8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist.

ABSTRACT

cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.