Ca2+ influx pathways mediated by swelling or stores depletion in mouse thymocytes

Abstract

We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of ≈70 s, these swelling-activated [Ca2+]i (SWAC) transients reached ≈650 nM from resting levels of ≈100 nM and declined with a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 μM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG-treated thymocytes, [Ca2+]i rose steadily for ≈100 s, peaked at ≈900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 μM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes, TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.