A new technique for simultaneous and in situ measurements of Ca²⁺ signals in arteriolar smooth muscle and endothelial cells

We report here the first local and global Ca²⁺ measurements made from in situ terminal arterioles. The advantages of the method are that there is minimal disturbance to the vessels, which retain their relationship to the tissue they are supplying (rat ureter) and the small size of vessel that can be studied. Good loading with the Ca²⁺ indicator, Fluo-4 was obtained, and confocal sectioning through the tissue enabled vascular smooth muscle and endothelial cells to be clearly seen, along with red blood cells, nerve endings and the ureteric smooth muscle cells. We find the terminal arterioles to be extremely active, both spontaneously and in response to nor-adrenaline stimulation, with Ca²⁺ sparks occurring in the vascular myocytes and Ca²⁺ puffs in the endothelial cells. Even under resting conditions, endothelial cells produced oscillations and waves, which could pass from cell to cell, whereas the vascular myocytes only produced waves in response to agonist stimulation, and with no increase in the frequency of Ca²⁺ sparks, and no spread from cell to cell. We compare our data to those obtained in dissected intact vessels and single cells. We conclude that this approach is a convenient and useful method for studying inter- and intracellular Ca²⁺ signalling events and communication between cell types, particularly in very small vessels.