Dynamics of ATP-induced Calcium Signaling in Single Mouse Thymocytes

Extracellular ATP (ATP(o)) elicits a robust change in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) in fura-2–loaded mouse thymocytes. Most thymocytes (60%) exposed to ATP(o) exhibited a biphasic rise in [Ca(2+)](i); [Ca(2+)](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 [Ca(2+)](i). Experiments performed in the absence of extracellular [Ca(2+)](o) abolished the rise in thymocyte [Ca(2+)](i), indicating that Ca(2+) influx, rather than the release of stored Ca(2+), is stimulated by ATP(o). ATP(o)- mediated Ca(2+) influx was potentiated as the [Mg(2+)](o) was reduced, confirming that ATP(4−) is the active agonist form. In the absence of Mg(2+) (o), 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(o) 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 ATP(o)-mediated [Ca(2+)](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(o) depending upon the degree of maturation and suggest that ATP(o) may be involved in processes that control cellular differentiation within the thymus.