Aquaporin-8-facilitated mitochondrial ammonia transport

Aquaporin-8 (AQP8) is a membrane channel permeable to water and ammonia. As AQP8 is expressed in the inner mitochondrial membrane of several mammalian tissues, we studied the effect of the AQP8 expression on the mitochondrial transport of ammonia. Recombinant rat AQP8 was expressed in the yeast Saccharomyces cerevisiae. The presence of AQP8 in the inner membrane of yeast mitochondria was demonstrated by subcellular fractionation and immunoblotting analysis. The ammonia transport was determined in isolated mitochondria by stopped flow light scattering using formamide as ammonia analog. We found that the presence of AQP8 increased by threefold mitochondrial formamide transport. AQP8-facilitated mitochondrial formamide transport in rat native tissue was confirmed in liver (a mitochondrial AQP8-expressing tissue) vs. brain (a mitochondrial AQP8 non-expressing tissue). Comparative studies indicated that the AQP8-mediated mitochondrial movement of formamide was markedly higher than that of water. Together, our data suggest that ammonia diffusional transport is a major function for mitochondrial AQP8.     

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin-8

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15 N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis.     

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.     

Lack of aquaporin-4 water transport inhibition by antiepileptics and arylsulfonamides

Inhibitors of brain glial water channel aquaporin-4 (AQP4) are of potential clinical utility, as they are predicted to modulate brain edema, neuroexcitation and glial scarring. Recently, Huber et al. (Bioorg. Med. Chem.2007, 17, 1270-1273; in press) reported that a series of arylsulfonamides, antiepileptics, and related small molecules strongly inhibited AQP4 water transport with IC(50)s down to 1 microM. We retested the compounds with greatest reported potencies, including acetylsulfanilamide, acetazolamide, 6-ethoxy-benzothiazole-2-sulfonamide, topiramate, zonisamide, phenytoin, lamotrigine, and sumatriptan, in AQP4-transfected mammalian cells and primary cultures of brain glial cells, using several sensitive assays of osmotic water permeability. Contrary to the findings of Huber et al., in our studies we found no significant inhibition of AQP4 water permeability by any of the compounds at concentrations up to 100 microM.     

Ammonium recruitment and ammonia transport by E. coli ammonia channel AmtB

To investigate substrate recruitment and transport across the Escherichia coli Ammonia transporter B (AmtB) protein, we performed molecular dynamics simulations of the AmtB trimer. We have identified residues important in recruitment of ammonium and intraluminal binding sites selective of ammonium, which provide a means of cation selectivity. Our results indicate that A162 guides translocation of an extraluminal ammonium into the pore lumen. We propose a mechanism for transporting the intraluminally recruited proton back to periplasm. Our mechanism conforms to net transport of ammonia and can explain why ammonia conduction is lost upon mutation of the conserved residue D160. We unify previous suggestions of D160 having either a structural or an ammonium binding function. Finally, our simulations show that the channel lumen is hydrated from the cytoplasmic side via the formation of single file water, while the F107/F215 stack at the inner-most part of the periplasmic vestibule constitutes a hydrophobic filter preventing AmtB from conducting water.     

Purification and functional characterization of aquaporin-8

BACKGROUND INFORMATION: Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported. RESULTS: We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes. CONCLUSIONS: In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium.     

Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport

Aquaporin (AQP)8-facilitated transport of NH₃ has been suggested recently by increased NH₃ permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH₃ transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH₃ transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH₃/NH₄⁺) gradients. Relative AQP8 single-channel NH₃-to-water permeability was 0.03. AQP8-facilitated NH₃ and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH₃ transport but provide evidence against physiologically significant AQP8-facilitated NH₃ transport in mice. water transport; transgenic mouse; liver     

Fast measurement of galactoside transport by lactose permease

Lactose permease of Escherichia coli was reconstituted into vesicles of dimyristoylphosphatidylcholine, and the rate of galactoside counterflow was measured in the millisecond time range. The turnover number and the half-saturation constant for transport agree with the values known for cells. This result demonstrates that lactose permease is the sole protein necessary for galactoside transport. Furthermore, lactose permease seems not to require a high level of negatively charged lipids or a certain degree of unsaturation of the lipid hydrocarbon chains. However, the lipids must be in the fluid state, because the transport rate drastically decreases below the lipid ordered fluid phase transition.     

Methylamine and ammonia transport in Saccharomyces cerevisiae.

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.     

Water transport and the distribution of aquaporin-1 in pulmonary air spaces

Effros, R. M., C. Darin, E. R. Jacobs, R. A. Rogers, G. Krenz, and E. E. Schneeberger. Water transport and thedistribution of aquaporin-1 in pulmonary air spaces.J. Appl. Physiol. 83(3): 1002-1016, 1997.Recent evidence suggests that water transport between the pulmonary vasculature and air spaces can be inhibited byHgCl₂, an agent that inhibitswater channels (aquaporin-1 and -5) of cell membranes. In the presentstudy of isolated rat lungs, clearances of labeled(³HOH) and unlabeled water werecompared after instillation of hypotonic or hypertonic solutions intothe air spaces or injection of a hypotonic bolus into the pulmonaryartery. The clearance of ³HOHbetween the air spaces and perfusate after intratracheal instillation and from the vasculature to the tissues after pulmonary arterial injections was invariably greater than that of unlabeled water, indicating that osmotically driven transport of water is limited bypermeability of the tissue barriers rather than the rate of perfusion.Exposure to 0.5 mM HgCl₂ in theperfusate and air-space solution reduced the product of the filtrationcoefficient and surface area(P_fS)of water from the air spaces to the perfusate by 28% afterinstillation of water into the trachea. In contrast, perfusion of 0.5 mM HgCl₂ in air-filled lungs reducedP_fSof the endothelium by 86% after injections into the pulmonary artery, suggesting that much of the action of this inhibitor is on the endothelial surfaces. Confocal laser scanning microscopy demonstrated that aquaporin-1 is on mouse pulmonary endothelium. No aquaporin-1 wasfound on alveolar type I cells with immunogold transmission electronmicroscopy, but small amounts were present on some type II cells.

     

Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient (P(f)) of in vivo perfused colon of +/+ mice, measured using the volume marker (14)C-labeled polyethylene glycol, was 0.016 +/- 0.002 cm/s. P(f) of proximal colon was greater than that of distal colon (0.020 +/- 0.004 vs. 0. 009 +/- 0.003 cm/s, P < 0.01). P(f) was significantly lower in -/- mice when measured in full-length colon (0.009 +/- 0.002 cm/s, P < 0. 05) and proximal colon (0.013 +/- 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 +/- 0.01 vs. 0.81 +/- 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 +/- 0.01 vs. 0.65 +/- 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 +/- 9 vs. 51 +/- 8 microl. min(-1). g(-1)). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.     

Fast axonal transport by neurons from the jellyfish Cyanea capillata

Neurons of the motor nerve net of Cyanea capillata were examined using video-enhanced DIC optics. A variety of organelles were visible within the axons and many were mobile. To quantify the movement organelles were divided into three classes (large, medium, and small) and the rates, direction, and types of movement displayed by the different particle types examined. The overall behavior and rates of movement of transported particles were comparable with those in axons from other species. The largest particles, mainly mitochondria were the slowest moving but were the only particles to reverse their direction of movement or to undergo interactions with other particles. The fastest movement was by the small particles, but both they and medium sized particles were transported continuously. In addition, the linear elements in these axons underwent considerable lateral movement.     

Modulation of electroneutral Na transport in sheep rumen epithelium by luminal ammonia

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as NH3 or NH4+ should modulate cytosolic pH and sodium-proton exchange via Na+/H+ exchanger (NHE). Transport rates of Na+, NH4+, and NH3 across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for NH4+. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, approximately 70% of ammonia is absorbed in the form of NH4+, whereas at pH 7.4, uptake of NH3 exceeds that of NH4+ by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH4+ and K+ but not Na+. Conductance was 135 +/- 12 pS in symmetrical NH(4)Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with P(K) > P(NH4) at high and P(NH4) > P(K) at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH4+ stimulate and nonionic diffusion of NH3 blocks NHE, thus contributing to sodium transport and regulation of pH.     

Fast and robust detection of ancestral selective sweeps

There are few methods tailored for detecting signals of positive selection in populations directly ancestral to multiple descendent populations. We introduce the ancestral branch statistic (ABS), a four‐population summary statistic for identifying selective sweeps occurring in the direct ancestor of a pair of populations. Simulations show that ABS performs at least as well as, and often better under model violations, than the complementary likelihood approach of 3P‐CLR across diverse selection scenarios and parameter values. We first applied ABS to contemporary human genomic data to identify genes that may have been adaptive in ancestral East Asian populations, uncovering the well‐established candidate EDAR, as well as a novel candidate SLC35F3, which encodes a putative thiamine transporter that may have been involved in adaptation to eating polished grains. Next, we performed scans with ancient European genomic data to reexamine evidence of recent positive selection in ancestral Europeans. The MCM6/LCT cluster and the SLC45A2 and HERC2 genes are strong outliers, agreeing with previous studies. Novel candidates, such as SLC30A9 and CYP1A2, may have been involved in adaptation to local nutrient sufficiency and lifestyle changes. Finally, we provide open‐source software, CalcABS, which can perform genomic scans of ancestral sweeps with ABS from population allele frequency data.     

Fluid transport by human nonpigmented ciliary epithelial layers in culture: a homeostatic role for aquaporin-1

We report for thefirst time that cultured nonpigmented human ciliary epithelial (NPE)cell layers transport fluid. Cells were grown to confluence onpermeable membrane inserts, and fluid transport across the resultingcell layers was determined by volume clamp at 37°C. These cell layerstranslocated fluid from the apical to the basal side at a steady rateof 3.6 µl · h¹ · cm²(n = 4) for 8 h. This fluid movement wasindependent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuricchloride, a nonspecific but potent blocker ofHg²⁺-sensitive aquaporins, and aquaporin-1 antisenseoligonucleotides both partially inhibited fluid transport across thecell layers, which suggests that water channels have a role in NPE cellhomeostasis. In addition, these results suggest that of the two ciliaryepithelial layers in tandem, the NPE layer by itself can transportfluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacologicalcharacterization of ciliary epithelial fluid secretion.

     

Testing of an ammonia ion selective electrode for ammonia nitrogen measurement in the methanogenic sludge

Summary The ammonia ion selective electrode method was the most practical and the most convenient one for measuring the ammonia nitrogen in methanogenic sludge. This is because of its simplicity, rapidity, high precision and accuracy, freedom from interference, small sample size, and possibility for continuous monitoring.