Faculty Collaboration Database

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta. J Vis Exp 2015 Jun 10(100):e52734

Date

07/02/2015

Pubmed ID

26132549

Pubmed Central ID

PMC4545029

DOI

10.3791/52734

Scopus ID

2-s2.0-84941241987 (requires institutional sign-in at Scopus site)   4 Citations

Abstract

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca(2+)]i) and nitric oxide (NO). In order to understand how [Ca(2+)]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Author List

Endres BT, Staruschenko A, Schulte M, Geurts AM, Palygin O

Author

Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Aorta
Calcium
Endothelial Cells
Microscopy, Fluorescence, Multiphoton
Muscle, Smooth
Nitric Oxide
Rats