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.     

Ectocellular CD38-catalyzed synthesis and intracellular Ca2+-mobilizing activity of cyclic ADP-ribose

CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD⁺ to nicotinamide and cyclic ADP-ribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca²⁺-mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca²⁺ response to KCl-induced depolarization, a process that can be qualified as a Ca²⁺-induced Ca²⁺ release (CICR) mechanism. On the other hand, in the CD38⁺ human Namalwa B lymphoid cells, NAD⁺ (and thiol compounds as well) induced a two-step process of self-aggregation followed by endocytosis of CD38, which resulted in a shift of cADPR metabolism from the cell surface to the cytosol. Both distinctive types of cellular responses to extracellular NAD⁺ seem to be suitable to elicit changes in the intracellular Ca²⁺ homeostasis.     

Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca(2+) signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.     

CD38/cyclic ADP-ribose signaling: role in the regulation of calcium homeostasis in airway smooth muscle

The contractility of airway smooth muscle cells is dependent on dynamic changes in the concentration of intracellular calcium. Signaling molecules such as inositol 1,4,5-trisphosphate and cyclic ADP-ribose play pivotal roles in the control of intracellular calcium concentration. Alterations in the processes involved in the regulation of intracellular calcium concentration contribute to the pathogenesis of airway diseases such as asthma. Recent studies have identified cyclic ADP-ribose as a calcium-mobilizing second messenger in airway smooth muscle cells, and modulation of the pathway involved in its metabolism results in altered calcium homeostasis and may contribute to airway hyperresponsiveness. In this review, we describe the basic mechanisms underlying the dynamics of calcium regulation and the role of CD38/cADPR, a novel pathway, in the context of airway smooth muscle function and its contribution to airway diseases such as asthma.     

Novel inhibitors of poly(ADP-ribose) synthetase.

In a search for new inhibitors of the nuclear enzyme poly(ADP-ribose) synthetase, it was found that various benzamides substituted in the 3-position were the most inhibitory compounds found to date. Two of the benzamides, 3-aminobenzamide and 3-methoxybenzamide, were found to be competitive inhibitors, with Ki values or less than 2 microM.     

CD38 signaling in B lymphocytes is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose

CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.     

Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis

Menaquinone (vitamin K(2)) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC(50) value of 5.7microM.     

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.     

Flavonoids as inhibitors of human CD38

CD38 is a multifunctional enzyme which is ubiquitously distributed in mammalian tissues. It is involved in the conversion of NAD(P)⁺ into cyclic ADP-ribose, NAADP⁺ and ADP-ribose and the role of these metabolites in multiple Ca²⁺ signaling pathways makes CD38 a novel potential pharmacological target. The dire paucity of CD38 inhibitors, however, renders the search for new molecular tools highly desirable. We report that human CD38 is inhibited at low micromolar concentrations by flavonoids such as luteolinidin, kuromanin and luteolin (IC₅₀ <10 μM). Docking studies provide some clues on the mode of interaction of these molecules with the active site of CD38.     

Hydrolysis of NADP+ by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2'-phosphate.

CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD+ and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP+ by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP+ resulted in the rapid and time-dependent accumulation of ADP-ribose 2'-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2'-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP+ glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2'-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2'-phosphate did not result in the formation of ADP-ribose 2'-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2'-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD+, displays exclusively a NADP+ glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2'-phosphate.     

Metabolism of cyclic ADP-ribose: Zinc is an endogenous modulator of the cyclase/NAD glycohydrolase ratio of a CD38-like enzyme from human seminal fluid

CD38, a bifunctional enzyme capable of both synthesis and hydrolysis of the second messenger cyclic ADP-ribose (cADPR). Using the natural substrate of the enzyme, NAD+, the ratio of ADP-ribosyl cyclase/NAD glycohydrolase of CD38 is about 1/100. Here we describe that human seminal fluid contain a soluble CD38 like enzyme with an apparent M.W. of 49 kDa. When purified this enzyme has a cyclase/NAD glycohydrolase ratio of about 1/120. However, the in situ cyclase/NAD glycohydrolase ratio measured in seminal plasma approaches 1/1. We also found that physiological concentrations of zinc present in the seminal fluid, in the range of 0.6 to 4 mM, are responsible for the modulation of the cyclase/NAD glycohydrolase ratio. This new information indicates that the cyclase/NAD glycohydrolase ratio can be modified in vivo.     

Mechanism-based irreversible inhibitors of cytokinin dehydrogenase

The effects of three N(6)-substituted aminopurine derivatives containing either allenic or acetylenic side-chains on in vitro and in vivo cytokinin dehydrogenase (CKX; EC 1.5.99.12) activities were determined. At concentrations < or = 100 microM, the acetylenic derivative (HA-2) had no effect on in vitro CKX activity. In contrast, the two allenic derivatives (HA-1, HA-8) inhibited in vitro CKX activity in a dose-dependent manner with 50% inhibition occurring at HA-1 and HA-8 concentrations of 9.0 and 0.4 microM (respectively). HA-8 inhibited the degradation of both the free bases and ribosides of N6-(2-isopentenyl)adenine and zeatin. Pretreatment with HA-8 inhibited CKX activity in both a time- and concentration-dependent manner. In contrast to the reversible phenylurea inhibitor N-(chloro-4-pyridyl)-N'-phenylurea, inhibition of CKX activity by HA-8 was not relieved by 24 h of dialysis. Both HA-1 and HA-8 (but not HA-2) inhibited the metabolism of exogenous [3H]-N(6)-(2-isopentenyl)adenosine in excised aseptic potato (Solanum tuberosum) leaves. These results demonstrate that HA-8 is a mechanism-based irreversible (suicide) inhibitor of CKX and indicate that it may be useful in determining the role of CKX in cytokinin homeostasis in planta.     

A minimal structural analogue of cyclic ADP-ribose: synthesis and calcium release activity in mammalian cells

Cyclic ADP-ribose (cADPR) is an endogenous Ca(2+)-mobilizing second messenger in many cell types and organisms. Although the biological activity of several modified analogues of cADPR has been analyzed, most of these structures were still very similar to the original molecule. Recently, we have introduced simplified analogues in which the northern ribose (N(1)-linked ribose) was replaced by an ether strand. Here we also demonstrate that the southern ribose (N(9)-linked ribose) can be replaced by an ether strand resulting in N(1)-[(phosphoryl-O-ethoxy)-methyl]-N(9)-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthinecyclic pyrophosphate (cIDP-DE). This minimal structural analogue of cyclic ADP-ribose released Ca(2+) from intracellular stores of permeabilized Jurkat T lymphocytes. In intact T lymphocytes initial subcellular Ca(2+) release events, global Ca(2+) release, and subsequent global Ca(2+) entry were observed. Cardiac myocytes freshly prepared from mice responded to cIDP-DE by increased recruitment of localized Ca(2+) signals and by global Ca(2+) waves.     

Mechanism of Cyclizing NAD to Cyclic ADP-ribose by ADP-ribosyl Cyclase and CD38

Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca²⁺ messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2′F-2′deoxynicotinamide adenine dinucleotide (ribo-2′-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.Cyclic ADP-ribose is a novel cyclic nucleotide with Ca²⁺-mobilizing activity targeting the endoplasmic reticulum. Its activity was first described in sea urchin eggs (1, 2), and cADPR³ has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions, from fission in the dinoflagellate (3) to social behavior in mice (Ref. 4 and reviewed in Refs. 5 and 6). The Aplysia ADP-ribosyl cyclase (cyclase) was the first protein identified that uses NAD, a linear substrate, and ligates its two ends to produce cADPR, with the release of the terminal moiety, nicotinamide (7). The cyclase is a soluble protein of 30 kDa and is present in large amounts in Aplysia ovotestis (7). It is also present in the neurons of the Aplysia buccal ganglion, where it is responsible for the synthesis of endogenous cADPR and the regulation of the evoked synaptic transmission (8). Recently, it is shown that depolarization of Aplysia neurons induces the translocation of the cyclase from the cytosol into the nucleus, providing a mechanism for fine tuning of nuclear Ca²⁺ signals in neurons (9).CD38 is the major mammalian homolog of the cyclase and is responsible for regulating a wide range of physiological functions. Deletion of the CD38 gene in mice produces multiple defects, including impairment of insulin secretion (10), neutrophil chemotaxis (11), and oxytocin release (4). Catalytically, CD38 is quite different from the cyclase. Although both use NAD as substrate, CD38 produces only a small amount of cADPR, whereas the major product is ADP-ribose (ADPR) instead (12–15) (Fig. 1a). It can also use cADPR as a substrate and hydrolyze it to ADPR (12–15). Ablation of the CD38 gene in mice, nevertheless, results in a large reduction in endogenous cADPR in many tissues (10, 11). CD38 is thus responsible for both the synthesis and the hydrolysis of cADPR in mammalian cells.Open in a separate windowFIGURE 1.Crystal structure of the complex of cyclase with ribo-2′F-NAD. a, chemical structure of the substrate ribo-2′F-NAD and the reactions catalyzed by CD38 and the cylase. b, crystal structure of the cyclase dimer with the intermediates at each of the active sites of the monomer. The color scheme for the secondary structures is: red, α-helix; yellow, β-sheet; gray, coil. The color scheme for the residues is: cyan, Tyr-81; beige, Phe-174; blue, Glu-179; magenta, Glu-98; purple, Phe-175. The intermediates are colored by their elements: green, carbon; red, oxygen; orange, phosphorus; blue, nitrogen; light green, fluorine. c, stereo view of the folded conformation with electron density from an omit F_o − F_c map contoured at 2.7 σ and shown as blue wire mesh. Other color schemes are the same as in b. The average B-factor is 76 Ų for the folded intermediate.In fact, both CD38 and the cyclase are multifunctional enzymes that can also use NADP as a substrate and, in the presence of nicotinic acid, produce nicotinic acid adenine dinucleotide phosphate (NAADP) via a base-exchange reaction (16). NAADP was first shown to have Ca²⁺-mobilizing activity in sea urchin eggs (17) and has since been established as another Ca²⁺ messenger molecule targeting yet another intracellular Ca²⁺ store, the lysosome, in a variety of cell types (18–20).To elucidate the mechanism of cyclizing a long linear substrate such as NAD to a compact cyclic product, cADPR, here we present the crystal structure of a stable complex of the cyclase with a substrate analog of NAD. The structure clearly identified critical residues for the cyclization process, which were verified by site-directed mutagenesis. The results demonstrate that catalysis by CD38 or the cyclase is controlled by one or two critical residues and that mutating them can interconvert the reactivities of the two enzymes. This study sets the stage for engineering enzymes with specific activity toward cADPR for expression in cells, which should be valuable tools for manipulating the function and metabolism of this novel Ca²⁺ messenger.     

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

Effects of deuterium oxide (D2O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of gamma rays. Growth of irradiated and unirradiated cells was inhibited by 45% D2O but not by 3-aminobenzamide at 10 mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. The D2O did not inhibit the radiation-induced increase in poly(ADP-ribose) synthesis as measured by the incorporation of [14C]NAD into the acid insoluble fraction, contrary to 3-aminobenzamide. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of gamma rays. Possible mechanisms underlying the inhibition are discussed.     

Catalysis-based inhibitors of the calcium signaling function of CD38

CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.     

Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase.

Two classes of enzymes, poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferases, catalyze covalent attachment of multiple or single residues, respectively, of the ADP-ribose moiety of NAD+ to various proteins. In order to find good inhibitors of poly(ADP-ribose) synthetase free of side actions and applicable to in vivo studies, we made a large scale survey using an in vitro assay system, and found many potent inhibitors. The four strongest were 4-amino-1,8-naphthalimide, 6(5H)- and 2-nitro-6(5H)-phenanthridinones, and 1,5-dihydroxyisoquinoline. Their 50% inhibitory concentrations, 0.18-0.39 microM, were about two orders of magnitude lower than that of 3-aminobenzamide that is currently most popularly used. A common structural feature among all potent inhibitors, including 1-hydroxyisoquinoline, chlorthenoxazin, 3-hydroxybenzamide, and 4-hydroxyquinazoline, in addition to the four mentioned above, was the presence of a carbonyl group built in a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Most of the inhibitors exhibited mixed-type inhibition with respect to NAD+. Comparative studies of the effects on poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase from hen heterophils revealed high specificity of most of the potent inhibitors for poly(ADP-ribose) synthetase. On the other hand, unsaturated long-chain fatty acids inhibited both enzymes, and saturated long-chain fatty acids and vitamin K1 acted selectively on mono(ADP-ribosyl)transferase. The finding of many inhibitors of ADP-ribosyltransferases, especially poly(ADP-ribose) synthetase, supports the view that ADP-ribosylation of proteins may be regulated by a variety of metabolites or structural constituents in the cell.     

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).