SYNCHRONIZED CA2-DARBY CANINE KIDNEY-CELLS BY BRADYKININ AND THROMBINBUT NOT BY ATP( OSCILLATIONS INDUCED IN MADIN)

In an earlier report, we described synchronous Ca2+ oscillations in gl obally stimulated, subconfluent MDCK cells [Rottingen J-A., Enden T., Camerer E., Iversen J-G., Prydz H. Binding of human factor VIIa to tis sue factor induces cytosolic Ca2+ signals in J82 cells, transfected CO S-1 cells, Madin-Darby canine kidney cells and in human endothelial ce lls induced to synthesize tissue factor, J Biol Chem 1995; 270: 4650-4 660]. In order to elucidate the mechanisms behind these oscillations, we have analyzed the fluctuations in cytosolic Ca2+ in single, Fura-2 loaded, MDCK cells grown to subconfluence, after stimulation with brad ykinin, thrombin and ATP. All three agonists gave rise to an initial C a2+ spike followed by oscillations or transients, Both the initial and subsequent spikes appeared to be due mainly to release of Ca2+ from i nternal stores, since they remained after Ca2+ influx was impeded by e ither La3+ or by chelation of extracellular Ca2+ with EGTA. The second ary spikes were apparently synchronized when the cells were (permanent ly and globally) stimulated with bradykinin or thrombin, but each cell seemed to oscillate independently when stimulated in the same way wit h ATP. Synchronized secondary spikes arose with a constant frequency a nd amplitude, independent of agonist concentration in contrast to most Ca2+ oscillations observed. Pretreatment of the cells with octanol to block gap junctions, or with EGTA or La3+ to inhibit Ca2+ influx, abo lished the synchronization induced by bradykinin or thrombin, We obser ved that in the MDCK cell layer there are some 'pacemaker' cells and h ypothesize that these have a higher sensitivity for the agonists than their neighboring cells, From these pacemakers, an intercellular Ca2wave can be seen to spread to adjacent cells in the presence of intact gap junctions, thereby initiating concurrent transients in all cells, The Ca2+ wave is amplified by release from internal stores, probably owing to the bell-shaped Ca2+ activation curve of the IP3 receptor and by subsequent Ca2+ influx through Ca2+ release activated channels.