Laser microspectrofluorometry for measuring dynamic changes of intracellular free Ca2+ in human airway gland cells

摘要

Intracellular Ca$+2$PLU$/ is a ubiquitous second messenger that regulates a wide variety of cellular functions including secretion, transepithelial solute and fluid transport. Laser confocal microspectrofluorometry (DILOR, Lille, France) was applied to visualize fluorescence emission spectra of the Indo-1 for measuring the intracellular free Ca$+2$PLU$/ levels ($LB@Ca$+2$PLU$/$RB$-i$/) in a human tracheal gland immortalized cell line (MM39 cell line). Under a 351 nm laser excitation (0.5 $mu@W), the intracellular spectrum was analyzed as a ratio of the emission intensities at 420 and 500 nm. Previously, the intracellular Ca$+2$PLU$/ calibration has been performed to define the relation between the intensity ratio and $LB@Ca$+2$PLU$/$RB$-i$/. Dynamic changes of single-cell $LB@Ca$+2$PLU$/$RB$-i$/ were measured either from one substrate-attached cell or from different adjacent cells in monolayer culture. Measurements of $LB@Ca$+2$PLU$/$RB$-i$/ are taken successively in different subcellular locations (up to 10 measurement points). Each measurement cycle was repeated 60 times. To do so, an (X,Y) motorized stage coupled with a computer allowed us to store the (X,Y) positions of several chosen points for the laser radiation. Cells were monitored for about 10 min. After agonist stimulation. Upon stimulating with calcium ionophore, 4BrA23187 (1 $mu@M), $LB@Ca$+2$PLU$/$RB$-i$/ increased immediately up to 10 fold from a resting value of 31 plus or minus 6 nM (n equals 36). Histamine (1 to 100 $mu@M) increased $LB@Ca$+2$PLU$/$RB$-i$/ in a concentration dependent manner with levels of up to 88 nM and 140 nM for 1 $mu@M and 100 $mu@M concentration, respectively, followed by a smooth decay back to baseline. Removal of extracellular Ca$+2$PLU$/ did not abolish the histamine-stimulation $LB@Ca$+2$PLU$/$RB$-i$/ rise, suggesting that a part of Ca$+2$PLU$/ mobilization comes from intracellular Ca$+2$PLU$/ stores. These results show that the combined use of the UV microspectrofluorometry and Indo-1 is well adapted and straight forward for the measurement of rapid responses of substrate-attached cells during experiments of long duration.