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Inhibition of Ca²⁺ signaling and glucagon secretion in mouse pancreatic -cells by extracellular ATP and purinergic receptors

¹Institute of Bioengineering, Miguel Hernandez University, Elche, Spain; ²CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Spain; and ³Department of Physiology and Biophysics, Institute of Biology, Unicamp, Campinas São Paulo, São Paulo, Brazil

Submitted 3 October 2007 ; accepted in final form 14 March 2008

	ABSTRACT

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Glucagon secreted from pancreatic -cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since -cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and β-cell secretory granules. Using immunocytochemistry, we identified in -cells the P2 receptor subtype P2Y₁, as well as the P1 receptors A₁ and A_2A. In contrast, only P2Y₁ and A₁ receptors were localized in β-cells. To analyze the role of purinergic receptors in -cell function, we studied their participation in Ca²⁺ signaling. At low glucose concentrations, mouse -cells exhibited the characteristic oscillatory Ca²⁺ signals that lead to secretion. Application of ATP (1–10 µM) abolished these oscillations or reduced their frequency in -cells within intact islets and isolated in culture. ATPS, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1–10 µM) was also found to reduce Ca²⁺ signals. ATP-mediated inhibition of Ca²⁺ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y₁ and A_2A were likely involved in the inhibitory effect on Ca²⁺ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the -cell function.

confocal microscopy; islets; paracrine communication

Despite the importance of this hormone in controlling glycemia, many important gaps remain in the understanding of -cell physiology (19). Most of the data concerning -cells derive from pancreas and islet secretion studies, yet there is not much information at the cellular level. This is partly due to the scarcity of this cell type in the islet, the lack of identification patterns, and also the technical limitations of conventional methods. In recent years, novel technical approaches based on imaging and electrophysiology have allowed further studies (15, 33, 38, 45) of -cell function, especially within the intact islet, a study model the behavior of which is closer to the physiological scenario compared with isolated islet cells or cell lines. Electrical activity, Ca²⁺ signaling, and glucagon release are all stimulated with low glucose concentrations in the -cell within the islet. However, these cellular events are inhibited when glucose levels rise (15, 31, 33, 38). This inhibitory effect has been attributed to both a direct action of the sugar and to paracrine mechanisms (19, 21, 29, 31). In this regard, several paracrine factors from β-cells have been revealed as potent suppressors of glucagon secretion. Although with interspecies differences, insulin, zinc, and GABA affect -cells at different levels inhibiting glucagon release (13, 27, 42, 51).

In addition to the above-mentioned paracrine signaling molecules, other extracellular messengers could also regulate -cell secretion. ATP is highly accumulated in synaptic vesicles of nerve terminals within the islet (up to 10 mM) as well as in insulin secretory granules (up to 1 mM; Refs. 4, 12, 26, 30, 41). Once released, extracellular ATP can reach concentrations of tens of micromolar or higher on the islet cells surface (23). Additionally, ATP molecules can be converted by plasma membrane ectonucleotidases into ATP metabolites or adenosine, subsequently activating multiple purinergic receptors and inducing a plethora of effects (12). While ATP binds to plasma membrane P2 receptors, adenosine activates P1 receptors (12, 41). The role of these extracellular messengers in the islet of Langerhans has been emphasized by the presence of several purinergic receptors and the rich innervation within the islet (3, 7, 32). Additionally, it has been proposed that neural ATP release could be involved in the coordination of islet function and the pulsatility of insulin release (16, 24, 44). The regulatory effect of extracellular ATP on the electrical activity, Ca²⁺ signals, and insulin secretion has been proven in mouse, rat, and human β-cells (11, 24, 36, 44). In the case of the -cell, secretion experiments with perfused rat pancreas indicated that, whereas ATP does not have a direct effect, adenosine can promote glucagon release (6, 35). However, the effect of purinergic stimulation on -cell function has not been analyzed at the cellular level. In the present study, we identify the presence of the ATP and adenosine purinergic receptors P2Y₁, A₁, and A_2A in mouse -cells, as well as indicate their possible function. We demonstrate that extracellular ATP and adenosine are important inhibitors of Ca²⁺ signaling in the -cell and also that, unlike adenosine, ATP produces a significant suppression of glucagon release. All these results reveal a complex purinergic signaling pathway in this islet cell type and indicate an important role in the regulation of glucagon secretion, which could be of therapeutic interest in diabetes management.

	MATERIALS AND METHODS

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Islet isolation and probe loading. All protocols were approved by the Animal Care Committee of the Miguel Hernandez University in accordance with the Spanish regulations and laws for animal experimentation. Swiss albino OF1 mice (8- to 10-wk-old) were killed by cervical dislocation, and pancreatic islets were then isolated by collagenase digestion as described previously (33, 37, 40). As previously reported (33, 38), single islets were loaded with 5 µM of the acetoxymethyl form of the Ca²⁺ probe fluo 3 (Molecular Probes; Leiden, The Netherlands) for at least 1 h at room temperature in a medium containing (mM): 115 NaCl; 10 NaHCO₃; 5 KCl; 1.1 MgCl₂; 1.2 NaH₂PO₄; 2.5 CaCl₂; 25 HEPES; 1 % BSA; and 5 D-glucose, pH 7.4. All experiments were carried out at 37°C.

In some experiments, isolated islets were dispersed into single cells and clusters by enzymatic digestion in the presence of 0.05% trypsin and 0.02% EDTA for 2 min (37, 40). Isolated cells were plated onto poly-L-lysine-treated coverslips and cultured at 37°C in RPMI 1640 (Sigma, Madrid, Spain), supplemented with 10% FCS, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 5.6 mM D-glucose (37, 40). After 24 h, cells were loaded with 4 µM fluo 3 for 1 h at 37°C.

Imaging Ca²⁺ signals by confocal microscopy. For imaging experiments, islets were placed on a perfusion chamber mounted on the microscope stage and attached onto poly-L-lysine-treated coverslips for 10 min before the experiments were commenced. Islets were then perfused at a rate of 1.5 ml/min with a modified Ringer solution containing (mM): 120 NaCl, 5 KCl, 25 NaHCO₃, 1.1 MgCl₂, and 2.5 CaCl₂ pH 7.4, gassed with 95% O₂-5% CO₂. Ca²⁺ signals were monitored in individual cells within the islets using a Zeiss LSM 510 laser confocal microscope equipped with a x40 oil immersion objective (numerical aperture = 1.3). The system configuration was set to excite the Ca²⁺ probe at 488 nm and collect the emission with a bandpass filter at 505–530 nm from an optical section of 8 µm. Images were collected at 2-s intervals. Temporal series were treated with a low pass filter and processed using the digital image software of the confocal microscope (33, 38, 39). Individual cells loaded with fluo 3 were easily identified at the periphery of the islet (see Fig. 2A). It has been previously reported (33, 38, 39) in islets as well as in other specimens that the fluorescent probes have difficulties penetrating the center of thick samples. However, this is not a problem since all the cell types are represented in the peripheral layers of the islet (5, 33, 39, 43). Pancreatic -cells were functionally identified by their characteristic oscillatory Ca²⁺ signal at low glucose concentrations (2, 31, 33, 38, 39, 42, 47). In culture experiments, single -cells were further identified by their response to epinephrine (18, 29).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Extracellular ATP and adenosine inhibit Ca2+ signals induced by low glucose concentrations in -cells. A: confocal image of an intact mouse islet loaded with the Ca2+-sensitive fluorescent dye fluo 3. Images were acquired from an optical section (8 µm) close to the equatorial plane. Several individual cells were easily identified at the periphery of the islet (white arrows). Color scale: blue is low [Ca2+]i and red is high [Ca2+]i. Scale bar = 50 µm. B–D: records of fluorescence intensity vs. time illustrate the effect of extracellular ATP (B, C) and adenosine (D) on the characteristic -cell oscillatory Ca2+ response at 0.5 mM glucose. ATP and adenosine markedly reduced the frequency of these oscillations or totally abolished the Ca2+ signal. E: mean frequency values of Ca2+ signals in control conditions (black column) and after stimuli application (gray column). F: frequency (%) of Ca2+ signals after stimuli compared with control conditions. Controls in E and F were 0.5 mM glucose for all experiments except 0.5 mM glucose plus suramin (100 µM) for suramin experiments. Data in E and F are means ± SE. *Statistically significant (P < 0.05) compared with controls. ns, Nonsignificant (P > 0.05); G, glucose; Aden, adenosine; Sur, suramin; F0 fluorescence signal at beginning of a record; F, F–F0.

Glucagon secretion. Batches of 15 islets were preincubated for 60 min at 37°C in 0.5 ml of Krebs-Ringer bicarbonate buffer supplemented with 15 mM HEPES, 0.5% BSA, and 5.6 mM glucose, pH 7.4 (25). Then, islets were incubated at 37°C for 60 min with Krebs-Ringer bicarbonate buffer supplemented with 0.5 mM glucose and additional reagents as indicated in RESULTS (see Figs. 3 and 4). At the end of the incubation, the medium was aspirated and assayed for glucagon using a commercial radioimmunoassay kit (GL-32K; Linco Research, St. Charles, MO). Glucagon release from intact islets was represented as described previously (25).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Glucagon secretion from intact islets after 1 h of incubation with different test agents. A: glucagon release in the presence of ATP, adenosine, Co2+ (5 mM), and 10 and 20 mM glucose was compared with control 0.5 mM glucose (n = 20–28 for each condition). B: glucagon release in the presence of adenosine and adenosine plus H-89 (10 µM; n = 5). Effect of glucose is also shown. H-89 was present throughout the experiment. Data are means ± SE. *Statistically significant (P < 0.05) compared with 0.5 mM glucose; ns, nonsignificant (P > 0.05) compared with 0.5 mM glucose; #significant (P < 0.05) compared with adenosine.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of ADPβS (A), CGS-21680 (B), and 2-chloro-N6-cyclopentyl adenosine (CCPA; C) on -cell Ca2+ signals induced by low glucose levels. D: mean frequency values of Ca2+ signals in control conditions (black bars) and after stimuli application (gray bars). Effect of the endogenous ligands ATP and adenosine are also shown. E: frequency (%) of Ca2+ signals after stimuli compared with control conditions. F: glucagon release in the presence of 10 and 20 mM glucose, adenosine, and CGS-21680 was compared with secretion at 0.5 mM glucose. Controls in D–F were 0.5 mM glucose for all experiments. Data in D–F are means ± SE. *Statistically significant (P < 0.05) compared with controls (ns; P > 0.05).

	RESULTS

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Multiple purinergic receptors coexist in the -cell. Among the extensive group of purinergic receptor subtypes described so far (12, 41), there is evidence that P2Y₁, A₁, and A_2A receptors may modulate islet function and/or glucagon release (6, 17, 35, 36). However, the existence of these three receptors at the protein level and their cellular location have not been provided. To study the presence of these receptors in the -cell, we analyzed cultures of isolated islet cells by immunocytochemistry and confocal microcopy (Fig. 1). Identification of the P2 receptor subtype P2Y₁ and the P1 receptors A₁ and A_2A was all demonstrated in glucagon-containing cells (Fig. 1A). As for β-cells, only P2Y₁ and A₁ but not A_2A receptors were identified (Fig. 1B) in agreement with previous pharmacological studies on β-cell function (1, 17, 35, 36). To prove the specificity of this staining, we performed a control experiment using blocking peptides for the antibodies against purinergic receptors (Supplemental Fig. S1). This procedure resulted in a marked decrease of the staining intensity, indicating that cell labeling was highly specific. Additionally, further experiments with different antibodies gave similar results about the presence of these purinergic receptor subtypes in the -cell (Supplemental Fig. S2).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Immunocytochemical analysis of purinergic receptors in isolated islet cells. Insulin (Insul) and glucagon (Gluc) staining is shown in red while labeling of the different receptors is shown in green. Corresponding transmitted-light image is also displayed at right. The 3 receptor subtypes were localized in glucagon-containing -cells (A) while only P2Y1 and A1 receptors were found in cells containing insulin (B). Scale bar = 10 µm.

Extracellular ATP and adenosine inhibit Ca²⁺ signaling in -cells.

Involvement of P2Y₁, A₁, and A_2A receptors in -cell Ca²⁺ signaling. In addition to the identification of P2Y₁, A₁, and A_2A receptors in the pancreatic -cell, we investigated whether these receptors were functional and if they were involved in the effect of ATP and adenosine on Ca²⁺ signaling. Although pharmacological tools are not very selective with purinergic signaling (12, 41), we used some agonists with higher affinity for the receptors identified by immunocytochemistry. ADPβS has been previously used in β-cells and other cell types to characterize the P2Y₁ receptor due to its major affinity for this receptor with respect to other subtypes (12, 35, 49). 2-Chloro-N6-cyclopentyl adenosine (CCPA) and CGS-21680 are relatively selective agonists for A₁ and A_2A receptors, respectively (12, 41). As shown in Fig. 4, A and B, both ADPβS and CGS-21680 produced a reduction in the frequency of Ca²⁺ oscillations. Although they were not as efficient as the endogenous ligands ATP or adenosine, both pharmacological agonists mimicked the inhibitory effect (Fig. 4, D and E). On the contrary, CCPA had no significant effect on Ca²⁺ signals (Fig. 4, C–E). Therefore, we show that, unlike A₁ receptors, P2Y₁ and A_2A receptors may be functional in -cells and involved in the regulation of Ca²⁺ signaling. Since adenosine exhibited a different effect on Ca²⁺ signals and secretion, we also tested the effect of CGS-21680 on glucagon release. As shown in Fig. 4F, activation of A_2A receptors by CGS-21680 stimulated glucagon secretion at 0.5 mM glucose, which points to a dual action as well.

Purinergic stimulation directly regulates -cells. The presence of purinergic receptors in the -cell and their involvement in Ca²⁺ signaling (Figs. 1 and 4) supported the idea that ATP and adenosine act directly on -cells rather than through paracrine mechanisms. Additionally, it has been demonstrated that both extracellular ATP and adenosine inhibit β-cell secretion in mouse islets (1, 6, 22, 36). In the case of ATP, this effect results from a strong interaction with the exocytotic machinery and occurs independently of the generation of a Ca²⁺ transient (36). Thus, the ATP and adenosine actions on -cell Ca²⁺ signals should not be due to a β-cell paracrine mechanism. Regardless of this ATP blocking effect on the β-cell, we further inhibited its secretory response using clonidine. Although this adrenergic agonist prevents insulin secretion at different levels, particularly in the exocytotic process (8, 28, 34), it has no effect on mouse -cells (25, 50). In the presence of clonidine, ATP (10 µM) reduced the frequency of Ca²⁺ oscillations to 53.84 ± 9.93% in -cells, similar to the effect obtained by ATP alone (Fig. 5, A, B, and C; Table 1). Finally, to further prove the direct effect on -cells, we performed some experiments with cultures of single isolated cells at low densities that were perfused at high flow rates (5 ml/min). In these conditions, paracrine interactions are negligible (42). As shown in Fig. 5D, ATP (10 µM) inhibited Ca²⁺ signals in single -cells as well (n = 5). Similar results were also obtained with 100 µM ATP (n = 5; not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 5. A: islets were preincubated for 10 min with clonidine (clon; 1 µM), a potent inhibitor of insulin release without effect on mouse -cells, and then continuously perfused throughout the experiment with this 2-adrenoreceptor agonist. Under these conditions, ATP produced a very similar effect. B: mean frequency values of Ca2+ signals in control conditions (black bars) and after stimuli application (gray bars). Stimuli were ATP (10 µM) and ATP (10 µM) plus clonidine (1 µM). C: frequency (%) of Ca2+ signals after stimuli compared with control conditions. D: ATP effect (10 µM) on Ca2+ signals in isolated single -cells (n = 5). The typical response of -cells to 5 µM adrenaline is also shown (21, 50). Controls in B and C were 0.5 mM glucose or 0.5 mM glucose plus clonidine (1 µM). Data in B and C are means ± SE. In C, effects of ATP and ATP plus clonidine were not found different (P > 0.05). *Statistically significant (P < 0.05) compared with controls.

	DISCUSSION

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There are nineteen purinergic receptors subtypes widely distributed in various tissues, the activation of which initiates a plethora of signaling cascades and effects (12, 41). Purinergic receptors for ATP (P2) involve ionotropic (P2X) and metabotropic (P2Y) receptors, whereas P1 receptors are adenosine specific. While ATP is released from nerve terminals and insulin-secretory granules, adenosine is mainly produced by ATP hydrolytic reactions in the extracellular space. Given that several purinergic receptor subtypes can coexist in the same cell and that current pharmacology is not selective for all these receptors, the study of purinergic signaling is complex (12). Here, we have shown that P2Y₁, A₁, and A_2A are all present in the pancreatic -cell (Figs. 1 and 4). Although A₂ and P2Y₁ receptors could be involved in glucagon secretion (6, 17, 35), the expression and localization of both receptors in the -cell have not been shown previously. In any case, we do not discard the presence of other subtypes. Actually, genes encoding for P2Y₁, P2Y₂, P2Y₄, and P2Y₆ have been detected in rat islets (2), while P2X₇ receptors have been identified by immunochemistry in -cells from streptozotocin-diabetic rats (7). However, P2X₇ receptors are unlikely to mediate the Ca²⁺-signaling blockade shown here, since activation of these ionotropic receptors is coupled to extracellular Ca²⁺ entry, which increases [Ca²⁺]_i (7, 12). Activation of P2Y₁, A₁, and A_2A receptors triggers multiple effects in addition to the classical signaling pathways involving adenylate cyclase and phospholipase C (41). Among other signal-transduction mechanisms, these three receptors participate in the regulation of K⁺ and voltage-gated Ca²⁺ channels, which could affect Ca²⁺ signals (1, 14, 36, 41). In the pancreatic β-cell, extracellular ATP leads to multiple actions, including effects on the exocytotic machinery or K_ATP channels and activation of phospholipase A2 and serine/threonine protein phosphatase calcineurin pathways (36). Thus, since multiple receptors coexist in the -cell, a complex purinergic signaling system may take place.

Purinergic receptors have been associated with either the activation or inhibition of Ca²⁺ signals in multiple systems (41). In β-cells, ATP triggers a Ca²⁺ transient by release from intracellular stores (48) but at the same time induces a subsequent inhibitory effect on voltage-gated Ca²⁺ channels in the plasma membrane, reducing Ca²⁺ currents and allowing a negative feedback loop (14, 44). Additionally, ATP decreases the activity of ATP-dependent K⁺ (K_ATP) channels, allowing a small depolarization in β-cells (36). It has also been documented that Ca²⁺ currents are reduced by adenosine in this cell type (1). Given that -cells exhibit Ca²⁺ oscillations at low glucose concentrations due to the activity of both K_ATP and voltage-gated Ca²⁺ channels (15, 21), it is reasonable to suggest that purinergic stimulation could inhibit -cell Ca²⁺ signals through interaction with similar targets as those reported in β-cells (1, 36). This inhibitory effect by ATP and adenosine indicates an important role of purinergic signaling in the regulation of Ca²⁺-dependent functions in -cells.

Additionally, extracellular ATP was found to be an inhibitory messenger for mouse -cell secretion (Fig. 3). A different situation has been reported in rats (17). These discrepancies are probably due to interspecies differences, given that rat and mouse -cells possess several divergences in the stimulus-secretion coupling and in the kind of channels involved (19). Since -cell exocytosis is a Ca²⁺-dependent process, the inhibitory effect of ATP on Ca²⁺ signals (Fig. 2) should be accompanied by a decrease in glucagon secretion, as we observed with cobalt, a general Ca²⁺-channel blocker, and with high glucose concentrations (Fig. 3; Ref. 21). In addition, ATP could also reduce glucagon release by a direct interaction with the exocytic process, as it has been shown in mouse β-cells (36).

Previous studies (6, 35) with perfused rat pancreas indicated that adenosine elevates glucagon secretion at low glucose levels through the activation of A₂ receptors. In a recent study (17) with rat islets, adenosine was also found to favor glucagon release at low glucose concentrations although this effect changed at 20 mM. Our results in mice indicated that glucagon levels in the presence of adenosine were similar to those at 0.5 mM glucose (Fig. 3A) in spite of a decrease in Ca²⁺ signaling. This dual effect may be explained by the fact that A_2A receptors are coupled to adenylate cyclase activation (4, 6, 12). Since Ca²⁺-dependent exocytosis in -cells is highly sensitive to activation of the cAMP/PKA pathway (18), it is reasonable to expect that an increase in cAMP levels through A_2A receptor activation could compensate for the effect of a reduced Ca²⁺ signal on glucagon secretion. Actually, it has been reported in -cells that cAMP-elevating agents can stimulate glucagon secretion notably in conditions of reduced Ca²⁺ currents (18, 20). In agreement with this idea, glucagon secretion in the presence of adenosine was markedly decreased by the PKA inhibitor H-89 (Fig. 3B). Furthermore, activation of A_2A receptors by CGS-21680 also produced a dual effect on Ca²⁺ signaling and glucagon secretion (Fig. 4). In this case, CGS-21680 increased glucagon secretion at 0.5 mM glucose compared with the action of adenosine. This effect probably results from the higher affinity of CGS-21680 on A_2A receptors. Although we found A₁ and A_2A receptors in the -cell, the experiments shown in Fig. 4 suggested that only A_2A receptors may be functional in agreement with previous studies (6, 35). Also, it has been indicated in other cell models that A_2A function may predominate over A₁ receptors at the adenosine concentrations used in these experiments (12). We do not discard, however, that adenosine may produce additional effects on other molecular targets.

Coexistence of P2 and P1 receptors in the same cell has been documented in multiple systems including the β-cell, although its functional significance is still under study (12, 35, 41). Integration of the responses from different receptors depends on many factors, including the local concentrations of ATP and adenosine, density and affinity of receptor subtypes, and activity of plasma membrane ectonucleotidases (12, 41). This integration can also change during pathological conditions. In our experiments, we observed that ATP affects -cell secretion in contrast to adenosine (12). In this case, the effect of ATP binding to P2 receptors may predominate over a potential action on P1 receptors induced by ATP conversion into adenosine. This idea was supported by the efficient inhibitory effect of the nonhydrolyzable ATPS on Ca²⁺ signaling and the lack of ATP effect in the presence of suramin (Fig. 2, E and F). Additionally, it has been indicated that high concentrations of ATP could inhibit several ectonucleotidases, decreasing adenosine production and then reducing the ATP effect through P1 receptors (12). Given that ATP is highly concentrated in β-cell secretory granules (1–3 mM) and, especially, in synaptic vesicles of islet nerve terminals (up to 10 mM; Refs. 26, 41), its inhibitory effect on -cell secretion should take place in physiological conditions. Actually, neural ATP has been proposed as a key signal responsible for the coordination of β-cell function (17, 24, 44). On the other hand, the effect of adenosine binding to P1 receptors may have a role in pathological situations, since during energy-deficient states such as hypoxia, ischemia, and fasting, adenosine is released from tissues, increasing its plasma levels (35).

The mechanisms that regulate glucagon secretion are still poorly understood. Here we demonstrate that extracellular ATP is an important inhibitor of -cell function and glucagon secretion. Given that the control of the suppression of glucagon release could be important for the treatment of hyperglycemia in diabetic patients (9, 10, 46), purinergic signaling may also be a therapeutic target of clinical interest.

	GRANTS

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This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paolo (2004/14016-7 to E. M. Carneiro), Ministerio de Educación y Ciencia (BFU2004-07283; BFU2007-67607/BFI and PCI2005-A7-0131 to I. Quesada), and the Instituto de Salud Carlos III (RD06/0015/0010).

	ACKNOWLEDGMENTS

 
We thank F. Almagro and I. Piqueras for expert technical assistance and A. Nadal and A. B. Ropero for critical reading of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Ivan Quesada, Institute of Bioengineering, Miguel Hernandez Univ., Avenida de la Universidad, s/n, 03202 Elche, Spain (e-mail: ivanq{at}umh.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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