THROMBIN-DEPENDENT CALCIUM SIGNALING IN SINGLE HUMAN ERYTHROLEUKEMIA-CELLS

1. A combination of single cell fluorescence and patch clamp technique s were used to study the mechanisms underlying thrombin-evoked Ca2+ si gnals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increas e in intracellular Ca2+ ([Ca2+](i)), consisting of both release of Ca2 + from intracellular stores and influx of extracellular Ca2+. Mn2+ que nch studies indicated that the thrombin-evoked divalent cation-permeab le pathway was activated during, but not prior to, release from intern al stores. 3. Thapsigargin (1 mu M) irreversibly released internal Ca2 + from the same store as that released by thrombin and continuously ac tivated a Ca2+-influx mechanism. The amplitude of the thrombin- and th apsigargin-induced Ca2+ influx displayed a marked single cell heteroge neity which showed no correlation with the size of the store Ca2+ tran sient. 4. In whole-cell patch clamp recordings, both thrombin and thap sigargin evoked an inwardly rectifying Ca2+ current which developed wi th little or no increase in current noise, showed no reversal in the v oltage range -110 to +6O mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ >> Ba2 + > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV var ied between 0.42 and 2.1 pA pF-(1) in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsig argin-induced inward current had fully developed. 5. These studies ind icate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca2+ store-release-activated Ca2+ current (I-crac) rather than via rec eptor-operated or second messenger-dependent Ca2+ channels. The level of expression of I-crac appears to be a major factor in determining th e duration of the thrombin-evoked [Ca2+](1) response and therefore rep resents a means by which cells can exert control over [Ca2+](i)-depend ent events.