Paracrine roles of NAD(+) and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyc lase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful unive rsal calcium mobilizer from intracellular stores. Recently, hexameric conne xin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guide, L., Zocchi, E., France, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide av ailable to the ectocellular active site of CD38. Here we investigated trans well co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca2+](i) levels a nd on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca2+](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibit able by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH2-cADPR. Addit ion of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders ind uced sustained [Ca2+](i) increases in CD38(-) target cells. Co-culture on C D38(+) feeders enhanced the proliferation of CD38(-) target cells over cont rol values and significantly shortened the S phase of cell cycle. These res ults demonstrate a paracrine process based on Cx43-mediated release of NAD( +), its CD38-catalyzed conversion to extracellular cADPR, and influx of thi s nucleotide into responsive cells to increase [Ca2+](i) and stimulate cell proliferation.