Fish oil-derived n-3 polyunsaturated fatty acids downregulate aquaporin 9 protein expression of liver and white adipose tissues in diabetic KK mice and 3T3-L1 adipocytes.

Aquaporin 9 (AQP9) is an integral membrane protein that facilitates glycerol transport in hepatocytes and adipocytes. Glycerol is necessary as a substrate for gluconeogenesis in the physiological fasted state, suggesting that inhibiting AQP9 function may be beneficial for treating type 2 diabetes associated with fasting hyperglycemia. The n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are rich in fish oil and lower the risk of metabolic syndrome; however, the effects of EPA and DHA on AQP9 expression in obese and type 2 diabetes are unclear. The KK mouse is an animal model of obesity and type 2 diabetes because of the polymorphisms on leptin receptor gene, which results in a part of cause for obese and diabetic conditions. In this study, we determined the effect of fish oil-derived n-3 PUFA on AQP9 protein expression in the liver and white adipose tissue (WAT) of KK mice and mouse 3T3-L1 adipocytes. The expression of AQP9 protein in the liver, epididymal WAT, and inguinal WAT were markedly decreased following fish oil administration. We also demonstrated that n-3 PUFAs, such as DHA, and to a lesser extent EPA, downregulated AQP9 protein expression in 3T3-L1 adipocytes. Our results suggest that fish oil-derived n-3 PUFAs may regulate the protein expressions of AQP9 in glycerol metabolism-related organs in KK mice and 3T3-L1 adipocytes.

Multivalent Display of Erythropoietin on Quantum Dots Enhances Aquaporin-4 Expression and Water Transport in Human Astrocytes In Vitro.

In mammalian cells, growth factor-induced intracellular signaling and protein synthesis play a critical role in cellular physiology and homeostasis. In the brain's glymphatic system (GS), the water-conducting activity of aquaporin-4 (AQPN-4) membrane channels (expressed in polarized fashion on astrocyte end-feet) mediates the clearance of wastes through the convective transport of fluid and solutes through the perivascular space. The glycoprotein erythropoietin (EPO) has been shown to induce the astrocyte expression of AQPN-4 via signaling through the EPO receptor and the JAK/STAT signaling pathway. Here, we self-assemble EPO in a multivalent fashion onto the surface of semiconductor quantum dots (QDs) (driven by polyhistidine-based self-assembly) to drive the interaction of the bioconjugates with EPOR on human astrocytes (HA). This results in a 2-fold augmentation of JAK/STAT signaling activity and a 1.8-fold enhancement in the expression of AQPN-4 in cultured primary HA compared to free EPO. This translates into a 2-fold increase in the water transport rate in HA cells as measured by the calcein AM water transport assay. Importantly, EPO-QD-induced augmented AQPN-4 expression does not elicit any deleterious effect on the astrocyte viability. We discuss our results in the context of the implications of EPO-nanoparticle (NP) bioconjugates for use as research tools to understand the GS and their potential as therapeutics for the modulation of GS function. More generally, our results illustrate the utility of NP bioconjugates for the controlled modulation of growth factor-induced intracellular signaling.

Chronic inhibition of astrocytic aquaporin-4 induces autistic-like behavior in control rat offspring similar to maternal exposure to valproic acid.

Social communication and interaction deficits, memory impairment, and anxiety-like behavior are characterized in many people identified with autism spectrum disorder (ASD). A thorough understanding of the specific aspects that contribute to the deficiencies associated with ASD can aid research into the etiology of the disorder while also providing targets for more effective intervention. As part of the ASD pathophysiology, alterations in synaptogenesis and abnormal network connections were seen in high-order brain areas, which control social behavior and communication. The early emergence of microglia during nervous system development may contribute to synaptic dysfunction and the pathobiology of ASD. Since aquaporin-4 (AQP4) appears to be required for the basic procedures of synapse activation, certain behavioral and cognitive impairments as well as disturbance in water homeostasis might likely arise from AQP4 deficiency. Here, through the measurement of the water content of the hippocampus and behavioral experiments we aim to explore the contribution of astrocytic AQP4 to the autism-like behavior induced by prenatal valproic acid (VPA) exposure and whether inhibition of AQP4 per se can induce autistic-like behavior in control rats. Microinjection of TGN-020 (10 µM, i.c.v), a specific AQP4 inhibitor, for 7 successive days before behavioral tasks from postnatal day 28 to 35 revealed that inhibition of AQP4 in the control offspring caused lower social interaction and locomotor activity, higher anxiety, and decreased ability to recognize novel objects, very similar to the behavioral changes observed in offspring prenatally exposed to VPA. However, VPA-exposed offspring treated with TGN-020, showed no further remarkable behavioral impairments than those detected in the autistic-like rats. Furthermore, both control offspring treated with TGN-020 and offspring exposed to VPA had a considerable accumulation of water in their hippocampi. But AQP4 inhibition did not affect the water status of the autistic-like rats. The findings of this study revealed that control offspring exhibited similar hippocampal water retention and behavioral impairments that were observed in maternal VPA-exposed offspring following inhibition of astrocytic AQP4, whereas, in autistic-like rats, it did not produce any significant change in water content and behaviors. Findings suggest that AQP4 deficiency could be associated with autistic disorder and may be a potential pharmaceutical target for treating autism in the future.

Effects of low nitrogen supply on biochemical and physiological parameters related to nitrate and water, involving nitrate transporters and aquaporins in Citrus macrophylla.

A reduction in chemical N-based fertillizer was investigated in Citrus plants. As N and water uptake are connected, the relationship between the physiological response to reductions in N was studied in relation to N metabolism and water. We examined the response of new and mature leaves and roots of Citrus macrophylla, grown under controlled conditions, and given different concentrations of N: 16, 8 or 4 mM. Differences in growth and development were determined for biochemical (mineral content, photosynthetic pigments, proteins and nitrate and nitrite reductase activity), physiological (photosynthesis and transpiration), and molecular (relative expression of nitrate transporters and aquaporins) parameters. Only plants given 4 mM N showed a reduction in growth. Although there were changes in NR activity, protein synthesis, and chlorophyll content in both 8 and 4 mM N plants that were highly related to aquaporin and nitrate transporter expression. The results revealed new findings on the relationship between aquaporins and nitrate transporters in new leaves of Citrus, suggesting a mechanism for ensuring growth under low N when new tissues are being formed.

Effects of a novel probiotic mixture on the modulation of brain and intestine Aquaporin-4 gene expression in rats exposed to Cadmium.

Cadmium (Cd) is a toxicant metal that risks human and animal health. Nowadays, the vital role of Aquaporin-4 (AQP-4) in brain and gut cell permeability has gathered too much attention to protecting against heavy metals. Studies have shown that heavy metals can harm the body due to oxidative stress. Probiotics are known for their health-beneficial effects and establish as dietary adjuncts mainly for their antioxidant properties. This study investigated the impact of a novel probiotic combination including Lactobacillus casei IBRC-M10783, Lactobacillus rhamnosus IBRC-M10782, and Lactobacillus helveticus TG-34 on the AQP-4 gene expression in CdCl2-induced Wistar rats. Rats were divided into three groups and received a specific dose of CdCl2 or probiotics. The AQP-4 expression level had estimated by Real-Time PCR in both the intestine and brain. These results showed a significant reduction in AQP-4 gene expression in the probiotic treatment group compared to the CdCl2 control group in the intestine and brain for the first time. Our research showed that consuming a probiotic mixture of L. casei, L. rhamnosus, and L. helveticus can reduce the expression of the aquaporin-4 gene in the brain and intestine of rats exposed to Cadmium, which can be promising in the field of aquaporin-4 regulation.

Production of Reactive Oxygen Species via Nanobubble Water Improves Radish Seed Water Absorption and the Expression of Aquaporin Genes.

Nanobubbles (NBs) stimulate seed germination; however, the mechanism of the promotion effect of NBs remains unclear. The impact of NBs on seed water absorption was investigated; we subsequently studied the genes associated with the response of radish seeds to NB water and used RNA sequencing to generate their expression profiles, especially those of aquaporin genes. NB water significantly promoted germination. The times at which 50% of the germinating seeds achieved germination (T50) for the submerged radish seeds in NB and control water were 11.6 and 17.4 h, respectively. NB water-germinated radish seeds showed a water uptake rate coefficient that was 15% higher than that of those germinated in control water. Through GO enrichment and cluster analyses, it was evident that NB water significantly increased the level of expression of the genes associated with the following activities: oxidoreductase, peroxidase, and antioxidant. Our results demonstrated that NB water increases the water uptake rate of radish seeds via two mechanisms. The NB water-produced exogenous hydroxyl radical (•OH) increases the seed coat's water permeability and enhances cell wall loosening, and NB water increases the aquaporin gene expression level of radish seeds.

Polystichum braunii ameliorates airway inflammation by attenuation of inflammatory and oxidative stress biomarkers, and pulmonary edema by elevation of aquaporins in ovalbumin-induced allergic asthmatic mice.

Asthma is a chronic inflammation of pulmonary airways associated with bronchial hyper-responsiveness. The study was aimed to validate the folkloric use of Polystichum braunii (PB) against ovalbumin (OVA)-induced asthmatic and chemical characterization OF both extracts. Allergic asthma was developed by intraperitoneal sensitization with an OVA on days 1 and 14 followed by intranasal challenge. Mice were treated with PB methanolic (PBME) and aqueous extract (PBAE) orally at 600, 300, and 150 mg/kg and using dexamethasone (2 mg/kg) as standard from day 15 to 26. High performance liquid chromatography-diode array detector analysis revealed the presence of various bioactive compounds such as catechin, vanillic acid, and quercetin. The PBME and PBAE profoundly (p < 0.0001-0.05) declined immunoglobulin E level, lungs wet/dry weight ratio, and total and differential leukocyte count in blood and bronchial alveolar lavage fluid of treated mice in contrast to disease control. Histopathological examination showed profoundly decreased inflammatory cell infiltration and goblet cell hyperplasia in treated groups. Both extracts caused significant (p < 0.0001-0.05) diminution of IL-4, IL-5, IL-13, IL-6, IL-1ß, TNF-α, and NF-κB and upregulation of aquaporins (1 and 5), which have led to the amelioration of pulmonary inflammation and attenuation of lung edema in treated mice. Both extracts profoundly (p < 0.0001-0.05) restored the activities of SOD, CAT, GSH and reduced the level of MDA dose dependently. Both extracts possessed significant anti-asthmatic action mainly PBME 600 mg/kg might be due to phenols and flavonoids and could be used as a potential therapeutic option in the management of allergic asthma.

Aquaporin 9 Represents a Novel Target of Chronic Liver Injury That May Antagonize Its Progression by Reducing Lipotoxicity.

In recent years, chronic liver injury has become a common disease that harms human health. Its clinical manifestations are hepatic steatosis and secondary chronic steatohepatitis, which can quickly transform into liver fibrosis and cirrhosis if not treated in time. Therefore, this study is aimed at searching for new therapeutic targets of chronic liver injury and clarifying the molecular mechanisms of the new targets involved in chronic liver injury. After aquaporin 9 was identified as a target by proteomics, Aqp9-/- mice were constructed using the CRISPR/Cas9 system. Biochemical and morphological tests were used to verify the effect of Aqp9 knockout on early chronic liver injury. Proteomics, molecular biology, and morphology experiments were used to screen and verify the effects of Aqp9 knockout on its downstream pathway. Through the above experiments, we demonstrated that aquaporin 9 could be used as an intervention target for antagonizing the development of early chronic liver injury and its gene knockout affected downstream inflammation, oxidative stress, apoptosis, and pyroptosis by alleviating hepatic lipotoxicity.

The herbal medicines Inchinkoto and Saireito improved hepatic fibrosis via aquaporin 9 in the liver of a rat bile duct ligation model.

OBJECTIVE: To determine if the administration of the Japanese herbal medicines Inchinkoto (ICKT) and Saireito (SRT) ameliorate hepatic fibrosis and derangement of hepatocyte aquaporins (AQPs) following bile duct ligation (BDL) in a rat model of obstructive cholestasis. MATERIALS AND METHODS: Five groups of Wistar rats were used, and the groups included sham surgery (Sham group), BDL with no treatment (NT group), BDL plus ICKT (ICKT group), BDL plus SRT (SRT group), and BDL plus ICKT and SRT (SRT/ICKT group). Each herbal medicine was administered at 1 g/kg/day on the first postoperative day. The serum levels and various clinical markers were measured with real-time polymerase chain reaction. Staining was used to evaluate the degree of fibrosis and the inflammatory responses. RESULTS: Serum aspartate aminotransferase and alanine aminotransferase in the ICKT and SRT/ICKT groups were significantly lower than those in the NT group. NF-κB mRNA expression was significantly decreased in the ICKT group and the SRT/ICKT group compared with the NT group. AQP9 mRNA expression was significantly increased in the ICKT group and the SRT/ICKT group compared with the NT group. The degree of Masson's trichrome staining in the SRT/ICKT group was significantly lower than that in the NT group. The degree of NF-κB staining in the SRT/ICKT group was significantly lower than that in the NT, ICKT, or SRT group. CONCLUSIONS: The postoperative administration of ICKT and SRT induced synergistic beneficial effects, resulting in the reduction of hepatic fibrosis via mechanisms involving the inhibition of NF-κB expression and the improvement of AQP9 downregulation.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) -aspirin, naproxen, nimesulide, and piroxicam- lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. RESULTS: Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. CONCLUSIONS: NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.

Aquaporin-assisted and ER-mediated mitochondrial fission: a hypothesis.

It is well established that the status of the endoplasmic reticulum (ER) and mitochondria, and the interactions between them, is critical to numerous cellular functions including apoptosis. Mitochondrial dynamics is greatly influenced by cell stress, and recent studies implicate ER in mitochondrial fission. Although a number of proteins have been identified to participate in ER-induced mitochondrial fission, the molecular mechanism of the process is little understood. In the current study, we confirm the involvement of ER in mitochondrial fission and hypothesize the involvement of water channels or aquaporins (AQP) in the process. Previous studies demonstrate the presence of AQP both in the ER and mitochondrial membranes. Mitochondrial swelling has been observed following mitochondrial calcium overload, and studies report that chelation of cytosolic calcium induces extensive mitochondrial division at ER contact sites. Based on this information, the involvement of ER in mitochondrial division, possibly via water channels, is hypothesized. Utilizing a multi-faceted imaging approach consisting of atomic force microscopy on aldehyde-fixed and semi-dry cells, transmission electron microscopy, and immunofluorescence microscopy on live cells, the physical interactions between the two organelles are demonstrated. Mitochondrial fission following ER stress was abrogated with exposure of cells to the AQP inhibitor mercuric chloride, suggesting the involvement of AQP(s) especially AQP8 and AQP9 known to be present in the mitochondrial membrane, in mitochondrial fission.

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function.

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.

[Aquaporins--a new element in the regulation of body water homeostasis]. / Akwaporyny--nowy element w regulacji gospodarki wodnej organizmu.

Aquaporins represent an ubiquitous class of integral membrane proteins that are serving in the passage of water across cell membrane. A subset of aquaporins may additionally facilitate transmembrane permeation of small neutral solutes such as glicerol (aquaglyceroporins). The widespread occurrence and an unique function of aquaporins give rise to the high degree of their intra- and interspecies homology, especially in their regions forming the internal wall of channel. The review presents current knowledge about the role of water channels in regulation of body homeostasis basing on results of experiments with mice lacking various aquaporins genes and studies on humans with inherited or acquired abnormalities in their function as well as about potential perspectives of pharmacological regulation of aquaporin activity.

Renal medullary gene expression in aquaporin-1 null mice.

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible approximately 12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-alpha were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

Long-term treatment with cyclosporine decreases aquaporins and urea transporters in the rat kidney.

The aim of this study was to evaluate the long-term effects of cyclosporine (CsA) treatment on urinary concentration ability. Rats were treated daily for 4 wk with vehicle (VH; olive oil, 1 ml/kg sc) or CsA (15 mg/kg sc). The influence of CsA on the kidney's ability to concentrate urine was evaluated using functional parameters and expression of aquaporins (AQP1-4) and of urea transporters (UT-A-1-3, and UT-B). Plasma vasopressin levels and the associated signal pathway were evaluated, and the effect of vasopressin infusion on urine concentration was observed in VH- and CsA-treated rats. Toxic effects of CsA on tubular cells in the medulla as well as the cortex were evaluated with aldose reductase (AR), Na-K-ATPase-alpha(1) expression, and by determining the number of terminal transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells. Long-term CsA treatment increased urine volume and fractional excretion of sodium and decreased urine osmolality and free-water reabsorption compared with VH-treated rats. These functional changes were accompanied by decreases in the expression of AQP (1-4) and UT (UT-A2, -A3, and UT-B), although there was no change in AQP2 in the cortex and outer medulla and UT-A1 in the inner medulla (IM). Plasma vasopressin levels were not significantly different between two groups, but infusion of vasopressin restored CsA-induced impairment of urine concentration. cAMP levels and Gsalpha protein expression were significantly reduced in CsA-treated rat kidneys compared with VH-treated rat kidneys. CsA treatment decreased the expression of AR and Na-K-ATPase-alpha(1) and increased the number of TUNEL-positive renal tubular cells in both the cortex and medulla. Moreover, the number of TUNEL-positive cells correlated with AQP2 or UT-A3) expression within the IM. In conclusion, CsA treatment impairs urine-concentrating ability by decreasing AQP and UT expression. Apoptotic cell death within the IM at least partially accounts for the CsA-induced urinary concentration defect.

[Effect of aquaporin-1 and sodium channel on pleural fluid transport in mice].

OBJECTIVE: To investigate the effect of sodium channel and aquaporin-1 on pleural fluid transport. METHODS: Mice were briefly anesthetized, and the pleural space was infused with 0.25 ml of hyperosmolar or isosmolar solution, with terbutaline or amiloride in instilled solution. The mice were treated with either intraperitoneal dexamethasone or intravenous HgCl(2). Mice were euthanized at specified times for measurement of pleural fluid osmolality, volume and (125)I-album in concentration. The rate of pleural fluid filtration and clearance was calculated, which was used to indicate the effect of sodium channel regulator (amiloride and terbutaline) and aquaporin regulator (dexamethasone and HgCl(2)) on pleural fluid transport. RESULTS: Amiloride decreased the osmotically driven water transport and isosmolar fluid clearance in pleural space (P < 0.05). Terbutaline increased osmotically driven water transport and isosmolar fluid clearance in pleural space (P < 0.05). HgCl(2) decreased, while dexamethasone increased, the osmotically driven water transport in pleural space (P < 0.05). HgCl(2) and dexamethasone had no effect on pleural isosmolar fluid clearance (P > 0.05). CONCLUSIONS: Sodium channel may play a role in osmotic and isosmolar fluid transport in pleural space. Aquaporin-1 may play a role in osmotic fluid transport in pleural space, while having no effect on isosmolar fluid clearance.

Aquaporin-1 and HCO3(-)-Cl- transporter-mediated transport of CO2 across the human erythrocyte membrane.

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO3(-)-Cl- transporter may be involved in CO2 transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO2 transport in human red blood cell ghosts at physiological temperatures (37 degrees C). Replacement of inert with CO2-containing gas above a stirred cell suspension caused an outside-to-inside directed CO2 gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO2 passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO2-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl2 at 65 microM) or the HCO3(-)-Cl- transporter (by DIDS at 15 microM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO2, nearly the entire CO2 transport across the red cell membrane is mediated by AQP1 and the HCO3--Cl- transporter. Therefore, these proteins may function as high affinity sites for CO2 transport across the erythrocyte membrane.

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation.

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.