Isaev D et al. (2023), Methylene Blue Inhibits Cromakalim-Activated K+...

XB-ART-59592

Membranes (Basel) 2023 Jan 18;132:. doi: 10.3390/membranes13020121.

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Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes.

Isaev D , Yang KS , Petroianu G , Lorke DE , Oz M .

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The effects of methylene blue (MB) on cromakalim-induced K+ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3-300 μM, MB inhibited K+ currents (IC50: 22.4 μM) activated by cromakalim, which activates KATP channels. MB inhibited cromakalim-activated K+ currents in a noncompetitive and voltage-independent manner. The respective EC50 and slope values for cromakalim-activation of K+ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 μM MB. The inhibition of cromakalim-induced K+ currents by MB was not altered by pretreatment with the Ca2+ chelator BAPTA, which suggests that MB does not influence Ca2+-activated second messenger pathways. K+ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the KATP ligand [3H]glibenclamide are not altered by MB in a concentration range between 1 μM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K+ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K+ currents in follicular cells of Xenopus oocytes.

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Species referenced: Xenopus laevis
GO keywords: ATP-sensitive potassium channel complex [+]

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	Figure 1. Cromakalim-induced outward currents are inhibited by MB in follicular cells of Xenopus oocytes. (A) Currents activated by 100 μM cromakalim (left), during co-administration of cromakalim and 30 μM MB following 20 min MB preincubation (middle), 20 min recovery (right). Administration times for cromakalim are shown with solid horizontal bars. Dotted lines represent continuous MB application during recordings. (B) Time course of the peak cromakalim-activated currents are shown in the absence (filled circles) and the presence of 30 µM MB (open circles). Each data point indicates the normalized means and S.E.M. of five to six experiments. The horizontal bar represents the duration of MB administration. (C) Concentration-response curve for the inhibitory effect of MB on cromakalim (100 μM)-activated currents. Data points are shown as means ± S.E.M. (n = 4–5). The curve is the best fit of the data to the logistic equation presented in the methods section. CKL: cromakalim; MB: methylene blue.
	Figure 2. Inhibition of cromakalim-induced K+ currents by MB is voltage-independent and not mediated through gap junctions. (A) Current-voltage relationship of cromakalim-activated currents recorded during 1 s voltage steps applied before (filled circles) and after application of 30 μM MB (open circles). (B) Different membrane potentials did not alter percentage inhibition of cromakalim-activated K+ currents by MB. Differences among the means of current inhibitions by 30 µM MB at different holding potentials were not statistically significant (p > 0.05, ANOVA, n = 5–7). (C) Inset to Figure 2C indicates the equivalent resistive-circuit diagram. The mean values for the sum of resistances through the oocyte gap junction (Rj), follicular cell membranes (Rf) in follicle-enclosed oocytes before (gray bars) and after 20 min administration of 30 μM MB (black bars) are shown on the right side (n = 14). Membrane resistance (Ro) of enzymatically defolliculated oocytes before and after MB treatment are presented on the left (n = 16). Values of resistances were calculated from current-voltage curves recorded in the range between –50 mV and +10 mV. (D) Effect of MB (30 μM) on cromakalim (100 μM)-induced K+ currents in control and in BAPTA-pretreated oocytes. CKL: cromakalim; MB: methylene blue.
	Figure 3. The effects of MB on the cromakalim dose-response curve and the specific binding of [3H]glibenclamide. (A) Concentration-response curves for cromakalim-activated currents in the absence (filled circles) and presence (open circles) of MB (30 μM). MB was administered for 20 min, and cromakalim and MB were then co-administered for 2 min. Data points represent the mean ± S.E.M. (n = 5–6; error bars not visible are smaller than the size of the symbols). Curves show the best fit of the data to the logistic equation presented in the methods section. The concentration-response curves are normalized to maximal control cromakalim response. (B) Specific binding as a function of the concentration of [3H]glibenclamide in the absence (filled circles) and presence of 30 μM MB (open circles). Data represent the means of four experimental measurements. The incubation time was 60 min at 22 °C, pH 7.5. In order to determine nonspecific binding, samples were incubated with 10 nM of unlabeled glibenclamide. (C) Increasing concentrations of MB do not alter the specific binding of [3H]glibenclamide (1 nM). Data represent the results of 4–5 experiments. Data points show means ± S.E.M. Microsomal membranes were incubated with 1 nM [3H]glibenclamide (0.3–0.5 mg/mL for 60 min) with increasing concentrations of MB in the medium. Free and bound [3H]glibenclamide were separated by filtration. MB: methylene blue.
	Figure 4. Effects of MB on K+ currents mediated through the C-terminally deleted Kir6.2 channels (KirΔC26) and the effects of cGMP modulating agents on MB inhibition of cromakalim-activated K+ currents. (A) Currents from inside-out patches in response to two s-voltage-ramps from –100 to +100 mV under symmetric conditions (140 mM K+) were recorded in the absence and presence of 30 μM MB. Leak currents, recorded in the presence of 10 mM ATP, were subtracted. (B) Time-course of the effect of 30 µM MB on K+ currents mediated through the C-terminally deleted Kir6.2 channels (KirΔC26) in the absence (filled circles) and presence (open circles) of MB (30 μM), respectively. The horizontal bar represents the administration time of MB. (C) The effects of 20 min preincubation with BAY 58–2667 (3 µM) and 8-Br-cGMP (100 µM) on inhibition of cromakalim (100 μM)-activated K+ currents by MB (30 µM) in follicle-enclosed oocytes (p > 0.05, n = 5–7, Student’s t-test). MB: methylene blue; Bay 58: BAY 58–2667.

References [+] :

Abi-Gerges, Methylene blue is a muscarinic antagonist in cardiac myocytes. 1997, Pubmed