Abstract

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.

Highlights

  • The ADP-ribosyl cyclase CD38 produces cyclic ADP-ribose from NADϩ in the extracellular space

  • We demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation induces calcium mobilization through three sequential steps, connexin 43 (Cx43)-mediated NAD؉ export, CD38-mediated generation of cyclic ADP-ribose (cADPR) and Cx43-mediated cADPR import in J774 cells

  • We demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation triggers intracellular Ca2ϩ mobilization using Cx43-mediated NADϩ export/cADPR import system

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Summary

Background

The ADP-ribosyl cyclase CD38 produces cyclic ADP-ribose from NADϩ in the extracellular space. It has been known that intracellular NAD؉ approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions It is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR CD38 itself or nucleoside transporter has been proposed to import cADPR. It has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD؉. We demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD؉ export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. We demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation triggers intracellular Ca2ϩ mobilization using Cx43-mediated NADϩ export/cADPR import system

EXPERIMENTAL PROCEDURES
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