CONFOCAL IMAGING ANALYSIS OF ATP-INDUCED CA2-CELLS OF THE ARTERY IN-SITU( RESPONSE IN INDIVIDUAL ENDOTHELIAL)

The mechanisms for mobilization of intracellular free Ca2+ have been s tudied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ( [Ca2+](i)) in individual cells loaded with the acetoxymethyl ester of flue 3 in situ, using laser scanning confocal microscopy. The method p ermitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a tra nsient increase in endothelial [Ca2+](i). It was concluded that the re sponse was induced via P-2Y purinoceptors, because adenosine 5'-O-(2-t hiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1- 10 mu M) and the peak amplitude of the transient increase in [Ca2+](i) were dose dependently increased. Using rapid xy-scanning and line-sca nning modes, we confirmed that 10 mu M ATP induced Ca2+ waves, at a ra te of 10-30 mu m/s, after a lag time of similar to 3 s. These results show that [Ca2+](i) waves within endothelial cells are physiologically induced by ATP via P-2Y purinoceptor, but not P-2Y purinoceptor, in a ortic strips in situ. The method should be of use in the study of vasc ular physiology and pathophysiology.