Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.     

Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates

Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers.     

Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells

BACKGROUND INFORMATION: Transmembrane water flow is aided by water-specific channel proteins, aquaporins. Plant genomes code for approx. 35 expressed and functional aquaporin isoforms. Plant aquaporins fall into four different subfamilies of which the PIPs (plasma membrane intrinsic proteins) constitute the largest and evolutionarily most conserved subfamily with 13 members in Arabidopsis and maize. Furthermore, the PIPs can be divided into two phylogenetic groups, PIP1 and PIP2, of which the PIP1 isoforms are most tightly conserved, sharing >90% amino acid sequence identity. As the nomenclature implies, the majority of PIPs have been shown to be localized at the plasma membrane. Recently, two highly abundant plasma membrane aquaporins, SoPIP2;1 and SoPIP1;2, have been purified and structurally characterized. RESULTS: We report the cloning of a cDNA encoding SoPIP1;2 and show that there are at least five additional sequences homologous with SoPIP2;1 and SoPIP1;2 in the spinach genome. To understand their role in planta, we have investigated the cellular localization of the aquaporin homologues SoPIP1;2 and SoPIP1;1. By Western- and Northern-blot analyses and by immunocytochemical detection at the light and electron microscopic levels, we show that SoPIP1;2 is highly expressed in phloem sieve elements of leaves, roots and petioles and that SoPIP1;1 is present in stomatal guard cells. CONCLUSIONS: Localization of the two abundant aquaporin isoforms suggests roles for specific PIPs of the PIP1 subgroup in phloem loading, transport and unloading, and in stomatal movements.     

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.     

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H2O2) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H2O2 also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H2O2 is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H2O2, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H2O2 across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H2O2 across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H2O2 in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H2O2.     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H₂O₂) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H₂O₂ also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H₂O₂ is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H₂O₂, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H₂O₂ across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H₂O₂ across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H₂O₂ in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H₂O₂.     

Effect of temperature on water transport through aquaporins

The mean effective water self-diffusion coefficient in maize root segments under the effect of aquaporin blocker (mercuric chloride, 0.1 mM) was measured using the spin-echo NMR method with pulsed magnetic field gradient within the temperature range from 10 to 35 °C. HgCl₂ caused the reduction in water diffusion by 30 % as compared to the control samples. Temperature dependences of water self-diffusion coefficients showed two linear regions with different values of Q₁₀ and activation energy, E_a. As the temperature reduced from 20 to 10 °C, E_a values calculated from the Arrhenius plots were close to those of bulk water (20 ± 3 kJ mol⁻¹) and slightly changed for the sample pretreated HgCl₂. Within the temperature range from 25 to 35 °C the slope of temperature dependences became steeper and E_a values were 31 ± 3 kJ mol⁻¹ for the control and 40 ± 4 kJ mol⁻¹ for the treated sample. In the vicinity of 20 °C, the temperature dependence of water diffusion via the mercury-sensitive water channels showed extreme value. In the region, the specific area of the mercury-sensitive aquaporins was 0.004 % of the total cell surface area. The data indicate that water transfer via aquaporins is sensitive to temperature, and the contributions of the transmembrane pathways (aquaporins, lipid bilayer) differ in different temperature ranges.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots

When chilling-sensitive plants are chilled, root hydraulic conductance (L(o)) declines precipitously; L(o) also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on L(o), and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H(2)O(2)) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H(2)O(2) accumulation we also determined the effects of a short H(2)O(2) treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of L(o) during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the L(o) recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling.     

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.     

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H₂O₂) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H₂O₂ to kill pathogens, and epithelial cells of large airways have also been reported to produce H₂O₂ for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H₂O₂ in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H₂O₂ is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H₂O₂ production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H₂O₂ production similar to that observed in plants.     

Differential responses of plasma membrane aquaporins in mediating water transport of cucumber seedlings under osmotic and salt stresses

It has long been recognized that inhibition of plant water transport by either osmotic stress or salinity is mediated by aquaporins (AQPs), but the function and regulation of AQPs are highly variable among distinct isoforms and across different species. In this study, cucumber seedlings were subjected to polyethylene glycol (PEG) or NaCl stress for duration of 2 h or 24 h. The 2 h treatment with PEG or NaCl had non‐significant effect on the expression of plasma membrane AQP (CsPIPs) in roots, indicating the decrease in hydraulic conductivity of roots (Lp_r) and root cells (Lp_rc) measured in these conditions were due to changes in AQP activity. After both 2 h and 24 h PEG or NaCl exposure, the decrease in hydraulic conductivity of leaves (K_leaf) and leaf cells (Lp_lc) could be attributed to a down‐regulation of the two most highly expressed isoforms, CsPIP1;2 and CsPIP2;4. In roots, both Lp_r and Lp_rc were further reduced after 24 h PEG exposure, but partially recovered after 24 h NaCl treatment, which were consistent with changes in the expression of CsPIP genes. Overall, the results demonstrated differential responses of CsPIPs in mediating water transport of cucumber seedlings, and the regulatory mechanisms differed according to applied stresses, stress durations and specific organs.     

Ca2+/H+ exchange through plasma membrane in the system of transport of calcium and hydrogen ions

The survey is aimed to review the data from literature, concerning possible mechanisms of Ca2+ and H+ transport through the plasma membrane of a cells, and also possibility of existence of Ca2+/H(+)-exchange in the plasma membrane of the muscle cells. It is known that the modification of pHl (delta pH) also can influence the work of the contractile system of muscle cells, and the transition of Ca2+ through the plasma membrane of the cells. Thus, one can suppose a direct relation between Ca2+ and H+ transport, through Ca2+/H+ exchange, and indirect relation through connection with other systems of transport of both Ca2+ (Ca(2+)-ATPase, Na+/Ca2+ exchange), and H+ (Na+/H(+)-exchange, H(+)-ATPase). For example it is shown, that the activator (inhibitor) of the Na+/H(+)-exchange through the plasma membrane of muscle cells, influence the work of the retractive system. And as is known, Ca2+ takes main part in involvement in the system excitation--contraction, and, thus, influencing the work of the Na+/H(+)-exchange, it is possible to regulate transport of Ca2+ through the plasma membrane of a muscle cell. The problem about a possibility of existence of Ca2+/H+ exchange, or functioning of Ca2+/H(+)-exchanger, is still far from the solution. Therefore, in the given review the attempt is made to analyze available information about possible connection between Ca2+ and H+ transport through the plasma cell membrane.     

Measurement of hydrogen peroxide in plasma and blood

Measurement of the oxygen metabolite hydrogen peroxide (H2O2) in biological fluids such as plasma could be of interest because it might indicate participation of toxic oxygen species in tissue injury. Recently several reports claimed to measure H2O2 using spectrophotometric and high pressure liquid chromatographic (HPLC) techniques that utilize oxidation of a substrate to a product by a peroxidase. In such a system it is crucial to perform two control experiments to verify whether the measured substance is H2O2. The specificity of the assay for H2O2 should be checked with catalase, and the degradation of H2O2 or inhibition of the assay system by the sample should be checked by determining the recovery of exogenously added H2O2. We performed both types of controls for HPLC and spectrophotometric determinations of H2O2 in plasma and blood. Our results indicate that contrary to previous reports in the literature the measured substance(s) in plasma or blood is not H2O2. Moreover, quantitative measurements of H2O2 in plasma or blood by HPLC was unreliable due to the irreversible binding of H2O2 to the column surface.