Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelial-dependent vasodilatation

The present study was aimed to evaluate the mechanisms involved in the vasorelaxant effects of red wine polyphenol compounds (RWPC) in small mesenteric rat arteries. RWPC produce relaxation in small mesenteric arteries. This relaxant effect was abolished by endothelial denudation, NO-synthase blockade with L-NAME and partial depolarization with KCl or L-NAME plus KCl. Incubation with the reactive oxygen species scavenger, superoxide dismutase (SOD) plus catalase, or inhibition of NAD(P)H-dependent oxidoreductases with diphenyleneiodonium also inhibited RWPC induced vascular relaxation. Application of RWPC elicited a transient increase in intracellular calcium concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC), which was attenuated by a mixture of SOD and catalase. Incubation of BAEC with RWPC increased the SOD inhibitable production of O2-. These results suggest the involvement of O2- in the [Ca2+]i increase evoked by RWPC, leading to the activation of enzymes involved in the release of endothelial relaxant factors and subsequent vasodilatation of resistance arteries.     

Regulation of vascular tone by plant polyphenols: role of nitric oxide

Vascular endothelium plays a crucial role in regulating blood flow and vascular tone. It can synthesize and release different relaxant factors including nitric oxide (NO). This article summarizes pharmacological properties of red wine polyphenol extracts (RWPC) with respect to endothelial NO. It is shown that RWPC produces endothelium-dependent relaxation as a result of enhanced NO synthesis rather than enhanced biological activity of NO or protection against breakdown by O2-. The mechanisms involve influx of Ca2+ and production of O2- within the endothelial cells. These results suggest that RWPC, by releasing endothelial NO, may have therapeutically relevant effects against cardiovascular diseases.     

Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2*- production in the intact coronary endothelium

The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.     

Ca2+/calmodulin-regulated nitric oxide synthases.

NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Transient increase of cytosolic free calcium in cultured human vascular endothelial cells by platelet-activating factor

The effect of platelet-activating factor (PAF-acether) on cytosolic free calcium, [Ca2+]i, in adherent human vascular endothelial cells in culture was directly determined using a new fluorescent calcium indicator, fura-2. It was found that PAF-acether but not lyso PAF-acether induced a rapid and transient increase in [Ca2+]i in endothelial cells. Restimulation with PAF-acether after the first challenge did not cause further response, while the cells were able to respond to thrombin. In the absence of extracellular calcium, PAF-acether evoked a similar transient increase, suggesting that PAF-acether raises [Ca2+]i mainly by discharging calcium from intracellular pools. PAF-acether-induced rise in [Ca2+]i was completely blocked by a specific antagonist, BN 52021. These results suggest the receptor-mediated increase in [Ca2+]i as an early event in PAF-acether activation of human vascular endothelial cells.     

Effect of crocin on experimental atherosclerosis in quails and its mechanisms

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg x L(-1)) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 microg x L(-1) Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca2+]i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca2+, crocin concentration-dependently inhibited the [Ca2+]i elevation induced by 120 mg x L(-1)Ox-LDL, In the absence of extracellular Ca2+, crocin could inhibit the [Ca2+]i elevation induced by CHCl3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca2+]i elevation in smooth muscle cell, Ca2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.     

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.     

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Cl-]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.     

Red wine polyphenols prevent endothelial damage induced by CCl4 administration

It became evident in the present study that carbon tetrachloride (CCl(4)), in addition to its known liver and renal toxicity, causes serious damage to endothelial cells. The preventive effect of red wine on cardiovascular diseases has been documented in a number of human population studies as well as in animal experimental models. In this study, the endothelium protective effect of polyphenolic compounds isolated from red wine was studied in rats administered 0.5 ml of CC(4)/kg body weight intraperitoneally twice a week for 8 weeks. Endothelemia (endothelial cells/10 microl of plasma) was used as the marker of endothelial cell injury in vivo. Chronic CCl(4) treatment for 8 weeks lead to a 3-fold increase of free endothelial cells circulating in the blood when compared to the baseline values (2.5+/-0.3). Parallel oral administration of polyphenols 40 mg/kg/day significantly decreased the endothelemia. Polyphenolic compounds alone did not produce significant changes. Three weeks of spontaneous recovery after the 8-week treatment with CCl(4) did not lead to a marked decrease of endothelemia, but the administration of red wine polyphenols during the 3-week period significantly decreased free endothelial cells in the blood. It can be concluded that long-term administration of CCl(4) may serve as a useful experimental model of endothelial damage. The red wine polyphenolic compounds exert a powerful protective effect on endothelial cells from the injury caused by CCl(4). This effect was documented by decreased endothelemia that corresponded to diminished endothelial cell swelling and detachment evaluated by histology of the vascular intima. The endothelium protective effect may be one of the key factors that contribute to the preventive action of red wine on cardiovascular diseases.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Staphylococcal enterotoxin A modulates intracellular Ca2+ signal pathway in human intestinal epithelial cells

We demonstrate here that staphylococcal enterotoxin A (SEA) induces an increase in intracellular calcium ([Ca2+]i) in human intestinal epithelial cells and the [Ca2+]i is released from intracellular stores. SEA-induced increase of [Ca2+]i was clearly inhibited by treatment with a nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine and guanidine. Intestinal epithelial cells express endothelial NOS in resting cell condition, and express inducible NOS after stimulating with tumor necrosis factor (TNF)-alpha. TNF-alpha-pretreated cells showed a significant increase in [Ca2+]i that was also inhibited by the NOS inhibitor. These results suggest that SEA modulated [Ca2+]i signal is dependent on NOS expression in human intestinal epithelial cells.     

Upregulation of endothelial cell Ca2+ signaling contributes to pregnancy-enhanced vasodilation of rat uteroplacental arteries

Normal pregnancy is characterized by an increased uterine blood flow due to growth and remodeling of the maternal uterine vasculature and enhanced vasodilation of the uterine arteries. The objective of the present study was to examine the role of endothelial cell Ca2+ signaling in augmented endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. We performed fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) in the cytoplasm of endothelial cells simultaneously with diameter in pressurized uterine arteries from nonpregnant (NP) and late-pregnant (LP) rats. Basal levels of endothelial cell [Ca2+]i were higher in arteries from LP rats compared with NP controls. Withdrawal of extracellular Ca2+ resulted in a decrease in the level of basal [Ca2+]i that was significantly larger in arteries of LP than NP rats. The rate of Mn2+ -induced quenching of fura-2 fluorescence was significantly elevated in late pregnancy, implicating augmented Ca2+ influx as a cause of increased basal levels of [Ca2+]i in endothelial cells. Elevation of intraluminal pressure resulted in a transient increase in endothelial [Ca2+]i that was markedly potentiated in late gestation. ACh-induced [Ca2+]i and vasodilator responses were significantly augmented in arteries of LP compared with NP rats and were abolished by BAPTA treatment, demonstrating a critical role of [Ca2+]i elevation in the production of endothelium-derived vasodilators. Together, these results indicate that late pregnancy is a state of enhanced basal and stimulated Ca2+ signaling in endothelial cells of uterine vessels, which may represent an important underlying mechanism for augmented vasodilation in the maternal uterine circulation.     

Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.     

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.