What is the significance of measuring membrane potential in endothelial cells?

Measuring membrane potential helps in understanding the functional status and signaling pathways of endothelial cells.

How does the calcium-sensitive dye work?

The dye binds to calcium ions, and its fluorescence intensity increases with higher calcium concentrations, allowing for quantification of intracellular calcium levels.

What are the implications of altered membrane potential?

Altered membrane potential can indicate changes in cellular activity, such as ion channel activation or signaling pathway engagement.

Can this method be applied to other cell types?

Yes, the methodology can be adapted for use with other cell types to study their membrane potential and calcium signaling.

What role do pharmacological agents play in this study?

Pharmacological agents are used to manipulate signaling pathways and assess their effects on calcium levels and membrane potential.

Is this technique suitable for live cell imaging?

Yes, the technique is designed for live cell imaging, allowing real-time observation of cellular dynamics.

Analysis of Calcium Dynamics and Membrane Potential Changes in a Mouse Arteriolar Endothelium

JoVE Encyclopedia of Experiments

Neuroscience

0 views • 2:38 min • April 28th, 2025

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Secure an arteriolar endothelial tube harvested from a mouse brain in a chamber with a continuous buffer flow.

Place the chamber in a recording setup.

Introduce a calcium-sensitive fluorescent dye. Incubate to allow cellular uptake of the dye.

Wash to remove any non-internalized dye.

Insert an electrode into an endothelial cell and record the resting membrane potential.

Raise the bath temperature to a physiological temperature to facilitate intracellular calcium binding to the dye.

Excite the dye and measure fluorescence to quantify baseline cellular calcium levels.

Introduce a drug that binds to specific G-protein-coupled receptors on the endothelial cells, initiating a signaling cascade.

This cascade releases calcium ions from the endoplasmic reticulum into the cytoplasm, enhancing calcium-dye binding and fluorescence.

Elevated cytoplasmic calcium levels also activate potassium channels, facilitating potassium ion efflux and decreasing membrane potential.

Record the data.

Increased fluorescence and decreased membrane potential indicate a functional endothelial tube.

To measure endothelial membrane potential, while viewing through the four times objective, carefully position the sharp electrode tip just over a cell of the arterial endothelial tube into the flowing physiological salt solution with a micromanipulator. Gradually, increase magnification to 400 times and reposition the electrode tip as needed. Using the micromanipulator, gently insert the tip of a sharp electrode into one of the cells of the endothelial tube, and start recording VM using an electrometer.

Once the endothelial resting VM is stable from minus 30 to minus 40 millivolts, apply the desired pharmacological agents per experimental objective. Load the endothelial tube with the fluorescence tracker for the plasma membrane or desired organelle at 37 degrees Celsius for 15 to 30 minutes. Wash the cells with fresh superfusion physiological salt solution, and image live cells under the microscope at the excitation wavelength of the respective dyes.