INTRACELLULAR CA2-CELLS( DISTRIBUTION IN MIGRATING TRANSFORMED RENAL EPITHELIAL)

Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depe nds on the polarized activity of a Ca2+-sensitive K+ channel. We teste d whether a gradient of intracellular Ca2+ concentration ([Ca2+](i)) u nderlies the horizontal polarization of K+ channel activity. [Ca2+](i) was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+](i) indicated that a horizontal gradient exists, with [Ca2+](i) being higher in the cell body than in the lamellipodium. Resting and m aximal levels during oscillations of [Ca2+](i) in the cell body were f ound to be 135 +/- 34 and 405 +/- 59 nmol/l, respectively, whereas the y were 79 +/- 18 and 307 +/- 102 nmol/l in the lamellipodium. This gra dient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusi on technique during migration experiments and measurements of [Ca2+](i ) to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charyb dotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migr ation to 47 +/- 10% and 9 +/- 5% of control, respectively. Local block ade of Ca2+ influx into the cell body and the lamellipodium with La3was followed by a decrease of [Ca2+](i) at both cell poles. This point s to Ca2+ influx occurring over the entire cell surface. This conclusi on was confirmed by locally superfusing Mn2+ over the cell body and th e lamellipodium. Fura-2 fluorescence was quenched in both areas, the d ecrease of fluorescence being two to three times faster in the cell bo dy than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+](i). We hypothesize tha t the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+](i).