Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na ϩ -K ϩ -2Cl Ϫ cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (Ն100 M). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC 50 ϳ20 M, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC 50 ϳ8 M), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 M had no effect, and 20 M caused 20% block of human Na ϩ -K ϩ -2Cl Ϫ cotransporter activity, in contrast to Ͼ90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl Ϫ currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Aquaporins (AQPs) in the major intrinsic family of proteins mediate fluxes of water and other small solutes across cell membranes. AQP1 is a water channel, and under permissive conditions, a nonselective cation channel gated by cGMP. In addition to mediating fluid transport, AQP1 expression facilitates rapid cell migration in cell types including colon cancers and glioblastoma. Work here defines new pharmacological derivatives of bumetanide that selectively inhibit the ion channel, but not the water channel, activity of AQP1. Human AQP1 was analyzed in the Xenopus laevis oocyte expression system by two-electrode voltage clamp and optical osmotic swelling assays. The aquaporin ligand bumetanide derivative AqB011 was the most potent blocker of the AQP1 ion conductance (IC 50 of 14 mM), with no effect on water channel activity (at up to 200 mM). The order of potency for inhibition of the ionic conductance was AqB011 . AqB007 .. AqB006 $ AqB001. Migration of human colon cancer (HT29) cells was assessed with a wound-closure assay in the presence of a mitotic inhibitor. AqB011 and AqB007 significantly reduced migration rates of AQP1-positive HT29 cells without affecting viability. The order of potency for AQP1 ion channel block matched the order for inhibition of cell migration, as well as in silico modeling of the predicted order of energetically favored binding. Docking models suggest that AqB011 and AqB007 interact with the intracellular loop D domain, a region involved in AQP channel gating. Inhibition of AQP1 ionic conductance could be a useful adjunct therapeutic approach for reducing metastasis in cancers that upregulate AQP1 expression.

SUMMARY1. Aquaporins (AQPs) are targets for drug discovery for basic research and medicine. Human diseases involving fluid imbalances and oedema are of major concern and involve tissues in which AQPs are expressed. The range of functional properties of AQPs is continuing to expand steadily with ongoing research in the field.2. Gating domains in AQPs are molecular sites for drug actions. Discovery of the arylsulphonamide AqB013 as an antagonist for AQP1 and AQP4 provided the first pharmacological agent with translational promise for the treatment of diseases in which AQPs have been implicated. The putative binding site for AqB013 in the internal vestibule of the AQP water pore involves amino acid residues that are located in the AQP loop D gating domain.3. Aquaporins have been proposed as novel targets in cancer and oedema and are associated with a surprising array of important processes in the brain and body, such as angiogenesis, cell migration, development and neuropathological diseases. Functions beyond their simple role as water channels are suggested by the subtype-specific regulation of AQP expression. In both cancer and brain oedema, current therapies are limited and new pharmacological approaches focused on AQPs offer exciting potential for clinical advances.

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by .20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

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