Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ.
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.
Ohata
H
H
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Ujike
Y
Y
Momose
K
K
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000814:Aniline Compounds; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; D014966:Xanthenes; C059715:Fluo-3; C018444:adenosine 5'-O-(1-thiodiphosphate); D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D014544:Uridine Triphosphate