TNF-alpha inhibits pregnancy-adapted Ca signaling in uterine artery endothelial cells.

Enhancement of vasodilation of uterine arteries during pregnancy occurs through increased connexin (Cx)43 gap junction (GJ) communication supporting more frequent and sustained Ca 'bursts'. Such adaptation is lacking in subjects with preeclampsia (PE). Here we show TNF-alpha, commonly increased in PE subjects, inhibits Cx43 function and Ca bursts in pregnancy-derived ovine uterine artery endothelial cells (P-UAEC) via Src and MEK/ERK phosphorylation of Cx43, and this can be reversed by PP2 or U0126.

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells.

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function.

Positive versus negative effects of VEGF165 on Ca2+ signaling and NO production in human endothelial cells.

Unlabelled: The role increased vascular endothelial growth factor (VEGF) plays in vascular function during normal vs. preeclamptic pregnancy has been a source of some controversy of late. In this study, we seek to understand how VEGF influences vasodilator production via Ca signaling mechanisms in human endothelial cells.

Changes in Ca2+ Signaling and Nitric Oxide Output by Human Umbilical Vein Endothelium in Diabetic and Gestational Diabetic Pregnancies.

Diabetes (DM) complicates 3%-10% of pregnancies, resulting in significant maternal and neonatal morbidity and mortality. DM pregnancies are also associated with vascular dysfunction, including blunted nitric oxide (NO) output, but it remains unclear why. Herein we examine changes in endothelial NO production and its relationship to Ca(2+) signaling in endothelial cells of intact umbilical veins from control versus gestational diabetic (GDM) or preexisting diabetic subjects.

Phosphorylation of Ser-279/282 and Tyr-265 positions on Cx43 as possible mediators of VEGF-165 inhibition of pregnancy-adapted Ca2+ burst function in ovine uterine artery endothelial cells.

Normal pregnancy requires increased uterine endothelial cell driven vasodilation that is related to increases in sustained Ca2+ signaling via increased connexin 43 (Cx43) gap junction function. Preeclampsia, a hypertensive disorder of pregnancy associated with endothelial dysfunction, is also linked with down regulation of Ca2+ driven vasodilator production and increased levels of vascular endothelial growth factor (VEGF). Cx43 function can be acutely down-regulated by phosphorylation of multiple inhibitory residues and VEGF is known to promote phosphorylation of Cx43.

Pregnancy programming and preeclampsia: identifying a human endothelial model to study pregnancy-adapted endothelial function and endothelial adaptive failure in preeclamptic subjects.

We have previously reported that the increase in vasodilator production in an ovine model pregnancy is underpinned by an increase in connexin 43 (Cx43) gap junction function, so allowing more uterine artery endothelial cells to produce a more sustained Ca(2+) burst response to agonist stimulation. Since activation of endothelial nitric oxide synthase (eNOS) requires elevated [Ca(2+)]i, it follows that the direct result of enhanced bursting in turn is an increase in nitric oxide (NO) production per cell from more cells, and for a longer period of time. Preeclampsia (PE) is associated with endothelial vasodilatory dysfunction, and the endocrine profile of women with PE includes an increase in a number of factors found in wound sites.

Local effects of pregnancy on connexin proteins that mediate Ca2+-associated uterine endothelial NO synthesis.

Unlabelled: Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth.

Hypothesis: local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P(635)eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle.

The loss of sustained Ca(2+) signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy.

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface.

[Ca2+]i signaling vs. eNOS expression as determinants of NO output in uterine artery endothelium: relative roles in pregnancy adaptation and reversal by VEGF165.

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase.

Pregnancy enhances sustained Ca2+ bursts and endothelial nitric oxide synthase activation in ovine uterine artery endothelial cells through increased connexin 43 function.

Endothelium-mediated vasodilation is specifically enhanced in uterine circulation during pregnancy, and production of nitric oxide (NO) is increased in response to a wide array of agonists. Uterine artery endothelial cells from nonpregnant (NP-UAECs) or pregnant (P-UAECs) ewes maintained in culture still show a pregnancy-enhanced difference in ATP-stimulated endothelial NO synthase (eNOS; official symbol NOS3) activation, even though NOS3 protein, purinergic receptors, and associated cell signaling proteins are expressed at equal levels. We have also shown that the pregnancy-enhanced endothelial cell NO response to ATP requires an enhanced and sustained capacitative entry phase that is likely mediated via canonical transient receptor potential protein/inositol 1,4,5-trisphosphate receptor type 2 interaction.

Pregnancy-enhanced store-operated Ca2+ channel function in uterine artery endothelial cells is associated with enhanced agonist-specific transient receptor potential channel 3-inositol 1,4,5-trisphosphate receptor 2 interaction.

We have previously shown that endothelial cells (EC) derived from the uterine artery (UA) of both pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes show a biphasic intracellular free Ca(2+) ([Ca(2+)](i)) response after ATP stimulation. In each case, the initial transient peak, caused by the release of Ca(2+) from the intracellular Ca(2+) stores, is mediated by purinergic receptor-Y2 and is very similar in both cell types. However, the sustained phase in particular, caused by the influx of extracellular Ca(2+), is heightened in the P-UAEC, and associates with an increased ability of the cells to demonstrate enhanced capacitative Ca(2+) entry (CCE) via store-operated channels (SOCs).

Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling.

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca(2+) concentration ([Ca(2+)](i)), but the P-UAEC show a heightened sustained phase.

Pregnancy-enhanced endothelial nitric oxide synthase (eNOS) activation in uterine artery endothelial cells shows altered sensitivity to Ca2+, U0126, and wortmannin but not LY294002--evidence that pregnancy adaptation of eNOS activation occurs at multiple levels of cell signaling.

During pregnancy, vascular remodeling and vasoactive agents such as nitric oxide (NO) increase blood flow to the uteroplacental unit. Using our uterine artery endothelial cell (UAEC) culture model, based on cells from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes, we investigate the relative physiological roles of Ca(2+) vs. kinase in the regulation of endothelial NO synthase (eNOS) activity.

Pregnancy-specific modulatory role of mitochondria on adenosine 5'-triphosphate-induced cytosolic [Ca2+] signaling in uterine artery endothelial cells.

Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization.

Activation of NAD(P)H oxidase by outward movements of H+ ions in renal medullary thick ascending limb of Henle.

The present study was designed to test the hypothesis that the production of superoxide (O(2)-* by NAD(P)H oxidase is coupled to tubular metabolic activity through ionic activation mediated by H(+) movement across cell membrane. Using dual fluorescent microscopic imaging analysis, intracellular O(2)-* levels and pH (pH(i)) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O(2)-* levels in these cells were increased in parallel to the elevation of pH(i) by outflow of H(+) induced via NH(4)Cl loading followed by rapid removal.

Simultaneous imaging of [Ca2+]i and intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy.

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca(2+) sensitive and be subject to regulation by Ca(2+)-independent kinases.

Urotensin II is a nitric oxide-dependent vasodilator and natriuretic peptide in the rat kidney.

Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and UNaV) in a dose-dependent manner.

Cyclic ADP-ribose contributes to contraction and Ca2+ release by M1 muscarinic receptor activation in coronary arterial smooth muscle.

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca(2+) release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 micromol/L) and oxotremorine (OXO; 2.

Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs.

Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries.

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration ([Ca(2+)](i), fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 microM) stimulated a rapid increase in [Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in [Ca(2+)](i) and a slow increase in intracellular NO levels.

Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries.

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-alpha (TNF-alpha) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-alpha (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of N(G)-nitro-L-arginine methyl ester, TNF-alpha produced no further inhibition on the vasorelaxation induced by these vasodilators.

Effects of homocysteine on intracellular nitric oxide and superoxide levels in the renal arterial endothelium.

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO](i)) and stimulates superoxide (O.) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 microM), and bradykinin (2 microM) were found to increase endothelial [NO](i) in freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 microM).

Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney.

We recently reported that NADH oxidase is one of the major enzymes responsible for superoxide (O(2)(-)*) production in the rat kidney. However, the functional significance of NADH oxidase-mediated O. production and the mechanisms regulating this enzyme activity are poorly understood.