hMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect.

In this study a combined analysis of osmotic injury and cytotoxic effect useful for the optimization of the cryopreservation process of a cell suspension is carried out. The case of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) in contact with dimethyl sulfoxide (DMSO) acting as Cryo-Protectant Agent (CPA) is investigated from the experimental as well as the theoretical perspective. The experimental runs are conducted by suspending the cells in hypertonic solutions of DMSO at varying osmolality, system temperature, and contact times; then, at room temperature, cells are pelleted by centrifugation and suspended back to isotonic conditions. Eventually, cell count and viability are measured by means of a Coulter counter and flow-cytometer, respectively. Overall, a decrease in cell count and viability results when DMSO concentration, temperature, and contact time increase. A novel mathematical model is developed and proposed to interpret measured data by dividing the cell population between viable and nonviable cells. The decrease of cell count is ascribed exclusively to the osmotic injury caused by expansion lysis: excessive swelling causes the burst of both viable as well as nonviable cells. On the other hand, the reduction of cell viability is ascribed only to cytotoxicity which gradually transforms viable cells into nonviable ones. A chemical reaction engineering approach is adopted to describe the dynamics of both phenomena: by following the kinetics of two chemical reactions during cell osmosis inside a closed system it is shown that the simultaneous reduction of cell count and viability may be successfully interpreted. The use of the Surface Area Regulation (SAR) model recently proposed by the authors allows one to avoid the setting in advance of fixed cell Osmotic Tolerance Limits (OTLs), as traditionally done in cryopreservation literature to circumvent the mathematical simulation of osmotic injury. Comparisons between experimental data and theoretical simulations are provided: first, a nonlinear regression analysis is performed to evaluate unknown model parameters through a best-fitting procedure carried out in a sequential fashion; then, the proposed model is validated by full predictions of system behavior measured at operating conditions different from those used during the best-fit procedure.

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes.

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca oscillation during the RVD response. Soft substrate significantly improves the Ca oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

A century of exercise physiology: key concepts in muscle cell volume regulation.

Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality. Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regulated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, beginning with consideration of osmosis more than 200 years ago and continuing through to the process of regulatory volume decrease and regulatory volume increase.

Stomatal responses in grapevine become increasingly more tolerant to low water potentials throughout the growing season.

The leaf of a deciduous species completes its life cycle in a few months. During leaf maturation, osmolyte accumulation leads to a significant reduction of the turgor loss point (ΨTLP ), a known marker for stomatal closure. Here we exposed two grapevine cultivars to drought at three different times during the growing season to explore if the seasonal decrease in leaf ΨTLP influences the stomatal response to drought. The results showed a significant seasonal shift in the response of stomatal conductance to stem water potential (gs ~Ψstem ), demonstrating that grapevines become increasingly tolerant to low Ψstem as the season progresses in coordination with the decrease in ΨTLP . We also used the SurEau hydraulic model to demonstrate a direct link between osmotic adjustment and the plasticity of gs ~Ψstem . To understand the possible advantages of gs ~Ψstem plasticity, we incorporated a seasonally dynamic leaf osmotic potential into the model that simulated stomatal conductance under several water availabilities and climatic scenarios. The model demonstrated that a seasonally dynamic stomatal closure threshold results in trade-offs: it reduces the time to turgor loss under sustained long-term drought, but increases overall gas exchange particularly under seasonal shifts in temperature and stochastic water availability. A projected hotter future is expected to lower the increase in gas exchange that plants gain from the seasonal shift in gs ~Ψstem . These findings show that accounting for dynamic stomatal regulation is critical for understanding drought tolerance.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Early transcriptional responses in Solanum peruvianum and Solanum lycopersicum account for different acclimation processes during water scarcity events.

Cultivated tomato Solanum lycopersicum (Slyc) is sensitive to water shortages, while its wild relative Solanum peruvianum L. (Sper), an herbaceous perennial small shrub, can grow under water scarcity and soil salinity environments. Plastic Sper modifies the plant architecture when suffering from drought, which is mediated by the replacement of leaf organs, among other changes. The early events that trigger acclimation and improve these morphological traits are unknown. In this study, a physiological and transcriptomic approach was used to understand the processes that differentiate the response in Slyc and Sper in the context of acclimation to stress and future consequences for plant architecture. In this regard, moderate (MD) and severe drought (SD) were imposed, mediating PEG treatments. The results showed a reduction in water and osmotic potential during stress, which correlated with the upregulation of sugar and proline metabolism-related genes. Additionally, the senescence-related genes FTSH6 protease and asparagine synthase were highly induced in both species. However, GO categories such as "protein ubiquitination" or "endopeptidase inhibitor activity" were differentially enriched in Sper and Slyc, respectively. Genes related to polyamine biosynthesis were induced, while several cyclins and kinetin were downregulated in Sper under drought treatments. Repression of photosynthesis-related genes was correlated with a higher reduction in the electron transport rate in Slyc than in Sper. Additionally, transcription factors from the ERF, WRKY and NAC families were commonly induced in Sper. Although some similar responses were induced in both species under drought stress, many important changes were detected to be differentially induced. This suggests that different pathways dictate the strategies to address the early response to drought and the consequent episodes in the acclimation process in both tomato species.

Rootstocks Overexpressing StNPR1 and StDREB1 Improve Osmotic Stress Tolerance of Wild-Type Scion in Transgrafted Tobacco Plants.

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

Quartz Crystal Microbalance with Dissipation Monitoring of Dynamic Viscoelastic Changes of Tobacco BY-2 Cells under Different Osmotic Conditions.

The plant cell mechanics, including turgor pressure and wall mechanical properties, not only determine the growth of plant cells, but also reflect the functional and structural changes of plant cells under biotic and abiotic stresses. However, there are currently no appropriate techniques allowing to monitor the complex mechanical properties of living plant cells non-invasively and continuously. In this work, quartz crystal microbalance with dissipation (QCM-D) monitoring technique with overtones (3-9) was used for the dynamic monitoring of adhesions of living tobacco BY-2 cells onto positively charged N,N-dimethyl-N-propenyl-2-propen-1-aminiumchloride homopolymer (PDADMAC)/SiO2 QCM crystals under different concentrations of mannitol (CM) and the subsequent effects of osmotic stresses. The cell viscoelastic index (CVIn) (CVIn = ΔD⋅n/ΔF) was used to characterize the viscoelastic properties of BY-2 cells under different osmotic conditions. Our results indicated that lower overtones of QCM could detect both the cell wall and cytoskeleton structures allowing the detection of plasmolysis phenomena; whereas higher overtones could only detect the cell wall's mechanical properties. The QCM results were further discussed with the morphological changes of the BY-2 cells by an optical microscopy. The dynamic changes of cell's generated forces or cellular structures of plant cells caused by external stimuli (or stresses) can be traced by non-destructive and dynamic monitoring of cells' viscoelasticity, which provides a new way for the characterization and study of plant cells. QCM-D could map viscoelastic properties of different cellular structures in living cells and could be used as a new tool to test the mechanical properties of plant cells.

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.

Endosperm cell size reduction caused by osmotic adjustment during nighttime warming in rice.

High night temperature (HNT) often reduces yield in field crops. In rice, HNT during the ripening stage diminishes endosperm cell size, resulting in a considerable reduction in final kernel weight; however, little is known about the underlying mechanisms at cell level. In this study, we performed picolitre pressure-probe-electrospray-ionization mass spectrometry to directly determine metabolites in growing inner endosperm cells of intact seeds produced under HNT conditions, combining with 13C feeding and water status measurements including in situ turgor assay. Microscopic observation in the inner zone suggested that approximately 24.2% of decrease in cell expansion rate occurred under HNT at early ripening stage, leading to a reduction in cell volume. It has been shown that HNT-treated plants were subjected to mild shoot water deficit at night and endosperm cell turgor was sustained by a decline in osmotic potential. Cell metabolomics also suggests that active solute accumulation was caused by a partial inhibition of wall and starch biosynthesis under HNT conditions. Because metabolites were detected in the single cells, it is concluded that a partial arrest of cell expansion observed in the inner endosperms was caused by osmotic adjustment at mild water deficit during HNT conditions.

The Xenopus Oocyte as an Expression System for Functional Analyses of Fish Aquaporins.

Aquaporins are membrane proteins present in all organisms that selectively transport water and small, uncharged solutes across biological membranes along an osmotic gradient. Recent gene editing technologies in zebrafish (Danio rerio) have started to uncover the physiological functions of the aquaporins in teleosts, but these approaches require methods to establish the effects of specific mutations on channel function. The oocytes of the South African frog Xenopus laevis are widely used for the expression of bacterial, plant, and animal aquaporins, and this heterologous system has contributed to numerous discoveries in aquaporin biology. This chapter focuses on techniques used for oocyte preparation and aquaporin expression and gives an overview of specific methods to determine water and solute permeability of the channels and their intracellular trafficking in oocytes.

Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome.

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

Plasma membrane vesicles from cauliflower meristematic tissue and their role in water passage.

BACKGROUND: Cauliflower (Brassica oleracea L. var. botrytis) inflorescences are composed mainly of meristematic tissue, which has a high cellular proliferation. This considerable cellular density makes the inflorescence an organ with a large proportion of membranes. However, little is known about the specific role of the lipid and protein composition of the plasma membrane present in this organ. RESULTS: In this work, we analyzed the lipids and proteins present in plasma membrane from two different stages of development of cauliflower inflorescence and compared them with leaf plasma membrane. For this purpose, plasma membrane vesicles were obtained by centrifugation for each sample and the vesicular diameter and osmotic permeability (Pf) were analyzed by dynamic light scattering and the stopped-flow technique, respectively. In addition, fatty acids and sterols were analyzed by gas chromatography and HPLC. The protein composition of the inflorescences and leaves was characterized by HPLC-ESI-QTOF-MS and the data obtained were compared with Brassicaceae proteins present in the UniProt database in relation to the presence of aquaporins determined by western blot analysis. The highest Pf value was found in 90 day inflorescences-derived plasma membrane vesicles (61.4 ± 4.14 µms- 1). For sterols and fatty acids, the concentrations varied according to the organ of origin. The protein profile revealed the presence of aquaporins from the PIP1 and PIP2 subfamilies in both inflorescences and leaves. CONCLUSION: This study shows that the composition of the sterols, the degree of unsaturation of the fatty acids, and the proteins present in the membranes analyzed give them high functionality for water passage. This represents an important addition to the limited information available in this field.

Cold-regulated gene LeCOR413PM2 confers cold stress tolerance in tomato plants.

Tomato (Lycopersicon esculentum Mill) is an important food plant that has been used as a model plant in genetic evolution and molecular biology research. The plant is originated from the tropics; thus, it is sensitive to cold. Its growth and development can be easily affected by cold stress. In this study, cold-regulated gene LeCOR413PM2 was cloned from tomato leaves and then used to generate two types of transgenic tomato plants: LeCOR413PM2-overexpressing transgenic plants and RNA-interference-expressing transgenic plants. The functions and expression of LeCOR413PM2 gene in response to cold stress were subsequently assessed. The results showed that LeCOR413PM2 localized in the plasma membrane. Expression of LeCOR413PM2 gene in the leaf of transgenic tomato plant was highest compared to that in other organs (i.e., root, stem, flower and fruit); it was elevated when plants were treated with cold stress. Overexpression of LeCOR413PM2 gene was found to not only reduce damage to cell membrane, accumulation of ROS, and photoinhibition of PSII, but also maintain high activity of antioxidant enzymes and content of osmotic regulators. The results also reveal that high activities of antioxidant enzymes were caused by the up-regulation of their gene expressions. This study demonstrates that the overexpression of LeCOR413PM2 could increase cold tolerance of transgenic tomato plants, while the suppressed expression of LeCOR413PM2 by RNA interference could increase the sensitivity of plants to cold.

Aquaporin 1 mediates early responses to osmotic stimuli in endothelial cells via the calmodulin pathway.

The aquaporins (AQPs) are a family of integral membrane proteins which play critical roles in controlling transcellular water movement in various tissues throughout the body. AQP1 helps mediate the cellular response to osmotic stress and tissue water permeability. However, the mechanism by which AQP1 mediates changes in cell volume is not completely clear. Here, we investigated how AQP1 responds to and controls cell volume upon osmotic stimuli during the early phase after the immediate response. Cells overexpressing AQP1 were exposed to hypotonic or hypertonic medium in the presence or absence of staurosporine or W-7 hydrochloride, and fluorescence imaging was performed at 0, 5, 10, and 15 min later. Osmotic stimuli induced redistribution of AQP1 into the cell membrane, hypotonic stimuli caused cell enlargement, and hypertonic stimuli induced a reduction in cell size, which was blocked by T157A/T239A mutations. Changes in cell size induced by osmotic stimuli were blocked by an antagonist of calmodulin kinase, W-7 hydrochloride, but not by the PKC inhibitor staurosporine. These results suggest that calmodulin kinase regulates AQP1 activity during the early response to osmotic stimuli.

Quaternary Ammonium-Based Ionosilica Hydrogels as Draw Solutes in Forward Osmosis.

In the last few years, forward osmosis (FO) has attracted increasing interest as a sustainable technique for water desalination and wastewater treatment. However, FO remains as an immature process principally due to the lack of efficient and easily recyclable draw solutes. In this work, we report that ionosilica hydrogels based on quaternary ammonium halide ionosilica are efficient draw solutes in FO. Fluidic ionosilica hydrogels were obtained via hydrolysis-polycondensation reactions of a trisilylated quaternary ammonium precursor in slightly acidic water/ethanol solvent mixtures. The liquid-to-gel transition of the precursor and the kinetics of the formation of hydrogels were monitored by liquid NMR measurements. The formed hydrogels were shown to generate osmotic pressure up to 10.0 atm, indicating the potential of these hydrogels as efficient draw solutes in FO. Our results suggest that iodide anions are the osmotically active species in the system. Regeneration of the hydrogels via ultrafiltration (UF) was successfully achieved, allowing the development of a closed FO-UF process. However, the osmotic performances of the ionosilica hydrogels irreversibly decreased along the successive FO-UF cycles, probably due to anion exchange processes.

A Plasma Membrane Nanodomain Ensures Signal Specificity during Osmotic Signaling in Plants.

In the course of their growth and development, plants have to constantly perceive and react to their environment. This is achieved in cells by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependent nanodomains within the plasma membrane (PM). These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS) that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, osmotic signal triggers interaction between ROP6 and two NADPH oxidases that subsequently generate ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensures signal specificity downstream of independent stimuli.

Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture.

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.

Energy and nitrogenous waste from glutamate/glutamine catabolism facilitates acute osmotic adjustment in non-neuroectodermal branchial cells.

Maintenance of homeostasis is one of the most important physiological responses for animals upon osmotic perturbations. Ionocytes of branchial epithelia are the major cell types responsible for active ion transport, which is mediated by energy-consuming ion pumps (e.g., Na+-K+-ATPase, NKA) and secondary active transporters. Consequently, in addition to osmolyte adjustments, sufficient and immediate energy replenishment is essenttableial for acclimation to osmotic changes. In this study, we propose that glutamate/glutamine catabolism and trans-epithelial transport of nitrogenous waste may aid euryhaline teleosts Japanese medaka (Oryzias latipes) during acclimation to osmotic changes. Glutamate family amino acid contents in gills were increased by hyperosmotic challenge along an acclimation period of 72 hours. This change in amino acids was accompanied by a stimulation of putative glutamate/glutamine transporters (Eaats, Sat) and synthesis enzymes (Gls, Glul) that participate in regulating glutamate/glutamine cycling in branchial epithelia during acclimation to hyperosmotic conditions. In situ hybridization of glutaminase and glutamine synthetase in combination with immunocytochemistry demonstrate a partial colocalization of olgls1a and olgls2 but not olglul with Na+/K+-ATPase-rich ionocytes. Also for the glutamate and glutamine transporters colocalization with ionocytes was found for oleaat1, oleaat3, and olslc38a4, but not oleaat2. Morpholino knock-down of Sat decreased Na+ flux from the larval epithelium, demonstrating the importance of glutamate/glutamine transport in osmotic regulation. In addition to its role as an energy substrate, glutamate deamination produces NH4+, which may contribute to osmolyte production; genes encoding components of the urea production cycle, including carbamoyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), were upregulated under hyperosmotic challenges. Based on these findings the present work demonstrates that the glutamate/glutamine cycle and subsequent transepithelial transport of nitrogenous waste in branchial epithelia represents an essential component for the maintenance of ionic homeostasis under a hyperosmotic challenge.