RESUMO
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. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O2 ) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca++ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-ß3 (PLCß3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCß3 under physiological chronic low oxygen.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Placenta/irrigação sanguínea , Permeabilidade Capilar/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Feminino , Humanos , GravidezRESUMO
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 VEGF165 influences vasodilator production via Ca2+ signaling mechanisms in human endothelial cells. We utilize human umbilical vein endothelial cells (HUVEC) as well as intact ex vivo human umbilical vein (HUV Endo) to address direct stimulation of Ca2+ and NO by VEGF165 alone, as well as the effect of VEGF165 on subsequent ATP-stimulated Ca2+ signaling and NO production. We show that VEGF165 stimulates Ca2+ responses in both HUVEC and HUV Endo, which results in a corresponding increase in NO production in HUV Endo. Longer-term VEGF165 pretreatment then inhibits sustained Ca2+ burst responses to ATP in HUVEC and HUV Endo. This is paralleled by a corresponding drop in ATP-stimulated NO production in HUV Endo, likely through inhibition of Cx43 gap-junction function. Thus, although VEGF165 makes a small initial positive impact on vasodilator production via direct stimulation of Ca2+ responses, this is outweighed by the greater subsequent negative impact on Ca2+ bursts and vasodilator production promoted by more potent agonists such as ATP. Overall, elevated levels of VEGF165 associated with preeclampsia could contribute to the endothelial dysfunction by preventing Ca2+ bursts to other agonists including but not limited to ATP. NEW & NOTEWORTHY: In this manuscript, we show that VEGF levels associated with preeclampsia are a net negative contributor to potential vasodilator production in both a human ex vivo and in vitro endothelial cell model. Therefore, pharmacological targeting of VEGF-stimulated signaling pathways could be a novel treatment modality for preeclampsia-related hypertension.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico/biossíntese , Pré-Eclâmpsia/metabolismo , Veias Umbilicais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Cálcio/metabolismo , Conexina 43/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Junções Comunicantes/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Técnicas In Vitro , Gravidez , Veias Umbilicais/metabolismoRESUMO
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. We have previously reported that endothelial cells of intact vessels show sustained Ca(2+) bursting in response to ATP, and these bursts drive prolonged NO production. Herein we show that in both GDM and DM pregnancies, the incidence of Ca(2+) bursts remains similar, but there is a reduction in overall sustained phase Ca(2+) mobilization and a reduction in NO output. Further studies show damage has occurred at the level of NOS3 protein itself. Since exposure to DM serum is known to impair normal human umbilical vein endothelial cell (HUVEC) function, we further studied the ability of HUVEC to signal through Ca(2+) after they were isolated from DM and GDM subjects and maintained in culture for several days. These HUVEC showed differences in the rate of Ca(2+) bursting, with DM > GDM = control HUVEC. Both GDM- and DM-derived HUVEC showed smaller Ca(2+) bursts that were less capable of NOS3 activation compared to control HUVEC. We conclude that HUVEC from DM and GDM subjects are reprogrammed such that the Ca(2+) bursting peak shape and duration are permanently impaired. This may explain why ROS therapy alone is not effective in DM and GDM subjects.
Assuntos
Sinalização do Cálcio , Diabetes Gestacional/metabolismo , Diabetes Gestacional/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Óxido Nítrico/biossíntese , Gravidez em Diabéticas/metabolismo , Gravidez em Diabéticas/fisiopatologia , Trifosfato de Adenosina/metabolismo , Adulto , Estudos de Coortes , Feminino , Humanos , Recém-Nascido , Ionomicina/farmacologia , Ionóforos/farmacologia , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Gravidez , Cultura Primária de Células , Estudos Prospectivos , Transdução de Sinais , Adulto JovemRESUMO
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. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 µM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 µM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.
Assuntos
Sinalização do Cálcio , Células Endoteliais da Veia Umbilical Humana/metabolismo , Óxido Nítrico/metabolismo , Pré-Eclâmpsia/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Ionóforos de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Feminino , Corantes Fluorescentes , Fura-2/análogos & derivados , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Ionomicina/farmacologia , Microscopia de Fluorescência , Imagem Molecular/métodos , Óxido Nítrico Sintase Tipo III/metabolismo , Pré-Eclâmpsia/fisiopatologia , Pré-Eclâmpsia/terapia , Gravidez , Fatores de Tempo , Adulto JovemRESUMO
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. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca(2+)](i)) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca(2+)](i) bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca(2+)](i) cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF(165). While treatment of luteal NP-UA Endo and P-UA Endo with VEGF(165) acutely stimulates a very modest [Ca(2+)](i) and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca(2+)](i) and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Canais de Cálcio/metabolismo , Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Artéria Uterina/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Ionomicina/farmacologia , Ionóforos/farmacologia , Óxido Nítrico Sintase Tipo III/biossíntese , Placenta/química , Placenta/efeitos dos fármacos , Placenta/metabolismo , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/metabolismo , Gravidez , Ovinos , Transdução de Sinais/efeitos dos fármacos , Artéria Uterina/citologia , Artéria Uterina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
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. In this study, we now show by simultaneous video imaging of individual Fura-2-loaded cells that the pregnancy-enhanced capacitative entry phase is not continuous and equal in all cells, but is in fact mediated as a series of periodic [Ca(2+)](i) bursts within individual cells. Not only does pregnancy increase the number of bursts over a longer time period in individual cells, but also a greater proportion of cells exhibit this burst activity, and at high cell density this occurs in a synchronous manner. The mediator of cell synchronization is connexin 43 (Cx43) gap junctions because 1) Cx43 is readily detectable by Western blot analysis in UAECs, whereas Cx40 and Cx37 are weakly detected or absent, and 2) pregnancy-specific enhancement of [Ca(2+)](i) bursts by ATP is blocked by inhibitory loop peptides selective to Cx43 ((43,37)GAP27) but not by a scrambled control peptide or (40)GAP27 or (40,37)GAP26 peptides, which are specific to Cx40 or Cx37. The relationship between Ca(2+) bursts and NOS3 activation is further established by the finding that (43,37)GAP27 inhibits ATP-stimulated NOS3 activation but has no effect on cell mitogenesis. We conclude that it is pregnancy-enhanced gap junction communication between cells that underlies pregnancy enhancement of capacitative entry via TRPC3 and, in turn, NOS3 activation. Such improved gap junction function allows greater and more sustained [Ca(2+)](i) responses to agents such as ATP within a single cell, as well as the additional recruitment of greater numbers of cells to the response in a coordinated and synchronous manner to support enhanced NO production.
Assuntos
Sinalização do Cálcio , Conexina 43/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Prenhez/metabolismo , Artéria Uterina/citologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Contagem de Células , Conexinas , Endotélio Vascular/metabolismo , Feminino , Junções Comunicantes/metabolismo , Oligopeptídeos , Gravidez , Ovinos , Canais de Cátion TRPC/metabolismo , Artéria Uterina/metabolismoRESUMO
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. When Ca(2+) mobilization is fully inhibited using inhibitors of phospholipase C (PLC) (U73122) and the inositol triphosphate (IP3) receptor (IP3-R) (2-APB), significant residual eNOS activity remains in both P- and NP-UAEC. No change in ATP-stimulated ERK2, Akt, or eNOS phosphorylation is observed with U73122 (0.01-1 microM) or 2-APB (1-50 microM). The MAPK kinase (MEK) 1/2 inhibitor U0126 (10 microM) did not alter ATP-stimulated eNOS activity in P-UAEC, but potentiated the ATP response in NP-UAEC. Using two phosphatidylinositol 3-kinase (PI3-K) inhibitors, we observed no effect with LY294002 (10 microM) on eNOS activity in P- and NP-UAEC, but wortmannin (10 microM) inhibited both P- and NP-UAEC eNOS activation. Expression of constitutively active Akt (ca-Akt) in UAEC resulted in slight elevation of basal eNOS activity, but relative ATP-stimulated eNOS activation was not altered by ca-Akt. Wortmannin continued to inhibit eNOS activation by ATP in the presence of ca-Akt; LY294002 still had no inhibitory effect. Our data indicate both [Ca(2+)](i) and multiple kinases are involved in the regulation of eNOS activity in our model. We report that pregnancy adaptation of eNOS activation includes the reduced sensitivity to ERK-mediated attenuation of eNOS activity and enhanced stimulation of eNOS activity through a wortmannin-sensitive, LY294002-insensitive, Akt-independent mechanism.
Assuntos
Androstadienos/farmacologia , Butadienos/farmacologia , Cálcio/metabolismo , Cromonas/farmacologia , Morfolinas/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Nitrilas/farmacologia , Útero/citologia , Útero/enzimologia , Trifosfato de Adenosina/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Western Blotting , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Corantes Fluorescentes/farmacologia , Fura-2/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Modelos Estatísticos , Óxido Nítrico Sintase/metabolismo , Fosforilação , Gravidez , Prenhez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ovinos , Transdução de Sinais , Fatores de Tempo , WortmaninaRESUMO
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. In order to establish whether this was due to an altered subclass of purinergic receptor (P2), both the dose dependency of [Ca(2+)](i) responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca(2+) signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca(2+) signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca(2+)is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca(2+) entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca(2+)](i) response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca(2+)signaling.
Assuntos
Trifosfato de Adenosina/farmacologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Animais , Artérias , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas de Ligação ao GTP/metabolismo , Microscopia de Fluorescência , Modelos Animais , Gravidez , Receptores Purinérgicos/metabolismo , Ovinos , Útero/irrigação sanguíneaRESUMO
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). Herein we demonstrated that the difference in the sustained [Ca(2+)](i) response is maintained for at least 30 min. When 2-aminoethoxydiphenyl borate (2APB) (an inhibitor of the inosital 1,4,5-trisphosphate receptor (IP3R) and possibly SOC) was used in conjunction with ATP, it was capable of completely inhibiting CCE. Since 2APB can inhibit SOC in some cell types and 2APB was capable of inhibiting CCE in the UAEC model, the role of SOC in CCE was first evaluated using the classical inhibitor La(3+). The ATP-induced sustained phase was inhibited by 10 microM La(3+), implying a role for SOC in the [Ca(2+)](i) response. Since canonical transient receptor potential channels (TRPCs) have recently been identified as putative SOCs in many cell types, including EC, the expression levels of several isoforms were evaluated in UAEC. Expression of TRPC3 and TRPC6 channels in particular was detected, but no significant difference in expression level was found between NP- and P-UAEC. Nonetheless, we were able to show that IP3R2 interacts with TRPC3 in UAEC, forming a protein complex, and that this interaction is considerably enhanced in an agonist sensitive manner by pregnancy. Thus, while IP3R and TRPC isoforms are not altered in their expression by pregnancy, enhanced functional interaction of TRPC3 with IP3R2 may underlie pregnancy-enhanced CCE in the UAEC model and so explain the prolonged [Ca(2+)](i) sustained phase seen in response to ATP.
Assuntos
Canais de Cálcio/metabolismo , Células Endoteliais/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Canais de Cátion TRPC/metabolismo , Útero/irrigação sanguínea , Trifosfato de Adenosina/farmacologia , Animais , Artérias , Compostos de Boro/farmacologia , Cálcio/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Imunoprecipitação/métodos , Receptores de Inositol 1,4,5-Trifosfato , Microscopia de Fluorescência , Modelos Animais , Gravidez , Ovinos , Estimulação QuímicaRESUMO
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. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10-20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12-15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Células Endoteliais/metabolismo , Mitocôndrias/fisiologia , Prenhez/fisiologia , Útero/irrigação sanguínea , Trifosfato de Adenosina/deficiência , Trifosfato de Adenosina/farmacologia , Animais , Artérias , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Eletrofisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Mitocôndrias/efeitos dos fármacos , Concentração Osmolar , Gravidez , Ovinos , Tapsigargina/farmacologiaRESUMO
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. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P(635)eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P(635)eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37-specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca(2+)-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca(2+)-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.
Assuntos
Cálcio/metabolismo , Conexinas/metabolismo , Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Prenhez , Artéria Uterina/fisiologia , Vasodilatação/fisiologia , Animais , Feminino , Gravidez , Ovinos , Transdução de SinaisRESUMO
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. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca(2+) concentration ([Ca(2+)](i)) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca(2+) pump inhibitor) were used to maximally elevate [Ca(2+)](i) and fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 microM) and TG (10 microM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 microM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca(2+)](i) response but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca(2+)](i)-insensitive pathways as well as through a greater eNOS sensitivity to [Ca(2+)](i).
Assuntos
Cálcio/fisiologia , Ciclo Estral/fisiologia , Óxido Nítrico/biossíntese , Prenhez/fisiologia , Útero/irrigação sanguínea , Trifosfato de Adenosina/farmacologia , Animais , Artérias/fisiologia , Compostos de Boro/farmacologia , Canais de Cálcio , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Endotélio Vascular/metabolismo , Feminino , Receptores de Inositol 1,4,5-Trifosfato , Ionomicina/farmacologia , Ionóforos/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Gravidez , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Ovinos , Tapsigargina/farmacologia , Útero/efeitos dos fármacos , Útero/fisiologiaRESUMO
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. This increase in intracellular O(2)-* levels was substantially blocked by an inhibitor of Na(+)/H(+) exchanger, methylisobutyl-amiloride (MIA; 100 microM), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 microM). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 microM) was used to produce H(+) conductance, leading to H(+) flux across cell membrane depending on extracellular pH. The efflux of H(+) increased both pH(i) and intracellular O(2)-* levels, but the influx of H(+) did not increase intracellular O(2)-* levels. The H(+) efflux-induced increase in intracellular O(2)-* levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 microM). In in invo experiments, renal medullary infusion of MIA (100 microM) was found to significantly decrease the concentrations of H(2)O(2) in the renal medullary interstitium. These results suggest that it is the outward movements of H(+) ions that activates NAD(P)H oxidase to produce O(2)-* in TALH cells. This H(+) outflow-associated activation of NAD(P)H oxidase importantly contributes to tissue levels of reactive oxygen species in the renal medulla.
Assuntos
Alça do Néfron/fisiologia , Espécies Reativas de Oxigênio/química , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Membrana Celular/fisiologia , Concentração de Íons de Hidrogênio , Transporte de Íons/fisiologia , Masculino , Oxirredução , Prótons , Ratos , Ratos Sprague-DawleyRESUMO
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. Using fluorescence microscopic imaging analysis, the present study demonstrated that thick ascending limbs of Henle's loop (TALHs) exhibited red fluorescence when incubated with dihydroethidium (DHE), suggesting that O(2)(-)* is produced in this tubular segment. Compared with other nephron segments, TALHs from both renal cortex and medulla showed the highest fluorescence intensity. By incubating cortical TALHs (cTALHs) with the substrates of NADH oxidase, xanthine oxidase, nitric oxide synthase, arachidonic acid-metabolizing enzymes, and intramitochondrial oxidases, NADH oxidase was found to be one of the most important enzymes for O(2)(-)* production in this tubular segment. The NADH oxidase inhibitor diphenyleneiodonium (DPI; 100 microM) completely blocked NADH-induced O(2)(-)* production in cTALHs. Exposure of cTALHs to low PO(2) (5-10 Torr) significantly increased O(2)(-)* production regardless of the absence or presence of NADH. Furthermore, angiotensin II (100 nM) increased NADH oxidase activity by 32%, which was completely blocked by DPI. These results suggest that NADH oxidase is a major enzyme responsible for O(2)(-)* production in the TALHs and that the production of O(2)(-)* via NADH oxidase may be regulated by renal tissue oxygenation and circulating hormones.
Assuntos
Rim/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Superóxidos/metabolismo , Angiotensina II/farmacologia , Animais , Hipóxia Celular/fisiologia , Quelantes/farmacologia , Ditiocarb/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Imuno-Histoquímica , Técnicas In Vitro , Rim/citologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Alça do Néfron/citologia , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/metabolismo , Masculino , Glicoproteínas de Membrana/biossíntese , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/genética , NADPH Oxidase 2 , Oniocompostos/farmacologia , Subunidades Proteicas , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/antagonistas & inibidoresRESUMO
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. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-alpha. Pretreatment of the arteries with desipramine (10 microM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-alpha on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-alpha activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-alpha inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.
Assuntos
Antineoplásicos/farmacologia , Ceramidas/metabolismo , Vasos Coronários/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Bovinos , Vasos Coronários/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Óxido Nítrico/metabolismo , Polietilenoglicóis/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Superóxido Dismutase/farmacologia , Vasodilatação/fisiologiaRESUMO
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. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.
Assuntos
Cálcio/metabolismo , Vasos Coronários , Endotélio Vascular/metabolismo , Líquido Intracelular/metabolismo , Óxido Nítrico/metabolismo , Animais , Arsenicais/farmacologia , Bradicinina/farmacologia , Cálcio/análise , Bovinos , Quelantes/farmacologia , Vasos Coronários/metabolismo , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Técnicas In Vitro , Líquido Intracelular/química , Ionóforos/farmacologia , Microscopia de Fluorescência/métodos , Óxido Nítrico/análise , Proteínas Tirosina Fosfatases/antagonistas & inibidoresRESUMO
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. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.
Assuntos
Medula Renal/metabolismo , Fatores de Transcrição/biossíntese , Animais , Northern Blotting , Western Blotting , Hipóxia Celular/fisiologia , Núcleo Celular/metabolismo , Separação Celular , Cobalto/farmacologia , DNA Complementar/biossíntese , DNA Complementar/genética , Sequestradores de Radicais Livres/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Medula Renal/citologia , Medula Renal/efeitos dos fármacos , Leupeptinas/farmacologia , Masculino , NADPH Oxidases/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional , Vitamina K 3/farmacologia , Xantina Oxidase/farmacologiaRESUMO
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). Preincubation of the arteries with L-Hcys (20-40 microM) significantly attenuated the increase in endothelial [NO](i). However, L-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [(3)H]arginine to [(3)H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting that L-Hcys reduces endothelial [NO](i) by its scavenging action. Because other thiol compounds such as L-cysteine and glutathione were also found to reduce [NO](i), it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O. levels using dihydroethidium trapping, we found that only L-Hcys among the thiol compounds studied markedly increased O. levels in the renal endothelium. These results indicate that L-Hcys inhibits the agonist-induced NO increase but stimulates O. production within endothelial cells. These effects of L-Hcys on [NO](i) and [O.] may contribute to endothelial injury associated with hHcys.
Assuntos
Endotélio Vascular/metabolismo , Homocisteína/farmacologia , Óxido Nítrico/metabolismo , Artéria Renal/metabolismo , Superóxidos/metabolismo , Animais , Bradicinina/farmacologia , Calcimicina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ionóforos/farmacologia , Masculino , Microscopia de Fluorescência , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Circulação Renal/efeitos dos fármacosRESUMO
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; 2.5-80 micromol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca(2+) ([Ca(2+)](i)) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca(2+)](i) in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca(2+)-free Hanks' solution. This OXO-induced rise in [Ca(2+)](i) was significantly reduced by pirenzepine (PIR; an M(1) receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from beta-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca(2+) release induced by activation of M(1) muscarinic receptors.
Assuntos
Cálcio/metabolismo , Vasos Coronários/fisiologia , ADP-Ribose Cíclica/fisiologia , Contração Muscular , Músculo Liso Vascular/fisiologia , Receptores Muscarínicos/fisiologia , ADP-Ribosil Ciclase/antagonistas & inibidores , Acetilcolina/farmacologia , Animais , Canais de Cálcio , Bovinos , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Fura-2 , Receptores de Inositol 1,4,5-Trifosfato , Agonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Niacinamida/farmacologia , Oxotremorina/farmacologia , Receptor Muscarínico M1 , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Espectrometria de Fluorescência , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasodilatadores/farmacologiaRESUMO
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. At a dose of 20 ng. kg-1. min-1, it increased RBF by 20% and UV and UNaV by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of approximately 200 microm, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 microM. l-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 microM) was shown to double the NO levels within the endothelium of freshly dissected small RA, and l-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA.