Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

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.     

A self-restricted CD38-connexin 43 cross-talk affects NAD+ and cyclic ADP-ribose metabolism and regulates intracellular calcium in 3T3 fibroblasts

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD(+) transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD(+) to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca(2+)](i) levels as a result of reduced intracellular conversion of NAD(+) to cADPR. A sharp inverse correlation emerged between [Ca(2+)](i) levels and NAD(+) transport (measured as influx into cells and as efflux therefrom), both in the CD38(+) cells (high [Ca(2+)](i), low transport) and in the CD38(-) fibroblasts (low [Ca(2+)](i), high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD(+) transport (CD38(-) cells) and vice versa (CD38(+) cells). Conversion of NAD(+)-permeable Cx43 to the phosphorylated, NAD(+)-impermeable form occurs via Ca(2+)-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38(+) fibroblasts whereby high [Ca(2+)](i) restricts further Ca(2+) mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD(+) from cells; (ii) depletion of intracellular NAD(+) by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca(2+)](i).     

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 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.     

Cyclic ADP ribose is a novel regulator of intracellular Ca2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca(2+) signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca(2+) (Ca(2+) (i) ) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca(2+) (i) oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca(2+) (i) oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca(2+) (i) oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca(2+) oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca(2+) messenger for mobilizing the internal Ca(2+) stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied.     

Cyclic ADP-ribose as a universal calcium signal molecule in the nervous system

beta-NAD(+) is as abundant as ATP in neuronal cells. beta-NAD(+) functions not only as a coenzyme but also as a substrate. beta-NAD(+)-utilizing enzymes are involved in signal transduction. We focus on ADP-ribosyl cyclase/CD38 which synthesizes cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer from intracellular stores, from beta-NAD(+). cADPR acts through activation/modulation of ryanodine receptor Ca(2+) releasing Ca(2+) channels. cADPR synthesis in neuronal cells is stimulated or modulated via different pathways and various factors. Subtype-specific coupling of various neurotransmitter receptors with ADP-ribosyl cyclase confirms the involvement of the enzyme in signal transduction in neurons and glial cells. Moreover, cADPR/CD38 is critical in oxytocin release from the hypothalamic cell dendrites and nerve terminals in the posterior pituitary. Therefore, it is possible that pharmacological manipulation of intracellular cADPR levels through ADP-ribosyl cyclase activity or synthetic cADPR analogues may provide new therapeutic opportunities for treatment of neurodevelopmental disorders.     

Signal transduction from bradykinin, angiotensin, adrenergic and muscarinic receptors to effector enzymes, including ADP-ribosyl cyclase

Muscarinic acetylcholine receptors in NG108-15 neuroblastoma x glioma cells, and beta-adrenergic or angiotensin II receptors in cortical astrocytes and/or ventricular myocytes, utilize the direct signaling pathway to ADP-ribosyl cyclase within cell membranes to produce cyclic ADP-ribose (cADPR) from beta-NAD+. This signal cascade is analogous to the previously established transduction pathways from bradykinin receptors to phospholipase Cbeta and beta-adrenoceptors to adenylyl cyclase via G proteins. Upon receptor stimulation, the newly-formed cADPR may coordinately function to upregulate the release of Ca2+ from the type II ryanodine receptors as well as to facilitate Ca2+ influx through voltage-dependent Ca2+ channels. cADPR interacts with FK506, an immunosuppressant, at FKBP12.6, FK506-binding-protein, and calcineurin, or ryanodine receptors. cADPR also functions through activating calcineurin released from A-kinase anchoring protein (AKAP79). Thus, some G(q/11)-coupled receptors can control cADPR-dependent modulation in Ca2+ signaling.     

Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR)

Cyclic ADP-ribose (cADPR) is a Ca2+ mobilizing second messenger found in various cell types, tissues and organisms. Receptor-mediated formation of cADPR may proceed via transmembrane shuttling of the substrate NAD and involvement of the ectoenzyme CD38, or via so far unidentified ADP-ribosyl cyclases located within the cytosol or in internal membranes. cADPR activates intracellular Ca2+ release via type 2 and 3 ryanodine receptors. The exact molecular mechanism, however, remains to be elucidated. Possibilities are the direct binding of cADPR to the ryanodine receptor or binding via a separate cADPR binding protein. In addition to Ca2+ release, cADPR also evokes Ca2+ entry. The underlying mechanism(s) may comprise activation of capacitative Ca2+ entry and/or activation of the cation channel TRPM2 in conjunction with adenosine diphosphoribose. The development of novel cADPR analogues revealed new insights into the structure-activity relationship. Substitution of either the northern ribose or both the northern and southern ribose resulted in much simpler molecules, which still retained significant biological activity.     

NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors

Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.     

Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signaling

Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+-dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and beta-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) and Ca2+ mobilization

Many physiological processes are controlled by a great diversity of Ca2+ signals that depend on Ca2+ entry into the cell and/or Ca2+ release from internal Ca2+ stores. Ca2+ mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca2+ release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP3 and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca2+ stores. Activation of the NAADP-sensitive Ca2+ channels evokes complex changes in cytoplasmic Ca2+ levels by means of channel chatter with other intracellular Ca2+ channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca2+ signaling.     

Amplification of depolarization-induced and ryanodine-sensitive cytosolic Ca2+ elevation by synthetic carbocyclic analogs of cyclic ADP-ribose and their antagonistic effects in NG108-15 neuronal cells

We synthesized analogs modified in the ribose unit (ribose linked to N1 of adenine) of cyclic ADP-ribose (cADPR), a Ca2+-mobilizing second messenger. The biological activities of these analogs were determined in NG108-15 neuroblastoma x glioma hybrid cells that were pre-loaded with fura-2 acetoxymethylester and subjected to whole-cell patch-clamp. Application of the hydrolysis-resistant cyclic ADP-carbocyclic-ribose (cADPcR) through patch pipettes potentiated elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) at the depolarized membrane potential. The increase in [Ca2+]i evoked upon sustained membrane depolarization was significantly larger in cADPcR-infused cells than in non-infused cells and its degree was equivalent to or significantly greater than that induced by cADPR or beta-NAD+. 8-Chloro-cADPcR and two inosine congeners (cyclic IDP-carbocyclic-ribose and 8-bromo-cyclic IDP-carbocyclic-ribose) did not induce effects similar to those of cADPcR or cADPR. Instead, 8-chloro-cADPcR together with cADPR or cADPcR caused inhibition of the depolarization-induced [Ca2+]i increase as compared with either cADPR or cADPcR alone. These results demonstrated that our cADPR analogs have agonistic or antagonistic effects on the depolarization-induced [Ca2+]i increase and suggested the presence of functional reciprocal coupling between ryanodine receptors and voltage-activated Ca2+ channels via cADPR in mammalian neuronal cells.     

Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.     

Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50?μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.     

The CD38-cyclic ADP-ribose signaling system in insulin secretion

Glucose induces an increase in the intracellular Ca2+ concentration in pancreatic -cells to secrete insulin. CD38 occurs in -cells and has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose (cADPR) from NAD+, and cADPR hydrolase, which converts cADPR to ADP-ribose. ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of the hydrolysis of cADPR and thereby causing cADPR accumulation in -cells. Cyclic ADP-ribose then binds to FK506-binding protein 12.6 in the ryanodine receptor Ca2+ channel (RyR), dissociating the binding protein from RyR to induce the release of Ca2+ from the endoplasmic reticulum. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylates RyR to sensitize and activate the Ca2+ channel. Ca2+, released from the RyR, further activates CaM kinase II and amplifies the process. Thus, cADPR acts as a second messenger for Ca2+ mobilization to secrete insulin. The novel mechanism of insulin secretion described above is different from the conventional hypothesis in which Ca2+ influx from extracellular sources plays a role in insulin secretion by glucose.     

Deficit of CD38/cyclic ADP-ribose is differentially compensated in hearts by gender

To elucidate whether myocardial CD38/cyclic ADP-ribose (cADPR) signaling plays a physiological role, we investigated the heart of CD38 knockout mice (CD38KO). In CD38KO, the myocardial cADPR content was reduced by 85% compared with wild-type mice (WT). Cardiac hypertrophy developed only in males. At 36 degrees C, none of the parameters for Ca(2+) transients and forces of the papillary muscles differed between WT and CD38KO. In contrast, at 27 degrees C, at which cADPR does not work, the peak [Ca(2+)](i) was increased and the decline in [Ca(2+)](i) was accelerated in CD38KO compared with WT. In CD38KO, the protein expression of SR Ca(2+) ATPase type2 (SERCA2) and the SERCA2-to-phospholamban ratio were increased compared with WT. The ryanodine receptor protein was increased only in female CD38KO compared with WT. These data suggest that the CD38/cADPR signaling plays an important role in intracellular Ca(2+) homeostasis in cardiac myocytes in vivo. Its deficiency was compensated differentially according to gender.     

Cyclic ADP ribose-mediated Ca2+ signaling in mediating endothelial nitric oxide production in bovine coronary arteries

The present study tested the hypothesis that cyclic ADP ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca2+ mobilization in coronary arterial endothelial cells (CAECs) and thereby contributes to endothelium-dependent vasodilation. In isolated and perfused small bovine coronary arteries, bradykinin (BK)-induced concentration-dependent vasodilation was significantly attenuated by 8-bromo-cADPR (a cell-permeable cADPR antagonist), ryanodine (an antagonist of ryanodine receptors), or nicotinamide (an ADP-ribosyl cyclase inhibitor). By in situ simultaneously fluorescent monitoring, Ca2+ transient and nitric oxide (NO) levels in the intact coronary arterial endothelium preparation, 8-bromo-cADPR (30 microM), ryanodine (50 microM), and nicotinamide (6 mM) substantially attenuated BK (1 microM)-induced increase in intracellular [Ca2+] by 78%, 80%, and 74%, respectively, whereas these compounds significantly blocked BK-induced NO increase by about 80%, and inositol 1,4,5-trisphosphate receptor blockade with 2-aminethoxydiphenyl borate (50 microM) only blunted BK-induced Ca2+-NO signaling by about 30%. With the use of cADPR-cycling assay, it was found that inhibition of ADP-ribosyl cyclase by nicotinamide substantially blocked BK-induced intracellular cADPR production. Furthermore, HPLC analysis showed that the conversion rate of beta-nicotinamide guanine dinucleotide into cyclic GDP ribose dramatically increased by stimulation with BK, which was blockable by nicotinamide. However, U-73122, a phospholipase C inhibitor, had no effect on this BK-induced increase in ADP-ribosyl cyclase activity for cADPR production. In conclusion, these results suggest that cADPR importantly contributes to BK- and A-23187-induced NO production and vasodilator response in coronary arteries through its Ca2+ signaling mechanism in CAECs.     

NADP+-Dependent internalization of recombinant CD38 in CHO cells

CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+ was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS-PAGE. Our data demonstrate that NADP+ can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.     

Elucidation of the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells: modulation by cyclic ADP-ribose and FK506

The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2+ release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2+ uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 microM) shifted the dose-response curve of ryanodine- or caffeine-induced 45Ca2+ release from the vesicles to the left. Preincubation with cADPR shifted the dose-response curve of the FK506-induced 45Ca2+ release upward. Preincubation with FK506 (3 microM) shifted the dose-response curve of the ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose-response curve of the cADPR-induced 45Ca2+ release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 microM)- or caffeine (10 mM)-induced 45Ca2+ release by the same extent as that in the case of cADPR alone or FK506 alone. These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same.     

Activation of CD38 by interleukin-8 signaling regulates intracellular Ca2+ level and motility of lymphokine-activated killer cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.