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

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.