Acute Hypo-osmotic Stress Research Articles

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

The dynamic response to hypo-osmotic stress reveals distinct stages of freshwater acclimation by a euryhaline diatom.

The salinity gradient separating marine and freshwater environments is a major ecological divide, and the mechanisms by which most organisms adapt to new salinity environments are poorly understood. Diatoms are a lineage of ancestrally marine microalgae that have repeatedly colonized and diversified in freshwaters. Cyclotella cryptica is a euryhaline diatom found in salinities ranging from fully freshwater to fully marine, thus providing a powerful system for understanding the genomic mechanisms for mitigating and acclimating to low salinity. To understand how diatoms mitigate acute hypo-osmotic stress, we abruptly shifted C.cryptica from seawater to freshwater and performed transcriptional profiling during the first 10h. Freshwater shock dramatically remodelled the transcriptome, with ~50% of the genome differentially expressed in at least one time point. The peak response occurred within 1h, with strong repression of genes involved in cell growth and osmolyte production, and strong induction of specific stress defence genes. Transcripts largely returned to baseline levels within 4-10h, with growth resuming shortly thereafter, suggesting that gene expression dynamics may be useful for predicting acclimation. Moreover, comparison to a transcriptomics study of C.cryptica following months-long acclimation to freshwater revealed little overlap between the genes and processes differentially expressed in cells exposed to acute stress versus fully acclimated conditions. Altogether, this study highlights the power of time-resolved transcriptomics to reveal fundamental insights into how cells dynamically respond to an acute environmental shift and provides new insights into how diatoms mitigate natural salinity fluctuations and have successfully diversified across freshwater habitats worldwide.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

In Silico Screen Identifies a New Family of Agonists for the Bacterial Mechanosensitive Channel MscL

MscL is a highly conserved mechanosensitive channel found in the majority of bacterial species, including pathogens. It functions as a biological emergency release valve, jettisoning solutes from the cytoplasm upon acute hypoosmotic stress. It opens the largest known gated pore and has been heralded as an antibacterial target. Although there are no known endogenous ligands, small compounds have recently been shown to specifically bind to and open the channel, leading to decreased cell growth and viability. Their binding site is at the cytoplasmic/membrane and subunit interfaces of the protein, which has been recently been proposed to play an essential role in channel gating. Here, we have targeted this pocket using in silico screening, resulting in the discovery of a new family of compounds, distinct from other known MscL-specific agonists. Our findings extended the study of this functional region, the progression of MscL as a viable drug target, and demonstrated the power of in silico screening for identifying and improving the design of MscL agonists.

Branchial transcriptional responses of spotted scat, Scatophagus argus, to acute hypo-osmotic stress

The spotted scat (Scatophagus argus) is a euryhaline marine fish with the capability to withstand different salinity levels and can tolerate abrupt transfer from salt water (SW) to fresh water (FW) or vice versa. Differentially expressed genes were identified by constructing suppression subtractive hybridization (SSH) cDNA libraries to explore branchial osmoregulatory mechanisms affected by salinity challenge. After trimming and blasting, 105 uniquely expressed sequence tags were identified. Among them, 24 candidate genes involved in the stress response, metabolism, and the respiratory chain were chosen for further validation by real-time polymerase chain reaction. More than two-fold expression differences were observed in ADP-ribosylation factor 1 (arf1), cyclic AMP-responsive element-binding protein 3-like 4 (creb3l4), EVI5-like protein (evi5l), protocadherin fat 2 (fat2), transferrin receptor protein 2 (tfr2), C-X-C chemokine receptor type 4 (cxcr4), aquaporin-3 (aqp3), sodium/potassium-transporting ATPase subunit beta-233 (nka β233), serum and glucocorticoid-regulated kinase 1 (sgk1), and ras GTPase-activating-like protein IQGAP1 (iqgap1). Among these genes, aqp and nka are important osmoregulatory factors. AQP3 protein was observed being localized to the membranes of mitochondrion-rich cells (MRCs) and pillar cells of the gill of S. argus, and NKA β233 subunit isoform was only found in MRC membranes by immunostaining. Significant differences in aqp3 and nka β233 expression occurred within 24h after being transferring into the freshwater, and nka β233 expression level continued to highly upregulated 2 and 7days post-transfer (dpt). These results suggest that branchial aqp3 and nka β233 play important roles in response to hypo-osmotic stress in S. argus.

Profiling of Up-Regulated Genes Response to Acute Hypo-Osmotic Stress in Hepatopancreas and Gill of the Pacific White Shrimps (Litopenaeus vannamei)

<p class="1Body">Suppression subtractive hybridization (SSH) was applied to screen responsively up-regulation genes in hepatopancreas and gill of Litopenaeus vannamei induced by acute hypo-osmotic stress. In the hepatopancreas, 196 clones were randomly selected and sequenced. 131 non-redundant transcripts, corresponding to 41 genes, were found with elevated expressions. They were functionally clustered into eight biological processes which were protein synthesis and processing, carbohydrate metabolism and energy production, transport, cell growth, apoptosis, cytoskeletal component, cell defense and homeostasis, signal transduction, accounting for 26.2%, 14.3%, 16.7%, 4.7%, 4.7%, 2.3%, 11.9% and 7.1%, respectively. When confirmed by real time qRT-PCR, the gene expression levels of MnSOD, glutathione and glutamine synthetase showed significant increases (2.64-folds, 3.44-folds and 2.16-folds, respectively) in hepatopancreas (<em>P</em><0.05). Totally 81 expressed sequence tags (ESTs) were obtained by random sequencing from the gill SSH cDNA library, and 52 unigenes, including 15 contigs and 37 singlets, after CP3 online matching were gained by clustering and assembling. The sequence alignment results revealed that 30 of them had significant homology to previously identified Genbank database sequences, while 22 unigenes did not match any sequence in the datebase and presumably represented unidentified cDNAs. Functionally, the unigenes were classified into six clusters, including ion channels and transport proteins (1); protein synthesis, translation and transcription factor (2); stress resistance and antioxidant factors (3); energy metabolism (4); signal receptor and transduction (5); cell fibrin and cytoskeletal proteins (6) with proportion of 23.3%, 20.0%, 20.0%, 16.7%, 10.0% and 10.0% respectively. Real time qRT-PCR confirmation revealed significant elevation of Arginine kinase, Carbonic anhydrase and NaK-ATPase-α-subunit expressions (3.73-folds, 2.55-folds and 5.83-folds, respectively) in the gill after acute low-osmotic stress (P < 0.05). These results provided insight into critical physiology processes and pathways constituting the hypo-osmotic stress adaption program in hepatopancreas and gill of <em>L. vannamei</em>.</p>

Molecular cloning and expression of the calreticulin gene of the Pacific white shrimp, Litopenaeus vannamei, in response to acute hypo-osmotic stress

Calreticulin is a ubiquitous and highly conserved calcium-binding protein that is located in the endoplasmic reticulum (ER). In the present study, the cDNA of the Litopenaeus vannamei calreticulin (LvCRT) gene was cloned using the rapid amplification of cDNA ends (RACE) technique. Its full-length cDNA was 1866bp in length, which included a 1221-bp open reading frame and encoded a 406-amino acid. In addition, a 222-bp 5′ untranslated region (UTR) and a 423-bp 3′ UTR with a polyadenylation [poly(A)] signal (AATAAA) located 27bp upstream of the poly(A) tail were identified. The predicted molecular weight and isoelectric point of the protein were 45.3kDa and 4.90, respectively. Protein analysis revealed that LvCRT contains a conserved calreticulin family signature sequence (KHEQNIDCGGGYLKVF), a signal peptide (MKTWVFLALFGVVLVES), and a conserved endoplasmic reticulum HDEL retention sequence. Furthermore, NCBI BLASTX and phylogenetic analyses showed that LvCRT has a high degree of sequence homology with CRT proteins from Fenneropenaeus chinensis and Penaeus monodon. In addition, qRT-PCR analysis indicated that the highest level of LvCRT expression was observed in the intestines, whereas the lowest was in the muscle. When the shrimps were transferred from salinity 30 to 2psu diluted seawater, the expression level of LvCRT in the hepatopancreas and hemocytes sharply increased at 6h and 12h (which were about 3.23-fold and 2.01-fold of that in the 0h control group), and then, significantly decreased at 96h and 24h (p<0.05). The expression of LvCRT in the intestines and gills was not affected by the low-salinity stress (p>0.05). In conclusion, the responses of L. vannamei to changes in salinity via the calreticulin protein facilitate a better understanding of its function in relation to stress.

Molecular cloning and expression of hepatopancreas glutamine synthetase in the Pacific white shrimp, Litopenaeus vannamei, induced by acute hypo-osmotic stress

Glutamine synthetase (GS) is a key enzyme involved in nitrogen metabolism and glutamine synthesis. A 1455bp cDNA clone encoding GS was isolated from Litopenaeus vannamei (LvGS, JN620540.1). The sequence consists of a 75bp 5′ untranslated region followed by a 1101bp open reading frame corresponding to 366 amino acids. The encoded protein has a 67% to 93% overall amino acid sequence identity with its counterparts in other various species. A phylogenetic analysis of the GS sequence showed that LvGS clustered with the invertebrate group as expected. qRT-PCR analysis indicated that LvGS was expressed in the stomach, gill, intestine, hepatopancreas, muscle, heart, and eyestalk. The profile of LvGS was up-regulated in the stomach, gills, intestines, and hepatopancreas after 48h of exposure to dilute seawater (4ppt). The mRNA levels of LvGS in the hepatopancreas significantly increased 2.9-fold after 3h to 3d of exposure to dilute seawater (P<0.05). The LvGS activity and glutamine in the hepatopancreas increased after 3h to 3d and then decreased after 7d to 20d of exposure to dilute seawater. However, the hemolymph total protein and ammonia decreased after 3h to 3d, and then increased after 7d to 20d. The hemolymph urea and uric acid significantly increased after 3h to 12h of exposure to dilute seawater. L. vannamei fully acclimated to a low salinity environment within a week. LvGS can participate in physiological osmotic adaptation to hypo-osmotic water and aid L. vannamei in resisting acute salinity changes via the manipulation of nitrogen metabolism.

Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the flatback mud crab Eurypanopeus depressus and designated as EdPrx-1 (GenBank accession no. EU684547). EdPrx-1 has a major open-reading frame of 594 bp and is capable of encoding a polypeptide of 198 amino acid residues. Like other 2-Cys Prxs, EdPrx-1 protein possesses two conserved cysteine residues that play an essential role for the antioxidant activity of the proteins. The EdPrx-1 protein, as deduced from the cDNA sequence, shows a high level (74-93%) of sequence similarity to the 2-Cys Prxs from other crustaceans as well as those from many arthropod species (73-76% similarity). It shares about 70% sequence similarity with homologs from mammalian species. EdPrx-1 gene is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition; however, its expression is elevated about three-fold in the gills under hypo-osmotic stress. This suggests a possible role in protecting against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation.

Real‐time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two‐photon microscopy

The brain swells over the several minutes that follow stroke onset or acute hypo-osmotic stress because cells take up water. Measuring the volume responses of single neurons and glia has necessarily been confined to isolated or cultured cells. Two-photon laser scanning microscopy enables real-time visualization of cells functioning deep within living neocortex in vivo or in brain slices under physiologically relevant osmotic and ischemic stress. Astrocytes and their processes expressing green fluorescent protein in murine cortical slices swelled in response to 20 min of overhydration (-40 mOsm) and shrank during dehydration (+40 or +80 mOsm) at 32-34 degrees C. Minute-by-minute monitoring revealed no detectable volume regulation during these osmotic challenges, particularly during the first 5 min. Astrocytes also rapidly swelled in response to elevated [K+](o) for 3 min or oxygen/glucose deprivation (OGD) for 10 min. Post-OGD, astroglial volume recovered quickly when slices were re-supplied with oxygen and glucose, while neurons remained swollen with beaded dendrites. In vivo, rapid astroglial swelling was confirmed within 6 min following intraperitoneal water injection or during the 6-12 min following cardiac arrest. While the astrocytic processes were clearly swollen, the extent of the astroglial arbor remained unchanged. Thus, in contrast to osmo-resistant pyramidal neurons (Andrew et al., 2007) that lack known aquaporins, astrocytes passively respond to acute osmotic stress, reflecting functional aquaporins in their plasma membrane. Unlike neurons, astrocytes better recover from brief ischemic insult in cortical slices, probably because their aquaporins facilitate water efflux.

Assessment of Potential Stimuli for Mechano-Dependent Gating of MscL: Effects of Pressure, Tension, and Lipid Headgroups

MscL is a mechanosensitive channel of large conductance that serves as an "emergency relief valve", protecting bacteria from acute hypoosmotic stress. Although it is well-accepted that the MscL protein and an adequate membrane matrix are necessary and sufficient for the function of this channel, the exact role of the membrane has yet to be elucidated. Here, we address the role of the membrane matrix through in vitro reconstitution of the MscL protein in defined lipid bilayers. We have applied Laplace's law to visualized membrane patches where we can measure patch curvature as described in previous studies. Here, by comparing patches with different curvatures, we demonstrate that the MscL channel senses tension within the membrane and that the pressure across it plays no detectable role as a stimulus. In addition, gating only occurs when the smallest radius of curvature is nearly achieved, suggesting that the lateral tension rather than membrane curvature is the important biophysical parameter. Finally, we have examined the contribution of specific headgroups by measuring their effect on the membrane tension required to gate the channel. We have found that the addition of neither anionic nor endogenous lipids to a non-native membrane effected a leftward shift in the activation curve. In fact, the major endogenous lipid of the Escherichia coli membrane, phosphatidylethanolamine, led to a channel activity at a higher tension threshold, suggesting that this lipid effects altered activity through changes in the biophysical properties of the membrane, rather than through an MscL-specific interaction.

An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating.

MscL is a mechanosensitive channel of large conductance that functions as an "emergency release valve," allowing bacteria to survive acute hypoosmotic stress. Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed. Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype. Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines. Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo. The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.

Compartmental responses to acute osmotic stress in Leishmania major result in rapid loss of Na + and Cl −

The elemental composition of the cytoplasm, electron dense vacuoles, and heterochromatin and euchromatin regions of the nucleus of Leishmania major promastigotes was measured by electron probe X-ray microanalysis under iso-osmotic conditions (305 mOsM) and shortly after a sudden increase (to 615 mOsM) or decrease (to 153 mOsM) in the osmolality of the buffer in which they were suspended. In response to acute hypotonicity a complete loss of Na from the electron dense vacuoles and an approximately threefold decrease in the Na concentrations in the cytoplasm and the nuclear regions occurred, together with an approximately threefold decrease in Cl content in each compartment and a smaller (approx. 1.2-fold) decrease in K content. Thus, in addition to the rapid change in shape and release of amino acids known to occur in response to acute hypo-osmotic stress, a major efflux of Na and Cl, and, to a lesser extent, of K, also occurs. In response to acute hypertonicity Na in the acidocalcisomes did not change but Na content of the cytoplasm decreased by 33%. A small increase in the S content of the cytoplasm and the electron dense vacuolar compartments occurred. No changes were detectable in Ca or Zn content in any of the compartments examined in response to hypotonicity or hypertonicity.

Neuronal and glial handling of glutamate and glutamine during hypoosmotic stress: a biochemical and quantitative immunocytochemical analysis using the rat cerebellum as a model.

Biochemical and immunocytochemical analyses were performed to resolve how glutamate and glutamine are handled in rat cerebellar cortex in acute hypoosmotic stress. Rats were subjected to a 15-20% reduction in plasma osmolality by intraperitoneal injection of distilled water and then perfusion fixed after 4 or 8 h survival. Some rats in the latter group had their plasma isoosmolality restored by injections of hypertonic saline 4 h prior to perfusion. Water loading caused a pronounced increase in the tissue level of glutamine and an equimolar decrease in the level of glutamate after 4 h survival. The increase in glutamine was transient, as judged by analyses at 8 h survival. Light microscopic immunocytochemistry revealed a pronounced enhancement of the glutamine immunolabelling of glial cells (Golgi epithelial cells and astrocytes), including their perivascular end feet, and quantitative immunogold analyses at the electron microscopic level showed that this enhancement reflected a 50% increase in the intracellular concentration of fixed glutamine. Since water loading was associated with glial swelling this change corresponded to a several-fold increase in the glial content of glutamine. There was a modest reduction in the overall staining intensity for glutamate. The biochemical and immunocytochemical changes were reversed upon restoration of plasma osmolality by hypertonic saline. These findings suggest that hypoosmotic stress causes an increased conversion of glutamate to glutamine in glial cells and that the latter amino acid is subsequently lost from the tissue. The flux of glutamate carbon skeletons through the glutamine synthetase pathway in glia, prior to an efflux to the systemic circulation, may explain how glutamate, and excitatory transmitter and potential toxin, can be used as an organic osmolyte in brain tissue.

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg 1) and pO 2 (95, 10, and 0% O 2) in the presence of 5% CO 2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O 2 sensor which was active under all osmolalities tested. The data also require that as pO 2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO 2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhy-drazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO 2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

Oxidation of Leucine by Leishmania donovani

The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.

The effects of pentachlorophenol (PCP) and 2,4-dinitrophenol (DNP) on the oxygen consumption of tissues from the blue crab, callinectes sapidus, under different osmotic conditions

1. 1. The effects of acclimation to 300 and 940 milliosmole seawater and an acute hypo-osmotic stress on the weight-specific oxygen consumption of the gills, muscle and hepatopancreas of the blue crab. Callinectes sapidus, were determined. 2. 2. The gill and muscle tissues exhibited an increase in oxygen consumption on acclimation of crabs to a low salinity or when the tissues were subjected to an acute hypo-osmotic stress. Similar treatments did not alter the oxygen consumption of the hepatopancreas. 3. 3. At a concentration of 5 × 10 −3 M, pentachlorophenol (PCP) and 2,4-dinitrophenol (DNP) caused inhibition of oxygen consumption in the tissues examined. The extent of this inhibition of oxygen consumption was independent of the metabolic activity of the tissues.