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Sci Rep
2018 Jun 29;81:9877. doi: 10.1038/s41598-018-27995-w.
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Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B.
Hampel P
,
Römermann K
,
MacAulay N
,
Löscher W
.
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The Na+-K+-2Cl- cotransporter NKCC1 plays a role in neuronal Cl- homeostasis secretion and represents a target for brain pathologies with altered NKCC1 function. Two main variants of NKCC1 have been identified: a full-length NKCC1 transcript (NKCC1A) and a shorter splice variant (NKCC1B) that is particularly enriched in the brain. The loop diuretic bumetanide is often used to inhibit NKCC1 in brain disorders, but only poorly crosses the blood-brain barrier. We determined the sensitivity of the two human NKCC1 splice variants to bumetanide and various other chemically diverse loop diuretics, using the Xenopus oocyte heterologous expression system. Azosemide was the most potent NKCC1 inhibitor (IC50s 0.246 µM for hNKCC1A and 0.197 µM for NKCC1B), being about 4-times more potent than bumetanide. Structurally, a carboxylic group as in bumetanide was not a prerequisite for potent NKCC1 inhibition, whereas loop diuretics without a sulfonamide group were less potent. None of the drugs tested were selective for hNKCC1B vs. hNKCC1A, indicating that loop diuretics are not a useful starting point to design NKCC1B-specific compounds. Azosemide was found to exert an unexpectedly potent inhibitory effect and as a non-acidic compound, it is more likely to cross the blood-brain barrier than bumetanide.
Figure 1. Chemical structures of clinically approved loop diuretics. According to their structures, the drugs were assigned to three groups. Several members of each group were tested for their potency to inhibit hNKCC1A vs. hNKCC1B.
Figure 2. Heterologous expression of hNKCC1A and hNKCC1B in Xenopus laevis oocytes, determined by Western blotting, and the effect on transport activity, measured by 86RB+ uptake in the absence of NKCC1 inhibitors. (A) Representative Western blots of hNKCC1A and hNKCC1B protein expression in injected oocytes as well as endogenous NKCC1 in uninjected oocytes determined with the anti-NKCC1 antibody ab59791. The membrane was cut at 70 kDa in order to perform anti-NKCC1 and anti-β-actin immunostainings in separate containers (for more details see Suppl. Figure 1). In B–D, data are shown as means ± SEM. Significant differences to uninjected controls is indicated by asterisks (*P < 0.05; *P < 0.01; ****P < 0.0001). (B) Quantitative data on protein expression of endogenous NKCC1, hNKCC1A, and hNKCC1B after normalization to the reference signals of ß-actin. Data are from 4 (uninjected controls) and 7 (hNKCC1A, hNKCC1B) independent experiments, with 1–4 oocytes per experiment. Protein expression of hNKCC1A vs. hNKCC1B was not significantly different. (C) Absolute values showing 86RB+ uptake in uninjected control oocytes (reflecting endogenous NKCC activity) vs. uptake in oocytes expressing either hNKCC1A or hNKCC1B. (D) Fold increase in 86RB+ uptake by oocytes expressing either hNKCC1A or hNKCC1B compared to uninjected oocytes. Data in (C) and (D) are from 22 (uninjected controls), 21 (hNKCC1A) and 20 (hNKCC1B) independent experiments, with 4–20 oocytes per experiment.
Figure 3. Effect of bumetanide on hNKCC1A- and hNKCC1B-mediated 86Rb+ uptake in Xenopus oocytes. (A) A representative experiment demonstrating the inhibitory effect of 0.03–100 µM bumetanide on 86Rb+ uptake (in CPM, counted for 10 minutes) in hNKCC1A- and hNKCC1B-expressing oocytes and on batch-matched uninjected oocytes. Data are shown as median of n = 5–10 oocytes per condition and error bars illustrate the interquartile range. (B) Dose-inhibition curve of bumetanide on hNKCC1A- and hNKCC1B-mediated 86Rb+ uptake (corrected for endogenous NKCC contribution in uninjected oocytes) normalized to control (0 µM bumetanide) and averaged across 7 experiments, with the IC50 calculated from each individual experiment prior to averaging.
Figure 4. Correlation between IC50 for inhibition of hNKCC1A vs. hNKCC1B in the Xenopus oocyte assay for the loop diuretics evaluated in this study (see Table 1). Note that log data were used for correlation analysis.
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