Dynamics of ATP-induced calcium signaling in single mouse thymocytes

Extracellular ATP (ATP(0)) elicits a robust change in the concentration of intracellular Ca2+ ([Ca2+](i)) in fura-2-loaded mouse thymocytes. Most thymocytes (60%) exposed to ATP, exhibited a biphasic rise in [Ca2+](i); [Ca2+](i) rose slowly at first to a mean value of 260 nM after 163 s and then increased rapidly to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the crest in [Ca2+](i). Experiments performed in the absence of extracellular [Ca2+](0) abolished the rise in thymocyte [Ca2+](i), indicating that Ca2+ influx, rather than the release of stored Ca2+, is stimulated by ATP(0). ATP(0)-mediated Ca2+ influx was potentiated as the [Mg2+](0) was reduced, confirming that ATP(4-) is the active agonist form. In the absence of Mg-0(2+), 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of those tested. The rank order of potency for adenine nucleotides was BzATP(4-)>ATP(4-)>MgATP(2-)>ADP(3-), suggesting purinoreceptors of the P2X(7)/P2Z class mediate the ATP(0) response. Phenotyping experiments illustrate that both immature (CD4(-)CD8(-), CD4(+)CD8(+)) and mature (CD4(+)CD8(-), CD4(-)CD8(+)) thymocyte populations respond to ATP. Further separation of the double-positive population by size revealed that the n ATP(0)-mediated [Ca2+](i) response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4(+)CD8(+) thymocytes. We conclude that thymocytes vary in sensitivity to ATP(0) depending upon the degree of maturation and suggest that ATP(0) may be involved in processes that control cellular differentiation within the thymus.