Abstract
CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.
Highlights
CD38, a type II transmembrane glycoprotein of 46 kDa, formerly known as a leukocyte activation antigen (1, 2), has attracted increasing attention since it proved to be a bifunctional ectoenzyme involved in the metabolism of two signal molecules, i.e. cyclic ADP-ribose1 and NAADPϩ (3, 4)
This dinucleotide transporter has been identified with connexin 43 hemichannels (12). (ii) Transmembrane CD38 is an active transporter of catalytically produced cyclic ADP-ribose (cADPR) across its oligomeric structure (13). (iii) A third, CD38-unrelated mechanism of permeation of extracellular cADPR across cell membranes has been postulated in selected cell types (14, 15)
We investigated the possible permeation of extracellular cADPR across the plasma membrane of native CD38Ϫ 3T3 fibroblasts
Summary
Materials—[32P]NADϩ (200 Ci/mmol) and [3H]NADϩ (40 Ci/mmol) were obtained by ICN (Milan, Italy) and PerkinElmer Life Sciences, respectively. cADPR, [32P]cADPR, and [3H]cADPR were prepared enzymatically from NADϩ, [32P]NADϩ, and [3H]NADϩ, respectively, with recombinant ADP-ribosyl cyclase from A. californica For determination of [Ca2ϩ]i in CD38Ϫ cells co-cultured with CD38ϩ/Ϫ feeders, target CD38Ϫ cells were harvested at different times from the transwells, washed twice in 1 ml of PBS for 30 s at 5,000 ϫ g, and resuspended in 1 ml of fresh complete medium. Determination of Extracellular cADPR—At various times of co-culture of CD38Ϫ over CD38ϩ/Ϫ 3T3 feeder cells in complete medium (without phenol red), the medium was collected and clarified by three repeated centrifugations at 300 ϫ g for 5 min. BrdUrd-labeled cells were detached with trypsin, washed twice in ice-cold PBS containing 2 mM EDTA, and prepared for flow cytometry-mediated analysis of BrdUrd and DNA contents as described previously (10). The Student’s t test was used to determine the statistical significance of the difference between the two cell populations in the four experiments performed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
