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RESEARCH COMMUNICATION |
a Institute of Biochemistry, University of Genova, Viale Benedetto XV/1, 16132, Genova; and
b Institute of Cybernetics and Biophysics, National Research Council, Via De Marini 6, 16149, Genova, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: pyridine nucleotides • cyclic ADP-ribose • intracellular calcium homeostasis • NAD+ transporter
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We recently described two different processes by which this topological paradox can be overcome: 1) the ligand (GSH or NAD+) -induced, vesicle-mediated internalization of CD38 in B-lymphocytic, human Namalwa cells, which is followed by an increase of intracellular cyclase activity and of intracellular cADPR concentration ([cADPR]i) (21); and 2) the cADPR-transporting function of membrane-bound CD38, which behaves as a catalytically active transporter responsible for generation and selective influx of cADPR across membranes (22). Both mechanisms could in fact cooperate to yield the same result: import of cADPR metabolism from the cell surface into the cytosol of CD38+ cells. Though it is still unknown whether the increased [cADPR]i produced by CD38 internalization also induces an increased cytosolic calcium concentration ([Ca2+]i), an unequivocal correlation between the two facts has been demonstrated during the opposite process of exocytotic transport of CD38 to the plasmamembrane (23). In this case, intracellularly produced cADPR was demonstrated to be cytosolic by the increase of basal [Ca2+]i it elicits in CD38+-transfected cells as compared to antisense-transfected controls.
These data prompted us to address the `subcellular' topological inconsistency of the CD38/cADPR system: the endocytotic vesicles mediating ligand-induced CD38 internalization derive from invaginations of the plasmamembrane and therefore the active site of CD38 is intravesicular, i.e., apparently not accessible to cytosolic NAD+. The same asymmetric organization shields the catalytic region of CD38 inside exocytotic vesicles (5, 19); however, these vesicles in fact produce functionally active cADPR during de novo expression of CD38 (23). To ensure production of cytosolic cADPR by the intravesicular catalytic domain of CD38, two subsequent events are required: NAD+ influx from the cytoplasm into the intravesicular space, and cADPR efflux into the cytoplasm to reach its microsomal receptors and release calcium (24–27). Whereas transport of intravesicularly produced cADPR across the vesicle membrane is the result of the channel activity of oligomeric CD38 (22), NAD+ fluxes across the plasma membrane of intact cells have not yet been reported.
Thus, the aim of this study was twofold: to ascertain whether cytosolic calcium is actually increased after ligand-induced CD38 internalization, as it proved to be during CD38 export to the cell membrane (23); and, on the other hand, to elucidate the mechanisms overcoming these topological problems. Results obtained demonstrate that CD38 internalization in various cell types is causally linked to increased [Ca2+]i. The mechanism underlying this process (also accounting for enhanced [Ca2+]i during CD38 exocytosis) was shown to involve influx of cytosolic NAD+ into the endocytotic CD38-containing vesicles, mediated by a hitherto unrecognized transmembrane pyridine dinucleotide transporter.
These data identify the occurrence of NAD+ and cADPR trafficking across cell membranes and demonstrate that internalization of CD38 ensures the intracellular production of functionally significant and topologically adequate (i.e., cytosolic) cADPR concentrations in CD38+ cells. Finally, the demonstration of NAD+ fluxes across the plasmamembrane of mammalian cells and of a functional effect of extracellular, micromolar NAD+ on [Ca2+]i, mediated by CD38 internalization and production of intracellular cADPR, imply a hitherto unrecognized extracellular, hormone-like function of NAD+ on CD38+ cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8-N3-NAD+ was chemically synthesized from 8-N3-AMP and ß-NMN (both purchased from Sigma), and was purified by high-pressure liquid chromatography (HPLC) as described (28, 29). All other chemicals were obtained from Sigma.
Cell cultures
Namalwa, Jurkat, and wild-type HeLa and NIH 3T3 cells (3T3) were
purchased from ATCC (Rockville, Md.). CD38 sense- and
antisense-transfected HeLa and 3T3 cells
(CD38+t and CD38-t,
respectively) were obtained and cultured as described (23). Peripheral
blood lymphocytes (PBL) were recovered after Ficoll gradient separation
from freshly drawn blood samples after informed consent from healthy
donors.
All cells were cultured in Dulbecco's modified Eagle's medium (without phenol red) (DME) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mM) in a humidified 5% CO2 atmosphere at 37°C.
Immunoflorescence microscopy
CD38+t HeLa cells were grown overnight
on glass coverslips. Incubations with the various internalizing agents
at millimolar concentrations were performed at 37°C in a petri dish
with a single addition at zero time, while micromolar NAD+
or 8-N3-NAD+ were continuously infused for 30
min at 25°C through a 200 µl chamber at a flow rate of 50 µl/s.
All solutions were in DME without FCS. Cells were fixed with the
Triton/paraformaldehyde method (30). Nonspecific protein binding to
cells was blocked by a 30 min incubation with 5% FCS. Cells were then
incubated with anti-CD38 or anti-HLA (histocompatibility leukocyte
antigen) class I (Sigma) mAb's (10 µg/ml). A FITC-conjugated
anti-mouse immunoglobulin G (IgG) mAb (Jackson Immunoresearch
Laboratories Inc., West Grove, Pa.) was used as secondary antibody, at
a concentration of 2.5 µg/ml.
Images of the samples were collected by confocal microscopy with a BioRad MRC 1024 Instrument (krypton/argon laser) on a Nikon DIAPHOT 200, x60 oil plan apo objective with NA1.4.
Cytofluorimetric analyses
CD38+t HeLa cells, either untreated or
treated with internalizing agents, were detached with trypsin and
incubated with the primary antibody against HLA class I (10 µg/ml) in
complete medium for 30 min at 4°C. Thereafter, cells were washed and
incubated with 10 µg/ml FITC-conjugated anti-mouse IgG antibody in
complete medium for 30 min at 4°C. Cells were then washed and
fluorescence intensity was quantitated by flow cytometry (21). Control
samples were incubated with the second antibody only.
Fluorimetric determination of the
[Ca2+]i
The experimental setting used for determination of
[Ca2+]i on Fura2-loaded, adherent cells has
been described in detail elsewhere (23). Briefly, cells were seeded on
glass coverslips 18 h before the experiment. After Fura2 loading
of the cells, the glass coverslip was mounted in a 200 µl recording
chamber, mounted on the stage of an inverted microscope (Zeiss IM35,
Stuttgart, Germany), continuously perfused at 25°C with solutions fed
by gravity through solenoid microvalves, and removed by a hydraulic
vacuum pump. Calcium measurements were performed on fields containing
2–10 cell bodies. At the beginning of each experiment, cells were
washed in saline zero calcium solution (135 mM NaCl, 5.4 mM KCl, 1 mM
MgCl2, 10 mM glucose, and 5 mM Hepes, pH 7.4). Intact cells
were either exposed to millimolar concentrations of internalizing
agents under conditions of stopped flow or to micromolar concentrations
under conditions of continuous perfusion in the zero calcium solution.
Calcium variations were recorded as described (23). Nonadherent cells
(Namalwa, Jurkat, and PBL) were treated similarly except that Fura2
loading and calcium measurements were performed in suspension in a 2 ml
cuvette (0.5x106 cells/ml).
Enzymatic assays
Ectocellular cyclase activity on NGD+ (23) and
protein content (31) were determined on the same samples used for
calcium measurements. Glucose 6-P dehydrogenase (G6PD) and hexokinase
activities were determined on sonicated cells and on centrifuged
supernatants as described (32). The percentage of cell lysis was
calculated as the percentage of total intracellular enzyme activity
released into the supernatant.
Determination of pyridine dinucleotide concentrations
The pyridine dinucleotide content of alkaline cell extracts and
of alkalinized, centrifuged cell culture supernatants was determined by
a sensitive enzymatic cycling assay procedure (33), as described (23).
Briefly, this assay enables the sensitive (
10 pmol per assay) and
specific determination of the amount of each pyridine dinucleotide
species (NAD+, NADH, NADP+, and NADPH). A
different assay mixture was used to detect NAD+/NADH or
NADP+/NADPH, the appropriate substrate and enzyme being
ethanol and alcohol dehydrogenase or glucose 6-phosphate and G6PD,
respectively. An aliquot of each sample was heated to 60°C for 30 min
to destroy the oxidized forms. The amount of the oxidized forms was
calculated from the difference between the unheated (oxidized +
reduced dinucleotides) and the heated (reduced form only) samples (33).
Assays were done in duplicate; in controls (run in parallel), the
enzyme was omitted from the assay mixture. Alternatively, aliquots of
each sample were incubated 60 min at 37°C with 1 U/ml of nucleotide
pyrophosphatase (Sigma): both controls gave comparable results and were
used alternatively. All assays were performed on freshly obtained (not
frozen) samples. Protein determination was obtained on aliquots of cell
extracts (31).
Influx of pyridine dinucleotides into cells
Two different experimental approaches were followed in the
studies investigating pyridine dinucleotide influx into cells. As the
transport proved to be bidirectional, a significant amount of the
internalized nucleotides could be lost from the cells during the
extensive washings necessary to remove the excess of extracellular
coenzymes. In one type of experiment, adherent HeLa or 3T3 cells
(approximately 106 cells per sample, seeded 24–36 h before
the experiment) were rapidly washed once in DME to remove the FCS
(which could be a source of dinucleotide pyrophosphatase; L. Guida,
unpublished data). Cells were incubated with the various pyridine
dinucleotides in DME at 37°C and subsequently washed at 0°C. A
control flask, without cells, was incubated and washed in parallel with
each sample. The number of washings was calibrated so as to reduce the
concentration of extracellular dinucleotide in the last washing of the
control flask to HPLC (34) -undetectable concentrations, i.e., <50 nM:
under these conditions, the total amount of extracellular dinucleotide
remaining after removal of the last washing accounted for 1% to 10%
(depending on the nucleotide type) of the intracellular amount of
dinucleotide in untreated cells. In a parallel experiment, cells were
incubated with each dinucleotide in suspension (approximately
106 cells per sample) and the extracellular nucleotides
were alternatively removed by centrifugation of the cells through 3 vol
of silicon oil (d=1.03–1.05 g/ml) for 15 s at 13,000 x
g at 25°C. Pellets were centrifuged again through 0.5 ml
of silicon oil, and cells were lysed in 200 µl of water and sonicated
for 30 s (Heat Systems-Ultrasonics, Inc. W-380) in ice. The
intracellular dinucleotide content was determined on alkaline cell
extracts with the cycling assay (33) or, in the experiments designed to
determine Km values, on TCA extracts by HPLC
(34).
In the kinetic experiments, which were performed on both CD38- and CD38+ cells, the ectocellular NADase activity of the latter cell types could interfere with the results by producing membrane-permeable nicotinamide and ADPR (22), from which intracellular NAD+ synthesis might occur. Thus, the incubation times were brief (from 30 s to 2 min) and control incubations were performed using nicotinamide and ADPR at the same concentrations as those actually produced during incubation of the CD38+ cells with NAD+. This treatment proved not to modify the [NAD+]i.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Similar results were obtained on CD38+t 3T3 cells, thus confirming and extending our previous observation of GSH-and NAD+-induced, vesicle-mediated CD38 internalization in human B-lymphocytic Namalwa cells (21). In these cells, CD38 internalization was found to be followed by an increase of the intracellular cADPR concentration (21). This prompted us to investigate the intracellular free calcium concentration ( [Ca2+]i) in CD38+ cells undergoing CD38 internalization.
CD38 internalization and increased
[Ca2+]i
After 3 h incubation of all cell types tested
(CD38+t HeLa and 3T3 cells and Namalwa B
lymphoid cells) with either NAD+ (2 mM) or GSH (10 mM), the
surface cyclase activity was reduced to approximately 35% of the
initial value, while the [Ca2+]i was markedly
increased up to approximately twice the basal concentration
(Table 1
).No further calcium increase was observed upon incubation of the cells
with either NAD+ or GSH for as long as 24 h. No
significant differences between the various cell lines were apparent.
Comparable results were obtained also on peripheral blood lymphocytes
(Table 1)
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Neither NAD+ nor GSH produced any increase in the [Ca2+]i in CD38-t HeLa and 3T3 cells (23), thus ruling out any direct effect of either CD38-internalizing agent on the intracellular calcium levels.
The time course of the calcium increase after addition of the CD38-internalizing agents to CD38+t HeLa cells was investigated. Upon addition of NAD+ (2 mM) or GSH (10 mM) to cells the [Ca2+]i increased to reach maximal values (approximately twice the initial concentration) within 40–45 min (Fig. 2 ).NAC, a stable thiol-containing molecule, elicited the same extent of calcium increase as GSH and NAD+ (Fig. 2) . After removal of any internalizing compound, the [Ca2+]i decreased progressively and almost linearly, with 190, 165, and 105% of the basal [Ca2+]i being recorded after 2, 6, and 24 h, respectively (mean of four experiments). The same kinetics and extent of [Ca2+]i increase were also observed on CD38+t 3T3 cells and on constitutively CD38+ Namalwa cells upon addition of each of the three internalizing agents. This calcium increase was not due to influx of Ca2+ from the extracellular medium because all experiments were performed in zero calcium saline solution (see Materials and Methods). Moreover, no increase in [Ca2+]i was detectable upon addition of either NAD+ or GSH to thapsigargin-pretreated CD38+t cells (not shown).
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To demonstrate that the increase in [Ca2+]i is specifically dependent on CD38 internalization, GSH was externally supplemented to cells where CD38 internalization had been prevented by prior covalent cross-linking of surface membrane proteins. In the absence of glutaraldehyde, exposure of CD38t+ HeLa cells to 10 mM GSH (40 min) resulted in an approximately 45% decrease of the ectocellular cyclase activity due to CD38 internalization (inset to Fig. 3 )and produced a marked (80%) and time-dependent increase in [Ca2+]i (Fig. 3) . After glutaraldehyde pretreatment of the cells, however, the surface cyclase activity remained constant upon addition of GSH (inset to Fig. 3 ). Thus, exposure of cells to glutaraldehyde effectively prevented CD38 internalization without interfering with its enzymatic activity. Under these conditions, no [Ca2+]i increase was observed in glutaraldehyde pretreated cells after addition of GSH (Fig. 3) .
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NAD+ efflux from intact CD38- cells
These results clearly demonstrated that the calcium increase is
dependent on ligand-induced transfer of CD38 into the cytoplasm. After
internalization, however, the catalytic domain of CD38 is secluded from
cytosolic NAD+ by the endocytotic vesicle membrane (21).
When internalization is triggered by exposure of cells to
NAD+, availability of NAD+ to the cyclase
activity of CD38 is ensured by endocytosed NAD+ inside the
vesicles. However, GSH and NAC induce the same extent and kinetics of
[Ca2+]i increase as NAD+ (Table 1
and Fig. 2
) on all cell types tested. This is consistent for the
occurrence of a transport mechanism for cytosolic NAD+ in
the membrane of the CD38 internalization vesicle. Subsequent efflux of
catalytically generated cADPR from the vesicle to reach its microsomal
receptors is made possible by the recently discovered
cADPR-transporting activity of CD38 itself (22).
The presence of a transport mechanism responsible for NAD+ influx into the endocytotic vesicles would also allow NAD+ to be released from the cell. Thus, we investigated NAD+ efflux from wild-type HeLa and 3T3 cells. Absence of any NAD+-hydrolyzing activity on the membrane of these cells was preliminarily checked as a necessary prerequisite for the detection of NAD+ efflux (S. Bruzzone, unpublished data). As illustrated in Fig. 4 ,repeated washings of adherent HeLa cells at 37°C in DME resulted in the progressive decrease of intracellular NAD+ and NADH content ([NAD++NADH]i), which was more extensive during the first three medium changes (Fig. 4) . The sum of the NAD++NADH released into the supernatants of the sequential washings closely paralleled the amount of pyridine dinucleotide lost from the cells (Fig. 4) . NAD+ was the most abundant species both in the cell extracts and in the supernatants, accounting for 90% of the total NAD+ + NADH pool.
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Approximately 25% of the intracellular NAD+ + NADH pool was consistently released from the cells into the supernatant. This NAD+ efflux was not due to cell lysis, the extent of which in each supernatant was below 0.5% (Fig. 4) . Comparable results were also obtained with wild-type 3T3 cells and with the CD38- murine fibroblast cell line M210B4 (not shown).
Susceptibility to inhibition is one of the key features of protein-mediated transport mechanisms. In preliminary experiments we attempted to inhibit efflux of metabolically labeled [14C-nicotinamide]-NAD+ from HeLa cells with extracellular unlabeled NAD+. We tested extracellular NAD+ concentrations of up to 0.25 mM without observing significant inhibitory effects. Higher extracellular concentrations of NAD+ were found to interfere with the HPLC separation of the excess [14C]-nicotinamide present in the cells from the NAD+ peak (34). The remarkable [NAD+]i in these cells (7.04 ±1.2 nmol NAD+ + NADH/mg protein in unwashed cells) may, however, require a much higher than 0.25 mM extracellular NAD+ concentration to create and maintain a gradient against NAD+ efflux. To circumvent these technical difficulties, the NAD+ analog 8-N3-NAD+ was synthesized (28, 29). Addition of the NAD+ analog to the supernatant, at progressively increasing concentrations (the highest being 1.2 mM), markedly inhibited efflux of metabolically labeled [14C] NAD+ from HeLa cells (Fig. 5 ).Upon removal of the analog, NAD+ efflux steadily resumed (Fig. 5) . This reversible inhibition of NAD+ efflux by 8-N3-NAD+, which was observed on 3T3 cells as well, rules out conclusively cell lysis as the source of extracellular NAD+ and indicates the existence of an inhibitable, bidirectional NAD+ transport in the membrane of these cells.
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These results suggested an experimental setting to maximize NAD+ efflux from these cell lines by maintaining a near-zero extracellular [NAD+]. Adherent HeLa cells were first washed to remove the intracellular exchangeable NAD+ and cells were subsequently cultured with or without medium changes for 7 h. As shown in Fig. 6 ,a much higher amount of NAD+ was released into the supernatant when the medium was changed every 2 h as compared to cell cultures where the medium was not replaced and the intracellular and extracellular NAD+ concentrations could equilibrate. Approximately 3% of the intracellular NAD+ was released into the medium per hour when the medium was changed. The [NAD+]i at the beginning and end of the 7 h incubation period was not significantly different: thus, biosynthesis apparently prevented NAD+ depletion inside the cells. In fact, incubation of the cells under the same culture conditions (medium changes every 2 h) but without nicotinamide in the medium resulted in a 50% reduction of NAD+ release into the supernatant.
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Influx of pyridine dinucleotides into intact cells
The results shown in Fig. 6
indicate that NAD+
transport depends on the concentration gradient across the
plasmamembrane. To demonstrate unequivocally the bidirectional nature
of dinucleotide transport and to investigate its kinetic properties, we
analyzed the influx of pyridine dinucleotides into HeLa and 3T3 cells.
A short incubation time was necessary to prevent intracellular
conversions between the various dinucleotide pools. As shown in
Table 2
,incubation of the cells with each of the four pyridine dinucleotide
species tested resulted in a two- to ninefold increase in intracellular
concentration without any significant modifications of the levels of
the other dinucleotides. Partial loss of the internalized nucleotides
during washings was confirmed, for each incubation experiment, by HPLC
analysis of the last washing, which indicated dinucleotide
concentrations 10- to 20-fold higher than that present in the last
washing of the control flask (see Materials and Methods). If this loss
was taken into account and added to the intracellular amount of
dinucleotides actually measured in cell extracts, the values of influx
were closely comparable to those obtained with the silicon oil
procedure. Similar results were also obtained with 3T3 cells.
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Finally, the kinetics of NAD+ influx were investigated both
in CD38- (HeLa and 3T3) cells and in CD38+
(Namalwa and Jurkat) cell lines. Influx of NAD+ into intact
HeLa cells followed hyperbolic saturation kinetics (not shown). The
apparent Km for NAD+ transport was
approximately 15 mM, similar for all cell types tested and close to
values previously recorded on resealed human erythrocyte membranes
(22). The initial rate of NAD+ influx was calculated to be
approximately 10 nmol/(min · mg cell protein) for HeLa and 3T3
cells. These values are the mean from results obtained with both
experimental settings described under Materials and Methods. Influx of
a number of NAD+ analogs was also investigated on both HeLa
and 3T3 cells: while modifications in the adenine moiety of the
molecule apparently did not affect transport
(8-N3-NAD+, NHD+ and
NGD+ were internalized similarly to NAD+),
modifications at the nicotinamide ring either decreased
(
-NAD+) or completely prevented (FAD) internalization
(not shown). These results seem to indicate some extent of specificity
of this pyridine dinucleotide transport system.
Role of cADPR in the increased [Ca2+]i
induced by CD38 internalization
8-N3-NAD+ is a substrate of both the
enzymatic and of the cyclic nucleotide-transporting activities of CD38
(L. Franco, unpublished data), but 8-N3-cADPR is a potent
antagonist of cADPR-induced calcium release (28, 29). We took advantage
of the fact that 8-N3-NAD+ is also internalized
by the pyridine dinucleotide-transporting system (see above) to devise
an experiment aimed to causally correlate endocytosis of CD38,
intracellular production of cADPR and [Ca2+]i
increase. Adherent CD38+t HeLa cells were first
depleted of their cytosolic free NAD+ by continuous
perfusion with DME in the recording chamber described in Materials and
Methods. Cells were incubated with either 10 µM NAD+ or
20 µM 8-N3-NAD+ for 30 min under continuous
flow, and the [Ca2+]i and the surface cyclase
activity on NGD+ were determined on the same sample. The
decrease of surface cyclase activity was closely comparable in
NAD+- and 8-N3-NAD+-incubated
cells, down to 48% and 50% of starting values, respectively. In
addition, confocal fluorescence microscopy confirmed a comparable
extent of CD38 internalization with both dinucleotides (not shown). The
[Ca2+]i, however, increased only in the cells
incubated with NAD+ while keeping fairly stable in the
cells exposed to the NAD+ analog (Fig. 7
).These results demonstrate a causal correlation between the
intracellular cADPR production by internalized CD38 and the
[Ca2+]i increase. Moreover, the experimental
setting of the continuous flow enabled us to demonstrate that
micromolar concentrations of NAD+ are sufficient to elicit
both internalization of CD38 and enhanced
[Ca2+]i, provided fresh NAD+ is
continuously supplied to the CD38+ cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The preliminary demonstration (Fig. 1 and Table 1 ) that CD38 internalization occurs in different cell types (Namalwa, CD38+t HeLa and 3T3 cells, and PBL), with a similar mechanism (NAD+- or GSH-induced, vesicle-mediated endocytosis) and with comparable kinetics (approximately 50% being internalized within 1 h incubation), supports the conclusion that internalization is an intrinsic property of CD38 itself and does not depend on cell type or membrane protein environment.
After CD38 internalization, the [Ca2+]i increased to maximal values (i.e., approximately twice the initial concentration) within 45–60 min incubation in all cell types tested and with either internalizing agent used—NAD+, GSH, or NAC (Table 1 and Fig. 2 ). These kinetics of calcium increase closely parallel the process of CD38 internalization and are different from the transient (seconds) and more limited (around 50%) increase of cytosolic calcium that immediately follows exposure of CD38+-transfected HeLa cells to NAD+ (22). The rapid elevation of [Ca2+]i triggered in the latter case is due to the recently demonstrated property of CD38 to behave as an active channel for generation and selective influx of cADPR across membranes (22). On the contrary, the sustained increase of [Ca2+]i after exposure of cells to NAD+, GSH, or NAC is mechanistically related to 1) specific CD38 internalization, as demonstrated by blockade of both processes by prior cross-linking of membrane proteins with glutaraldehyde (Fig. 3) , and 2) the consequently enhanced formation of intracellular cADPR, as shown by the experiments with 8-N3-NAD+ (Fig. 7) .
A significant outcome of the present investigation was the demonstration of a hitherto unrecognized transport system for NAD+ in cell membranes and of its role in the regulation of cytosolic calcium related to the ligand-induced CD38 internalization. Evidence for this transport emerged from the attempt to explain a topological inconsistency of the CD38/cADPR system during either internalization (this study) or export (23) of membrane-bound CD38. In fact, due to the reverse polarity of asymmetrically oriented CD38 molecules in the plasmamembrane, both endocytotic and exocytotic membrane vesicles display the carboxyl-terminal catalytic region of CD38 in the intravesicular space, therefore not directly accessible to its cytosolic substrate NAD+. Accordingly, for the endocytotic or exocytotic vesicles to behave as cADPR-dependent calcium-releasing systems in cells, as actually observed in different experimental conditions (21,23), two requirements should be met: 1) availability of NAD+ to the apparently secluded active site of CD38, and 2) release of catalytically generated cADPR from both types of vesicles into the cytosol, allowing the cyclic nucleotide to reach its receptor-operated calcium stores (24–27). As mentioned, membrane-embedded CD38 was shown to meet the latter requirement since it proved to be a catalytically active transporter of cADPR, but not of NAD+, across membranes (22), by virtue of its oligomeric (dimeric and tetrameric), channel-generating structure (22, 35). The need to identify an NAD+-translocating system prompted us to investigate NAD+ fluxes across the plasmamembrane, since the endocytotic vesicles derive from the plasmamembrane itself. These studies were carried out on intact cells, mostly using the wild-type human HeLa and murine 3T3 cells, which do not exhibit any NAD+-cleaving enzyme activity at their outer surface. Therefore, both influx and efflux of NAD+ from these cells could be investigated without any interference by NAD+ degradation.
The present findings, besides conclusively demonstrating that cell membranes are permeable to NAD+, also identify some properties of this novel transport system. This proves to be a passive transport, because the direction of pyridine dinucleotide flux depends on the concentration gradient only and no apparent energy source is required. The transport system is characterized by low dinucleotide specificity and follows hyperbolic saturation kinetics. No significant effect of temperature was observed in influx and efflux experiments (not shown), this suggesting that dinucleotide translocation occurs across a channel rather than involving a transporter oscillating between different conformations (36).
The demonstration of NAD+ fluxes through the plasmamembrane of intact mammalian cells is, to our knowledge, without precedent, although slow NAD+ leakage across the mitochondrial membranes has been described both in plant (37) and mammalian cells (38). We found that approximately 25% of intracellular NAD+ can be lost from HeLa or 3T3 cells upon repeated washings: this `exchangeable' NAD+ (over 90% oxidized) is probably the cytosolic, free (i.e., non enzyme-bound) dinucleotide (Fig. 4) . Efflux of NAD+ can be reversibly inhibited by the NAD+ analog 8-N3-NAD+ (Fig. 5) , but attempts to irreversibly inhibit NAD+ efflux from HeLa cells by photolysis were unsuccessful. However, photoaffinity labeling does not necessarily yield stoichiometric labeling: values between 0.01 and 1.0 mol to mol are usually reported (39).
Influx of all pyridine dinucleotides (NAD+, NADH, NADP+, and NADPH) occurs across the plasmamembrane of intact HeLa and 3T3 cells: although some degree of intracellular reequilibration between the various oxidized and reduced dinucleotide pools takes place at longer incubation times (not shown), selective increase of the intracellular concentration of each extracellularly added nucleotide was obtained by reducing the incubation time to 5 min (Table 2) . NAD+ influx into both CD38- HeLa and 3T3 cells and CD38+ Namalwa and Jurkat cells displays hyperbolic saturation kinetics: over the short incubation time sufficient for NAD+ influx (below 2 min), a very limited NAD+ degradation occurred with CD38+ cells. Incubation of these cells with concentrations of nicotinamide and ADPR as low as those produced ectocellularly from NAD+ during the incubation time failed to determine any increase of the [NAD+]i. This control confirms the existence of NAD+ transport also in the plasmamembrane of CD38+ lymphocytic cell lines.
It is significant that micromolar NAD+ concentrations were effective in inducing both CD38 internalization and calcium increase (Figs. 1, 7) . Although reported extracellular NAD+ concentrations are approximately 10 nM in rat cerebellar interstitial fluid (6) and 50–60 nM in human blood serum (T. F. Walseth, personal communication), these values represent average levels of the dinucleotide in the extracellular compartment: micromolar NAD+ concentrations in the extracellular fluids may be reached in specific conditions (e.g., apoptosis in lymphnodes; see ref 40 ) and in selected districts, also in view of the demonstrated ability of fibroblast and epithelial cell lines to steadily release NAD+ (Fig. 6) . It is likely that also micromolar concentrations of GSH or reducing agents can induce internalization of CD38 in vivo; a relevant observation is that oral administration of therapeutic doses of N-acetylcysteine for 3 days resulted in a 30% decrease of ectocellular ADP-ribosyl cyclase activity in PBL (L. Guida, E. Zocchi, and A. De Flora, unpublished data).
Extracellular GSH proved not to affect the efflux of [14C]-NAD+ from metabolically labeled HeLa cells (not shown). Accordingly, the presence of high GSH concentrations in the endocytotic vesicles generated by GSH-induced internalization does not restrict the influx of cytosolic NAD+ into these vesicles. The high intracellular concentrations of NAD+ in the cell lines investigated (HeLa and 3T3) and the high rate of NAD+-transporting activity are consonant with a remarkable efficiency of cytosolic NAD+ influx into the vesicles.
The identification of a still unrecognized dinucleotide transport system, responsible for bidirectional NAD+ flux across membranes, and the recently established nature of CD38 as a selective and unidirectional cADPR transporter (22) allow us to rationalize the complex interplay of intracellular trafficking of NAD+ and of cADPR as well. Both transport systems seem to operate sequentially inside cells so as to bypass apparent processes of compartmentalization and to produce functionally significant, cytosolic cADPR concentrations during endocytosis or exocytosis, effective on intracellular calcium levels. As previously postulated (22), this subcellular type of structural and functional organization centered on opposite polarity of NAD+ and cADPR transports may provide the dual advantage of preventing unrestricted NAD+ consumption, while representing a flexible means for finely tuning the NAD+-dependent regulation of [Ca2+]. Unequivocal confirmation of the regulatory role of NAD+ on [Ca2+]i should come from elucidation of the patterns of NAD+-induced, cADPR-related [Ca2+]i increases in specific cell types (e.g., oscillatory vs. sustained) and from identification of selected molecular targets of these calcium movements, e.g., expression of specific genes (41–43). Dissection between distinctive functional consequences of oscillatory and sustained increases of [Ca2+]i, as possibly elicited by different schedules of exposure and by different concentrations of internalizing compounds, will be important in this respect, since CD38 per se is suitable to elicit both types of effects as a steady cADPR channel (22) and as a catalytic component of slowly cADPR-generating vesicles, respectively.
The recognized efflux of NAD+ from the plasmamembrane of intact cells and the properties of transmembrane CD38 suggest that the CD38-related regulation of [Ca2+]i is not restricted to a single CD38+ cell. Rather, it may occur through transfer of NAD+ from any cell type to CD38+ cells, according to paracrine mechanisms whereby NAD+ could act as a hormone, involved in cell-to-cell interactions, and cADPR as its intracellular second messenger. These results, besides accounting for a hithertho unknown cross-talk of signal metabolites between different cell compartments or between different cells, also identify potential targets for the development of new drug molecules affecting intracellular calcium levels in various diseases characterized by imbalanced calcium homeostasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: 3T3 cells, NIH 3T3 cells; cADPR, cyclic adenosine diphosphate ribose; [Ca2+]i, cytosolic calcium concentration; mAb, monoclonal antibody; NAADP+, nicotinic acid adenine dinucleotide phosphate; GSH, glutathione (reduced form); NAC, N-acetylcysteine; 8-N3-NAD+, 8-azido-NAD+; 8-N3-AMP, 8-azido-AMP; PBL, peripheral blood lymphocytes; DME, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; Ig, immunoglobulin; NMN, nicotinamide mononucleotide; NGD+, nicotinamide guanine dinucleotide; NHD+, nicotinamide hypoxanthine dinucleotide; G6PD, glucose 6-P dehydrogenase; HPLC, high-pressure liquid chromatography; TCA, trichloroacetic acid; ADPR, adenosine diphosphate ribose; HLA, histocompatibility leukocyte antigen; FAD, flavine adenine dinucleotide.
Received for publication August 17, 1998.
Revision received October 16, 1998.
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), 10 mM GSH (
), or 10
mM N-acetylcysteine (
) at 25°C. Traces shown are representative of
4 consistent experiments. Each point is the mean of the values (no=200)
recorded in 2 min. Similar results were obtained with
CD38+t and CD38-t 3T3
cells. As reported (23), the basal [Ca2+]i is
higher in CD38+t cells compared with
CD38-t controls. The inset shows the
[Ca2+]i after removal of GSH by extensive
washing. Comparable data were observed upon removing either
NAD+ or NAC as internalizing agents.
) 0.25% glutaraldehyde for 10 min at 37°C and
washed. On duplicate incubations, the [Ca2+]i
was continuously monitored after addition of 10 mM GSH (arrow) at
25°C, while surface cyclase activity, measured on NGD+
(inset), was determined at the end of 40 min incubation with GSH (no=5)
at 25°C. Traces shown are representative of results obtained in 5
consistent experiments. Each point is the mean of the values (no=200)
recorded in 2 min.
) and supernatants
(
) was estimated by the enzymatic cycling procedure (33) and the
total amount of NAD+ + NADH released was calculated as the
sum of the pyridine dinucleotide present in each individual medium
change. The percentage of cell lysis in each washing was below 0.5% as
determined by assay of G6PD and hexokinase activities in the
corresponding supernatants. Results of a representative experiment are
shown. Similar results were obtained with wild-type 3T3 cells.
) HPLC. Control (
