels from HC. Furthermore, the Ca(2+) signaling in the perinuclear cytosol, which promotes cell contraction, and that of the subplasmalemmal region, which contributes to smooth muscle relaxation, were examined in freshly isolated smooth muscle cells. In cells from HC, increases in perinuclear Ca(2+) concentration ([Ca(2+)](peri)) in response to 30 mM KCl and 300 nM NE were increased by 67 and 93%, respectively. In contrast, the increase in the subplasmalemmal Ca(2+) concentration ([Ca(2+)](sub)) to 10 microM NE was reduced in cells from HC by 33%. No further differences in perinuclear and subplasmalemmal Ca(2+) signaling were found in cultured smooth muscle cells from CI and HC (primary culture 4-6 weeks after isolation). These data indicate a significant change in the subcellular Ca(2+) distribution in smooth muscle cells from HC. In addition, production of superoxide anions (O(2)(-)) was increased 3.8-fold in uterine arteries from HC. Treatment of smooth muscle cells with the O(2)(-)-generating mixture xanthine oxidase/hypoxanthine mimicked hypercholesterolemia on smooth muscle Ca(2+) signaling. From these findings, we conclude that during hypercholesterolemia, besides a reduced endothelium-dependent relaxation, changes in smooth muscle reactivity take place. Thereby, smooth muscle contractility is increased possibly due to the observed changes in subcellular Ca(2+) signaling. The observed increased O(2)(-) production in HC might play a crucial role in the alteration of smooth muscle function in hypercholesterolemia.

Selective stimulation of L-arginine uptake contributes to shear stress-induced formation of nitric oxide.

The aim of this study was to investigate the kinetics of L-arginine transport mechanisms and the role of extracellular L-arginine in nitric oxide formation during shear stress activation of endothelial cells. Porcine aortic endothelial cells were grown to confluence and were exposed to various amounts of shear stress for 40 min. Formation of nitric oxide was monitored by measuring elevation of endothelial cGMP. Activity of amino acid transport systems was determined by measuring the uptake of L-[3H]leucine (L system) and L-[3H]arginine (y+) under resting and shear stress condition. Shear stress-mediated nitric oxide formation critically depended on the presence of extracellular L-arginine, which increased shear stress-induced cGMP increases in a concentration dependent manner (EC50=123 microM). In addition, shear stress increased L-arginine uptake, while the transport capacity for neutral amino acids (L system) remained unchanged under shear stress conditions. Analysis of the kinetics of the uptake of L-arginine under resting and shear stress conditions indicate that shear stress increased velocity of the high affinity, low capacity transport (y+) without affecting affinity of this system. These data suggest that shear stress selectively activates uptake of L-arginine in endothelial cells and that the uptake of L-arginine might be important for shear stress-mediated nitric oxide formation.

Alterations in platelet Ca2+ signalling in diabetic patients is due to increased formation of superoxide anions and reduced nitric oxide production.

Increased aggregation of platelets might contribute to the development of vascular complication in diabetes mellitus. In this study release of superoxide anions, intracellular Ca2+ signalling and nitric oxide formation stimulated by the receptor-dependent agonist adenosine 5 '-diphosphate (ADP) and the receptor-independent stimulus thapsigargin, were compared in platelets isolated from patients with Type II (non-insulin-dependent) diabetes mellitus and healthy control subjects. Diabetes augmented intracellular Ca2+ release and Ca2+ entry to ADP by 40 and 44% (control subjects: n = 11; diabetic: n = 6), while the median effective concentration (EC50) of ADP to initiate Ca2+ signalling was similar in both groups. The effect of thapsigargin on Ca2+ concentration was increased by 69% in diabetic patients (control subjects: n = 22; diabetic patients: n = 9). In addition, release of superoxide anions was 70% greater in diabetic patients (control subjects: n = 9; diabetic patients: n = 6). Treatment of platelets from control subjects with the superoxide anion-generating mixture xanthine oxidase and hypoxanthine or buthioninesulphoximine (BSO) mimicked the effect of diabetes on platelet Ca2+ signalling. The antioxidant glutathione normalized enhanced Ca2+ response in the diabetic group (control subjects: n = 5: diabetic patients: n = 6). Basal and thapsigargin-evoked nitric oxide synthase activity was reduced in the diabetic group by 85 and 64%, respectively (control subjects: n = 13; diabetic subjects: n = 13). The nitric oxide-donor 2-(N,N-diethylamino)-diazenolate-2-oxide sodium (DEA/NO) normalized enhanced Ca2+ signalling in platelets preincubated with xanthine oxidase and hypoxanthine (n = 12) and in those from diabetics (control subjects: n = 6; diabetic patients: n = 6). Inhibition of nitric oxide synthase by N-nitro-L-arginine (L-NA) augmented thapsigargin-induced Ca2+ signalling by 51% (n = 8). These data indicate that in diabetes platelet Ca2+ signalling might be enhanced by excessive superoxide production and an attenuated negative direct or indirect feedback control by nitric oxide, due to its reduced production.

Effects of superoxide anions on endothelial Ca2+ signaling pathways.

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 (O2-) might affect endothelial signal transduction. In this study, we investigated the effects of preincubation with the O2(-)-generating system xanthine oxidase/hypoxanthine (XO/HX) on mechanisms for Ca2+ signaling in cultured porcine aortic endothelial cells. Incubation of cells with XO/HX yielded increased intracellular Ca2+ release and capacitative Ca2+ 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 Ca2+ entry induced by the receptor-independent stimulus 2,5-di-(tert-butyl)-1,4-benzohydroquinone or thapsigargin was enhanced in O2(-)-exposed cells (+38% and +32%, respectively). Increased Ca2+ release in response to bradykinin in XO/HX-pretreated cells might be due to enhanced formation of inositol-1,4,5-trisphosphate (+140%). Exposure to XO/HX also affected other signal transduction mechanisms involved in endothelial Ca2+ signaling, such as microsomal cytochrome P450 epoxygenase and membrane hyperpolarization to Ca2+ store depletion with thapsigargin (+103% and +48%, respectively) and tyrosine kinase activity (+97%). A comparison of bradykinin-initiated intracellular Ca2+ 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 Ca2+ release and membrane viscosity. Because intracellular Ca2+ is a main regulator of endothelial vascular function, our data suggest that O2- anions are involved in regulation of the vascular endothelium.

Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane.

1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ 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 Ca2+ elevations occur which are controlled by cell membrane Na(+)-Ca2+ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca2+ concentration ([Ca2+]b) was monitored using fura-2 or Calcium Green-1 and subplasmalemmal Ca2+ concentration ([Ca2+]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 3. Sodium fluoride (10 mmol l-1) and submaximal concentrations of bradykinin (Bk; 1 nmol l-1) stimulated Ca2+ entry with no increase in [Ca2+]b. Although inositol 1,4,5-trisphosphate formation and intracellular Ca2+ release in response to both stimuli were similar, Ca2+ entry in response to NaF exceeded that in response to 1 nmol l-1 BK by fourfold, suggesting additional effects of NaF on Ca+ entry pathways but stimulation via intracellular Ca2+ release. 4. Prevention of Na(+)-Ca2+ exchange activity by decreasing extracellular Na+ unmasked intracellular Ca2+ release in response to NaF and 1 nmol l-1 Bk, indicated by an increase in [Ca2+]b. Thereby, NaF depleted Bk-releasable Ca2+ pools, while mitochondrial Ca2+ content (released with FCCP or oligomycin) and the amount of Ca2+ stored within the cells (released with ionomycin) was increased compared with cells treated with NaF under normal Na+ conditions. The NaF-initiated increase in [Ca2+]b and depletion of Bk-releasable Ca2+ pool(s) in the low-Na+ condition was diminished by 25 mumol l-1 ryanodine, indicating the involvement of Ca(2+)-induced Ca2+ release (CICR). 5. In simultaneous recordings of [Ca2+]sp (with FFP-18) and [Ca2+]b (with Calcium Green-1), 1 nmol l-1 Bk or 10 mmol l-1 NaF yielded focal [Ca2+] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 mumol-1 nocodazole caused the SER to collapse and unmasked Ca2+ release in response to 1 nmol l-1 Bk and 10 mmol l-1 NaF, similar to low-Na+ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca2+ elevations in response to small or slow IP3 formation occur due to vectorial Ca2+ release from the SER towards the plasmalemma followed by Ca2+ extrusion by Na(+)-Ca2+ exchange. While these local Ca2+ elevations are not detectable with Ca2+ dyes for the determination of [Ca2+]b, prevention of Ca2+ extrusion or SER disruption yields increases in [Ca2+]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca2+ release and [Ca2+] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca2+ homeostasis, Ca2+ distribution and Ca2+ entry. The existence of such a subplasmalemmal Ca2+ control unit (SCCU) needs to be considered in discussions of Ca2+ signalling, especially when cytoplasmic Ca2+ dyes, such as fura-2 or Calcium Green-1, are used.

Antioxidants prevent high-D-glucose-enhanced endothelial Ca2+/cGMP response by scavenging superoxide anions.

Very recently we proposed that hyperactivity of endothelial Ca2+/cGMP signaling under hyperglycemic conditions is due to superoxide anion (O2-) release. The present study was designed to investigate changes in endothelial glutathione (GSH) levels in response to high D-glucose and possible prevention of the high-D-glucose-initiated changes in Ca2+/cGMP signal by antioxidants. Under hyperglycemic conditions, GSH content increased by 29% within 4 h. Co-incubation with 10 mM GSH during high-D-glucose treatment normalized the Ca2+/cGMP response associated with an increase in GSH content by 222%. Vitamin C (250 microM) markedly diminished the high-D-glucose-mediated hyperreactivity of endothelial Ca2+ entry (by 40%) and Ca2+ release (by 52%). Similar to GSH, co-incubation with vitamin E (alpha-tocopherol; 50 micrograms/ml) and probucol (50 microM) completely prevented the high-D-glucose-initiated hyperreactivity of the endothelial Ca2+/cGMP response. Vitamin E, probucol, GSH and vitamin C diminished the high-D-glucose-mediated O2- release by 78, 65, 89 and 46%, respectively. These data suggest that antioxidants prevent high-D-glucose-initiated changes in endothelial Ca2+/cGMP response by scavenging the overshoot of O2-.

High D-glucose-induced changes in endothelial Ca2+/EDRF signaling are due to generation of superoxide anions.

Pretreatment of porcine aortic endothelial cells with high D-glucose results in enhanced endothelium-derived relaxing factor (EDRF) formation (39%) due to increased endothelial Ca2+ release (57%) and Ca2+ entry (97%) to bradykinin. This study was designed to investigate the intracellular mechanisms by which high D-glucose affects endothelial Ca2+/EDRF response. The aldose-reductase inhibitors, sorbinil and zopolrestat, failed to diminish high D-glucose-mediated alterations in Ca2+/EDRF response, suggesting that aldose-reductase does not contribute to high D-glucose-initiated changes in Ca2+/EDRF signaling. Pretreatment of cells with the nonmetabolizing D-glucose analog, 3-O-methylglucopyranose (3-OMG), mimicked the effect of high D-glucose on Ca2+ release (41%) and Ca2+ entry (114%) to bradykinin, associated with elevated EDRF formation (26%). High D-glucose and 3-OMG increased superoxide anion (O2-) formation (133 and 293%, respectively), which was insensitive to inhibitors of cyclooxygenase (5,8,11,14-eicosatetraynoic acid [ETYA], indomethacin), lipoxygenase (ETYA, gossypol, nordihydroguaiaretic acid [NDGA]), cytochrome P450 (NDGA, econazole, miconazole), and nitric oxide (NO) synthase (L-omega N-nitroarginine), while it was diminished by desferal, a metal chelator. The gamma-glutamyl-cysteine-synthase inhibitor, buthioninesulfoximine (BSO), also increased formation of O2- by 365% and mimicked the effect of high D-glucose on Ca2+/EDRF signaling. The effects of high D-glucose, 3-OMG, and BSO were abolished by co-incubation with superoxide dismutase. Like high D-glucose, pretreatment with the O2(-)-generating system, xanthine oxidase/hypoxanthine, elevated bradykinin-stimulated Ca2+ release (+10%), Ca2+ entry (+75%), and EDRF (+73%). We suggest that prolonged exposure to pathologically high D-glucose concentration results in enhanced formation of O2-, possibly due to metal-mediated oxidation of D-glucose within the cells. This overshoot of O2- enhances agonist-stimulated Ca2+/EDRF signaling via a yet unknown mechanism.

Hyperkalemia alters EDHF-mediated hyperpolarization and relaxation in coronary arteries.

Hyperkalemic solutions are widely used to preserve organs for transplantation and for cardiac surgery. The present study was designed to test the hypothesis that hyperkalemia may alter endothelial function through a non-nitric oxide (NO) pathway, since preliminary studies have shown that the NO pathway may not be affected. Porcine coronary artery rings were studied in organ chambers. After incubation with 20 or 50 mM K+ for 1 h, the indomethacin- and NG-nitro-L-arginine+ (L-NNA)-resistant relaxation induced by A23187 or bradykinin, which could be further inhibited by tetraethylammonium but not glibenclamide, was significantly reduced. Incubation with hyperkalemia also significantly increased the concentration eliciting 50% of the maximal response to A23187 and bradykinin. A23187-induced hyperpolarization of the membrane potential was significantly reduced by hyperkalemic incubation. However, 1-h incubation with hyperkalemia does not affect the endothelial Ca2+ concentration. We conclude that exposure to hyperkalemia reduces the indomethacin- and L-NNA-resistant endothelium-dependent relaxation and endothelium-dependent hyperpolarization. This reduction in the relaxation and hyperpolarization is related to the endothelium-derived hyperpolarizing factor by affecting its effect on the smooth muscle cell, probably through partially depolarizing the membrane, and the Ca2(+)- activated K+ channels rather than by affecting its biosynthesis and/or release in the endothelial cell. Our study may suggest a new mechanism for coronary dysfunction after exposure to hyperkalemic cardioplegia and organ preservation solutions.

Intracellular mechanism of high D-glucose-induced modulation of vascular cell proliferation.

Development of atherosclerosis in diabetes patients is thought to be associated with high D-glucose-induced changes in vascular cell proliferation. This study was designed to investigate the intracellular mechanisms of altered proliferation in porcine aortic endothelial and smooth muscle cells under high D-glucose conditions. Two different technical approaches were used for determination of cell proliferation, a cell counting procedure and bromodeoxyuridine incorporation. D-Glucose diminished endothelial cell proliferation (30.3%) and increased smooth muscle cell proliferation (143%) in a dose-dependent manner. Neither D-mannitol, sucrose nor L-glucose mimicked the effect of D-glucose. Inhibition of D-glucose uptake into vascular cells by cytochalasin B prevented the effect of high D-glucose on cell proliferation. The aldose-reductase inhibitors, sorbinil and zopolrestat, little affected high D-glucose-attenuated endothelial cell proliferation, while the enhanced proliferation of smooth muscle cells was prevented by aldose-reductase inhibitors. Elevation of cellular glutathione levels yielded protection of both cell types from high D-glucose-mediated changes in cell proliferation, suggesting that high D-glucose may act via generation of oxidative species. Finally, aminoguanidine was shown to constitute a very potent inhibitor of D-glucose-induced dysfunction in vascular cell proliferation. These data suggest that high D-glucose-induced changes in cell proliferation of endothelial and smooth muscle cells are related to specific D-glucose uptake rather than hyperosmolality. Aldose-reductase seems to be mainly involved in the effect of high D-glucose only on smooth muscle cell proliferation, while in endothelial cells there is (are) other factor(s) in addition to the sorbitol pathway involved in high D-glucose-induced changes in cell proliferation.

Cytochrome P450 mono-oxygenase-regulated signalling of Ca2+ entry in human and bovine endothelial cells.

1. We tested the hypothesis that agonist-stimulated Ca2+ entry, and thus formation of endothelium-derived nitric oxide (EDNO) in vascular endothelial cells, is related to activation of microsomal P450 mono-oxygenase (P450 MO) and the biosynthesis of 5,6-epoxyeicosatrienoic acid (5,6-EET). 2. Several P450 inhibitors diminished the sustained [Ca2+]i plateau response to agonist or intracellular Ca2+ store depletion with ATPase inhibitors by 31-69% (fura-2 technique). Mn2+ influx stimulated by agonists or ATPase inhibitors was prevented by P450 inhibitors. 3. Histamine- or ATPase inhibitor-stimulated formation of EDNO was strongly attenuated (50-83%) by P450 inhibitors, without any effect on EDNO formation by the Ca2+ ionophore A23187, indicating that decreased EDNO synthesis is due specifically to the inhibition of Ca2+ entry by these compounds. 4. Induction of P450 MO by beta-naphthoflavone potentiated agonist-induced Ca2+ and Mn2+ influx by 60 and 53%, respectively. Intracellular Ca2+ release remained unchanged. 5. The P450 MO product, 5,6-EET (< 156 nmol l-1), activated Ca2+/Mn2+ entry without any depletion of intracellular Ca2+ stores. The 5,6-EET-stimulated Ca2+/Mn2+ entry was not affected by P450 inhibitors. 6. As with the bradykinin-stimulated Ca2+ entry pathway, the 5,6-EET-activated Ca2+ entry pathway was permeable to Mn2+ and Ba2+, sensitive to Ni2+, La3+ and membrane depolarization, and insensitive to the removal of extracellular Na+ or the organic Ca2+ antagonist, nitrendipine. 7. In the presence of 5,6-EET, stimulation with bradykinin only transiently increased [Ca2+]i. Vice versa, 5,6-EET failed to increase [Ca2+]i further in bradykinin-stimulated cells. The sustained [Ca2+]i plateau phase induced by a co-stimulation with bradykinin and 5,6-EET was identical to that observed with bradykinin or 5,6-EET alone. 8. These results demonstrate that Ca2+ entry induced by the P450 MO product, 5,6-EET, is indistinguishable to that observed by stimulation with bradykinin. 9. All data support our hypothesis that depletion of endothelial Ca2+ stores activates microsomal P450 MO which in turn synthesizes 5,6-EET. We propose that the arachidonic acid metabolite 5,6-EET or one of its metabolites is a second messenger for activation of endothelial Ca2+ entry.

Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.

Altered release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) has been proposed as a final common pathway underlying the abnormal vasodilator responses to gram-negative lipopolysaccharide (endotoxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarified. We evaluated direct effects of Escherichia coli endotoxin on agonist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cultured bovine and porcine aortic endothelial cells (ECs). Two methods were used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct quantitative measurement of NO release (chemiluminescence method). Cytosolic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence methodology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 microgram/mL, 1 hour plus 1-hour wash) significantly inhibited bradykinin (100 nmol/L)- and ADP (10 mumol/L)-mediated increases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase in cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mumol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation with endotoxin inhibited ADP-stimulated increases in NO release and EDRF bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivity. Endotoxin produced significant decreases in both transient and sustained [Ca2+]i responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+]i in response to bradykinin was reduced to 31% of the initial increases in control cells, and the secondary plateau phase was reduced to only 3% of respective control responses. Concentration-response relation to endotoxin (10(-3)) to 10(0) micrograms/mL) indicated high correlation and similar IC50 values (0.025 and 0.021 micrograms/mL, respectively) for inhibitory effects on cGMP and [Ca2+]i. Endotoxin had no effect on inositol trisphosphate formation ([3H]myo-inositol incorporation) and intracellular Ca2+ release ([Ca2+]i responses in Ca(2+)-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was significantly attenuated by endotoxin treatment. These studies demonstrate that endotoxin directly decreases agonist (bradykinin and ADP)-mediated biosynthesis and release of EDRF/NO from ECs. These effects can be explained by altered [Ca2+]i mobilization mechanisms, which in turn produce subsequent decreases in activity of the Ca(2+)-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of agonist-mediated NO release and endothelium-dependent vasodilation.

Intracellular mechanisms involved in D-glucose-mediated amplification of agonist-induced Ca2+ response and EDRF formation in vascular endothelial cells.

Prolonged treatment of vascular endothelial cells with pathologically high D-glucose amplifies autacoid-induced Ca2+ mobilization and thus formation of nitric oxide. This study investigated the Ca2+ source for the change in endothelial CA2+ response on agonist stimulation. Pretreatment with high D-glucose (44 vs. 5 mM) enhanced release of intracellular Ca2+ by bradykinin as a result of a 2.0-fold increased formation of inositol 1,4,5-trisphosphate. High D-glucose also amplified Ca2+ influx (2.0-fold). In high D-glucose preincubated cells, stimulation with bradykinin significantly increased transplasmalemmal 45Ca2+ flux (3.2-fold) and caused a 2.0-fold increase in permeability to Mn2+, a surrogate for endothelial plasma membrane Ca2+ channels. A significant 2.0-fold increase occurred in the maximal slope, suggesting a higher rate of Mn2+ (Ca2+) influx. Ca2+ influx, stimulated by an inositol phosphate-independent depletion of intracellular Ca2+ stores with 2,5-di-(tert-butyl)-hydroquinone was also significantly increased 2.4-fold by high D-glucose, with no effect on intracellular Ca2+ release. D-glucose failed to modulate resting or stimulated cAMP levels. We suggest that prolonged exposure to pathologically high D-glucose increases formation of inositol polyphosphates, thus increasing Ca2+ release. Ca2+ entry is increased by amplification of unknown signal transduction mechanisms triggered by Ca2+ store depletion.

Heterogeneity of caffeine- and bradykinin-sensitive Ca2+ stores in vascular endothelial cells.

The filling state of Ca2+ stores in endothelial cells regulates Ca2+ entry. The functional relationship between the major Ca2+ stores [i.e. Ins(1,4,5)P3-sensitive (= bradykinin-sensitive stores, 'BsS') and caffeine-sensitive stores] is unknown. In pig right-coronary-artery endothelial cells, caffeine failed to release Ca2+ in 68% of the cells (quiet-responders), but increased bradykinin (Bk)-induced Ca2+ release 2.5-fold. In Bk-pre-stimulated cells, caffeine increased Ca2+ release upon a second stimulation with Bk 3.2-fold. In quiet-responders caffeine alone did not affect net Ca2+ storage, whereas Bk or caffeine followed by Bk decreased the intracellular Ca2+ pool to 45% and 15%, respectively. Blockade of the endoplasmic-reticulum Ca2+ pump by thapsigargin unmasked the effect of caffeine in quiet-responders, resulting in a transient increase in intracellular free Ca2+ concentration ([Ca2+]i). In 37% of the cells caffeine alone transiently increased [Ca2+]i and depleted BsS. This study suggests a heterogeneity in functional organization of endothelial Ca2+ stores. In quiet-responders, caffeine translocates Ca2+ towards the BsS, whereas in overt-responders caffeine empties the BsS.