Frontiers in Marine Science (Jun 2014)
Crambescidin 816 induces calcium influx though glutamate receptors in primary cultures of cortical neurons
Abstract
INTRODUCTION Crambescins and crambescidins are guanidine alkaloids first reported in the early 90s. They are produced by the red encrusting marine sponge Crambe crambe widely distributed in the Western Mediterranean Sea but also in the Macaronesian archipelagos (Berlinck et al., 1990;Berlinck et al., 1993). There are few studies about the biological activity of these compounds and their pharmaceutical potential, mainly due to difficulties to obtain large quantities of pure compounds (Jares-Erijman et al., 1993). An antagonistic activity of crambescidin 816 (Cramb816) on voltage-sensitive calcium channels, higher than the one elicited by the selective blocker of L-type Ca+2 channels, nifedipine (NIF) has been reported (Berlinck et al., 1993). Moreover, several representative compounds of the crambescin family (norcrambescinA2, crambescinC1 and crambescinA2) partially blocked voltage gated potassium channels and Crambescin C1 (CrambC1) and Cramb816 partially blocked voltage gated Na+ channels (Martin et al., 2013). Furthermore, we pharmacologically isolated the two main fractions of neuronal high voltage activated (HVA) Ca+2 channels in cortical neurons and described that Cav1 or L-type calcium channels are the main target for Cramb816 in cortical neurons (Martin et al., 2013). In the present study, the effect of Cramb816 and CrambC on neuronal viability as well as the effect of Cramb816 in cytosolic calcium concentration was evaluated in order to get an approach of the possible mechanism by which Cramb816 is cytotoxic in cortical neurons. MATERIAL AND METHODS Primary cultures of cortical neurons. Primary cortical neurons were prepared from Swiss mice embryos (E15-18) as previously described (Martin et al., 2013). The cell suspension was seeded in 18 mm glass coverslips precoated with poly-D-lysine and incubated in 12 multiwell plates for 4-7 days in vitro in a humidified 5 % CO2/95 % air atmosphere at 37 ºC. Determination of cellular viability. Cell viability was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) test, in cultures grown in 96 well plates and exposed to different concentrations of C816 or CrambC1 for 24 h. Saponin was used as cellular death control. After the exposure time, cells were rinsed and incubated for 60 min with a solution of MTT dissolved in Locke’s buffer. After washing off excess MTT, cells were disaggregated with 5% SDS and the absorbance of the colored formazan salt was measured at 595 nM in a spectrophotometer plate reader. Measurement of cytosolic free Ca+2. Cell cultures of 7 days seeded onto 18-mm glass coverslips were washed twice with cold Locke’s buffer. Cells were loaded with the calcium sensitive dye Fura-2 AM (0.5 μM) for 8 min at 37ºC in Locke’s buffer containing 0.1% bovine serum albumin (BSA). After incubation, the loaded cells were washed three times with cold Locke’s buffer. The glass coverslips were inserted into a thermostated chamber at 37ºC and cells were viewed with a Nikon Diaphot 200 microscope, equipped with epifluorescence optics. The excitation wavelengths for Fura-2 AM were 340 and 380 nm, and emission was collected at 505 nm. The experiments were performed in triplicate. Toxins and drugs used. Cramb816 and CrambC were extracted and isolated from the Mediterranean sponge Crambe crambe (Bondu et al., 2012). Nifedipine (NIF) and D(-)-2-amino-5-phosphonopentanoic acid (APV) were purchased from Sigma- Aldrich and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was from Tocris. The final concentration of NIF, Cramb816 and CrambC solvent (DMSO), was less than 0.01%. APV and CNQX were solved in H2O. Statistical Analysis. All data are expressed as means ± SEM of n determinations. Statistical comparison was by paired Student’s t test. P-values <0.05 were considered statistically significant. RESULTS AND DISCUSSION Cytotoxic effect of Cramb816 and CrambC on cortical neurons. As shown in Figure 1A and Figure 1B, 24 hours exposure of 4-6 DIV cortical neurons to concentrations of Cramb816 and CrambC ranging from 10 to 1000 nM did not affect cellular viability. These results are in contrast with a previous report where Cramb816 produced an almost complete cell death at 1000 nM (Bondu et al., 2012). However in their experimental conditions they used 25 mM K+ in the media which could be neurotoxic. In order to clarify if this could explain the discrepancies with our results, neurons were treated with 25 mM K+ in the absence and presence of 1 μM Cramb816. As shown in Figure 2, incubation with 25 mM K+ produced an almost complete cell death reducing the cellular viability by 81.9 ± 5.2 % (n = 3, p < 0.001). A similar result was obtained in those cells coincubated with 25 mM K+ and 1 μM Cramb816 where neuronal viability decreased by 86.5 ± 4.4 % (n = 3, p < 0.001). This indicates that a media containing 25 mM K+ may exacerbate the cytotoxic effect of Cramb816 in cortical neurons. Since previously Bondu et al. had revealed that Cramb816 was cytotoxic in the same cortical neurons (Bondu et al., 2012), we increased the concentration of both, Cramb816 and CrambC. Therefore we studied the effects of both toxins at 10 μM obtaining that Cramb816 (Figure 3A) and CrambC (Figure 3B) reduced the cellular viability by 55.9 ± 3.3% (n = 3, p < 0.05) and 66.8 ± 5.5% (n = 3, p < 0.05), respectively. Effect of Cramb816 in cytosolic calcium concentation [Ca+2]c. Intracellular calcium levels are critical to regulate neuronal excitability (Torkkeli et al., 2012), release of transmitters (Augustine et al., 1987), synaptic plasticity (Dou et al., 2001;Neher, 2001;Baker et al., 2013;Catterall et al., 2013), and apoptosis (Tong et al., 1996;Gwag et al., 1999;Momeni and Jarahzadeh, 2012). The effect of 1, 5 and 10 µM Cramb816 on [Ca+2]c was evaluated first in 4-6 DIV neurons. In a Ca+2 free medium (Figure 4A), Cramb816 did not affect [Ca+2]c, thus indicating that the intracellular Ca+2 stores were not affected by the toxin. However, when 1 mM Ca+2 was added to the bath solution, Cramb816 induced Ca+2 influx in a concentration dependent manner. Thus, the average ratio of 340/380 wavelengths measured five minutes after the addition of the drugs indicated that 5 μM and 10 μM Cramb816 significantly enhanced the [Ca+2]c by 17.5 ± 0.9% (n = 4; p = 0.004) and 30.6 ± 4.3% (n = 4; p = 0.002), respectively, while at 1 μM Cramb816 did not affect [Ca+2]c. Since calcium homeostasis changes during neuronal development (Kortekaas and Wadman, 1997), we further elucidated whether the effect of Cramb816 was dependent on the neuronal development. Therefore, we compared the effect of 10 μM Cramb816 in immature (4-6 DIV) and mature cortical neurons (10-11 DIV). In a Ca+2 free media 10 µM Cramb816 did not affect [Ca+2]c neither in immature nor in mature neurons (Figure 4B). However, when 1 mM Ca+2 was added to the bath solution, 10 μM Cramb816 enhanced the [Ca+2]c by 26.5 ± 4.1% (n = 4, p = 0.001) in young neurons and only by 10.9 ± 1.7% (n = 4, p = 0.004) in mature cortical neurons, thus indicating that young neurons are more susceptible than mature neurons to the Cramb816 insult regarding its effect on intracellular calcium concentration. Taking into account that Cramb816 did not affect cortical neurons in the absence of Ca+2 in the extracellular media, the following experiments were performed in a media containing 1 mM Ca+2. We chose cortical neurons between 4-6 DIV for further experiments because of their higher sensibility to Cramb816. Thus, we then evaluated the effect of the toxin again at 1, 5 and 10 μM due to the fact that the presence of calcium during the whole recording could influence the basal fluorescence ratio. In the presence of calcium 5 μM and 10 μM Cramb816 significantly enhanced [Ca+2]c by 19.6 ± 7.3% (n = 4, p = 0.03) and 37.4 ± 9.0% (p = 0.009), respectively, (Figure 5). However this effect was not significant for the lowest concentration of Cramb816, 1 μM. In neurons, the entry of calcium from the outside is regulated mainly by voltage-gated channels and by receptor operated channels activated by glutamate. Therefore, next the mechanisms underlying the Cramb816-dependent Ca+2 influx in cortical neurons were evaluated. As shown in Figure 6, blockade of L-type voltage gated calcium channels with 10 μM NIF, added 3 minutes prior to the administration of 10 μM Cramb816, did not affect the calcium increase elicited by Cramb816, thus indicating that the calcium influx elicited by Cramb816 in cortical neurons was not through L-type voltage gated calcium channels. Since activation of N-methyl-D-aspartate receptors (NMDAr) is known to induce Ca+2 influx in neurons, the involvement of these receptors in the calcium increase elicited by Cramb816 was also evaluated. In order to do this, NMDA receptors were blocked by 100 μM APV added to the bath solution 30 minutes before Cramb816. In this condition, the Cramb816 dependent Ca+2 influx was partially reduced (Figure 7A). In addition to NMDA receptors, activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAr) also induces calcium increases in neurons. Therefore, in the next set of experiments both NMDAr and AMPA receptors were simultaneously blocked in the presence of 100 μM APV and 20 μM CNQX for 30 min before toxin addition. As shown in Figure 7B, the Ca+2 influx produced by Cramb816 was completely prevented by simultaneous blockade of AMPA and NMDA glutamate receptors, thus indicating that the calcium increase elicited by Cramb816 in cortical neurons was the result of glutamate receptors activation. In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in cortical neurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.
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