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A transport mechanism for NAADP in a rat basophilic cell line

DiSCAFF and DFB Center, Università del Piemonte Orientale, Novara, Italy

¹Correspondence: DiSCAFF, Via Bovio 6, Novara 28100, Italy. E-mail: billington{at}pharm.unipmn.it

SPECIFIC AIMS

The goals of this work were to investigate whether the second messenger NAADP could be translocated from the extracellular medium across the plasma membrane into the cytosol and have the potential to act as a paracrine/autocrine signal.

PRINCIPAL FINDINGS

1. RBL-2H3 cells possess a concentrative uptake mechanism for NAADP
Incubation of RBL-2H3 cells with [³²P]NAADP resulted in a substantial accumulation of the radioactive second messenger in cells (6.6±0.6 fmol in 1 h when 125 fmol of [³²P]NAADP were added extracellularly) (Table 1 ). This accumulation was due to a transporter since 1) it was significantly abrogated at 4°C; 2) it was unaffected by pre- or postincubation with an excess nonradioactive NAADP; 3) nonradioactive NAADP competed for the uptake mechanism dose-dependently, while NAAD competed only modestly at 10 µM and NAD did not compete; 4) the mechanism was time and concentration dependent (a rapid uptake phase was observed in the first 2 min of incubation, then a slower phase for up to at least 4 h); and 5) the mechanism was concentrative, resulting in an estimated concentration of this molecule inside cells far superior to that of the extracellular medium. For example, when cells were incubated with 250 pM [³²P]NAADP in the medium, the concentration inside cells was estimated to be 962 ± 101 pM(n=9) after 10 min and 3.93 ± 0.36 nM after 60 min (n=18).

2. Ion requirements and pharmacological characteristics of the uptake mechanism
NAADP transport was dependent on the presence of extracellular Na⁺ and Ca²⁺ ions, since experiments performed in the absence of either ion resulted in a marked decrease of uptake (50 and 80%, respectively). Removal of K⁺ ions, however, had no effect on transport. NAADP transport was partially blocked by 100 µM dipyridamole but was unaffected by 100 µM nitrobenzylthioinosine, uridine, or adenosine (Fig. 1 ). These data suggest that transport is not via either concentrative or equilibrative nucleoside transporters despite the partial block by dipyridamole. Experiments performed in the neuroblastoma cell line SK-N-BE suggest that this transport system is also present in neuronally derived cells and may be more efficient in accumulating NAADP.

View larger version (16K): [in this window] [in a new window]   Figure 1. A) Ion dependence of nucleotide transport (NAADP, filled bars; cADPR, open bars). Data are expressed as mean ± SE, n= 8–12. B) Pharmacology of nucleotide transport (NAADP filled bars, cADPR open bars). Data expressed as mean ± SE, n= 4–11. All experiments were carried out for 10 min.

3. cADPR uptake shows different characteristics compared with NAADP uptake
In RBL-2H3 cells, [³²P]cADPR was also transported, in line with previous reports in other cell models, with a similar rate compared with NAADP (2.0±0.2 fmol/10 min). Yet, differences were observed in the pharmacological profiles of transport of cADPR and NAADP: namely, cADPR transport was completely insensitive to removal of extracellular Ca²⁺, but was significantly reduced by the removal of extracellular Na⁺. However, it was significantly blocked by both dipyridamole and nitrobenzylthioinosine but, surprisingly, not by 100 µM uridine or 100 µM adenosine.

4. NAADP binding sites are present in homogenates of RBL-2H3 cells
In RBL-2H3 cell homogenates, a binding site could be detected with a K_d of 125 ± 20 nM and a B_max of 4.5 ± 0.8 pmol/mg. This binding site was specific for NAADP over NADP and was unaffected by NAD, ADPR, and NAAD in line with the characteristics displayed with the characterized binding sites in other systems. Incubation of cells with a fluorescent analog of NAADP (1,N⁶-etheno-aza-NAADP) resulted in uptake of this molecule in the cell cytosol and in the enrichment of fluorescence in the vicinity of granule-like structures that partially co-localize with lysotracker red staining, suggesting that uptake and binding to intracellular structures occurs sequentially. Incubation of cells with 100 µM NAADP after 1,N⁶-etheno-aza-NAADP displaced 1,N⁶-etheno-aza-NAADP from these structures with a consequent increase in cytosolic fluorescence.

5. Extracellular application of NAADP induces cytosolic Ca²⁺ transients
Application of 100 nM NAADP to the extracellular medium of Fluo-4/Fura-2-loaded RBL-2H3 cells stimulated transient rises in cytosolic Ca²⁺. Transients were observed in 33% of cells and were variable both in the latency, and the type of signal observed (slow rises, spikes, and oscillations). Application of 100 nM NADP did not stimulate Ca²⁺ rises.

CONCLUSIONS AND SIGNIFICANCE

NAADP has been identified as a Ca²⁺-releasing messenger in some model systems, ranging from echinoderms and plants to mammalian exocrine cells. In most systems investigated to date, it has been demonstrated that NAADP triggers Ca²⁺ release from intracellular stores via a channel that is distinct from those sensitive to ryanodine and IP₃. Although this clear-cut distinction has been challenged in some systems and independently of the number of channels used, the use of three distinct intracellular messengers allows cells to increase their repertoire of available Ca²⁺-signals to encode intracellular messages. Such increased complexity might also be due to the existence of multiple intracellular Ca²⁺ pools. NAADP signaling has also been shown to rely on an intracellular Ca²⁺ pool distinct from the endoplasmic reticulum, and this has been suggested to be an acidic organelle. Although controversy still exists on the exact nature of this store, it has been suggested to be a lysosomal compartment or a secretory vesicle.

Among the important and as yet unresolved issues in the field is the site of NAADP production in cells. NAADP has been shown to be present in cells and changes in its intracellular concentrations have been reported in both sea urchin and mammalian cells. Nonetheless, the only enzymes in mammals that have been shown to produce NAADP belong to the CD38/CD157 glycohydrolase family, which possess their catalytic domain on the extracellular side. Although some CD38 protein is present on intracellular membranes, this topological paradox poses difficulties in interpreting how NAADP signaling can be fast and efficient.

In the present paper, we have investigated whether extracellular NAADP, which per se is not freely diffusible across the plasma membrane because of its four negative charges at physiological pH, can be transported across the plasma membrane in a basophilic cell line to reach its intracellular target receptor. This is of particular relevance since it has been recently demonstrated that extracellular NAADP can trigger Ca²⁺ responses in both glia and neurons.

Cells incubated with [³²P]NAADP transported a substantial amount of radioactivity. Approximately 5% of total radioactivity was recovered in cells after 1 h incubation and 14% after 4 h. This accumulation of radioactivity was not due to binding to the plasma membrane, since it was not displaceable by subsequent additions of excess nonradioactive NAADP (Table 1) .

Our data demonstrate that extracellular NAADP can pass the plasma membrane and concentrate intracellularly. This could be due to endocytosis/pinocytosis or to a transport mechanism. A number of direct evidences can rule out endocytosis/pinocytosis: 1) the mechanism presented is concentrative, and concentrations in cells reach significantly higher levels (4-fold in 10 min) than in the extracellular medium, which is incompatible with pure endocytosis; 2) the uptake of [³²P]NAADP shows competition by unlabeled NAADP (if endocytosis were responsible for the effects observed, it would be expected that radioactive and nonradioactive compounds would not compete with each other); 3) in the experiments described above the amount of [³²P]NAADP that is recovered in cells in 1 h amounts to 5.3% of the total extracellular fluid (500 µL)—in a conservative estimate, this volume (25 µL) represents 30-fold the volume of the cells used, while no increase in cell volume is observed in phase contrast upon NAADP addition; 4) the mechanism is sensitive to a known blocker of nucleoside transport; and 5) the sensitivity of the mechanism to the calcium chelator EGTA and to the removal of extracellular Na⁺ tend to suggest that constitutive endocytosis cannot account for the results observed.

The rate of transport observed is compatible with the intracellular responses observed by Heidelmann et al. (2005), who reported intracellular Ca²⁺ increases upon 5 µM NAADP addition with a latency of 30 s. In RBL cells this protocol would lead to mid-nanomolar concentrations of NAADP intracellularly after 30 s, which fits well with the efficient concentrations reported for Ca²⁺ release in intact cells. Indeed, application of 100 nM NAADP to the extracellular medium of RBL-2H3 cells was able to induce Ca²⁺ transients in the cytosol after a latency.

Ionic dependency and pharmacological data suggest that, in RBL-2H3 cells, transport systems exist for NAADP and cADPR, which are distinct from the nucleoside transporter systems previously reported. We propose that either separate, unrelated, systems exist for the transport of cADPR and NAADP or that one transport mechanism that displays differential regulation with regard to Ca²⁺ and NBMPR for NAADP and cADPR transport is responsible for the phenomenon we observed (Fig. 1) .

If NAADP enters cells, it would be expected to be recognized by an intracellular receptor. Indeed, when radioligand binding was performed on cell homogenates on ice, a specific binding site was unmasked. The affinity of this binding site (125±20 nM) is compatible with the one reported in brain but has lower affinity to that reported in heart.

We have demonstrated that NAADP can be efficiently transported in rat basophilic cells and in neuroblastoma cells. This system is a high-affinity transporter sensitive to picomolar concentrations of NAADP but at the same time has a high capacity. This report, coupled with the recent evidence that NAADP, when administered extracellularly, is capable of eliciting an intracellular response and the long-standing paradox on the presence of ectoenzymes capable of synthesizing, raises the possibility that alongside a canonical second messenger NAADP might also be an autocrine or paracrine signal. Such findings open new possibilities on how NAADP might be used to transduce signals. For example, it has been shown recently that schistosomes possess an NAADP-generating enzyme on the outer surface. It would therefore be fascinating to speculate that NAADP synthesized by these enzymes could then be used by cells of the immune system to trigger a response or as a chemoattractant.

View larger version (24K): [in this window] [in a new window]   Figure 2. Hypothetical model on the role of NAADP as a paracrine/autocrine signal. CD38-like ectoenzymes present on either the same cell, neighboring cells, or microorganisms generate NAADP. In turn, this is taken up by the transporter described here. Once inside, NAADP binds to a specific intracellular Ca2+ channel and triggers Ca2+ release. A similar pathway is present for cADPR.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5058fje;

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