Personalized Treatment Response Assessment for Rare Childhood Tumors Using Microcalorimetry-Exemplified by Use of Carbonic Anhydrase IX and Aquaporin 1 Inhibitors.

We present a novel approach to a personalized therapeutic concept for solid tumors. We illustrate this on a rare childhood tumor for which only a generalized treatment concept exists using carbonic anhydrase IX and aquaporin 1 inhibitors. The use of microcalorimetry as a refined in vitro method for evaluation of drug susceptibility in organotypic slice culture has not previously been established. Rapid microcalorimetric drug response assessment can refine a general treatment concept when it is applied in cases in which tumors do not respond to conventional chemo-radiation treatment. For solid tumors, which do not respond to classical treatment, and especially for rare tumors without an established protocol rapid microcalorimetric drug response testing presents an elegant novel approach to test alternative therapeutic approaches. While improved treatment concepts have led to improved outcome over the past decades, the prognosis of high risk disease is still poor and rethinking of clinical trial design is necessary. A small patient population combined with the necessity to assess experimental therapies for rare solid tumors rather at the time of diagnosis than in relapsed or refractory patients provides great challenges. The possibility to rapidly compare established protocols with innovative therapeutics presents an elegant novel approach to refine and personalize treatment.

Bumetanide-Derived Aquaporin 1 Inhibitors, AqB013 and AqB050 Inhibit Tube Formation of Endothelial Cells through Induction of Apoptosis and Impaired Migration In Vitro.

AqB013 and AqB050 compounds inhibit aquaporin 1 (AQP1), a dual water and ion channel implicated in tumour angiogenesis. We tested AqB013 and AqB050 either as monotherapy or in combination on tube formation of murine endothelial cells (2H-11 and 3B-11) and human umbilical vascular endothelial cells (HUVECs). The mechanism underlying their anti-tubulogenic effect was explored by examining cell viability, induction of apoptosis and migration using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, Annexin V/propidium iodide apoptosis assay and scratch wound assay. Tube formation of all the cell lines was inhibited by AqB013, AqB050 and the combination of the two compounds. The inhibition of 2H-11 and 3B-11 was frequently accompanied by impaired migration, whereas that of HUVEC treated with AqB050 and the combination was associated with reduced cell viability due to apoptosis. AqB013 and AqB050 exhibited an anti-tubulogenic effect through inhibition of AQP1-mediated cell migration and induction of apoptosis. Together with previously reported anti-tumour cell effect of AqB013 and AqB050, our findings support further evaluation of these compounds as potential cancer therapeutics.

The blocking of aquaporin-3 (AQP3) impairs extravillous trophoblast cell migration.

Several aquaporins (AQPs) are expressed in extravillous (EVT) and villous trophoblast cells. Among them, AQP3 is the most abundant AQP expressed in chorionic villi samples from first trimester, followed by AQP1 and AQP9. Although AQP3 expression persists in term placentas, it is significantly decreased in placentas from preeclamptic pregnancies. AQP3 is involved in the migration of different cell types, however its role in human placenta is still unknown. Here, we evaluated the role of AQP3 in the migration of EVT cells during early gestation. Our results showed that Swan 71 cells expressed AQP1, AQP3 and AQP9 but only the blocking of AQP3 by CuSO4 or the silencing of its expression by siRNA significantly attenuates EVT cell migration. Our work provides evidence that AQP3 is required for EVT cell migration and suggests that an altered expression of placental AQP3 may produce failures in placentation such as in preeclampsia.

Therapeutic effect of acetazolamide, an aquaporin 1 inhibitor, on adjuvant-induced arthritis in rats by inhibiting NF-κB signal pathway.

OBJECTIVES: Previous studies have shown that aquaporin 1 (AQP1) is up-regulated in synovium and cartilage of rheumatoid arthritis (RA) patients and that AQP1 may be involved in joint swelling and synovial inflammation. This study was aimed to investigate the potential therapeutic effect of acetazolamide (AZ, an AQP1 inhibitor) on rat adjuvant-induced arthritis (AIA) and explore its related mechanisms. MATERIALS AND METHODS: Rat AIA was induced by complete Freund's adjuvant. The effect of AZ on rat AIA was evaluated by secondary hind paw swelling, arthritis index, TNF-α and IL-1ß serum levels and histological examination of ankle joint. Proteoglycans expression and mRNA levels of type-II collagen (COII) and aggrecan in cartilage were measured by alcian blue staining and real-time PCR, respectively. The protein levels of AQP1, IκBα, phospho-IκBα (p-IκBα), NF-κB p65 and phospho-NF-κB p65 (p-NF-κB p65) in synovial tissues were detected by western blot. RESULTS: AZ treatment could inhibit secondary hind paw swelling and arthritis index, reduce serum levels of TNF-α and IL-1ß, and ameliorate pathological changes of ankle joint in AIA rats. AZ increased proteoglycans production and mRNA levels of COII and aggrecan in cartilage tissues. Moreover, AZ decreased AQP1 protein level and suppressed the activation of NF-κB pathway in synovium, indicated by inhibiting the degradation and phosphorylation of IκBα and reducing p-NF-κB p65 protein level. CONCLUSIONS: AZ as an AQP1 inhibitor has a powerful therapeutic effect on rat AIA via inhibiting NF-κB activation, suggesting AQP1 inhibition might be of potential clinical interest in RA treatment.

The Aquaporin 1 Inhibitor Bacopaside II Reduces Endothelial Cell Migration and Tubulogenesis and Induces Apoptosis.

Expression of aquaporin-1 (AQP1) in endothelial cells is critical for their migration and angiogenesis in cancer. We tested the AQP1 inhibitor, bacopaside II, derived from medicinal plant Bacopa monnieri, on endothelial cell migration and tube-formation in vitro using mouse endothelial cell lines (2H11 and 3B11) and human umbilical vein endothelial cells (HUVEC). The effect of bacopaside II on viability, apoptosis, migration and tubulogenesis was assessed by a proliferation assay, annexin-V/propidium iodide flow cytometry, the scratch wound assay and endothelial tube-formation, respectively. Cell viability was reduced significantly for 2H11 at 15 µM (p = 0.037), 3B11 at 12.5 µM (p = 0.017) and HUVEC at 10 µM (p < 0.0001). At 15 µM, the reduced viability was accompanied by an increase in apoptosis of 38%, 50% and 32% for 2H11, 3B11 and HUVEC, respectively. Bacopaside II at ≥10 µM significantly reduced migration of 2H11 (p = 0.0002) and 3B11 (p = 0.034). HUVECs were most sensitive with a significant reduction at ≥7.5 µM (p = 0.037). Tube-formation was reduced with a 15 µM dose for all cell lines and 10 µM for 3B11 (p < 0.0001). These results suggest that bacopaside II is a potential anti-angiogenic agent.

The Effect of Aquaporin 1-Inhibition on Vasculogenic Mimicry in Malignant Mesothelioma.

Malignant mesothelioma (MM) is an aggressive malignancy of the serosal membranes, with poor overall survival and quality of life. Limited targeted treatment strategies exist due to restricted knowledge of pathogenic pathways. Vasculogenic mimicry (VM) is a newly described phenomenon associated with increased aggressiveness in other malignancies, and has been characterized in MM. Normal mesothelium expresses aquaporin 1 (AQP1) and retained expression has been associated with improved survival in MM. AQP1 is expressed by normal vascular endothelium and is involved in mediating MM cell motility and proliferation. We investigated the role of AQP1 in VM, and its interaction with the pro-angiogenic factor vascular endothelial growth factor A (VEGFA), which is variably expressed in MM. Matrigel VM assays were performed using NCI-H226 and NCI-H28 MM cell lines and primary cells in hypoxia and normoxia. The synthetic blocker AqB050 and siRNA were used to inhibit AQP1, and bevacizumab was used to inhibit VEGF. Inhibition of AQP1 resulted in increased VEGFA secretion by MM cells and reduced VM in MM cell lines in hypoxia but not normoxia. No change in VM was seen in MM primary cells. Combined inhibition of AQP1 and VEGF had no effect on VM in normoxia. In a heterotopic xenograft mouse model, AqB050 treatment did not alter vessel formation. AQP1 may interact with VEGFA and play a role in VM, especially under hypoxic conditions, but the heterogeneity of MM cells may result in different dominant pathways between patients.

Bumetanide Derivatives AqB007 and AqB011 Selectively Block the Aquaporin-1 Ion Channel Conductance and Slow Cancer Cell Migration.

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 (IC50 of 14 µM), with no effect on water channel activity (at up to 200 µM). 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.

Differential Inhibition of Water and Ion Channel Activities of Mammalian Aquaporin-1 by Two Structurally Related Bacopaside Compounds Derived from the Medicinal Plant Bacopa monnieri.

Aquaporin-1 (AQP1) is a major intrinsic protein that facilitates flux of water and other small solutes across cell membranes. In addition to its function as a water channel in maintaining fluid homeostasis, AQP1 also acts as a nonselective cation channel gated by cGMP, a property shown previously to facilitate rapid cell migration in a AQP1-expressing colon cancer cell line. Here we report two new modulators of AQP1 channels, bacopaside I and bacopaside II, isolated from the medicinal plant Bacopa monnieri Screening was conducted in the Xenopus oocyte expression system, using quantitative swelling and two-electrode voltage clamp techniques. Results showed bacopaside I blocked both the water (IC50 117 µM) and ion channel activities of AQP1 but did not alter AQP4 activity, whereas bacopaside II selectively blocked the AQP1 water channel (IC50 18 µM) without impairing the ionic conductance. These results fit with predictions from in silico molecular modeling. Both bacopasides were tested in migration assays using HT29 and SW480 colon cancer cell lines, with high and low levels of AQP1 expression, respectively. Bacopaside I (IC50 48 µM) and bacopaside II (IC50 14 µM) impaired migration of HT29 cells but had minimal effect on SW480 cell migration. Our results are the first to identify differential AQP1 modulators isolated from a medicinal plant. Bacopasides could serve as novel lead compounds for pharmaceutic development of selective aquaporin modulators.

Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1.

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

A generic high-throughput assay to detect aquaporin functional mutants: Potential application to discovery of aquaporin inhibitors.

BACKGROUND: The discovery of stable, yet functional, protein mutants is a limiting factor in the development of biotechnological applications, structural studies or in drug discovery. Rapid detection of functional mutants is especially challenging for water channel aquaporins, as they do not have a directly measurable enzymatic or binding activity. Current methods available are time consuming and only applicable to specific aquaporins. METHODS: Herein we describe an assay based on the protective effect of aquaporins on yeast S. cerevisiae in response to rapid freezing. RESULTS: Yeast overexpressing a functional water-permeable aquaporin of choice are rescued after the challenge, while inactive or blocked aquaporins confer no protection and lead to cell death. The potential of this assay is shown by screening a small number of E. coli aquaporin Z (AQPZ) mutants. Additionally, a library of ~10,000 drug-like compounds was tested against human AQP1 (hAQP1). CONCLUSIONS: Since rescue is only dependent on transmembrane water flux, the assay is applicable to water-permeable aquaporins of any origin. GENERAL SIGNIFICANCE: Mapping of permissive mutations on the aquaporin structure can help delineate the minimal requirements for effective water transport. Alternatively, the assay can be potentially used to discover compounds that inhibit aquaporin water transport. When additionally screened for thermostability, functional aquaporin mutants can be useful in the development of biomimetic membranes for water purification, or to improve the likelihood of producing well-diffracting crystals, enabling rational design of much needed aquaporin inhibitors.

Influence of AQP1 on cell adhesion, migration, and tumor sphere formation in malignant pleural mesothelioma is substratum- and histological-type dependent.

Malignant pleural mesothelioma (MPM) is an aggressive cancer. MPM cells express aquaporin-1 (AQP1) that in other cancers has been shown to participate in the tumor metastasis processes. However, in MPM patients AQP1 overexpression is an independent prognostic factor favoring survival. In this study we aimed at evaluating the role of AQP1 in cell adhesion, migration, and tumor sphere formation in nonmalignant mesothelial cells (MeT-5A) and in epithelioid (M14K) and sarcomatoid (ZL34) MPM cell lines. We used fibronectin (FN) or homologous cell-derived extracellular martrix (ECM) substratum to investigate the role of AQP1 in these experimental phenotypes, inhibiting AQP1 by 10(-5) M mercury chloride (MC). Deposited ECM during cell culture exhibited significant concentration differences among cell types. ZL34 cell adhesion was significantly higher than MeT-5A or M14K cells on FN and ECM. MeT-5A and M14K cell adhesion on FN was sensitive to AQP1 inhibition, whereas AQP1 inhibition on ECM was limited to M14K cells. Wound healing in ZL34 cells was significantly higher than MeT-5A and M14K cells on FN and ECM. AQP1 inhibition significantly lowered cell migration in ZL34 cells on FN and ECM. Sphere formation was not dependent on FN or ECM in the media. AQP1 inhibition in FN media reduced sphere formation in M14K cells, whereas, in ECM, all three cell types were sensitive to AQP1 inhibition.

Fragment Screening of Human Aquaporin 1.

Aquaporins (AQPs) are membrane proteins that enable water transport across cellular plasma membranes in response to osmotic gradients. Phenotypic analyses have revealed important physiological roles for AQPs, and the potential for AQP water channel modulators in various disease states has been proposed. For example, AQP1 is overexpressed in tumor microvessels, and this correlates with higher metastatic potential and aggressiveness of the malignancy. Chemical modulators would help in identifying the precise contribution of water channel activity in these disease states. These inhibitors would also be important therapeutically, e.g., in anti-cancer treatment. This perceived importance contrasts with the lack of success of high-throughput screens (HTS) to identify effective and specific inhibitors of aquaporins. In this paper, we have screened a library of 1500 "fragments", i.e., smaller than molecules used in HTS, against human aquaporin (hAQP1) using a thermal shift assay and surface plasmon resonance. Although these fragments may not inhibit their protein target, they bound to and stabilized hAQP1 (sub mM binding affinities (KD), with an temperature of aggregation shift ΔTagg of +4 to +50 °C) in a concentration-dependent fashion. Chemically expanded versions of these fragments should follow the determination of their binding site on the aquaporin surface.

Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells.

The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1.

The Effects of Aquaporin-1 in Pulmonary Edema Induced by Fat Embolism Syndrome.

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). In vivo, edema was more serious in FES mice compared with the control group. The expression of AQP1 and the wet-to-dry lung weight ratio (W/D) in the FES group were significantly increased compared with the control group. At the same time, inhibition of AQP1 decreased the pathological damage resulting from pulmonary edema. Then we performed a study in vitro to investigate whether AQP1 was induced by FFA release in FES. The mRNA and protein level of AQP1 were increased by FFAs in a dose- and time-dependent manner in PMVECs. In addition, the up-regulation of AQP1 was blocked by the inhibitor of p38 kinase, implicating the p38 MAPK pathway as involved in the FFA-induced AQP1 up-regulation in PMVECs. Our results demonstrate that AQP1 may play important roles in pulmonary edema induced by FES and can be regarded as a new therapy target for treatment of pulmonary edema induced by FES.

The water channel aquaporin-1a1 facilitates movement of CO2 and ammonia in zebrafish (Danio rerio) larvae.

The present study tested the hypothesis that zebrafish (Danio rerio) aquaporin-1a1 (AQP1a1) serves as a multi-functional channel for the transfer of the small gaseous molecules, CO2 and ammonia, as well as water, across biological membranes. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to AQP1a1. Knockdown of AQP1a1 significantly reduced rates of CO2 and ammonia excretion, as well as water fluxes, in larvae at 4 days post fertilization (dpf). Because AQP1a1 is expressed both in ionocytes present on the body surface and in red blood cells, the haemolytic agent phenylhydrazine was used to distinguish between the contributions of AQP1a1 to gas transfer in these two locations. Phenylhydrazine treatment had no effect on AQP1a1-linked excretion of CO2 or ammonia, providing evidence that AQP1a1 localized to the yolk sac epithelium, rather than red blood cell AQP1a1, is the major site of CO2 and ammonia movements. The possibility that AQP1a1 and the rhesus glycoprotein Rhcg1, which also serves as a dual CO2 and ammonia channel, act in concert to facilitate CO2 and ammonia excretion was explored. Although knockdown of each protein did not affect the abundance of mRNA and protein of the other protein under control conditions, impairment of ammonia excretion by chronic exposure to high external ammonia triggered a significant increase in the abundance of AQP1a1 mRNA and protein in 4 dpf larvae experiencing Rhcg1 knockdown. Collectively, these results suggest that AQP1a1 in zebrafish larvae facilitates the movement of CO2 and ammonia, as well as water, in a physiologically relevant fashion.

A general anaesthetic propofol inhibits aquaporin-4 in the presence of Zn²âº.

AQP4 (aquaporin-4), a water channel protein that is predominantly expressed in astrocyte end-feet, plays an important role in the brain oedema formation, and is thereby considered to be a potential therapeutic target. Using a stopped-flow analysis, we showed that propofol (2,6-diisopropylphenol), a general anaesthetic drug, profoundly inhibited the osmotic water permeability of AQP4 proteoliposomes in the presence of Zn²âº. This propofol inhibition was not observed in AQP1, suggesting the specificity for AQP4. In addition, the inhibitory effects of propofol could be reversed by the removal of Zn²âº. Other lipid membrane fluidizers also similarly inhibited AQP4, suggesting that the modulation of protein-lipid interactions plays an essential role in the propofol-induced inhibition of AQP4. Accordingly, we used Blue native PAGE and showed that the profound inhibition caused by propofol in the presence of Zn²âº is coupled with the reversible clustering of AQP4 tetramers. Site-directed mutagenesis identified that Cys²5³, located at the membrane interface connecting to the C-terminal tail, is responsible for Zn²âº-mediated propofol inhibition. Overall, we discovered that propofol specifically and reversibly inhibits AQP4 through the interaction between Zn²âº and Cys²5³. The findings provide new insight into the functional regulation of AQP4 and may facilitate the identification of novel AQP4-specific inhibitors.

The involvement of AQP1 in heart oedema induced by global myocardial ischemia.

Aquaporin-1 (AQP1) is a member of aquaporin family that was previously proven to be involved in myocardial dysfunction; however, the role of AQP1 in myocardial stunning is less clear. To determine the change of AQP1 expression level in the heart and its effect on oedema after global myocardial ischemia, 40 adult goats underwent cardiopulmonary bypass (CPB) with an aortic cross-clamp time of 2 h and total bypass time of 6, 12, 24, 48 and 72 h followed by subsequent reperfusion. AQP1 function of eight goats was inhibited by HgCl(2) during the 24 h on CPB. All groups were compared with eight sham bypass control goats. Myocardial water content was measured, and the APQ1 mRNA and protein levels were detected by RT-PCR and immunoblotting, respectively. The results showed that the degree of myocardial oedema increased significantly at 6, 12, 24 and 48 h of reperfusion after CPB as compared with the control and recovered at 72 h of subsequent reperfusion. Expression levels of AQP1 mRNA and protein began to increase at 12 h and peaked at 24 h of CPB following reperfusion. Furthermore, myocardial oedema was reduced in the HgCl(2) group compared with the time-matched CPB and control groups. These data suggested that AQP1 expression increases in CPB and AQP1 plays an important role in myocardial oedema during CPB.

Co action of CFTR and AQP1 increases permeability of peritoneal epithelial cells on estrogen-induced ovarian hyper stimulation syndrome.

BACKGROUND: Ovarian hyper stimulation syndrome (OHSS) is an iatrogenic complication associated with fertility drugs. It is characterized by increased vascular permeability and substantial fluid shift with accumulation in the body cavity. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. RESULTS: Using western blot and short-circuit current (Isc) techniques, we investigate the potential coactions of analysis in cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin 1 (AQP1) on the hyper permeability of body cavity peritoneal epithelial cells in the pathogenesis of OHSS. The rats develop OHSS symptoms, with the up regulation of both CFTR and AQP1 expression and enhanced CFTR channel activity in peritoneal epithelial cells, can also be mimicked by administration of estrogen, alone in ovariectomized rats. Administration of progesterone suppresses CFTR activity, OHSS symptoms as well as CFTR and AQP1 expression. Besides, AQP1 inhibitor, HgCl(2), can suppress CFTR channel activity. Therefore, antisera against CFTR or AQP1 to OHSS animals may result in alleviation of the symptom. CONCLUSION: This study confirms the coactions of CFTR and AQP1 play a critical role in the development and progression of increased peritoneal epithelial permeability in severe OHSS. These findings may provide grounds for ameliorating assisted reproduction treatment strategy to reduce the risk of OHSS in in vitro fertilization (IVF).

Reinvestigation of drugs and chemicals as aquaporin-1 inhibitors using pressure-induced hemolysis in human erythrocytes.

Recently, we have found that pressure-induced hemolysis is enhanced by inhibiting water transport via aquaporin-1 (AQP1), as seen in p-chloromercuribenzoate (pCMB)-treated erythrocytes. So, using this method we reinvestigated the functions as AQP1 inhibitors of drugs and chemicals such as acetazolamide, sodium nitroprusside, tetraethylammonium ions (TEA(+)), and dimethylsulfoxide (DMSO). The values of hemolysis at 200 MPa were almost unaffected by acetazolamide or sodium nitroprusside, decreased by TEA(+), and increased significantly by DMSO. Furthermore, the erythrocytes were exposed to pCMB in the presence of TEA(+) or DMSO. The enhancement effect of pCMB on pressure-induced hemolysis was unaffected by TEA(+) but attenuated by DMSO. Taken together, these results suggest that, of drugs and chemicals examined here, DMSO only is an AQP1 inhibitor, but the effect of DMSO is small compared with pCMB.

Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients.

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport-produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors-were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg (W) (-1) . These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.