Altered VEGF-stimulated Ca²⁺ signaling in part underlies pregnancy-adapted eNOS activity in UAEC

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   Figure 1

   Descriptive characterization of VEGF165-stimulated Ca²⁺ responses in UAEC. UAEC from non-pregnant animals (NP-UAEC) (n=8 dishes) and UAEC from pregnant animals (P-UAEC) (n=8 dishes) were treated with 10 ng/ml VEGF165 for 30 min. (A and B) Typical variations in Ca²⁺ response shapes. Percentages and line thickness correspond to the proportion of responding cells that can be roughly placed into each category. (C) Response delay time between agonist addition and initiation of a detectable Ca²⁺ response, reaching double the basal levels. (D) Mean Ca²⁺ responses, aligned at the point of response origin and plotted throughout the course of the experiment (n=64 responding cells for UAEC from non-pregnant animals n=58 cells for UAEC from pregnant animals).
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   Figure 2

   VEGF165 dose–response stimulation of Ca²⁺. UAEC from non-pregnant and pregnant animals were treated with 0.1 ng/ml (n=5 dishes for each condition), 1 ng/ml (n=5 dishes for each condition), or 10 ng/ml VEGF165 (n=8 dishes for each condition) and imaged for 30 min. Results are the mean percentages of cells giving a detectable Ca²⁺ response per dish at each dose±s.e.m. *P<0.05 versus 0.1 ng/ml of same cell type, ^#P<0.05 versus 1 ng/ml of same cell type, and ⁺P<0.05 versus equivalent dose in UAEC from non-pregnant animals.
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   Figure 3

   VEGF165-stimulated Ca²⁺ signals through the VEGFR2 kinase domain and is dependent on Src but not ERK activity. UAEC from non-pregnant animals (n=5 dishes) and UAEC from pregnant animals (n=5 dishes) were treated for 30 min with 10 ng/ml VEGF165, which was preceded by a 25-min treatment with 1 μM VEGFR2 kinase inhibitor (VEGFRi), or a 20-min pretreatment with 10 μM PP2 or 10 μM U0126. Cells were grown to 70% density, loaded with Fura-2, and 50–80 cells/dish were imaged for 30 min. Results are the mean percentages of cells responding±s.e.m. *P<0.05 versus control.
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   Figure 4

   Sustained Ca²⁺ responses to VEGF165 depend on extracellular Ca²⁺ influx. Ca²⁺ was removed from the experimental buffer and UAEC were treated with 10 ng/ml VEGF165 (A, P<0.001 for UAEC from non-pregnant animals (n=5 dishes) versus UAEC from pregnant animals (n=5 dishes) in the first 300 s, analyzed by paired t-test and one-way ANOVA). In B, quantification of AUC for UAEC from both non-pregnant and pregnant animals in the presence of Ca²⁺-free buffer versus normal buffer (n=5 dishes per cell type, per treatment) is shown. Data are represented as dish means±s.e.m. (*P<0.05 versus normal buffer of same cell type and ^#P<0.01 versus UAEC from non-pregnant animals of Ca²⁺ free buffer). Significance was determined using Student's t-test for B.
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   Figure 5

   Both initial and sustained Ca²⁺ responses to VEGF165 are IP3-dependent. The dose responses to inhibitors of the IP3 signaling pathway for VEGF165-stimulated Ca²⁺ responses are shown. In A and B, dose responses for pretreatment for 20 min with the PLC inhibitor, U73122, for both UAEC from non-pregnant animals (n=5 dishes) and UAEC from pregnant animals (n=5 dishes), with 10 ng/ml VEGF165 stimulation, are shown (A, *P<0.05 versus control). Too few cells responded to VEGF165 at the 1 μM dose of U73122 to permit statistical analysis for area under the curve (AUC, (B) indicated by NA). In C and D, dose responses for pretreatment for 5 min with the IP3R antagonist, 2-APB, with 10 ng/ml VEGF165 in UAEC from non-pregnant animals (n=5 dishes) and UAEC from pregnant animals (n=5 dishes) are shown (C, *P<0.05 versus control, ^#P<0.05 versus same dose of 2-APB in UAEC from pregnant animals; D, *P<0.05 versus control). Data presented as mean±s.e.m. for all panels.
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   Figure 6

   Reducing the VEGF165-stimulated Ca²⁺ response signal reveals Ca²⁺ bursts. Using a submaximal dose of VEGF165 (1 ng/ml, cells 3 and 4) or 10 μM 2-APB pretreatment with 10 ng/ml VEGF165 (cells 1 and 2) revealed that Ca²⁺ burst responses may be fused in continuous Ca²⁺ responses to 10 ng/ml VEGF165 alone in UAEC from pregnant animals. Burst activity is also observed in UAEC from non-pregnant animals under similar conditions.
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   Figure 7

   Inhibitors of VEGF165-stimulated Ca²⁺ responses inhibit eNOS activity. eNOS activity was measured by arginine-to-citrulline conversion assay. UAEC from non-pregnant animals (n=4/dose) and UAEC from pregnant animals (n=4/dose) were pretreated with inhibitor for 5 min, followed by a 10-min treatment with 10 ng/ml VEGF165. (A) Cells were treated with 0.1, 1, or 10 μM U73122. (B) Cells were treated with 1, 10, or 50 μM 2-APB. Data for both panels presented as mean±s.e.m. (*P<0.05 versus control).

• © 2014 Society for Endocrinology