Central pontine myelinolysis and the osmotic demyelination syndromes: an open and shut case?

Central pontine myelinolysis and extrapontine myelinolysis are collectively called the osmotic demyelination syndromes. Despite being described in 1959, there are several aspects of the disorder that remain an enigma. Animal models and neuroimaging techniques have allowed us to understand the condition better. From being a universally fatal disorder that was diagnosed post mortem, increased awareness, neuroimaging techniques and supportive care have enabled us to make the diagnosis ante-mortem. This has also led to a significant drop in associated mortality. The aim of this review is to highlight the clinical spectrum, neuroimaging findings, and recent developments.

Adrenomedullin Inhibits Osmotic Water Permeability in Rat Inner Medullary Collecting Ducts.

Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.

Reactive oxygen species scavenging mechanisms associated with polyethylene glycol mediated osmotic stress tolerance in Chinese potato.

Influence of polyethylene glycol (PEG) mediated osmotic stress on reactive oxygen species (ROS) scavenging machinery of Chinese potato (Solenostemon rotundifolius (Poir.) J. K. Morton) was investigated. Five genotypes of Chinese potato were raised in Murashige and Skoog (MS) basal medium containing 6-benzylaminopurine (BAP, 1 mg L-1) along with various concentrations of PEG-6000 mediated stress conditions (0, -0.2 and -0.5 MPa) and evaluated for osmotic stress tolerance in vitro. The medium containing PEG-6000 had a detrimental effect on plantlet growth and development while compared with the control. Accumulation of H2O2 was lower in Sreedhara and Subala and higher in Nidhi under PEG stress, which was evident by in situ detection in leaves. Lipid peroxidation product such as malondialdehyde (MDA) content was increased due to PEG stress which was more in susceptible genotype than that in tolerant ones. An enhanced ROS-scavenging antioxidant enzyme was observed under stress with respect to the control. The enzymes of ascorbate-glutathione cycle showed an important role in scavenging ROS. The imposition of PEG stress also increased the non-enzymatic antioxidants viz., the ascorbate and reduced glutathione content which was prominent in tolerant genotypes in comparison to susceptible. The present study indicated that, Sreedhara and Subala showed more tolerance to osmotic stress with better ROS scavenging machineries which would be the lines of interest for augmenting future breeding strategies in this climate resilient minor tuber crop.

Differential effects of NaCl and Na2SO4 on the halophyte Prosopis strombulifera are explained by different responses of photosynthesis and metabolism.

Prosopis strombulifera (Lam.) Benth. is a halophytic shrub found in highly saline soils in Argentina, with high tolerance against NaCl but strong growth inhibition by Na2SO4. In the present study, the differences in the physiological responses caused by these salts and an iso-osmotic combination thereof on photosynthesis, mineral composition and metabolism were analyzed. Na2SO4 treated plants were the most affected by salinity, showing a significant decrease in several photosynthetic parameters. Proline and cysteine accumulated significantly in the plants in response to salt stress. These results show by the first time that the SO42- anion is triggering damage in the photosynthetic apparatus and consequently affecting the photosynthetic process, which may explain the strong growth inhibition in these plants at high salinity. Moreover, the SO42- anion provoke challenges in the incorporation of nutrients, decreasing the levels of K, Ca, P and Mg, and inducing a strong antioxidant activity in P. strombulifera.

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea.

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Suppression of fatty acid ß-oxidation and energy deficiency as a cause of inhibitory effect of E. coli lipopolysaccharide on osmotic water transport in the frog urinary bladder.

Previously we showed that arginine-vasotocin (AVT)-stimulated osmotic water permeability (OWP) of the frog urinary bladder was decreased if the mucosal side of the bladder has been naturally colonized by Gram-negative bacteria, or if bacterial lipopolysaccharide (LPS) was introduced into the lumen of the isolated bladder (J. Exp. Zool., 2013, 319, 487-494). Taking into account that in different tissues and cell types, challenge with LPS causes significant metabolic shift and energy deficiency, we hypothesized that an LPS-induced decrease of AVT-stimulated OWP could depend on the reduction of fatty acid oxidation (FAO), which is important for generation of ATP in epithelia. Using an isolated frog Rana temporaria urinary bladder we showed that the AVT-induced increase of OWP did not depend on the external glucose, but was inhibited by oligomycin, an ATP-synthase inhibitor, and by etomoxir, an inhibitor of carnitine palmitoyltransferase-1. In primary cultured epithelial cells isolated from the bladder mucosa, LPS E. coli (25 µg/ml, 21 h), as well as etomoxir (100 µM), decreased FAO accompanied by triacylglycerol accumulation. Both drugs impaired mitochondrial functions demonstrated by decreased ATP production and a reduced maximal oxygen consumption rate (OCR) and OCR directed at ATP synthesis. Additionally, we found that LPS decreased the expression of peroxisome proliferator-activated receptor alpha, a key player in the regulation of FAO. These data indicate that the impairment of AVT-induced water transport in osmoregulatory epithelium caused by LPS depends at least partly on defects in FAO and FAO-dependent energy production.

Copper accumulation and toxicity in earthworms exposed to CuO nanomaterials: Effects of particle coating and soil ageing.

Engineered nanomaterials (ENMs) may be functionalised with a surface coating to enhance their properties, but the ecotoxicity of the coatings and how hazard changes with ageing in soil is poorly understood. This study determined the toxic effect of CuO ENMs with different chemical coatings on the earthworm (Eisenia fetida) in fresh soil, and then after one year in aged soil. In both experiments, earthworms were exposed for 14 days to the CuO materials at nominal concentrations of 200 and 1000 mg Cu kg-1 dry weight and compared to CuSO4. In the fresh soil experiment, CuO-COOH was found to be the most acutely toxic of the nanomaterials (survival, 20 ±â€¯50%), with tenfold increase of total Cu in the earthworms compared to controls. Sodium pump activity was reduced in most CuO ENM treatments, although not in the CuSO4 control. There was no evidence of glutathione depletion or the induction of superoxide dismutase (SOD) activity in any treatment. Histology showed a mild hypoplasia of mucous cells in the epidermis with some nanomaterials. In the aged soil, the CuO-NH4+ was the most acutely toxic ENM (survival 45 ±â€¯3%) and Cu accumulation was lower in the earthworms than in the fresh soil study. Depletion of tissue Mn and Zn concentrations were seen in earthworms in aged soil, while no significant effects on sodium pump or total glutathione were observed. Overall, the study showed some coating-dependent differences in ENM toxicity to earthworms which also changed after a year of ageing the soil.

Sensitivity to near-future CO2 conditions in marine crabs depends on their compensatory capacities for salinity change.

Marine crabs inhabit shallow coastal/estuarine habitats particularly sensitive to climate change, and yet we know very little about the diversity of their responses to environmental change. We report the effects of a rarely studied, but increasingly prevalent, combination of environmental factors, that of near-future pCO2 (~1000 µatm) and a physiologically relevant 20% reduction in salinity. We focused on two crab species with differing abilities to cope with natural salinity change, and revealed via physiological and molecular studies that salinity had an overriding effect on ion exchange in the osmoregulating shore crab, Carcinus maenas. This species was unaffected by elevated CO2, and was able to hyper-osmoregulate and maintain haemolymph pH homeostasis for at least one year. By contrast, the commercially important edible crab, Cancer pagurus, an osmoconformer, had limited ion-transporting capacities, which were unresponsive to dilute seawater. Elevated CO2 disrupted haemolymph pH homeostasis, but there was some respite in dilute seawater due to a salinity-induced metabolic alkalosis (increase in HCO3- at constant pCO2). Ultimately, Cancer pagurus was poorly equipped to compensate for change, and exposures were limited to 9 months. Failure to understand the full spectrum of species-related vulnerabilities could lead to erroneous predictions of the impacts of a changing marine climate.

Disturbance in Na+ regulation in cells rich in mitochondria isolated from gills of the yellow clam Mesodesma mactroides exposed to copper under different osmotic conditions.

Cells rich in mitochondria were isolated from gills of the seawater clam Mesodesma mactroides, incubated in isosmotic saline solution (840 mOsmol/kg H2O), and exposed (3 h) to environmentally realistic Cu concentrations (nominally: 0, 5, 9 and 20 µg/L). In cells exposed to 20 µg Cu/L, Cu accumulation, Na+ content reduction and carbonic anhydrase (CA) activity inhibition were observed, without significant changes in cell viability and Na+,K+-ATPase (NKA) activity. In the absence of Cu, cell viability and Cu content were reduced in hyposmotic media respect with the control, without changes in Na+ content and enzyme (CA and NKA) activities. In the presence of 5 or 9 µg/L Cu, cell Cu content was increased, especially at 670 mOsmol/kg H2O. Cell Na+ content and NKA activity were reduced after exposure to 20 µg/L Cu at 670 mOsmol/kg H2O. In turn, CA activity was dependent on Cu concentration, being significantly reduced in cells exposed to 9 and 20 µg/L Cu in both hyposmotic conditions. These findings indicate that Cu also negatively affects Na+ regulation in gill cells of the seawater clam M. mactroides, with Cu toxicity increasing at hyposmotic conditions. Also, they indicate that physiology is more important than water chemistry in predicting Cu toxicity in environments of changing salinity, pointing out CA activity as a potential biomarker of Cu exposure.

Jasmonic acid-induced tolerance to root-knot nematodes in tomato plants through altered photosynthetic and antioxidative defense mechanisms.

Plant parasitic nematodes cause severe damage to cultivated crops globally. Management of nematode population is a major concern as chemicals used as nematicides have negative impact on the environment. Natural plant products can be safely used for the control of nematodes. Among various plant metabolites, plant hormones play an essential role in developmental and physiological processes and also assist the plants to encounter stressful conditions. Keeping this in mind, the present study was designed to evaluate the effect of jasmonic acid (JA) on the growth, pigments, polyphenols, antioxidants, osmolytes, and organic acids under nematode infection in tomato seedlings. It was observed that nematode inoculation reduced the growth of seedlings. Treatment with JA improved root growth (32.79%), total chlorophylls (71.51%), xanthophylls (94.63%), anthocyanins (37.5%), and flavonoids content (21.11%) when compared to inoculated seedlings alone. The JA application enhanced the total antioxidant capacity (lipid- and water-soluble antioxidants) by 38.23 and 34.37%, respectively, in comparison to infected seedlings. Confocal studies revealed that there was higher accumulation of glutathione in hormone-treated seedlings under nematode infection. Treatment with JA increased total polyphenols content (74.56%) in comparison to nematode-infested seedlings. JA-treated seedlings also enhanced osmolyte and organic acid contents under nematode stress. Overall, treatment with JA improved growth, enhanced pigment levels, modulated antioxidant content, and enhanced osmolyte and organic acid content in nematode-infected seedlings.

Cell membrane permeability coefficients determined by single-step osmotic shift are not applicable for optimization of multi-step addition of cryoprotective agents: As revealed by HepG2 cells.

HepG2 cells have a number of research applications and cryopreservation of these cells would improve supply and thus facilitate the study. Development of effective cryopreservation protocols relies on knowledges of the fundamental mass transport characteristics of HepG2 cell membrane. Currently, the permeability parameters estimated from single-step addition are routinely used to predict the osmotic responses of the cells in multistep protocols, as well as used for prediction of optimal cooling rates. However, the reasonability of this approach has not been rigorously studied. Here we measured the hydraulic conductivity (Lp) and the permeability coefficient (Ps) of HepG2 cells in the absence/presence of dimethyl sulfoxide (Me2SO) at various temperatures with single and multistep addition of Me2SO. We found that the permeability yielded via one-step addition of the Me2SO cannot exactly predict the volume change of the cells when the CPA was added in multiple steps.

Effects of waterborne copper delivered under two different exposure and salinity regimes on osmotic and ionic regulation in the mudflat fiddler crab, Minuca rapax (Ocypodidae, Brachyura).

The effects of exposure to copper (Cu) on tissue Cu accumulation, on hemolymph osmotic, Na+ and Cl- regulation, and on gill Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) activities were evaluated in the fiddler crab Minuca rapax. Waterborne copper was delivered to the crabs at one of three salinities (seawater at 25‰ salinity [S] = isosmotic control; distilled water [<0.1‰ S] = hypo-osmotic medium; or 60‰ S = hyper-osmotic seawater) either for 5 days in a 0.5-cm water film containing 0, 50, 150, 250 or 500µg Cu/L with free access to a dry surface, or in crabs fully submerged for 5h at 0, 250 or 500µg Cu/L. In the crabs with free access to a dry surface, the highest Cu concentrations were found in the hemolymph and hepatopancreas with some accumulation in the gills; accumulation in the hemolymph and gills was enhanced in low salinity but was salinity independent in the hepatopancreas. Osmotic regulation was unaffected by Cu exposure; however Na+ and Cl- hypo- regulation was impaired by Cu in 25 and 60‰ S. Gill NKA activity was stimulated 2-fold at 50µg Cu/L and markedly inhibited at 150µg Cu/L and above in 0 and 25‰ S. Gill CA was inhibited in <0.1‰ S but stimulated in 25 and 60‰ S; an inverse concentration-CA activity response was seen above 150µg Cu/L for all salinities. In the submerged crabs, Cu accumulated in all tissues in 60‰ S; however, there was no clear-cut Cu concentration-accumulation relationship evident in any tissue for either exposure regime, likely owing to the crabs' ability to regulate Cu. Copper exposure diminished osmotic, [Na+] and [Cl-] hypo-regulatory ability, especially in higher salinities. Gill NKA activity was markedly inhibited by Cu overall, and particularly above 250µg Cu/L in <0.1‰ S. Gill CA activity was inhibited in 25‰ S but inconsistently affected in 0 and 60‰ S. These findings show that Minuca rapax is affected both physiologically and biochemically by Cu contamination, although to different degrees, depending on the delivery regime, salinity, copper concentration and target tissue.

Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions.

Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin, the antiapoptotic marker B cell lymphoma 2, as well as the cell surface adhesion molecules CD106 (vascular cell adhesion molecule 1) and CD54 (intracellular adhesion molecule 1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

Assessment of the carcinogenic potential of high intense-sweeteners through the test for detection of epithelial tumor clones (warts) in Drosophila melanogaster.

High intensity-sweeteners (HIS) are natural or synthetic substances, sweeter than sugar, providing sweetness without calories. Sweeteners are mainly used as an aid in losing weight, preventing obesity and controlling blood sugar levels for diabetics. The objective of this study was to evaluate the carcinogenic potential of the sweeteners aspartame, sucralose, sodium saccharin and steviol glycoside, using the test for detection of epithelial tumor clones in Drosophila melanogaster. Larvae of 72 ± 4h, obtained from wts/TM3 female mated with mwh/mwh males, were treated for approximately 48h with different concentrations of aspartame (0.85, 1.7, 3.4, 6.8 or 13.6 mM ); sucralose (0.5, 1.25, 2.5, 5.0 or 10 mM); sodium saccharin (25; 50; 100; 200 or 400 mM) and steviol glycoside (2.5; 5.0; 10; 20 or 40 mM). Water (Reverse Osmosis) and doxorubicin (DXR 0.4 mM) were used as negative and positive controls, respectively. No statistically significant differences were observed (p > 0.05) in tumor frequencies in individuals treated with all concentrations of these sweeteners when compared to negative control. It was therefore concluded that, in these experimental conditions, aspartame, sucralose, sodium saccharin and steviol glycoside have no carcinogenic effect in D. melanogaster.

Water Permeability Adjusts Resorption in Lung Epithelia to Increased Apical Surface Liquid Volumes.

The apical surface liquid (ASL) layer covers the airways and forms a first line of defense against pathogens. Maintenance of ASL volume by airway epithelia is essential for maintaining lung function. The proteolytic activation of epithelial Na+ channels is believed to be the dominating mechanism to cope with increases in ASL volumes. Alternative mechanisms, in particular increases in epithelial osmotic water permeability (Posm), have so far been regarded as rather less important. However, most studies mainly addressed immediate effects upon apical volume expansion (AVE) and increases in ASL. This study addresses the response of lung epithelia to long-term AVE. NCI-H441 cells and primary human tracheal epithelial cells, both cultivated in air-liquid interface conditions, were used as models for the lung epithelium. AVE was established by adding isotonic solution to the apical surface of differentiated lung epithelia, and time course of ASL volume restoration was assessed by the deuterium oxide dilution method. Concomitant ion transport was investigated in Ussing chambers. We identified a low resorptive state immediately after AVE, which coincided with proteolytic ion transport activation within 10-15 minutes after AVE. The main clearance of excess ASL occurred during a delayed (hours after AVE) high resorptive state, which did not correlate with ion transport activation. Instead, high resorptive state onset coincided with an increase in Posm, which depended on aquaporin up-regulation. In summary, our data demonstrate that, aside from ion transport activation, modulation of Posm is a major mechanism to compensate for long-term AVE in lung epithelia.

Osmotic Effects Induced by Pore-Forming Agent Nystatin: From Lipid Vesicles to the Cell.

The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of "cell-vesicles" and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell's behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena.

Suppression of Reactive Oxygen Species Accumulation in Chloroplasts Prevents Leaf Damage but Not Growth Arrest in Salt-Stressed Tobacco Plants.

Crop yield reduction due to salinity is a growing agronomical concern in many regions. Increased production of reactive oxygen species (ROS) in plant cells accompanies many abiotic stresses including salinity, acting as toxic and signaling molecules during plant stress responses. While ROS are generated in various cellular compartments, chloroplasts represent a main source in the light, and plastid ROS synthesis and/or elimination have been manipulated to improve stress tolerance. Transgenic tobacco plants expressing a plastid-targeted cyanobacterial flavodoxin, a flavoprotein that prevents ROS accumulation specifically in chloroplasts, displayed increased tolerance to many environmental stresses, including drought, excess irradiation, extreme temperatures and iron starvation. Surprisingly, flavodoxin expression failed to protect transgenic plants against NaCl toxicity. However, when high salt was directly applied to leaf discs, flavodoxin did increase tolerance, as reflected by preservation of chlorophylls, carotenoids and photosynthetic activities. Flavodoxin decreased salt-dependent ROS accumulation in leaf tissue from discs and whole plants, but this decline did not improve tolerance at the whole plant level. NaCl accumulation in roots, as well as increased osmotic pressure and salt-induced root damage, were not prevented by flavodoxin expression. The results indicate that ROS formed in chloroplasts have a marginal effect on plant responses during salt stress, and that sensitive targets are present in roots which are not protected by flavodoxin.

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.

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-ß1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-ß1 from Müller cells.

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response.

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca(2+) uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H(+))/K(+) symporter that mediates a high-affinity uptake during K(+) deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K(+) efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K(+) loss. Taken together, our results position ILK1 as a link between plant defense pathways and K(+) homeostasis.