Intracellular Ca2+ pools in Jurkat T-lymphocytes

Abstract

Jurkat T-lymphocytes comprise at least four intracellular Ca2+ pools. Pool I was agonist-sensitive and contained 23 +/- 8% (n = 18) of the total Ca(2+)-storage capacity, as shown in intact cells in the presence of EGTA. The time courses of the agonist-induced formation of Ins(1,4,5)P3 and of the Ca2+ release from pool I were nearly superimposable, indicating that the agonist-sensitive pool I is emptied by Ins(1,4,5)P3. Likewise, in permeabilized cells, the size of the Ins(1,4,5)P3-sensitive Ca2+ pool I was 27 +/- 11% (n = 14). Pool II contained 26 +/- 5% (n = 9) of intracellularly stored Ca2+ and was liberated by thapsigargin, an inhibitor of the endoplasmic-reticulum (ER) Ca(2+)-ATPase. Addition of thapsigargin before addition of agonist abolished the agonist-induced Ca2+ release in both intact and permeabilized cells, indicating that pool I is a subcompartment of the ER Ca2+ pool. The content of this ER Ca2+ pool (pools I and II) amounted to 51 +/- 15% (n = 9) in intact cells and 49 +/- 16% (n = 16) in permeabilized cells. Caffeine released Ca2+ even when the ER pool (pools I and II) was emptied by previous addition of thapsigargin, indicating the presence of a third pool independent of pools I and II. Pool III contained 23 +/- 6% (n = 8) in intact cells, but 41 +/- 8% (n = 5) in permeabilized cells. The remaining intracellularly stored Ca2+ was released by addition of the Ca2+ ionophore ionomycin. This fourth pool contained 27 +/- 8% (n = 9) in intact cells, but less than 10% in permeabilized cells. The size of pool III was increased when pools I and II were emptied before addition of caffeine, whereas the size of pool IV was decreased under such conditions. In conclusion, this first comprehensive description of intracellular Ca2+ pools in Jurkat T-lymphocytes demonstrates the presence of four different Ca2+ pools, provides estimates of their sizes and describes relationships between each other. Release of Ca2+ from pool I [Ins(1,4,5)P3-sensitive] has previously been shown to play a major role in T-cell activation, whereas the physiological role of pools II-IV remains to be established.

Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity

“…IP4 is a precursor for several higher order inositol polyphosphates, some of which are generated in TCR-stimulated T cells (49,50). Their physiological roles are largely undefined (9), but recent studies in yeast suggest functions in regulating chromatin remodeling (51,52).…”

NAADP-mediated Ca 2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist

“…Rat antigen-specific T cell clones were obtained from lymph node preparations of immunized Lewis rats. Stimulation, expansion, and culture of specific rat T cells were conducted under conditions as described (44). Details concerning generation of MBP-specific CD4 ϩ T cells (TMBP) retrovirally engineered to express the marker gene EGFP are provided in the SI Methods.…”

“…Both Jurkat T lymphoma and rat TMBP cells were loaded with Fura-2/AM (Calbiochem) as described (44) and kept in the dark at room temperature until use. Intracellular Ca 2ϩ ([Ca 2ϩ ]i) was determined in suspensions of Fura-2-loaded rat T MBP cells which had been preincubated with BZ194 or DMSO at the indicated concentrations for at least 8 h in a Hitachi F-2000 fluorometer as described previously (46).…”

The Structural and Functional Versatility of Inositol Phosphates

“…The major pathway of InsP 6 dephosphorylation in mammals is not well-characterized, but several InsP 5 isomers are generated (757), and Ins(l,2,3,4)P 4 / Ins(l,2,3,6)P 4 , Ins(l,2,3)P 3 and ïns(l,2)P 2 / Ins(2,3)P 2 also appear to be formed during the degradation to free Ins (93,150,158). There is some evidence that the activity of this catabolic pathway may change both during the cell cycle, or upon cell differentiation (131,(150)(151)(152), but the cause/effect relationship between these events is not known. Some workers have reported that InsP 6 , or one of its metabolites, has an anti-neoplastic action (759).…”

“…Cellular levels of InsP 6 do not respond dramatically to short-term receptor activation (see above). However, they do rise and fall at certain points in the cell-cycle and during cellular differentiation (131,(150)(151)(152), when there are also dramatic changes in vesicle trafficking processes (753). It is possible that extracellular agonists might also act upon InsP 6 in a more subtle manner, by perhaps altering its distribution between cellular membranes (see above) and the cytosol.…”