Pregnancy-Specific Enhancement of Agonist-Stimulated ERK-1/2 Signaling in Uterine Artery Endothelial Cells Increases Ca2+ Sensitivity of Endothelial Nitric Oxide Synthase as well as Cytosolic Phospholipase A2*

Di, Tao; Sullivan, Jeremy A.; Magness, Ronald R.; Zhang, Li; Bird, Ian M.

doi:10.1210/endo.142.7.8278

Cited by 30 publications

(39 citation statements)

References 25 publications

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“…Figure 1 shows representative images of cells at a density of 20%-100% and recordings of 100 lM ATP-induced [Ca 2þ ] i responses in NP-UAECs and P-UAECs at these same cell densities. Of note, the initial response in the first few minutes (less than 5 min) agreed with that described previously [7]-namely, that the initial rapid [Ca 2þ ] i release peak was of similar size in NP-UAECs and P-UAECs-but repeated [Ca 2þ ] i bursts occurred for a longer time in P-UAECs than in NP-UAECs. Some additional superimposed rapid oscillations (see inset) were also seen in P-UAECs but not NP-UAECs.…”

Section: Atp Induces Cell Density-dependent and Pregnancyenhanced Sussupporting

confidence: 89%

“…Several investigations have confirmed that UA endothelium is more capable of agonist-dependent nitric oxide (NO) and prostacyclin production during pregnancy [2,5,6]. Our own studies have further revealed that this adaptive response of UA endothelium includes profound reprogramming at the level of postreceptor cell signaling, specifically at the level of both kinase activation and Ca 2þ signaling, particularly during the sustained phase of Ca 2þ signaling [3,4,7].…”

Section: Introductionmentioning

confidence: 72%

“…We have previously shown in single UAECs from pregnant ewes (by photometry one cell at a time) that ATP can stimulate a more prolonged [Ca 2þ ] i response than in cells from NP ewes, and that this can also include a period of superimposed rapid oscillations within the first 5 min [7]. Imaging of multiple cells either freshly isolated [4] or on the luminal surface of intact vessels [14] confirmed that the more sustained nature of the [Ca 2þ ] i response occurs in vivo and specifically defined its direct association with more sustained NO production during pregnancy.…”

Section: Atp Induces Cell Density-dependent and Pregnancyenhanced Susmentioning

confidence: 99%

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Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Feng

Boeldt

Gifford

et al. 2010

Biology of Reproduction

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100

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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.

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Section: Atp Induces Cell Density-dependent and Pregnancyenhanced Sussupporting

confidence: 89%

Section: Introductionmentioning

confidence: 72%

Section: Atp Induces Cell Density-dependent and Pregnancyenhanced Susmentioning

confidence: 99%

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Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Feng

Boeldt

Gifford

et al. 2010

Biology of Reproduction

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100

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show abstract

“…Pretreatment of UAECs with DUP 753 (10 µM), PD 123319 (10 µM), or the cotreatment of both inhibitors for 20 min showed an observable decrease in ERK2 phosphorylation to a level not significantly different from control and not significantly different from AII treatment alone. In view of this ambiguous result, we wished to confirm that AII was indeed activating ERK2 via Mitogen Activated Protein Kinase-Kinase (MEK) as previously suggested Di et al 2001). Implication of a MEK-mediated ERK2 phosphorylation event following AII stimulation was achieved using the MEK1/2 inhibitor, PD 98059 (20 µM, 20-min pretreatment) that exhibited a significant decrease in ERK2 phosphorylation (Figure 9).…”

Section: Resultsmentioning

confidence: 77%

“…Our functional data concerning the second messenger signaling responses to AII raise further questions concerning the possible role of another receptor to AII. We have previously established that AII stimulates a small but significant PLC-mediated response in NP-UAECs and P-UAECs but that there is still a pregnancy-specific enhancement of AII-mediated activation of the ERK1/2 signaling pathway (Bird et al , 2003Di et al 2001). In order to gain more insight into the possible roles of AT 1 R, AT 2 R, and perhaps previously unidentified AII receptors, we chose to look more closely at the aforementioned functional responses, particularly the dose responses.…”

Section: Discussionmentioning

confidence: 99%

Pregnancy and Ovarian Steroid Regulation of Angiotensin II Type 1 and Type 2 Receptor Expression in Ovine Uterine Artery Endothelium and Vascular Smooth Muscle

et al. 2005

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Although pregnancy is clearly associated with refractoriness to infused angiotensin II (AII) in the uteroplacental unit, there is still dispute over the mechanism by which angiotensin type 1 and type 2 receptors (AT1R and AT2R) may mediate this response in the uterine artery. This is in large part due to incomplete knowledge of levels of AT1R and AT2R expression and function in uterine artery endothelium (UA Endo) in the nonpregnant (NP) and pregnant (P) states, combined with the disagreement on whether AII may act through release of adrenomedullary catecholamines. The authors have previously described an increase in AT1R in UA Endo but not UA vascular smooth muscle (VSM) during pregnancy as compared to the nonpregnant intact ewe. Herein they report that the pregnancy-associated increase in AT(1)R expression in UA Endo is regulated by ovarian steroids. Using a recently developed antibody to AT2R, the authors now show there is no change in AT2R in UA Endo or VSM associated with ovarian function, and although AT2R is not changed in UA Endo by pregnancy, there is a significant decrease observed in UA VSM at that time. The authors also examined changes in receptors in UA Endo and VSM in estrogen (E2beta)-primed ewes in view of the common use of this model as a control for physiologic studies. In contrast to their findings in nonprimed nonpregnant or pregnant animals, the authors observed a significant increase in both AT1R and AT2R in UA Endo in response to the supraphysiologic priming with E2beta. In order to address the possible functionality of AT1R or AT2R in UA Endo, the authors used the uterine artery endothelial cell (UAEC) model of UA endothelial cells maintained in culture to passage 4. Differences in expression of AT1R or AT2R were normalized at passage 4 in P-UAECs and NP-UAECs. Treatment with AII activated phospholipase C (PLC) in both NP- and P-UAECs but signaling through the extracellular signal-regulated kinase (ERK) pathway was dramatically enhanced in P-UAECs compared to NP-UAECs. Surprisingly, both phosphoinositol turnover and ERK2 phosphorylation responses failed to display the expected dose-responses. Inhibition of AII-stimulated ERK2 phosphorylation with antagonists DUP 753 (AT1R, 10 microM) and PD 123319 (AT2R, 10 microM) failed to selectively inhibit ERK2 phosphorylation. The authors conclude that (a) the net effect of pregnancy may be an increase in the AT1R/AT2R ratio in both UA Endo and VSM but through apparently distinct mechanisms, (b) the ovariectomized animal model is similar to the luteal state for AT1R and AT2R expression, while the E2beta-primed model does not resemble the nonpregnant or pregnant state, and (c) there is a real possibility that AII may mediate its effects either through a complex AT1R-AT2R interaction or via an as-yet unidentified non-AT1, non-AT2 receptor.

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Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells

Cale

Tsoi

Toppe

et al. 2005

Journal of the Society for Gynecologic Investigation

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While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca(2+)/calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca(2+)](i)). The sheep, however, is widely used as a model for cardiovascular adaptation to pregnancy and ovine uterine artery endothelial cell (UAEC) eNOS undergoes pregnancy-specific (P) enhancement of activity associated with increased Ca(2+) and protein kinase signaling in response to a number of agonists, including adenosine triphosphate (ATP). The degree of homology between the ovine and human full-length cDNAs was not previously known and yet is necessary to determine the validity in using an ovine model to study human physiology. The objectives of this study were to isolate and validate the clone of ovine eNOS cDNA and investigate ovine eNOS activation when expressed in COS-7 cells. The ovine eNOS cDNA has high homology to published human and bovine sequences and shares identity with the bovine amino acid sequence. When ovine eNOS was transiently expressed in COS-7 cells (COS-7/oeNOS), A23187 increased specific catalytic activity in a dose- and time-dependent manner. A23187-stimulated activation of eNOS was, however, also accompanied by phosphorylation of eNOS S1179 and dephosphorylation of T497, demonstrating that an increase in [Ca(2+)](i) may not be the sole mechanism of activation. The physiologic relevance of this was further underscored by the finding that ATP dose-dependently increased peak [Ca(2+)](i) and eNOS activity in COS-7/oeNOS, but also increased eNOS p-S1179 and decreased p-T497. This finding was similar to those in ovine P-UAEC treated with the Ca(2+)-mobilizing agonist ATP, wherein activation of eNOS was again concomitant with a rise p-S1179 as well as a slight decrease in p-T497. In conclusion, we describe the full-length ovine eNOS cDNA sequence and show that both physiologic and nonphysiologic calcium-mobilizing agents, which activate ovine eNOS in COS-7 and P-UAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca(2+) elevation and specific phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction.

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Pregnancy-Specific Enhancement of Agonist-Stimulated ERK-1/2 Signaling in Uterine Artery Endothelial Cells Increases Ca2+ Sensitivity of Endothelial Nitric Oxide Synthase as well as Cytosolic Phospholipase A2*

Cited by 30 publications

References 25 publications

Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Pregnancy and Ovarian Steroid Regulation of Angiotensin II Type 1 and Type 2 Receptor Expression in Ovine Uterine Artery Endothelium and Vascular Smooth Muscle

Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells

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