Two‐Photon Imaging of Intracellular Ca 2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of Isolated Rat Vessels

Abstract

In order to understand how intracellular calcium ([Ca2+]i) and nitric oxide (NO) are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies two photon microscopy to measure calcium handling (or NO production) in individual cells of freshly isolated blood vessels. To validate the procedure, endothelial cell [Ca2+]i and NO activity were measured in response to application of acetylcholine (ACh) in a freshly isolated aorta of a Dahl salt-sensitive rat loaded with the intracellular [Ca2+]i ionophore, Fluo-4 AM, or with DAF-FM to indicate NO production. Application of ACh produced a detectable transient increase of the selected ionophore within individual endothelial cells of the aorta. To determine whether the protocol was sufficient to measure [Ca2+]i within small resistance arteries, renal microvessels were isolated, incubated in Fluo-4 AM dye, and imaged. Following addition of Endothelin-1, the smooth muscle cells (SMC) responded by increasing [Ca2+]i and emitting a strong fluorescent signal, which allowed us to calculate calcium transients within individual SMCs of the microvessels. This approach was further used to assess Ca2+ handling and NO production in vessels isolated from Plekha7 and p66 Shc knockout rats. Using this experimental procedure along with gene-editing resources, we have now begun testing the contribution of endothelial and SMC to vascular dysfunction and other pathological states within isolated vessels.