Imaging cytoplasmic free calcium in histamine stimulated endothelial cells and in fMet-Leu-Phe stimulated neutrophils.
Intracellular free [Ca2+] ([Ca2+]i) was imaged in Fura-2 loaded human umbilical vein endothelial cells, using dual excitation light, an inverted epifluorescence microscope and a silicon intensified target camera. Oscillations of [Ca2+]i were induced in these cells by 0.5 microM histamine. Because the image processor contained a large random access memory of 32 Mbyte, it was possible to follow these oscillations over a period of several minutes with a time resolution of approximately 2 s. Plots of the variation of average [Ca2+]i measured from these images in neighbouring cells showed that not only were the oscillations asynchronous but also that they occurred at different frequencies. This indicates that there is no strong coupling of [Ca2+]i in adjacent endothelial cells. The rise in [Ca2+]i stimulated by 0.5 microM histamine was qualitatively imaged at 80 ms intervals by recording images at 380 nm only. [Ca2+]i-independent variations in image intensity were removed by ratioing these images against a 380 nm image obtained before the stimulation by histamine. Such images revealed that [Ca2+]i rises first locally and then propagates across the cell at approximately 50 microns.s-1. Neutrophils were imaged following stimulation by fMet-Leu-Phe, also with a time resolution of 2 s. Three responses were determined from the images: (a) the rise in [Ca2+]i; (b) the cell spreading; and (c) the chemokinesis. Measuring these responses in single cells clearly showed the temporal relationships with the responses occurring in the order listed above.
Jacob
R
R
Smith Kline & French Research Ltd, Welwyn, Hertfordshire, UK.
D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*
D001572:Benzofurans; D005456:Fluorescent Dyes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006632:Histamine; D002118:Calcium; D016257:Fura-2