Related Experiment Video
Updated: Sep 28, 2026
Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium
Published on: January 20, 2019
Altered calcium dynamics do not account for attenuation of endothelium-derived hyperpolarizing factor-mediated
Elke M Golding1, Dorota M Ferens, Sean P Marrelli
1Department of Anesthesiology, Baylor College of Medicine, Houston, Texas, USA. egolding@bcm.tmc.edu
Insights
Endothelium-derived hyperpolarizing factor (EDHF) mediated dilations are reduced in female rats due to estrogen. This study found the issue is not calcium levels but a coupling factor affecting smooth muscle cells.
Area of Science:
- Vascular biology
- Neuroscience
- Endocrinology
Background:
- Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in regulating vascular tone.
- ATP-mediated dilations, which involve EDHF, are attenuated in female rat middle cerebral arteries.
- Estrogen's role in this attenuation is suspected, potentially linked to endothelial calcium levels.
Purpose of the Study:
- To investigate the contribution of EDHF to ATP-mediated dilations in the rat middle cerebral artery.
- To test the hypothesis that reduced endothelial intracellular calcium ([Ca(2+)](i)) underlies attenuated EDHF responses in females.
- To determine if estrogen mediates this effect.
Main Methods:
- Concomitant measurement of vascular diameter and endothelial cell [Ca(2+)](i) in perfused rat middle cerebral artery segments.
- Utilized videomicroscopy and fura 2 fluorescence for measurements.
- Administered N(G)-nitro-L-arginine methyl ester and indomethacin to isolate EDHF-mediated responses.
Main Results:
- ATP-mediated dilation was significantly reduced in intact and estrogen-treated ovariectomized (OVX) females compared to males and vehicle-treated OVX females.
- Endothelial cell [Ca(2+)](i) levels were comparable across all groups, refuting the initial hypothesis.
- Smooth muscle cell [Ca(2+)](i) decreased more in males than females, and less in estrogen-treated OVX females compared to vehicle-treated OVX females.
Conclusions:
- The attenuated EDHF-mediated dilations in the female middle cerebral artery are not due to insufficient endothelial cell [Ca(2+)](i).
- A loss of a coupling factor between EDHF and smooth muscle cell [Ca(2+)](i) reduction is implicated.
- Estrogen may influence this coupling factor, contributing to reduced vasodilation in females.
Background And Purpose:
The contribution of endothelium-derived hyperpolarizing factor (EDHF) to ATP-mediated dilations is significantly attenuated in the rat middle cerebral artery of intact and estrogen-treated ovariectomized (OVX) females compared with males and vehicle-treated OVX females. Since an increase in endothelial calcium appears to be a critical prerequisite in the EDHF response, we tested the hypothesis that endothelial cell intracellular calcium ([Ca(2+)](i)) fails to reach sufficient levels to elicit robust EDHF-mediated dilations in females and that this effect is mediated by estrogen.
Methods:
Vascular diameter and [Ca(2+)](i) were measured concomitantly in perfused middle cerebral artery segments with the use of videomicroscopy and fura 2 fluorescence, respectively.
Results:
In the presence of N(G)-nitro-L-arginine methyl ester and indomethacin, the dilation to 10(-5) mol/L ATP was significantly reduced (P<0.05) in intact females (42+/-8%; n=6) and estrogen-treated OVX females (25+/-6%; n=9) compared with intact males (89+/-5%; n=6) and vehicle-treated OVX females (92+/-2%; n=7). Contrary to our initial hypothesis, endothelial cell [Ca(2+)](i) increased to comparable levels in intact females (461+/-116 nmol/L), estrogen-treated OVX females (417+/-50 nmol/L), intact males (421+/-77 nmol/L), and vehicle-treated OVX females (530+/-92 nmol/L). In response to luminal ATP (10(-5) mol/L), smooth muscle cell [Ca(2+)](i) decreased to a greater degree in males (37+/-4%; n=8) compared with females (21+/-5%; n=7) and in vehicle-treated OVX females (18+/-7%; n=7) compared with estrogen-treated OVX females (3+/-5%; n=9).
Conclusions:
Our data suggest that loss of a factor coupling EDHF to reduction of ionized smooth muscle cell [Ca(2+)](i) accounts for the attenuated EDHF-mediated dilations in the female middle cerebral artery.
