Aquaporin Inhibitors Research Articles

Diuretic effect of Lagopsis supina fraction in saline-loaded rats is mediated through inhibition of aquaporin and renin-angiotensin-aldosterone systems and up-regulation of atriopeptin

Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. has been used as a diuretic agent in China for centuries with limited scientific evidence. This study investigated the diuretic efficacy and underlying mechanism of a macroporous adsorption resin with 30% ethanol elution fraction from L. supina (LSC) in saline-loaded rats and to identify its phytochemicals by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS). As a result, 18 phenylpropanoids, 14 flavonoids and 15 others were identified in LSC, among which stachysoside A and acteoside could be the main bio-active constituents responsible for the diuretic effect. In parallel, the daily administration of LSC (16, 32 and 64 mg/kg) markedly promoted urinary excretion after 2 h of treatment. Moreover, LSC had no effect on urinary Na+ and K+ concentrations, as well as on serum Na+-K+-ATPase activity. Meanwhile, LSC significantly decreased the serum levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), aldosterone (ALD), aquaporin (AQP) 1, AQP2 and AQP3, suppressed renal AQP1, AQP2, and AQP3 mRNA expressions, down-regulated AQP1, AQP2 and AQP3 protein levels, and up-regulated serum atriopeptin (ANP) level in a dose-dependent manner. These findings suggest that LSC has acute and prolonged diuretic effects by inhibiting the AQPs, RAAS, and upregulation of atriopeptin in saline-loaded rats, and this finding support LSC as a novel diuretic agent.

Inhibition of aquaporins as a potential adjunct to breast cancer cryotherapy.

Cryoablation is an emerging type of treatment for cancer. The sensitization of tumors using cryosensitizing agents prior to treatment enhances ablation efficiency and may improve clinical outcomes. Water efflux, which is regulated by aquaporin channels, contributes to cancer cell damage achieved through cryoablation. An increase in aquaporin (AQP) 3 is cryoprotective, whereas its inhibition augments cryodamage. The present study aimed to investigate aquaporin (AQP1, AQP3 and AQP5) gene expression and cellular localization in response to cryoinjury. Cultured breast cancer cells (MDA-MB-231 and MCF-7) were exposed to freezing to induce cryoinjury. RNA and protein extracts were then analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Localization of aquaporins was studied using immunocytochemistry. Additionally, cells were transfected with small interfering RNA to silence aquaporin gene expression and cell viability was assessed using the Sulforhodamine B assay. Cryoinjury did not influence gene expression of AQPs, except for a 4-fold increase of AQP1 expression in MDA-MD-231 cells. There were no clear differences in AQP protein expression for either cell lines upon exposure to frozen and non-frozen temperatures, with the exception of fainter AQP5 bands for non-frozen MCF-7 cells. The exposure of cancer cells to freezing temperatures altered the localization of AQP1 and AQP3 proteins in both MCF-7 and MDA-MD-231 cells. The silencing of AQP1, AQP3 and AQP5 exacerbated MDA-MD-231 cell damage associated with freezing compared with control siRNA. This was also observed with AQP3 and AQP5 silencing in MCF-7 cells. Inhibition of aquaporins may potentially enhance cryoinjury. This cryosensitizing process may be used as an adjunct to breast cancer cryotherapy, especially in the border area targeted by cryoablation where freezing temperatures are not cold enough to induce cellular damage.

Osmotic adaptation of nucleus pulposus cells: the role of aquaporin 1, aquaporin 4 and transient receptor potential vanilloid 4.

The microenvironment of the nucleus pulposus is hyperosmotic and fluctuates diurnally due to mechanical loading. Changes in extracellular osmolality result in cell volume alterations, responsiveness to such changes is essential for cellular homeostasis. Aquaporins allow movement of water across cell membranes and control water permeability in response to osmotic gradients. Furthermore, transient receptor potential vanilloid 4 has been shown to sense osmotic and mechanical stimuli resulting in changes to intracellular Ca2+. It has been shown previously that aquaporin 1 and 4 expression decreases during disc degeneration. Here, the expression of transient receptor potential vanilloid 4 by human nucleus pulposus cells during disc degeneration, and the roles of aquaporin 1, 4 and transient receptor potential vanilloid 4 in regulating responses to osmotic gradients was investigated. Transient receptor potential vanilloid 4 was expressed by the majority of human nucleus pulposus cells and not affected by disc degeneration. Aquaporin 4 staining co-localised with primary cilia. Nucleus pulposus cells modulated their rate of volume change, water permeability and Ca2+ influx in response to extracellular osmolality. These responses were inhibited by chemical inhibition of aquaporin 4, transient receptor potential vanilloid 4, and to a lesser extent aquaporin 1; suggesting that both aquaporins and transient receptor potential vanilloid 4 play important roles in the fundamental adaptation of nucleus pulposus cells to their osmotic environment. Co-localisation with primary cilia indicates these proteins may function synergistically to achieve adaptation, which may be lost during disc degeneration, when aquaporin 1 and 4 expression is reduced.

Secondary mechanisms of injury and viable pathophysiological targets in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) can be divided into a primary and secondary phase. In the primary phase, hematoma volume is evaluated and therapies are focused on reducing hematoma expansion. In the secondary, neuroprotective phase, complex systemic inflammatory cascades, direct cellular toxicity, and blood-brain barrier disruption can result in worsening perihematomal edema that can adversely affect functional outcome. To date, all major randomized phase 3 trials for ICH have targeted primary phase hematoma volume and incorporated clot evacuation, intensive blood pressure control, and hemostasis. Reasons for this lack of clinical efficacy in the major ICH trials may be due to the lack of therapeutics involving mitigation of secondary injury and inflexible trial design that favors unilateral mechanisms in a complex pathophysiology. Potential pathophysiological targets for attenuating secondary injury are highlighted in this review and include therapies increasing calcium, antagonizing microglial activation, maintaining macrophage M1 versus M2 balance by decreasing M1 signaling, aquaporin inhibition, NKCCl inhibition, endothelin receptor inhibition, Sur1-TRPM4 inhibition, matrix metalloproteinase inhibition, and sphingosine-1-phosphate receptor modulation. Future clinical trials in ICH focusing on secondary phase injury and, potentially implementing adaptive trial design approaches with multifocal targets, may improve insight into these mechanisms and provide potential therapies that may improve survival and functional outcome.

Aquaporins (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

Aquaporins and aquaglyceroporins are membrane channels that allow the permeation of water and certain other small solutes across the cell membrane, or in the case of AQP6, AQP11 and AQP12A, intracellular membranes, such as vesicles and the endoplasmic reticulum membrane [17]. Since the isolation and cloning of the first aquaporin (AQP1) [21], 12 additional mammalian members of the family have been identified, although little is known about the functional properties of one of these (AQP12A; Q8IXF9) and it is thus not tabulated. The other 12 aquaporins can be broadly divided into three families: orthodox aquaporins (AQP0,-1,-2,-4,-5, -6 and -8) permeable mainly to water, but for some additional solutes [5]; aquaglyceroporins (AQP3,-7 -9 and -10), additionally permeable to glycerol and for some isoforms urea [16], and superaquaporins (AQP11 and 12) located within cells [14]. Some aquaporins also conduct ammonia and/or H2O2 giving rise to the terms 'ammoniaporins' ('aquaammoniaporins') and 'peroxiporins', respectively. Aquaporins are impermeable to protons and other inorganic and organic cations, with the possible exception of AQP1 [16]. One or more members of this family of proteins have been found to be expressed in almost all tissues of the body [reviewed in Yang (2017) [27]]. AQPs are involved in numerous processes that include systemic water homeostasis, adipocyte metabolism, brain oedema, cell migration and fluid secretion by epithelia and loss of function mutations of some human AQPs, or their disruption by autoantibodies further underscore their importance [reviewed by Verkman et al. (2014) [24], Kitchen et al. (2105) [16]]. Functional AQPs exist as homotetramers that are the water conducting units wherein individual AQP subunits (each a protomer) have six transmembrane helices and two half helices that constitute a seventh 'pseudotransmembrane domain' that surrounds a narrow water conducting channel [17]. In addition to the four pores contributed by the protomers, an additional hydrophobic pore exists within the center of the complex [17] that may mediate the transport of gases (e.g. O2, CO2, NO) and cations (the central pore is the proposed transport pathway for cations through AQP1) by some AQPs [8, 15]. Although numerous small molecule inhibitors of aquaporins, particularly APQ1, have been reported primarily from Xenopus oocyte swelling assays, the activity of most has subsequently been disputed upon retesting using assays of water transport that are less prone to various artifacts [6] and they are therefore excluded from the tables [see Tradtrantip et al. (2017) [23] for a review].

Key factors affect selenite absorption in wheat leaf blades: pH, temperature, light intensity and leaf position

Foliage-spraying selenite solution is an effective measure to enhance selenium (Se) concentration in wheat grains. However, how pH, temperature, light intensity, and leaf position affects selenite absorption in wheat leaf blades is not fully understood. In this study, the effects of pH, temperature, light intensity, and leaf position on selenite absorption in wheat leaf blades were investigated. The results indicated that the selenite absorption rate dramatically decreased with increasing pH. Further study revealed that aquaporin inhibitors such as HgCl<sub>2</sub> and AgNO<sub>3</sub> strongly inhibited selenite absorption at pH 3.0. Light and higher temperatures significantly promoted selenite absorption. Newly expanded leaf blades had higher rates of selenite absorption than younger and older leaf blades. Thus, higher rates of selenite absorption in leaf blades should attribute to the entrance of selenite into mesophyll cells via aquaporins in the form of H<sub>2</sub>SeO<sub>3</sub> at low pH values. Foliage-spraying selenite solution on upper leaf blades at lower pH values benefited to increase the selenite absorption rate in wheat leaf blades.

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Uptake, translocation and biotransformation of selenium nanoparticles in rice seedlings (Oryza sativa L.)

BackgroundSelenium (Se) in soil mainly consists of selenite, selenate, and elemental Se. However, little is known about the mechanism involved in the uptake and biotransformation of elemental Se by plants.ResultsIn this study, the uptake, translocation, subcellular distribution and biotransformation of selenium nanoparticles (SeNPs) in rice (Oryza sativa L.), and a comparison with selenite and selenate, were investigated through hydroponic experiments. The study revealed that SeNPs could be absorbed by rice plants; and aquaporin inhibitor was responsible for a 60.4% inhibition of SeNP influx, while metabolic inhibitor was ineffective. However, the SeNPs uptake rate of rice roots was approximately 1.7 times slower than that of selenite or selenate. Under the SeNPs or selenite treatment, Se was primarily accumulated in roots rather than in shoots, whereas an opposite trend was observed with selenate treatment. Additionally, most of the absorbed Se was distributed in cell wall of the SeNPs or selenite treated-rice plants, while its proportion was the highest in soluble cytosol of the selenate treated-rice plants. The absorbed SeNPs or selenite was rapidly assimilated to organic forms, with SeMet being the most predominant species in both shoots and roots of the rice plants. However, following selenate treatment, Se(VI) remained as the most predominant species, and only a small amount of it was converted to organic forms.ConclusionTherefore, this study provides a deeper understanding of the mechanisms associated SeNPs uptake and biotransformation within plants.

Transpiration difference under high evaporative demand in chickpea (Cicer arietinum L.) may be explained by differences in the water transport pathway in the root cylinder.

Terminal drought substantially reduces chickpea yield. Reducing water use at vegetative stage by reducing transpiration under high vapor pressure deficit (VPD), i.e. under dry/hot conditions, contributes to drought adaptation. We hypothesized that this trait could relate to differences in a genotype's dependence on root water transport pathways and hydraulics. Transpiration rate responses in conservative and profligate chickpea genotypes were evaluated under increasing VPD in the presence/absence of apoplastic and cell-to-cell transport inhibitors. Conservative genotypes ICC 4958 and ICC 8058 restricted transpiration under high VPD compared to the profligate genotypes ICC 14799 and ICC 867. Profligate genotypes were more affected by aquaporin inhibition of the cell-to-cell pathway than conservative genotypes, as measured by the root hydraulic conductance and transpiration under high VPD. Aquaporin inhibitor treatment also led to a larger reduction in root hydraulic conductivity in profligate than in conservative genotypes. In contrast, blockage of the apoplastic pathway in roots decreased transpiration more in conservative than in profligate genotypes. Interestingly, conservative genotypes had high early vigour, whereas profligate genotypes had low early vigour. In conclusion, profligate genotypes depend more on the cell-to-cell pathway, which might explain their higher root hydraulic conductivity, whereas water-saving by restricting transpiration led to higher dependence on the apoplastic pathway. This opens the possibility to screen for conservative or profligate chickpea phenotypes using inhibitors, itself opening to the search of the genetic basis of these differences.

Exogenous melatonin alleviates PEG-induced short-term water deficiency in maize by increasing hydraulic conductance

BackgroundWater deficiency is likely to become more frequent and intense as a result of global climate change, which may severely impact agricultural production in the world. The positive effects of melatonin (MEL) on alleviation drought or osmotic stress-induced water deficiency in plants has been well reported. However, the underlying mechanism of MEL on the detailed process of plant water uptake and transport under water deficiency condition remains largely unknown.ResultsApplication of 1 μM MEL led to enhanced tolerance to water deficiency stress in maize seedlings, as evidenced by maintaining the higher photosynthetic parameters, leaf water status and plant transpiration rate. The relatively higher whole-plant hydraulic conductance (Kplant) and root hydraulic conductance (Lpr) in MEL-treated seedlings suggest that exogenous MEL alleviated water deficiency stress by promoting root water absorption. HgCl2 (aquaporin inhibitor) treatment inhibit the transpiration rate in MEL-treated plants greater than those of MEL-untreated; after recovery by dithiothreitol (DTT, anti-inhibitor), the transpiration rate in MEL-treated plants increased much higher than those of untreated plants. Moreover, under water deficiency, the transcription level of aquaporin genes was up-regulated by MEL application, and the H2O2 was less accumulated in MEL-treated root.ConclusionsExogenous MEL promoted aquaporin activity, which contributed to the maintaining of Lpr and Kplant under short-term water deficiency. The increased water uptake and transport lead to improved water status and thus increased tolerance to PEG-induced short-term water deficiency in maize seedlings.

Aquaporin3 expression and the potential role of aquaporins in motility and mitochondrial membrane potential in human spermatozoa.

Aquaporins are water selective channels which play important roles in cell volume regulation during the transmission of spermatozoa to female tract. This study investigated the expression of aquaporin3 and determined the role of aquaporins in human sperm motility and mitochondrial membrane potential (MMP). RT-PCR and flow cytometry analysis were done to investigate aquaporin3 expression levels, and immunolocalisation of aquaporin3 in the spermatozoa was detected using immunocytochemical analysis. The sperm suspension was divided into four groups of spermatozoa: (a) Spermatozoa at 0hr, (b) spermatozoa in control group, (c) spermatozoa treated with HgCl2 (as an aquaporin inhibitor) and (d) spermatozoa treated with HgCl2 + and 2-mercaptoethanol. The sperm samples were examined in terms of sperm motility and mitochondrial membrane potential. Results confirmed aquaporin3 expression in human spermatozoa and immunocytochemistry results showed an intense immunoreactivity in whole sperm tail. After 60min, HgCl2 showed a significant decrease in motility and MMP compared to the control group. At this time point, 2-mercaptoethanol in the HgCl2 + 2-mercaptoethanol group reversed the effects of HgCl2 as compared to the HgCl2 group. Present study showed the expression and immunolocalisation of AQP3 in human spermatozoa and the potential role of AQPs in the sperm motility and MMP.

Genomic Profiling and Physiological Approaches to Understand Aquaporins and their Role in ROS Signalling within Skeletal Muscle

Aquaporin (AQP) channels are a family of integral membrane proteins shown to aid the movement of hydrogen peroxide (H202) across membranes [1]. Their role in skeletal muscle is of key importance since intracellular generation of H202 and other reactive oxygen species (ROS) occurs in response to muscle contractions. These ROS initiate signalling processes and activate of a number of transcription factors [2, 3]. During ageing, excessive levels of ROS have pathological effects, inducing muscle degradation, loss of neuromuscular junction integrity and loss of neuromuscular transmission.Our studies [4], as well as those of others, has identified the sources, sinks and functions of H202 in muscle but exactly how H202 is transported from its site of origin within muscle has never been examined. We hypothesize that (AQPs) are the primary means of regulating H202 movement through skeletal muscle membranes. This study has therefore examined expression of AQP isoforms in muscle and how they may change with age.RNA transcripts from Tibialis Anterior (TA) muscle from control adult (6–8 months), and old (26month) mice, and from mice following peroneal nerve crush, were examined by RNAseq (150bp‐read depth >280 M clusters per lane, n=5). Individual muscle myotubes were also treated pharmacologically with aquaporin inhibitors and transfected with AAV6‐HyPer2 to determine the role of different AQP isoforms on intracellular H202 content and H20 permeability was determined using the swell assay [5, 6].RNAseq data identified 8 AQP mRNA transcripts present in TA muscle. A decrease in AQP4 and AQP1 and an increase in AQP3 contents were found following nerve crush (n=5, P<0.01) and this was confirmed by immunological techniques. The localisation and roles of these aquaporins is currently being investigated to determine whether they have protective functions or contribute to the age‐related decline skeletal muscle.An exponential increase in intracellular H2O2 levels was recorded when AAV6‐Hyper2 transfected muscle fibres were exposed to 10μM H2O2. Pre‐treatment of muscle cells with AQP blockers HgCl2 (30μM), TEA (3mM) or Bumetanide (5μM) substantially decreased the rate of increase in intracellular H202 content. These data demonstrate the ability of aquaporins to regulate skeletal muscle H2O2 permeability. Challenging cells with an 80mOsm solution in conjunction with pharmacological AQP1 inhibitors also demonstrated a significant reduction in the rate of increase in cell volume.In summary, these data demonstrate a potential important role for AQPs in muscle. Further work using AAV‐shRNA to knockdown specific AQPs is being utilised to identify the major AQPs that function in skeletal muscle.Support or Funding InformationThis study was supported by the MRC and NIA (AG051442).

AqF026 may act as a cancer therapeutic agent via inducing cancer cell oncosis

Cancer is one of the leading causes of death worldwide, and metastasis is the major cause of death in cancer, therefore, treatments to attenuate metastasis are urgently needed. Cell migration is indispensable in metastatic cascade, and aquaporins (AQPs) promote cell migration by facilitating water influx at cell front (lamellipodia). In fact, AQPs overexpressed widely among many cancer types. Accordingly, previous efforts of targeting AQPs as strategies of cancer treatments were based on AQP inhibitors, yet the efficacy of AQP inhibition was limited based on recent surveys. On the contrary, whether AQP agonist has role in cancer treatments has not been explored. AqF026, an AQP1 agonist, was initially applied to a mouse model of peritoneal dialysis. Herein, we aimed to apply AqF026 to magnify the water influx into lamellipodia of migrating cancer cells so as to induce oncosis by causing overloaded cancer cell swelling in advance of metastatic cascade. Cell swelling is a characteristic of oncosis. With impairment or insufficient regulatory volume decrease (RVD), cell swelling can lead to oncosis. Cancer cells with metastatic potentials shared the same population of cancer cells with multidrug resistance (MDR) lineage, and the impairment or insufficient RVD is shown in cancer cells with MDR. Taken together, the author hypothesized that given appropriate concentration or dose of AQP1 agonist AqF026, the AqF026 may induce oncosis of cancer cells preferentially rather than normal cells by causing overloaded water influx via AQP1 and consequent irreversible cell swelling.

The intracellular water volume modulates the accumulation of cadmium in Euglena gracilis

In order to describe a relatively novel mechanism associated to heavy metal resistance in Euglena gracilis, the osmotic responses during cadmium accumulation were analyzed. Several physiological/biochemical parameters were assessed in E. gracilis exposed to 50 and 200 μM Cd2+, concentrations well within the range found in polluted soils and aquatic environments. The Cd2+ accumulation attained after only 24 h exposure induced marked increases in both the intracellular water volume and cellular size, which were maintained for several days; these changes correlated with (i) an increased intracellular osmolarity (i.e. osmotic pressure) driven by a generalized increase in the osmo-metabolites (trehalose, phosphate-molecules, amino acids, thiol-molecules, betaines and polyamines) content; and (ii) the triggering of antioxidant defenses (increased GSH/GSSG ratios and GPx, GR and APx activities). In contrast, no changes in intracellular volume, cellular size and antioxidant status were observed under hyperaccumulation of Zn2+. Furthermore, E. gracilis cultured in a hypoosmotic medium promoted greater Cd2+ accumulation and water volume, compared to a hyperosmotic medium. These changes were significantly decreased by the aquaporin inhibitors Hg2+ and pentamidine. The results suggested that the cell responses to Cd2+ and osmotic stress share biochemical mechanisms in this algae-like protist, in which Cd2+ accumulation closely correlates with the intracellular water volume changes in an oxidative stress-mediated process.

Water relations responses of the pea (Pisum sativum L.) mutant SGECdt to mercury

Mercury (Hg) is one of the most toxic heavy metals and has multiple impacts on plant growth and physiology, including disturbances of plant water status. The impact of Hg on water relations was assessed by exposing the unique Hg-sensitive pea (Pisum sativum L.) mutant SGECdt and its wild-type (WT) line SGE in hydroponic culture. When the plants were grown in the presence of 1 or 2 µM HgCl2 for 11 days, the SGECdt mutant had lower whole plant transpiration rate and increased leaf temperature, indicating stomatal closure. Shoot removal of Hg-untreated plants resulted in greater root-pressure induced xylem sap flow in the SGECdt mutant than WT plants. Treating these plants with 50 µM HgCl2 (an inhibitor of aquaporins) for 1 h decreased xylem sap flow of both genotypes by about 5 times and eliminated differences between WT and mutant. Adding 1 mM dithiothreitol (the reducing thiol reagent used for opening aquaporins) to the nutrient solution of Hg-treated plants partially restored xylem sap flow in SGECdt roots only, suggesting genotypic differences in aquaporin function. Thus root water uptake is important in mediating sensitivity of SGECdt to toxic Hg.

Alleviation of Cerebral Infarction of Rats With Middle Cerebral Artery Occlusion by Inhibition of Aquaporin 4 in the Supraoptic Nucleus.

In ischemic stroke, vasopressin hypersecretion is a critical factor of cerebral swelling and brain injury. To clarify neural mechanisms underlying ischemic stroke-evoked vasopressin hypersecretion, we observed the effect of unilateral permanent middle cerebral artery occlusion (MCAO) in rats on astrocytic plasticity and vasopressin neuronal activity in the supraoptic nucleus (SON) as well as their associated cerebral injuries. MCAO for 8 hr caused cerebral infarction in the MCAO side where water contents also increased. Immunohistochemical examination revealed that the percentage of phosphorylated extracellular signal-regulated protein kinase 1/2 (pERK1/2)-positive vasopressin neurons in the SON of MCAO side was significantly higher than that in non-MCAO side and in sham group. In the cortex, pERK1/2 and aquaporin 4 expressions increased significantly in the infarction area, while glial fibrillary acidic protein (GFAP) reduced significantly compared with the noninfarction side in brain cortex. Microinjection of N-(1,3,4-Thiadiazolyl)nicotinamide-020 [TGN-020, a specific blocker of aquaporin 4] into the SON blocked MCAO-evoked increases in pERK1/2 in the SON as well as the reduction of GFAP and the increase in pERK1/2 and aquaporin 4 in the infarction area of the cortex. Finally, oxygen and glucose deprivation reduced GFAP expression and the colocalization and molecular association of GFAP with aquaporin 4 in the SON in brain slices. These effects were blocked by TGN-020 and/or phloretin, a blocker of astrocytic volume-regulated anion channels. These findings indicate that blocking aquaporin 4 in the SON may reduce the activation of vasopressin neurons and brain injuries elicited by vasopressin during ischemic stroke.

Effect of AQP Inhibition on Boar Sperm Cryotolerance Depends on the Intrinsic Freezability of the Ejaculate.

Aquaporins (AQPs) are transmembrane channels with permeability to water and small solutes that can be classified according to their structure and permeability into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs. In boar spermatozoa, AQPs are related to osmoregulation and play a critical role in maturation and motility activation. In addition, their levels differ between ejaculates with good and poor cryotolerance (GFE and PFE, respectively). The aim of this work was to elucidate whether the involvement of AQPs in the sperm response to cryopreservation relies on the intrinsic freezability of the ejaculate. With this purpose, two different molecules: phloretin (PHL) and 1,3-propanediol (PDO), were used to inhibit sperm AQPs in GFE and PFE. Boar sperm samples were treated with three different concentrations of each inhibitor prior to cryopreservation, and sperm quality and functionality parameters were evaluated in fresh samples and after 30 and 240 min of thawing. Ejaculates were classified as GFE or PFE, according to their post-thaw sperm viability and motility. While the presence of PHL caused a decrease in sperm quality and function compared to the control, samples treated with PDO exhibited better quality and function parameters than the control. In addition, the effects of both inhibitors were more apparent in GFE than in PFE. In conclusion, AQP inhibition has more notable consequences in GFE than in PFE, which can be related to the difference in relative levels of AQPs between these two groups of samples.

Molecular Modeling and Docking of Aquaporin Inhibitors to Reveal New Insights into Schistosomiasis Treatment.

Schistosomiasis (snail fever/bilharzia), a disease caused by parasitic flatworms (schistosomes), infects millions of people worldwide. Aquaporins from these organisms were found to be a potent drug target. We investigate the possible mechanism of inhibition of Aquaporin (AQP) from S.mansoni by 5 drug molecules (Praziquantel, Metrifonate, Artimisinin, Albendazole, and Amoscanate). 3D molecular structure of Aquaporin was obtained through homology modeling and further protein-ligand docking and MD simulation were performed. VAL-75, ASN-91, ALA-220, ASN-222, ARG-225 amino acids were found to play crucial role in ligand binding. TRP-71 and other important residues play major role in hydrophobic interactions stabilizing protein-ligand complexes. We hope that this study (with the newly identified aquaporin target) will support the development of structure and pharmacophore-based novel S. mansoni drugs to control and curb Schistosomiasis.

Aquaporins (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Aquaporins and aquaglyceroporins are membrane channels that allow the permeation of water and certain other small solutes across the cell membrane, or in the case of AQP6, AQP11 and AQP12A, intracellular membranes, such as vesicles and the endoplasmic reticulum membrane [9]. Since the isolation and cloning of the first aquaporin (AQP1) [10], 12 additional mammalian members of the family have been identified, although little is known about the functional properties of one of these (AQP12A; Q8IXF9) and it is thus not tabulated. The other 12 aquaporins can be broadly divided into three families: orthodox aquaporins (AQP0,-1,-2,-4,-5, -6 and -8) permeable mainly to water, but for some additional solutes [2]; aquaglyceroporins (AQP3,-7 -9 and -10), additionally permeable to glycerol and for some isoforms urea [8], and superaquaporins (AQP11 and 12) located within cells [5]. Some aquaporins also conduct ammonia and/or H2O2 giving rise to the terms 'ammoniaporins' ('aquaammoniaporins') and 'peroxiporins', respectively. Aquaporins are impermeable to protons and other inorganic and organic cations, with the possible exception of AQP1 [8]. One or more members of this family of proteins have been found to be expressed in almost all tissues of the body [reviewed in Yang (2017) [13]]. AQPs are involved in numerous processes that include systemic water homeostasis, adipocyte metabolism, brain oedema, cell migration and fluid secretion by epithelia and loss of function mutations of some human AQPs, or their disruption by autoantibodies further underscore their importance [reviewed by Verkman et al. (2014) [12], Kitchen et al. (2105) [8]]. Functional AQPs exist as homotetramers that are the water conducting units wherein individual AQP subunits (each a protomer) have six transmembrane helices and two half helices that constitute a seventh 'pseudotransmembrane domain' that surrounds a narrow water conducting channel [9]. In addition to the four pores contributed by the protomers, an additional hydrophobic pore exists within the center of the complex [9] that may mediate the transport of gases (e.g. O2, CO2, NO) and cations (the central pore is the proposed transport pathway for cations through AQP1) by some AQPs [4, 6]. Although numerous small molecule inhibitors of aquaporins, particularly APQ1, have been reported primarily from Xenopus oocyte swelling assays, the activity of most has subsequently been disputed upon retesting using assays of water transport that are less prone to various artifacts [3] and they are therefore excluded from the tables [see Tradtrantip et al. (2017) [11] for a review].

Aquaporins and their function in root water transport under salt stress conditions in Eutrema salsugineum

Eutrema salsugineum is considered as extremophile model species. To gain insights into the root hydraulic conductivity and the role played by aquaporins in E. salsugineum, we investigated the aquaporin family profiles, plant water status and root hydraulic conductivity under standard (salt-free) and salt stress conditions. We found that there was no variation in the relative electric conductivity of the leaves when the salt concentration was less than 200 mM NaCl, and the transpiration rate dropped to 60.6% at 100 mM NaCl for 14 days compared to that at standard conditions. The pressure chamber techniques indicated that the root hydraulic conductivity of E. salsugineum was repressed by salt stress. However, propionic acid, usually used as an aquaporin inhibitor, unexpectedly enhanced the root hydraulic conductivity of E. salsugineum. The aquaporin family in E. salsugineum was profiled and the PIP aquaporin expression was investigated at the transcriptional and translational levels. Finally, two EsPIPs were identified to play a role in salt stress. The overall study provides evidence on how halophytes maintain their water status and aquaporin regulation pattern under salt stress conditions.