Autocrine and paracrine effects of endothelium-derived relaxing factor on intracellular Ca2+ of endothelial cells and vascular smooth muscle cells. Identification by two-dimensional image analysis in coculture.
To elucidate the effects of endothelium-derived relaxing factor (EDRF) released from vascular endothelial cells (ECs) on handling of intracellular calcium ion (Ca2+i) in ECs themselves and vascular smooth muscle cells (VSMCs), we measured the Ca2+i by two-dimensional digital image analysis of fura-2-loaded ECs and VSMCs in tissue culture. In isoculture of one cell type, adenosine triphosphate (ATP, 1 microM) transiently increased the Ca2+i of both ECs and VSMCs. High-K+ depolarization or angiotensin II also elevated the Ca2+i of VSMCs, whereas neither stimulants changed the Ca2+i of ECs. In coculture of ECs with VSMCs, the same dose of ATP rapidly increased the Ca2+i of ECs and then transiently decreased the Ca2+i of VSMCs to below the resting level. The maximal Ca2+i-modulating effects of ATP on both cell types were reproducible after the second application of ATP. Three kinds of EDRF blockers (L-NG-monomethylarginine, methemoglobin, or methylene blue) potentiated the ATP-induced Ca2+i rise in ECs and attenuated the Ca2+i reduction in VSMCs, suggesting the autocrine and paracrine effects of EDRF on ECs and VSMCs, respectively. However, neither indomethacin, superoxide dismutase, nor neutralizing monoclonal antibody to endothelin-1 altered the second responses. Thus, two-dimensional Ca2+i image analysis of ECs and VSMCs in coculture enabled direct visualization of the EDRF actions in ECs and VSMCs and their modifications.
Shin
W S
WS
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Sasaki
T
T
Kato
M
M
Hara
K
K
Seko
A
A
Yang
W D
WD
Shimamoto
N
N
Sugimoto
T
T
Toyo-oka
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002478:Cells, Cultured; D016232:Endothelins / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D011464:Epoprostenol / Q000502:physiology; D007091:Image Processing, Computer-Assisted; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
D002413:Cations, Divalent; D016232:Endothelins; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D011464:Epoprostenol; D002118:Calcium