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P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases.
The importance of ATP in biological systems is universally known. In addition to being a source of energy for the cell, extracellular ATP can act as a neurotransmitter in the nervous system. Recently, it has been observed that the purinergic system can induce cell death within the central nervous system (CNS). In the retina, purines are involved not only in signaling between the different cell types , but high levels of extracellular ATP also produce retinal neurodegeneration [2–4]. Different studies demonstrated that both P2X and P2Y purinoceptors are expressed in various retinal cell types, such as photoreceptors, retinal ganglion cells, bipolar cells, and Muller cells [5–7]. P2Y are transmembrane G protein-coupled receptors while the P2X receptors form ATP-gated ion channels and are distributed in many cell types and tissues. To date, seven different P2X subunits (P2X1-7), which assemble into homotrimeric and/or heterotrimeric proteins, have been found . Among these subtypes, P2X7 receptor (P2X7R) plays an important role. This receptor is present in immune cells and in the CNS, including the retina [9–12], and it has been proposed as a potential therapeutic target in CNS disorders, such as Alzheimer’s disease, neuropathic pain, and spinal cord injury by regulating the levels of intracellular calcium ([Ca2+]i) and interleukin-1β, and by activating multiple caspases (see for review ). This receptor is characterized by two states of permeability , both inducing a [Ca2+]i elevation that could be critical for the biology of the cell. Zhang and colleagues  demonstrated in in vitro experiments that stimulation of P2X7R by the potent agonist 3′-O-(4-benzoylbenzoyl) ATP (BzATP) produced robust elevations in the Ca2+ level inside rat retinal ganglion cells, leading the cells to apoptosis. Furthermore, they showed that this damage was inhibited by the selective P2X7R antagonist Brilliant Blue G (BBG). Similarly, Notomi and co-authors  suggested that photoreceptor cell apoptosis induced by ATP was prevented by BBG.
Saffron (Crocus sativus L.) is a species belonging to the Iridaceae family and has been widely used as an herbal medicine and spice since ancient times . Chemically, saffron stigmas contain more than 150 volatile and many non-volatile active components, including vitamins, sugars, minerals, various α- and ß-carotenes, carotenoids (zeaxanthin and crocetin), and crocins derived by crocetin esterification with sugars. Its flavor arises from safranal, which is present in the glycoside picrocrocin. However, saffron cultivars coming from different areas all over the world may differ in their characteristics which may lead to pronounced differences in the general composition of extracts. This could explain the variety of effects and discrepancies found in the literature. Pharmacological studies have demonstrated that saffron (Saf) and its constituents protect against damage, exerting anti-ischemic , anxiolytic , anti-inflammatory , and antitumor  properties. Maccarone et al.  provided data showing that Saf is protective in a rat model of light-induced retinal degeneration. A proof-of-principle clinical trial in age-related macular degeneration (AMD) patients confirmed the potentiality of Saf treatment in neurodegenerative diseases and its consistency in time [23, 24] and in patients carrying genetic mutations . The biological mechanisms underlying neuroprotection are thus far unknown even though a direct control of gene expression was suggested [26, 27]. Despite the large number of causes of photoreceptor degeneration, the final pathways leading to photoreceptor death are similar. Elevations in [Ca2+]i originate the activation of degradative proteases, such as calpains, which can induce photoreceptor apoptosis. In photoreceptors of a mouse model of retinal degeneration (rd1), intracellular Ca2+ levels increased to approximately 190 % compared with control photoreceptors .
The aim of this work was to investigate whether one of the possible ways of Saf neuroprotective action may be related to the modulation of purinergic receptors. To avoid using saffrons coming from different cultivars that may present different composition of the extract, in this paper, we used only saffron derived from Hortus Novus (L’Aquila, Italy), whose chemical characteristics has been analytically examined in previous studies. We tested Saf on two different cell models, primary mouse retinal cells and retina-derived mouse 661W cell line, stressed by relatively high ATP concentrations. The last cell line derived from retinal tumors of a transgenic mouse line and showed biochemical and cellular properties of retinal photoreceptors. Moreover, these cells have been shown to respond to a variety of toxic stimulation and to oxidative and light stresses by activating the same apoptotic program of normal retinal photoreceptor cells . We evaluated the possible interaction between Saf and purinergic P2X receptors, particularly P2X7R. Different experimental approaches were used to analyze the Saf effect on retina-derived mouse 661W cell line and HEK293 cell lines stably expressing rat P2X7 (HEK-P2X7R) and P2X2 (HEK-P2X2R) receptors. Interestingly, Saf increased the cell viability after ATP treatment and inhibited the ATP-mediated [Ca2+]i elevation on 661W cells and on HEK-P2X7R but not on HEK-P2X2R cells. Patch-clamp experiments confirmed the block induced by Saf on HEK-P2X7R cationic currents.
Primary retinal cell cultures were prepared using 2-week-old C57BL/6 mice. The experiments were performed in compliance with the Animal Care and Use Committee guidelines and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The retina was detached, suspended in cold Hanks balanced salt solution (HBSS; Gibco—Life Technologies, Italy), and incubated at 37 °C in activated papain solution (0.06 mg/ml [Sigma-Aldrich, Milan, Italy] prepared in Neurobasal-A medium (NB; Gibco). After 20 min, 2 ml of 2 % fetal bovine serum (FBS) in NB was added for 5 min to stop the enzymatic reaction and tissue aggregates were eliminated by the addition of 200 μl DNase I (Sigma-Aldrich). FBS was aspirated and NB supplemented with 2 % B27 (Gibco) and 0.5 mM l-glutamine (Gibco) was added to tissue. To dissociate the cells, the papain-treated retina was gently manually pipetted (50 times) using a Pasteur pipette. Tissues were allowed to settle for 2 min and supernatant containing the dissociated cells was collected. After the dissociation, the cells were seeded at 1×104 cells in 96-well plates and incubated for 4–5 days before experiments.
The mouse retinal photoreceptor-derived 661W cell line (kindly provided by Dr. Muayyad Al-Ubaidi (University of Oklahoma Health Sciences Center, OK, USA)) was cultured in Dulbecco minimum essential medium supplemented with 10 % FBS, 10 %l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco).
Human embryonic kidney cell line HEK293 was stably transfected with a pcDNA3 plasmid containing the full length rat P2X7-GFP cDNA, as described previously . HEK293 cells stably transfected with rat P2X2 were kindly provided by Prof. Surprenant (University of Manchester, UK). Cells were maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham supplemented with 10 % FBS, 5 mg/ml gentamycin and 200 mM glutamine. Confluent cells were re-plated on 20-mm glass coverslips at a density of 5×103 for Ca2+ and current measurements and in 96-well plates for viability tests.
Confluent cultures were subcultured in 96-well culture plates at a density of 5×103 cells/well and allowed to attach for 24 h. Saffron (L’Aquila Saffron, Italy) used in the present paper has the same chemical characteristics (defined by HPLC analysis) as the saffron tested in animal models and human AMD patients  and has been patented (Hortus Novus S.r.l.). Stigmas of saffron were crushed and dissolved in water at a concentration of 5 mg/ml 18–24 h before cell treatment. Cells were incubated with different Saf and ATP or BzATP concentrations. Cell viability was assessed by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) after 24 h of ATP treatment. MTT solution (5 mg/ml in phosphate-buffered saline (PBS)) was added for 2 h resulting in formazan formation, which was solubilized with DMSO (100 μl). The absorption at 570 nm was measured using a FLUOstar Omega micro-plate reader.
Apoptosis/necrosis tests were done on 661W cells using the GFP-Certified Apoptosis/Necrosis detection kit (Enzo LifeSciences). Cells were seeded on glass slides at 80 % confluency and treated for 24 h with ATP (5 or 10 mM) with and without the addition of 25 μg/ml Saf. Slides were washed with PBS and incubated for 15 min in the dual detection reagent containing apoptosis (Annexin V) and necrosis (7-AAD) detection reagent in 1× binding buffer. The samples were incubated at room temperature in the dark. After staining, the cells were washed with binding buffer and observed under a Leica SP2 confocal microscope with dual filter set for Annexin V (Ex/Em 550/570) and for 7-ADD (Ex/Em 546/647).
The mouse retinal photoreceptor-derived 661W cell, the control HEK293 cells, and HEK293 cells stably transfected with P2X7R-EGFP were lysed in a buffer containing 62.5 mM Tris, 2 % sodium dodecyl sulfate (SDS), and a cocktail of protease inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.8 μM aprotinin, 0.2 μM leupeptin, 40 μM bestatin, 15 μM pepstatin A, 14 μM E-64). Samples were sonicated for 2 s and heated at 98 °C for 5 min. Protein concentration was determined using the method of Lowry  with bovine serum albumin as the standard. Equal amounts of proteins (15 μg) were subjected to SDS polyacrylamide gel electrophoresis analysis. Separated proteins were transferred to PVDF membrane (Millipore, Billerica, Massachusetts, USA) for 1 h. The blots were then incubated with a polyclonal antibody against an intracellular epitope of P2X7R (1:500; Alomone Labs, Jerusalem, Israel) as primary antibody and with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000, Santa Cruz, Santa Cruz, CA, USA) as secondary antibody. Immunodetection was performed using Amersham ECL PLUS detection reagents and the images were captured by using Amersham Hyperfilm ECL. Developed films (Kodak, Rochester, NY, USA) were scanned using a flat-bed scanner with a resolution of 600 dpi. In order to confirm the homogeneity of the loaded proteins, immunoblots were stripped by incubating them with stripping buffer (62.5 mM Tris pH 6.8, 10 % SDS, and 1 % β-mercaptoethanol for 30 min at 55 °C) and reprobed with a polyclonal anti-actin primary antibody (1:3000, Sigma-Aldrich).
Biotinylated 661W cell surface samples were incubated with the anti-intracellular epitope of P2X7 antibody (1:500; Alomone Labs, Jerusalem, Israel) as primary antibody and with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000, Santa Cruz, Santa Cruz, CA, USA) as secondary antibody. Anti-pan cadherin antibody, which recognizes the plasma-membrane marker cadherin, was used as a gel loading control. In order to confirm the specificity of the anti-P2X7 primary antibody, whole lysate and membrane surface samples of 661W cells were pre-incubated in excess control antigen and analyzed by Western blot. Each experiment was conducted in triplicate.
Intracellular calcium measurements were performed by using the fluorescent Ca2+ indicator fura-2 AM. Cells were loaded with 5 μM fura-2 AM dissolved in extracellular solution for 45 min at 37 °C. Pluronic acid (0.1 %) was added to improve dye uptake. Cell coverslip was placed on the stage of an inverted fluorescence microscope Nikon TE200 (Nikon, Tokyo, Japan) equipped with a dual excitation fluorometric calcium imaging system (Hamamatsu, Sunayama-Cho, Japan). The external standard solution was composed of (mM) 135 NaCl, 5.4 KCl, 1 CaCl2, 5 Hepes, and 10 glucose at pH 7.3. Ca2+-free standard solution was similar but Ca2+ was replaced with 5 mM EGTA. Crocins from Sigma (Sigma-Aldrich) were dissolved in water at a concentration of 10 mg/ml. To obtain crocetin, 1 ml HCl (12 M) was added to a solution containing crocins or saffron for 30 min at 37 °C; pH was increased to 7 with NaOH.
Fluorescence emission was acquired with a digital CCD camera (Hamamatsu C4742-95-12ER). Monochromator settings, chopper frequency, and data acquisition were controlled by dedicated software (Aquacosmos/Ratio U7501-01, Hamamatsu). The sampling rate was 0.25 or 0.5 Hz. Fura-2 AM-loaded cells were excited at 340 and 380 nm and fluorescence emission measured at 510 nm. The fluorescence ratio F340/F380 was used to monitor [Ca2+]i changes. In some experiments, [Ca2+]i was calculated according to Grynkiewicz , using a KD of 140 nmol/l for the Ca2+/fura-2 complex.
The electrophysiological studies were performed at room temperature (20–22 °C) using the whole-cell configuration of the patch-clamp technique. Patch pipettes were pulled from borosilicate glass capillaries (Clark Electromedical Instruments) and heat-polished to obtain a resistance of 2–4 MΩ when filled with pipette solution. The external standard solutions were the same as those used for intracellular calcium measurements. The standard pipette solution contained (mM) 135 NMDG-Cl, 1 MgCl2, 5 Hepes, 5 EGTA, and 5 glucose at pH 7.3. Membrane currents were recorded using an AXOPATCH 200A patch-clamp amplifier (Axon Instruments) and were filtered at 1 kHz before acquisition. Both voltage stimulation and data acquisition were obtained using a 12-bit interface (Axon Instruments) and a microcomputer equipped with pClamp 10.2 software. Experiments were performed at a holding potential of −60 mV with stimulation protocol of 90-ms length.
All data are given as means±SEM from at least four experiments. The statistical significance of differences between mean values was assessed using Student’s t test. Differences were regarded as statistically significant for *p<0.05 and **p<0.01.
We evaluated in in vitro experiments the efficiency of saffron extract (Saf) in mitigating ATP stress by using two different mouse retinal cell cultures, primary retinal cells, and mouse retinal photoreceptor-derived 661W cells. In previous reports, 661W cells have been used as a cellular model to investigate the mechanisms involved in photoreceptor degeneration . Both cell cultures were stressed by using ATP concentrations ranging from 1 to 10 mM. The neuroprotective effects of Saf were tested using a MTT assay (see Fig. 1). Cells were incubated with Saf 25 μg/ml and ATP at concentrations of 1, 5, and 10 mM for 24 h. As observed from experiments, Saf alone did not produce any significant difference in cell viability (97 %) with respect to control (Fig. 1a); moreover, increasing Saf concentrations from 5 to 500 μg/ml in the presence of extracellular ATP did not increase cell viability (Fig. 1c), whereas the effect of ATP was concentration dependent in both cell types (Fig. 1a, b). On primary retinal cultures, 1 mM ATP had no effect on cell viability, while at 5 mM the percentage of live cells was reduced to about 57 % compared to untreated control cells (Fig. 1a). Saffron was significantly protective against 5 mM ATP stress, shifting the cell viability to about 77 %, respectively (p<0.01). A smaller but still significant effect of Saf was seen when the primary culture of retinal cells was incubated with a higher concentration of ATP (10 mM): treatment with Saf increased cell viability from about 34 to 43 % (p<0.01).
The mouse retinal photoreceptor-derived 661W cells presented a higher sensitivity to stress induced by external ATP; cell viability decreased to 79 % at 1 mM, to 44 % at 5 mM, and to 8 % at 10 mM ATP (Fig 1b). In the presence of 1 mM ATP, the analysis showed a mild, but statistically significant, effect of Saf (p<0.01). Viability values of 661W cell with Saf treatment (Saf plus ATP 5 mM) significantly increased to 54 % (p<0.05). When the cells were incubated with a higher concentration of ATP (10 mM), Saf showed also a significant protection (p<0.01). The number of living cells after treatment of 5 mM ATP and Saf plus ATP was evaluated also by using fluorescence microscopy. Cells were stained with DAPI to visualize the nuclei of living cells (Fig. 1d). In the presence of ATP alone (Fig. 1d—middle panel), cells were less numerous than those treated with Saf plus ATP (right panel). The number of fluorescent nuclei was evaluated in each condition and normalized to the number of stained nuclei of control cells, and results were plotted in Fig. 1d (last panel).
It is known that ATP induces apoptosis in retinal cells ; therefore, Annexin V assays (see “Materials and methods” section) were used on 661W cells in order to distinguish whether ATP treatment produced necrosis or apoptosis in this cellular model. In addition, we evaluated the efficiency of the incubation with Saf on mitigating cell apoptosis induced by ATP. Figure Figure22 shows the images of 661W cells in control condition (a), stressed by 10 mM ATP (b), and in the presence of Saf (25 μg/ml) plus ATP (10 mM) (c). As observed with confocal fluorescence microscopy, treatment with extracellular ATP induced apoptosis (in early and late stage) in 661W cells (Fig. 2b–d). On the contrary, no significant necrosis was observed at both ATP concentrations (less than 2 % in cells treated with 5 and 10 mM ATP). Moreover, the incubation with Saf decreased significantly the number of apoptotic cells (Fig. 2c), but had no effect on the number of necrotic cells. Figure Figure2e2e summarizes the results obtained in the different conditions (5 and 10 mM ATP with and without Saf incubation). As expected, increasing the amount of ATP augmented the number of apoptotic cells.
Several studies reported the critical involvement of ATP-activated P2X7 purinoceptors in retinal neurodegeneration [15, 16, 35]. As previously mentioned, P2X7Rs are expressed in the majority of retinal cell types, such as photoreceptors, ganglion cells, and bipolar cells. On the contrary, until now there is no evidence for the presence of this receptor on 661W cells. We verified the expression of P2X7Rs in 661W cell line by using Western blot analysis. In order to validate the functionality of the antibody, the HEK293 cell line either untransfected or stably transfected with the rat P2X7R-GFP was used. Figure Figure3a3a reveals a band of 68 kDa in 661W cells and a band of 95 kDa in HEK293-P2X7R-GFP cells, which correspond to the expected molecular weight of the P2X7 receptor subunit alone and plus GFP, respectively. Vice versa, this band was completely absent in untransfected HEK293 cell line; actin was used as a control housekeeping protein for the different cell lines. Moreover, cell surface biotinylation assay on 661W cells allows us to recognize the presence of the full-glycosylated P2X7 protein on the plasma membrane of this cell line (Fig. 3b). In both kinds of experiments, the specificity of the antibody for its target was further demonstrated by the complete obliteration of staining when the antibody was pre-incubated in excess control antigen. After proving the presence of P2X7R on the plasma membrane of 661W cells, we investigated if saffron protected from stress induced by the potent P2X7 agonist BzATP (3′-O-(4-benzoyl)benzoyl-ATP). Cells were exposed to 400 μM BzATP for 24 h with saffron (25 μg/ml). The results in Fig. 3c show that 400 μM BzATP decreased 661W cell viability to about 60 % compared to the control. This result further confirms that P2X7 receptors are expressed in the 661W cell line. As observed for ATP, saffron significantly protected from BzATP damage, increasing cell viability to 76 % (p<0.01).
Furthermore, we evaluated the P2X7R involvement on Saf protection from ATP stress by measuring the intracellular calcium [Ca2+]i variations induced by external ATP in mouse retinal photoreceptor-derived 661W cells. Because 1 mM ATP produced a change of [Ca2+]i only in 27 % of the cells (17/62), we increased the agonist concentration to 2 mM in these experiments. In fura-2-loaded 661W cells bathed with an extracellular control solution, a subsequent application of 2 mM ATP caused a relatively rapid increase in [Ca2+]i that slowly attenuated to a lower level (Fig. 4a; n=87 cells). On the contrary, when 2 mM ATP was applied in a nominally Ca2+-free solution (no Ca2+, 5 mM EGTA added), cells generally did not show a significant [Ca2+]i elevation but a typical intracellular Ca2+ increase was observed switching to an extracellular solution containing 1 mM Ca2+ (Fig. 4b). Calcium imaging analysis showed that exposure of cultured 661W cells to 2 mM ATP in external 0 Ca2+ promoted a Ca2+ release from intracellular stores only in 18 % of the cells examined (10/54 cells). We then tested the effect of Saf on the ATP-induced [Ca2+]i. increase. The addition of Saf alone (25 μg/ml) did not induce any variations of the intracellular Ca2+ level (Fig. 4c), but a subsequent application (after 5 min) of 2 mM ATP elicited a lower [Ca2+]i rise than that evoked by individual application of ATP (Fig. 4d). The results summarized in Fig. 4f showed that the ATP-induced [Ca2+]i maximal response was inhibited by 43 % in the presence of Saf. Similar experiments were performed in the presence of the specific P2X7R antagonists A438079 (0.5 and 1 μM), oxidized ATP (oATP; 100 μM; pre-incubation for 2 h), or Brilliant Blue G (BBG; 1 μM) [36–38]. A438079 and BBG were applied 5 min before the addition of 2 mM ATP. The analysis showed that the ATP-induced [Ca2+]i increase was inhibited in the presence of all three P2X7R antagonists (Fig. 4g). In particular, the calcium signal fully inhibited in the presence of 1 μM A438079 (Fig. 4e; first part of the trace) and completely recovered after the removal of the antagonist (Fig. 4e; the last part of the trace). These data indicate that P2X7R is critically implicated in ATP-induced [Ca2+]i variations in retinal photoreceptor-derived 661W cells.
To confirm the involvement of P2X7Rs in Saf protection against ATP stress, we extended our analysis on HEK293 cells stably expressing rat P2X7Rs (HEK293-P2X7R). In parallel experiments, these cells were exposed to either the natural agonist ATP or the synthetic one BzATP. It is known that both ATP and BzATP responses differ between rat and mouse P2X7R: rat P2X7R is 10 times more sensitive to ATP and 100 times more sensitive to BzATP than mouse P2X7R . Based on these considerations and on previous results on rat HEK293-P2X7R , we used 250 μM ATP and 10 μM BzATP to reduce cell viability to approximately 60 %. In Fig. 5a, HEK293-P2X7R cells in control condition are shown and compared to cells treated for 24 h with ATP alone and with the addition of Saf. The number of viable cells was estimated in the different conditions and normalized to that obtained in control condition. Similar to retinal cells, Saf protected from damage when it was incubated for 24 h with ATP; cell viability increased from 57 % with 250 μM ATP alone to 69 % with ATP plus Saf (p<0.01; Fig. 5b). An analogous behavior was observed in the presence of BzATP. Figure Figure5c5c shows that the percentage of living cells exposed at 10 μM BzATP decreased to 66 % compared to control. Saf treatment for 24 h increased cell viability to 82 % (p<0.01). Saf modulation was also compared to the activity of 1 μM of BBG, which selectively inhibits P2X7Rs . The BBG inhibitor alone had only a minor effect reducing the cell viability to about 95 %, whereas when applied with BzATP, the viability value was about 88 % compared to the 82 % in the presence of BzATP plus Saf. This experiment demonstrated that the inhibitory effect of Saf on the P2X7R was similar to that of the specific antagonist BBG.
To better elucidate the specificity of Saf protection through its effects on P2X7R, we used HEK293 cells stably expressing the full-length rat P2X2 receptor, which in some properties is similar to P2X7R. Cells were exposed to 250 μM ATP, 30 μM BzATP, and ATP or BzATP plus Saf using the same experimental protocol used in the previous experiments. Both purines induced a decrease of cell viability compared to control similar to that observed for HEK293-P2X7R (Fig. 5d), but, differently from HEK293-P2X7R, Saf was not able to increase the cell viability of HEK-P2X2R. This experiment confirmed that Saf interacts with the P2X7R and not with P2X2R.
In order to get insights into the mechanism of interaction between Saf and the ionotropic P2X7R, we analyzed the effect of Saf on BzATP-evoked intracellular calcium variations. It is known that HEK293 cells transfected with the full-length rat P2X7R challenged with micromolar concentrations of the relatively selective agonist BzATP exhibited a [Ca2+]i signal elicited by Ca2+ influx through the P2X7R itself . First of all, we verified the effect of Saf and BzATP, applied alone, on [Ca2+]i variations. In fura-2-loaded HEK293-P2X7R, the application of 5 μg/ml Saf did not induce any Ca2+ increase (Fig. 6), whereas 3 μM BzATP promoted a biphasic [Ca2+]i rise with a rapid and a slow [Ca2+]i elevation. In the presence of Saf, a subsequent (after 5 min) BzATP application (3 μM) elicited a lower [Ca2+]i rise than that evoked by individual application of BzATP, particularly affecting the slower component of the [Ca2+]i elevation (Fig. 6 last trace). Similar experiments performed with the specific P2X7R antagonist BBG and by using different Saf concentrations were summarized in Fig. 6b. The results show that 100 nM BBG inhibited the BzATP-induced [Ca2+]i increase by 90 %, whereas the maximal inhibitory effect of Saf was about 30 %. We tried different Saf concentrations: 1, 5, 10, 25 μg/ml. As shown in the histogram 5, 10, and 25 μg/ml gave the same effect on the BzATP-induced Ca2+ signal, so in all subsequent experiments, we used 5 μg/ml Saf. The effects of Saf were tested on different agonist concentrations. Figure Figure6c6c shows representative traces of [Ca2+]i response induced by 20 μM BzATP alone and when the agonist was applied in the presence of 5 μg/ml Saf. The dose-response curves demonstrate that the maximal [Ca2+]i elevation was obtained at 20 μM BzATP, whereas in the presence of Saf, the same concentration did not induce the maximum effect (Fig. 6d). The EC50 value was 1.1 μM and the Hill coefficient 2.1 in control conditions and 1.7 μM with a Hill coefficient of 1.5 in the presence of BzATP plus Saf. These results indicate that the values of the two EC50 were not significantly different, but the maximal [Ca2+]i elevation in the presence of Saf was strongly reduced, suggesting a non-competitive block of P2X7Rs.
Finally, we tested the effects of two potent antioxidant ingredients of saffron: crocins (CRN) and crocetin (CRT), a carotenoid dicarboxylic acid which forms the core of crocins. We found that both compounds interfered with calcium signaling of P2X7Rs. Figure Figure77 shows a representative trace of the [Ca2+]i response induced by 3 μM BzATP alone (a) and in co-presence of 10 μg/ml CRN (b) and 10 μg/ml CRT (c). We tested the two carotenoids at two different concentrations, 10 and 25 μg/ml. As demonstrated by the histogram, both two carotenoids were more active at 25 μg/ml concentration and crocetin was found to be more active than crocin (Fig. 7d).
Patch-clamp experiments were carried out on HEK293-P2X7R cells to verify the modulation induced by Saf on P2X7 channels. Cells were clamped at a holding potential of −60 mV and whole-cell inward currents were elicited in response to 3 μM BzATP (Fig. 8a). Upon repeated (every 3 min, 30 s long) applications of 3 μM BzATP, the evoked currents increased in magnitude and reached their maximal levels after four applications. After 5-min washout and in the presence of 5 μg/ml Saf (3 to 5 min), a subsequent application of 3 μM BzATP plus Saf induced a smaller inward current compared to the control condition (Fig. 8b). The current trace in panel c was representative of the recovery obtained after 5-min washout of BzATP and Saf (Fig. 8c). The summarized results demonstrate that Saf was able to reduce the inward current amplitude to about 30 %, analyzed during 3 μM BzATP application (Fig. 8d).
In this study, we show that low amounts of saffron extract positively influence the cell viability of two different retinal cell cultures: mouse primary retinal cells and mouse retinal photoreceptor-derived 661W cells, both stressed by relatively high concentrations of ATP. Previous studies have demonstrated that extracellular ATP contributes to photoreceptor degeneration in rodents through excessive activation of P2 purinoreceptors. The involvement of P2X7Rs in retinal degeneration has been also documented in BALBCrds (retinal degeneration slow) mice . In this work, we provide direct evidence of P2X7R expression in the mouse retinal photoreceptor-derived 661W cell line. Using the Western blotting technique, we identified the receptor with an apparent molecular weight of 68 kDa; a small extra band immediately beneath the denser band is consistent with the notion that the receptor can appear with different glycosylation patterns linked to its maturation process . This fact is further demonstrated by the Western blot analysis of the P2X7 biotinylated fraction of 661W cells, where only one band, corresponding to the P2X7 glycosylated membrane fraction, is observable.
The presence of P2X7Rs in 661W cells was confirmed by calcium imaging experiments. ATP application in the absence of extracellular Ca2+ induced a transient [Ca2+]i elevation in only about 18 % of the cells indicating that extracellular Ca2+ entry was the main mechanism responsible for the ATP-induced [Ca2+]i sustained signal. In addition, we observed that millimolar concentrations of ATP are necessary to induce [Ca2+]i variations in mouse-retinal photoreceptor-derived cells, which is consistent with the low ATP affinity of P2X7R compared to other purinoreceptors (EC50≈1 mM ATP for mouse P2X7R ). Finally, we analyzed [Ca2+]i variation in the presence of BBG, oATP, and the selective P2X7R antagonist A438079. BBG is known as a potent antagonist with a 30- to 50-fold greater sensitivity for rat versus human or mouse P2X7Rs. oATP is an irreversible P2X7Rs inhibitor (1–2-h incubation) that also acts on other purinoreceptors, P2X1Rs and P2X2Rs, as well as on nuclear factor NF-kB and cytokine release. Differently, A438079 is a reversible antagonist, selective for P2X7Rs over other P2 receptors . Additionally, this last compound exhibited little or no activity on many other membrane receptors and ion channels. Our results showed that all these antagonists were able to block the [Ca2+]i increase in 661W cells. Moreover, removing the reversible A438079 restored the ability of ATP to induce [Ca2+]i increase. Interestingly, Saf significantly reduced [Ca2+]i level in 661W cells indicating that its protective action involves P2X7Rs.
Similar results were observed in HEK293 cells stably transfected with recombinant rat P2X7Rs. Saf treatment increased the cell viability and decreased [Ca2+]i level induced by stimulations with ATP or BzATP. Whereas in vivo the ATP4− form of ATP is the physiological ligand, it is well recognized that in vitro BzATP is a stable and potent P2X7R activator, with a 100 times higher sensitivity in rat than in mouse . The differences in the properties observed between the P2X7Rs of different species are important. Many pharmacological results have been obtained by heterologous expression of the rat P2X7Rs, restricting the knowledge of the effects of antagonists in other species. In this work, we observed a good agreement on Saf properties between 661W cells (of mouse origin) and HEK293-P2X7R (of rat origin): cell viability increased ≈10 % whereas [Ca2+]i level decreased ≈40 % in the presence of Saf. Moreover, we noted that Saf mainly affected the slow component of the calcium rise. The significance of the biphasic behavior and the blocking effect of one component alone involves important considerations on various assumptions concerning the receptor structure and/or the high and low affinity binding sites [30, 41]. This topic is very interesting and deserves to be treated separately in future studies. We also verified that Saf modulated cationic currents in HEK293-P2X7R cells. Rat P2X2 receptors were used to examine the specificity of Saf to reduce P2X7-mediated Ca2+ entry. To our knowledge, this is the first report describing the possible neuroprotective effect of Saf through the regulation of P2X7R-mediated neuronal damage. The findings that the ATP-induced cell damage was almost abolished by the use of specific antagonists in both 661W cells and HEK293-P2X7R and the fact that Saf did not influence the recombinant P2X2-mediated response strongly suggest that P2X7R is a molecular target of Saf action. However, because of the partial effect of Saf on cell viability and P2X7R-mediated Ca2+ entry, we cannot consider the inhibiting effect of Saf on P2X7R as an exclusive mechanism of action.
The contribution of Saf in regulating the severity of neurodegeneration is under investigation. Saffron has been tested in an animal model of light-induced photoreceptor degeneration (light-induced damage (LD)) and in AMD patients. In both LD and AMD rats, oxidative stress plays a relevant role and a direct antioxidant activity of its main active components, crocins and crocetin, can be hypothesized. In this paper, we tested the efficiency of these components as antagonists of P2X7Rs. Calcium imaging experiments showed an effect of both crocins and crocetin in reducing Ca2+ entry on HEK293-P2X7R cells. However, results present in the literature suggest that the action of saffron can be more complex  and includes the modulation of gene expression [26, 27], e.g., the downregulation of genes involved in neuroinflammation (chemokines) and reduce microglia activation (paper in preparation). Recently, the identification of purinergic receptors on microglia has driven a strong debate on the pivotal role played by microglia and local ATP release as an injury signal in the process of aging and age-related diseases such as AMD [42, 43]. In this scenario, our data showing that Saf directly reduces the [Ca2+]i response evoked by purinergic P2X7R stimulation unravels a novel mechanism through which Saf may exert its protective role in neurodegeneration. The next step will be to test saffron efficiency on the modulation of P2X7R in animal models like LD or AMD mice.
Another important issue that remains to be addressed in detail is the physiological significance of these results. In the brain, the P2X7R has been proposed to be involved in the signaling cascade leading to neuroinflammation and neurodegeneration [42, 44]. How the receptor contributes to these processes is not yet unequivocally verified. There is in vitro evidence that P2X7R contributes to regulate the inflammatory and degenerative responses by stimulating the synthesis and release of various cytokines and chemokines. Given the importance of [Ca2+]i elevation in the biochemical cascades leading to apoptotic cell death , and because of the correlation of P2X7R-mediated cell death with the pore configuration of the receptor , it is tempting to speculate that the ability of Saf to inhibit the P2X7R-mediated [Ca2+]i rise could interact with the structure of P2X7 pore involved in apoptotic cell death.
We wish to thank Dr. Maria Maggi (HN s.r.L.) and Lab of Analytical Chemistry, University of L’Aquila, for performing the chemical analysis. We thank Dr. Joachim Scholz-Starke (IBF-CNR, Italy) for critical comments on the manuscript. The technical assistance of Francesca Quartino and Alessandro Barbin (IBF-CNR, Italy) was highly appreciated. This study was supported by MIUR-PRIN (2010–2011) research grant to SB.
The experiments were performed in compliance with the Animal Care and Use Committee guidelines and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Prof. Bisti holds a non-remunerative relationship with “Hortus Novus s.r.l.,” the company that provided the saffron used in this study. All the other authors declare that they have no competing interests.