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Acute Effects of 17-Estradiol and ATP on Endothelial Cells; Effects of Genistein and PMA on Calcium Response
Various studies have shown that 17-estradiol (E2), has acute effects on cardiovascular system in addition to its genomic effect. Acute administration of E2 had been shown to increase intracellular free calcium concentration (Ca+2)i in human umbilical vein endothelial cells (HUVEC). The present study investigates the signalling pathway responsible for Ca+2 response to E2. In the study, the effect of E2 on phosphoinositide turnover was investigated by use of Dowex-1 anion-exchange columns after labeling cells with myo(3H)inositol. Additionally, the effects of tyrosine phosphorylation inhibitor genistein and protein kinase C activator 4-phorbol-12-myristate-13-acetate (PMA) on the Ca+2 response to E2 and ATP were investigated and compared in fura-2 loaded HUVEC. The data demonstrates that E2 treatment causes 45% increase in inositol phosphate production in parallel to increases in (Ca+2)i. Genistein and PMA inhibit the Ca+2 response to E2 ~75%, 49%, while they inhibit the response to ATP ~62%, 73% respectively. Our data suggests the involvement of PLC in the signaling stimulated by E2 and indicate the involvement of tyrosine phosphorylation and PKC. Differences in the effect of the inhibitors on E2- and ATPinduced responses suggest that there may be differences in the upstream signaling initiated by E2 and ATP, such as different roles for tyrosine phosphorylation.

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Authors: A. Serpil Bilsel, Hadi Moini, Kaya Emerk

Keywords:

  • Year: 2002
  • Number: 2
  • Referance No: 37630

  • Figure 1. Effect of E2 and ATP application on (Ca+2)i of HUVEC. Tracings showing the changes in (Ca+2)i of HUVEC evoked by application of 10 M E2 and 100 M ATP. Cultures were deprived of E2 for 48 hours. Fluorescence was recorded from fura-2 loaded monolayers grown on glass coverslips, at room temperature in HBS containing 0.1% BSA, 1.8 mM CaCl2, pH 7.4. Tracings represent the mean of increases in (Ca+2)i obtained from 6 experiments.
  • Figure 2. Anion-exchange elution profiles of lysates from myo-[2-3H]inositol prelabelled HUVEC: Cells were incubated with only 10 L ethanol (basal) or 10 M estradiol or genistein + estradiol for 5 minutes. Cell lysates were applied to Dowex-1 columns. After washing out myo-(2-3H) inositol with water, (3H) inositolphosphates were eluted with stepwise addition of solutions containing increasing amounts of formate. Elution profile exibits 4 distinct peaks corresponding to glycerophosphoinositol (GroIP), inositol-1-phosphate (IP), inositolbisphosphate (IP2), inositoltrisphosphate (IP3) and demonstrates that treatment of endothelial cells with E2 increases phosphoinositide formation.
  • Figure 3. (3H) Inositol labelled endothelial cells were stimulated for 5 minutes with E2 or E2 vehicle ethanol (basal) or with E2 after incubation with genistein. Cells were lyzed and applied to Dowex-1 columns. After washing out (3H) inositol with water, total inositolphosphates were eluted by addition of 1 M ammonium formate/0.1 M formic acid. Radioactivity in eluates was measured by liquid scintillation counting. The results (mean SEM of 4 seperate experiments) show the percent increases in total inositolphosphates. The graph demonstrates that E2 causes 45% increase in phoshoinositide turnover which can be significantly inhibited by genistein pretreatment in HUVEC
  • Figure 4. The role of tyrosine phosphorylation in E2- stimulated signaling pathway in HUVEC. The tracing demonstrates that genistein attenuates the peak increase in (Ca+2)i evoked by E2 and ATP 75% and 62% respectively compared to controls and markedly decreases the second phase of ATP response. The tracing represent the mean of data obtained from 4 experiments. Cells were treated with 100 M genistein for 12 minutes before stimulation with E2 (10 M) and ATP (100 M). After genistein treatment E2 vehicle ethanol caused nearly 20% increase in (Ca+2)i. The extracellular Ca+2 concentration was 1.8 mM during the experiments.
  • Figure 5. The role of PKC on E2-evoked Ca+2 response in HUVEC. Tracing demonstrates that PMA attenuates the peak increase in (Ca+2)i stimulated by E2 and ATP compared to controls as shown in Figure 1. E2- deprived HUVEC were incubated with 100 nM PMA for 2.5 hours before stimulation with E2 (10 M) and ATP (100 M). Tracing represent the mean of data obtained in 9 experiments.

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