Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ

Abstract

The mechanisms for mobilization of intracellular free Ca2+ have been studied 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 fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

Purinergic Signaling and Blood Vessels in Health and Disease

“…ADP and ATP-mediated endothelium-dependent relaxations in guinea pig aorta were inhibited by the P2Y 1 receptor-selective antagonist MRS2179; UTP relaxations were insensitive to MRS2179 and UDP was inactive, suggesting an involvement of P2Y 4 receptors (Kaiser and Buxton, 2002). Ca 2+ waves within endothelial cells are induced by ATP, but not UTP, via P2Y receptors in guinea pig aortic strips (Ohata et al, 1997), consistent with a likely expression of P2Y 1 and P2Y 4 but not P2Y 2 receptors in this vessel. In mouse thoracic aorta, P2Y 1 , P2Y 2 , and P2Y 6 receptors are present on endothelial cells and mediate vasorelaxation (Bény, 2004;Guns et al, 2005).…”

“…It has been shown that Ca 2+ waves within endothelial cells are induced by ATP via P2Y (P2Y 1 ), but not P2U (P2Y 2 and/or P2Y 4 ), receptors in guinea pig aortic strips (Ohata et al, 1997). ATP stimulates L-arginine uptake and NO release from porcine aortic endothelial cells (Bogle et al, 1991).…”

Calcium Spots

“…Use of a confocal imaging system would allow us to discriminate between the endothelial cells and the smooth muscle cells, and, furthermore, the real-time imaging of the [Ca 2ϩ ] i dynamics in these cell layers along the z-axis would be possible if scanning were conducted using a piezoelectric actuator added to the real-time scanning system. In practice, Ohata et al 21 previously demonstrated the confocal images of [Ca 2ϩ ] i in endothelial cells in situ with clear discrimination from the smooth muscle layer.…”

Optical Bioimaging: From Living Tissue to a Single Molecule: Calcium Imaging in Blood Vessel In Situ Employing Two-Photon Excitation Fluorescence Microscopy

“…In practice, we have established an imaging analysis protocol with respect to [Ca 2+ ] i in individual endothelial cells in situ with LSCM (12,13). However, LSCM led to increased photodamage with total time necessary to acquire images, especially during continuous imaging.…”