Thrombin-dependent calcium signalling in single human erythroleukaemia cells.
1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]i), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 microM) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca(2+)-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +60 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 varied 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 thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca(2+)-store-release-activated Ca2+ current (Icrac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of Icrac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i-dependent events.
Somasundaram
B
B
Physiological Laboratory, Cambridge, UK.
Mason
M J
MJ
Mahaut-Smith
M P
MP
D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D004594:Electrophysiology; D005453:Fluorescence; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D005456:Fluorescent Dyes; C049925:fura-2-am; D008345:Manganese; D019284:Thapsigargin; D013917:Thrombin; D002118:Calcium; D016257:Fura-2