Cheikh A et al. (2019), Xenopus Oocyte's Conductance for Bioactive Comp...

XB-ART-55916

Int J Mol Sci 2019 Apr 27;209:. doi: 10.3390/ijms20092083.

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Xenopus Oocyte's Conductance for Bioactive Compounds Screening and Characterization.

Cheikh A , Tabka H , Tlili Y , Santulli A , Bouzouaya N , Bouhaouala-Zahar B , Benkhalifa R .

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BACKGROUND: Astaxanthin (ATX) is a lipophilic compound found in many marine organisms. Studies have shown that ATX has many strong biological properties, including antioxidant, antiviral, anticancer, cardiovascular, anti-inflammatory, neuro-protective and anti-diabetic activities. However, no research has elucidated the effect of ATX on ionic channels. ATX can be extracted from shrimp by-products. Our work aims to characterize ATX cell targets to lend value to marine by-products. METHODS: We used the Xenopus oocytes cell model to characterize the pharmacological target of ATX among endogenous Xenopus oocytes' ionic channels and to analyze the effects of all carotenoid-extract samples prepared from shrimp by-products using a supercritical fluid extraction (SFE) method. RESULTS: ATX inhibits amiloride-sensitive sodium conductance, xINa, in a dose-dependent manner with an IC50 of 0.14 µg, a maximum inhibition of 75% and a Hill coefficient of 0.68. It does not affect the potential of half activation, but significantly changes the kinetics, according to the slope factor values. The marine extract prepared from shrimp waste at 10 µg inhibits xINa in the same way as ATX 0.1 µg does. When ATX was added to the entire extract at 10 µg, inhibition reached that induced with ATX 1 µg. CONCLUSIONS: ATX and the shrimp Extract inhibit amiloride-sensitive sodium channels in Xenopus oocytes and the TEVC method makes it possible to measure the ATX inhibitory effect in bioactive SFE-Extract samples.

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	Graphical Abstract.
	Figure 1 Xenopus oocytes current recordings obtained in different conditions of bathing media and holding potentials. The same oocyte was used for the experiments in each panel. Representative current traces of Xenopus oocytes held at −60 mV in BAMS medium (Na+ = 50 mM) (a), in OR2 medium (Na+ = 82.5 mM) (b), and in OR2 medium at Vh = −20 mV (c). Current-voltage relationships constructed using the current sizes of a, b and c families at different potential steps (d).
	Figure 2 ATX inhibits Endogenous currents in Xenopus oocytes. TEVC currents recorded with [Na+] (82.5 mM) in the bath. Current families traces in control conditions (a), in the presence of ATX (b) and after washout (c). Current-voltage relationships constructed using the current sizes of a, b and c families at different potential steps (d).
	Figure 3 ATX inhibits voltage gated sodium channels that are sensitive to amiloride in Xenopus oocytes. TEVC currents were recorded with [Na+] (82.5 mM) in the bath. In (A) Current traces families in control conditions (a) in the presence of ATX (b) with ATX removed and replaced by TTX (c). Current-potentials relationships constructed from a, b and c show that ATX inhibits the current amplitude contrary to TTX (d). In (B) Current families trace in control conditions (a) in the presence of ATX (b) and then in presence of amiloride (c). Current-potentials relationships constructed from a, b and c show that ATX inhibits the current amplitude in the same manner as amiloride (d).
	Figure 4 Reversible inhibitory effect of SFE-Extract on endogenous currents in Xenopus oocytes in OR2 and BAMS media. In OR2, the currents were recorded according to the protocol above in the control conditions (a) in the presence of SFE-Extract (10 µg) (b) after washout (c). I–V relationships are plotted in (d). Oocytes held at −80 mV in BAMS solution containing CsOH, 2 mM to block potassium currents. The oocytes were then depolarized from −40 to +100 mV during 300 ms. Current time course inhibition of SFE-Extract, 10 μg. Maximal effect occurs about 2 min after the beginning of the perfusion. A partial recovery from the inhibitory effect was observed after the washout of the Extract (e).
	Figure 5 Analysis of ATX and SFE-Extract inhibitory effects on amiloride-sensitive sodium currents in Xenopus oocytes. In (A) Doses-response curve for the M1 inhibitory effect on Xenopus endogenous current traces recorded during a 250 ms voltage clamp step at 60 mV from a HP of −60 mV. (B) Histograms illustrating the ATX inhibitory effect of the current peak at different concentrations. In (C) Histograms of the percentage of inhibition in the presence of SFE-Extract (10 µg) alone and enriched by ATX 1 µg.
	Figure 6 Characterization of ATX and SFE-Extract effects on sodium currents activation in Xenopus oocytes. In (A) Time course inhibition in the presence of SFE-Extract alone, the extract enriched with ATX, and then the ATX alone. Maximal effect is obtained in the presence of ATX and the washout allows a partial recovery of the current (a). Representative traces of xINa recorded at +60 mV in control conditions and in the presence of SFE-Extract and SFE-Extract enriched with ATX (1 µg) (b). In (B) Mean steady state activation curves of xINa in control conditions, in the presence of ATX (a) SFE-Extract (10 µg) (b) and SFE-Extract/ATX (c). Data are means ± SEM (n = 4 to 6) and fitted with a standard Boltzmann function.
	Figure 1. Xenopus oocytes current recordings obtained in different conditions of bathing media and holding potentials. The same oocyte was used for the experiments in each panel. Representative current traces of Xenopus oocytes held at −60 mV in BAMS medium (Na+ = 50 mM) (a), in OR2 medium (Na+ = 82.5 mM) (b), and in OR2 medium at Vh = −20 mV (c). Current-voltage relationships constructed using the current sizes of a, b and c families at different potential steps (d).
	Figure 2. ATX inhibits Endogenous currents in Xenopus oocytes. TEVC currents recorded with [Na+] (82.5 mM) in the bath. Current families traces in control conditions (a), in the presence of ATX (b) and after washout (c). Current-voltage relationships constructed using the current sizes of a, b and c families at different potential steps (d).
	Figure 3. ATX inhibits voltage gated sodium channels that are sensitive to amiloride in Xenopus oocytes. TEVC currents were recorded with [Na+] (82.5 mM) in the bath. In (A) Current traces families in control conditions (a) in the presence of ATX (b) with ATX removed and replaced by TTX (c). Current-potentials relationships constructed from a, b and c show that ATX inhibits the current amplitude contrary to TTX (d). In (B) Current families trace in control conditions (a) in the presence of ATX (b) and then in presence of amiloride (c). Current-potentials relationships constructed from a, b and c show that ATX inhibits the current amplitude in the same manner as amiloride (d).
	Figure 4. Reversible inhibitory effect of SFE-Extract on endogenous currents in Xenopus oocytes in OR2 and BAMS media. In OR2, the currents were recorded according to the protocol above in the control conditions (a) in the presence of SFE-Extract (10 µg) (b) after washout (c). I–V relationships are plotted in (d). Oocytes held at −80 mV in BAMS solution containing CsOH, 2 mM to block potassium currents. The oocytes were then depolarized from −40 to +100 mV during 300 ms. Current time course inhibition of SFE-Extract, 10 μg. Maximal effect occurs about 2 min after the beginning of the perfusion. A partial recovery from the inhibitory effect was observed after the washout of the Extract (e).
	Figure 5. Analysis of ATX and SFE-Extract inhibitory effects on amiloride-sensitive sodium currents in Xenopus oocytes. In (A) Doses-response curve for the M1 inhibitory effect on Xenopus endogenous current traces recorded during a 250 ms voltage clamp step at 60 mV from a HP of −60 mV. (B) Histograms illustrating the ATX inhibitory effect of the current peak at different concentrations. In (C) Histograms of the percentage of inhibition in the presence of SFE-Extract (10 µg) alone and enriched by ATX 1 µg.
	Figure 6. Characterization of ATX and SFE-Extract effects on sodium currents activation in Xenopus oocytes. In (A) Time course inhibition in the presence of SFE-Extract alone, the extract enriched with ATX, and then the ATX alone. Maximal effect is obtained in the presence of ATX and the washout allows a partial recovery of the current (a). Representative traces of xINa recorded at +60 mV in control conditions and in the presence of SFE-Extract and SFE-Extract enriched with ATX (1 µg) (b). In (B) Mean steady state activation curves of xINa in control conditions, in the presence of ATX (a) SFE-Extract (10 µg) (b) and SFE-Extract/ATX (c). Data are means ± SEM (n = 4 to 6) and fitted with a standard Boltzmann function.

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