Towards phenotyping adaptive traits in camels: A study of the influence of hypotonic saline solutions on blood cell area.

Single-humped camels are livestock of physical, physiological, and biochemical adaptations to hot desert environments and to water scarcity. The tolerance of camels to water deprivation and their exceptional capacity for rapid rehydration requires blood cells with membranes of specialized organization and chemical composition. The objectives of this study are to examine the changes in the area (a proxy for volume) of camel blood cells in solutions with decreasing concentrations of NaCl and consequently identify the conditions under which blood cells can be phenotyped in a large population. Whole-blood samples from three healthy adult female camels were treated with four different concentrations of NaCl and examined at six incubation-periods. Observationally, red blood cells in all treatments remained intact and maintained their elliptical shape while white blood cells experienced some damage, lysing at concentrations below 0.90%. Average basal (in 0.90% NaCl) RBC area was ~15 µm² and swelled in the various treatments, in some cases reaching twice its original size. Excluding the damaged cells, the average area of combined WBCs, ~32.7 µm², expanded approximately three times its original size. We find that camel WBCs, like their RBCs, are adapted to hypotonic environments, and are capable of expanding while maintaining their structural integrity.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines.

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4-/- knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4-/- cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Mechanosensitive cation currents through TRPC6 and Piezo1 channels in human pulmonary arterial endothelial cells.

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.

Effect of mechanical properties of Jurkat cell on adhesion properties of Jurkat integrin and VCAM-1: An AFM study.

Mechanical properties play key roles in the immune system, especially the activation, transformation and subsequent effector responses of immune cells. As transmembrane adhesion receptors, integrins mediate the adhesion events of both cells and cell-extracellular matrix (ECM). Integrin affinity would influence the crosslinking of cytoskeleton, leading to the change of elastic properties of cells. In this study, the cells were treated with F-actin destabilizing agent Cytochalasin-D (Cyt-D), fixed by Glutaraldehyde, and cultivated in hypotonic solution respectively. We used Atomic force microscopy (AFM) to quantitatively measure the elasticity of Jurkat cells and adhesion properties between integrins and vascular cell adhesion molecule-1 (VCAM-1), and immunofluorescence to study the alteration of cytoskeleton. Glutaraldehyde had a positive effect on the adhesion force and Young's modulus. However, these mechanical properties decreased in a hypotonic environment, confirming the findings of cellular physiological structure. There was no significant difference in the bond strength and elasticity of Jurkat cells treated with Cytochalasin-D, probably because of lower importance of actin in suspension cells. All the treatments in this study pose a negative effect on the adhesion probability between integrins and VCAM-1, which demonstrates the effect of structural alteration of the cytoskeleton on the conformation of integrin. Clear consistency between adhesion force of integrin/VCAM-1 bond and Young's modulus of Jurkat cells was shown. Our results further demonstrated the relationship between cytoskeleton and integrin-ligand by mechanical characteristics.

Impaired Regulatory Volume Decrease and Characterization of Underlying Volume-Activated Currents in Cystic Fibrosis Human Cholangiocyte Cell Line.

The volume-activated chloride channel (VACC) serves vital cellular functions in secretion and cell volume regulation via regulatory volume decrease (RVD) in various epithelia. Previously, we have shown that RVD in primary CF mouse cholangiocytes is impaired. Thus, the effect of CFTR defect on VACC and RVD in CF human immortalized cholangiocyte cell (HBDC) was examined in comparison with those in normal HBDC by using cell volume measurement and whole-cell patch clamp techniques, respectively. The CF HBDC had an impaired RVD, which was not further inhibited by removing the extracellular calcium or administering BAPTA-AM, NPPB, or DIDS. When exposed to a hypotonic solution, CF HBDC exhibited large, outwardly rectified currents with time-dependent inactivation at a positive potential. The amplitude of the outward currents was about three times that of the inward currents. The amplitude and reversal potential of VACC was dependent on chloride concentration. The VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, or acetate in the hypotonic solution as well as by an administration of NPPB or tamoxifen, classical VACC inhibitors. Surprisingly, the VACC amplitude is greater in CF HBDC than in normal HBDC, suggesting that the channel density or open probability of VACC is increased, thus CFTR may have inhibitory effects on VACC. On the contrary, the amplitude of the volume-activated potassium current is lower in CF HBDC, suggesting the potassium channel density or open probability is decreased in CF cholangiocytes and/or CFTR may have regulatory effects on volume-activated potassium current. In conclusion, RVD is impaired in CF human cholangiocytes. The VACC of CF human cholangiocytes has similar electrophysiological characteristics as that of normal cholangiocytes but its activity is augmented in CF cholangiocytes, while volume-activated potassium current is decreased in CF human cholangiocytes, providing a fundamental underlying pathophysiologic mechanism for the impaired RVD in CF cholangiocytes.

Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Real-time confocal microscopy imaging of corneal cytoarchitectural changes induced by different stresses.

Maintenance of the corneal refractive power and tissue transparency is essential for normal vision. Real-time characterization of changes in corneal cells during suffering stresses or wound healing may provide a way to identify novel targets, whose therapeutic manipulation can improve the outcome of this response induced by injury. Here we describe a novel user friendly and effective confocal real-time confocal microscopy attachment that monitors the effects of anisoosmotic stress on cell morphology and corneal thickness in situ. Corneal epithelial nuclei gradually became highly reflective in the isotonic group and the corneal stroma was slightly thickened as compared with that seen prior to 60 min exposure to a hypotonic solution. After 30 min of exposure to hypertonic stress, the corneal stromal cells became crenate and shriveled. The hyper-reflective area of the corneal stroma in the hypo-osmotic group was significantly larger than that in the other two groups, as demonstrated by 3D reconstruction imaging. The hypotonic fresh chlorinated pool water was observed to cause atrophy of corneal epithelial nuclei, while the isosmotic bee venom solution caused high reflection of the corneal stroma layer and corneal endothelial cell damage. With the microscopic attachment, the inward movement of corneal epithelial cells toward the denuded central region was detected in the serum-treated group. The microscopy attachment is an effective system for obtaining a more detailed understanding of the time dependent losses in the corneal cell structure and tissue architecture of full thickness corneas induced by osmotic stress or cytotoxic agents.

Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes.

PURPOSE: We studied the quality differences between the different hypo-osmotic swelling test (HOST) classes, as measured by criteria of DNA fragmentation, DNA decondensation, and nuclear architecture. The aim was to find particular HOST classes associated with good-quality metrics, which may be potentially used in ICSI (intra-cytoplasmic sperm injection). METHODS: Ten patients from the Department of Reproductive Medicine at Tenon Hospital (Paris, France) were included. Their semen samples were collected and divided into two fractions: one was incubated in a hypo-osmotic solution as per HOST protocol and sorted by sperm morphology, and a second was incubated without undergoing the HOST protocol to serve as an unsorted baseline. Three parameters were assessed: DNA fragmentation (TUNEL assay), DNA decondensation (chromomycin A3 assay), and nuclear architecture (FISH, with telomeric and whole chromosome painting probes). The different HOST classes were evaluated for these three parameters, and statistical analysis was performed for each class versus the unsorted non-HOST-treated sperm. Results with p<0.05 were considered statistically significant. RESULTS: For each of the parameters evaluated, we found significant differences between HOST-selected spermatozoa and non-selected spermatozoa. Overall, spermatozoa of HOST classes B and B+ exhibited the highest quality based on four metrics (low DNA fragmentation, low DNA decondensation, short inter-telomeric distance, and small chromosome 1 territory area), while spermatozoa of HOST classes A and G exhibited the poorest quality by these metrics. CONCLUSION: In addition to their pathophysiological interest, our results open possibilities of sperm selection prior to ICSI, which may allow for optimization of reproductive outcomes in heretofore unstudied patient populations.

SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway.

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

The Cation Channel TMEM63B Is an Osmosensor Required for Hearing.

Hypotonic stress causes the activation of swelling-activated nonselective cation channels (NSCCs), which leads to Ca2+-dependent regulatory volume decrease (RVD) and adaptive maintenance of the cell volume; however, the molecular identities of the osmosensitive NSCCs remain unclear. Here, we identified TMEM63B as an osmosensitive NSCC activated by hypotonic stress. TMEM63B is enriched in the inner ear sensory hair cells. Genetic deletion of TMEM63B results in necroptosis of outer hair cells (OHCs) and progressive hearing loss. Mechanistically, the TMEM63B channel mediates hypo-osmolarity-induced Ca2+ influx, which activates Ca2+-dependent K+ channels required for the maintenance of OHC morphology. These findings demonstrate that TMEM63B is an osmosensor of the mammalian inner ear and the long-sought cation channel mediating Ca2+-dependent RVD.

Hypotonic, Acidic Oxidizing Solution Containing Hypochlorous Acid (HClO) as a Potential Treatment of Hailey-Hailey Disease.

Hailey-Hailey disease (HHD) is a rare, chronic and recurrent blistering disorder, characterized by erosions occurring primarily in intertriginous regions and histologically by suprabasal acantholysis. Mutation of the Golgi Ca2+-ATPase ATP2C1 has been identified as having a causative role in Hailey-Hailey disease. HHD-derived keratinocytes have increased oxidative-stress that is associated with impaired proliferation and differentiation. Additionally, HHD is characterized by skin lesions that do not heal and by recurrent skin infections, indicating that HHD keratinocytes might not respond well to challenges such as wounding or infection. Hypochlorous acid has been demonstrated in vitro and in vivo to possess properties that rescue both oxidative stress and altered wound repair process. Thus, we investigated the potential effects of a stabilized form of hypochlorous acid (APR-TD012) in an in vitro model of HHD. We found that treatment of ATP2C1-defective keratinocytes with APR-TD012 contributed to upregulation of Nrf2 (nuclear factor (erythroid-derived 2)-like 2). Additionally, APR TD012-treatment restored the defective proliferative capability of siATP2C1-treated keratinocytes. We also found that the APR-TD012 treatment might support wound healing process, due to its ability to modulate the expression of wound healing associated cytokines. These observations suggested that the APR-TD012 might be a potential therapeutic agent for HHD-lesions.

Effect of low temperature on the regulation of cell volume after hypotonic shock in gastric cancer cells.

Although peritoneal lavage with distilled water performed after surgery prevents peritoneal seeding, cancer cells may avoid rupture under mild hypotonicity through regulatory volume decrease (RVD), which is the homeostatic regulation of ion and water transport. The aim of the present study was to investigate the effect of low temperature on cell volume and cell death under hypoosmolal conditions and determine the underlying molecular mechanisms in gastric cancer (GC). Three human GC cell lines (NUGC4, KATO­III and MKN45) were exposed to hypotonic solutions, and the effects of low temperature on cell volume and viability were examined. Low temperature­induced changes in membrane transporters were evaluated, and knockdown and overexpression experiments were conducted to determine their effects on cell volume during hypotonic stimulation. Low temperature (24˚C) during hypotonic stimulation inhibited RVD and enhanced the cytocidal effects on GC cells. The expression of leucine­rich repeat containing protein A (LRRC8A), a component of a Cl­ channel, was decreased, and aquaporin 5 (AQP5) expression was increased at low temperatures. LRRC8A knockdown markedly slowed the decrease in cell volume following cell swelling by hypotonic shock. AQP5 overexpression enhanced initial cell swelling after hypotonic shock and increased the final cell volume. These results suggest that a hypotonic solution at low temperature increased initial water influx via activation of AQP5 and decreased Cl­ efflux via inhibition of LRRC8A. Therefore, low temperature enhanced the hypotonicity­induced cytocidal effects on GC cells, and these results may contribute to the development of a novel lavage method effective in reducing peritoneal recurrence in GC.

Hypoosmotic stress induces R loop formation in nucleoli and ATR/ATM-dependent silencing of nucleolar transcription.

The contribution of nucleoli to the cellular stress response has been discussed for over a decade. Stress-induced inhibition of RNA polymerase I-dependent transcription is hypothesized as a possible effector program in such a response. In this study, we report a new mechanism by which ribosomal DNA transcription can be inhibited in response to cellular stress. Specifically, we demonstrate that mild hypoosmotic stress induces stabilization of R loops in ribosomal genes and thus provokes the nucleoli-specific DNA damage response, which is governed by the ATM- and Rad3-related (ATR) kinase. Activation of ATR in nucleoli strongly depends on Treacle, which is needed for efficient recruitment/retention of TopBP1 in nucleoli. Subsequent ATR-mediated activation of ATM results in repression of nucleolar transcription.

Application of continuous-wave photoacoustic sensing to red blood cell morphology.

The feasibility of continuous wave laser-based photoacoustic (CWPA) response technique in detecting the morphological changes in cells during the biological studies, through the features extracted from CWPA signal (i.e., amplitude) is demonstrated here. Various hematological disorders (e.g., sickle cell anemia, thalesemia) produce distinct changes at the cellular level morphologically. In order to explore the photoacoustic response technique to detect these morphological changes, we have applied CWPA technique onto the blood samples. Results of our preliminary study show a distinct change in the signal amplitude of photoacoustic (PA) signal due to a change in the concentration of blood, which signifies the sensitivity of the technique towards red blood cell (RBC) count (related to hematological disease like anemia). Further hypotonic and hypertonic solutions were induced in blood to produce morphological changes in RBCs (i.e., swollen and shrink, respectively) as compared to the normal RBCs. Experiments were performed using continuous wave laser-based photoacoustic response technique to verify the morphological changes in these RBCs. A distinct change in the PA signal amplitude was found for the distinct nature of RBCs (swollen, shrink, and normal). Thus, this can serve as a diagnostic signature for different biological studies based on morphological changes at cellular level. The experiments were also performed using conventional pulsed laser photoacoustic response technique which uses nano-second pulsed laser and the results obtained from both PA techniques were validated to produce identical changes. This demonstrates the utility of continuous wave laser-based photoacoustic technique for different biological studies related to morphological cellular disorders.

Action of Protein Tyrosine Kinase Inhibitors on the Hypotonicity-Stimulated Trafficking Kinetics of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells: Analysis Using a Mathematical Model.

BACKGROUND/AIMS: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. METHODS: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. RESULTS: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. CONCLUSION: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling.

BACKGROUND/AIMS: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. METHODS: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. RESULTS: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. CONCLUSION: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission.

The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs) in the organum vasculosum laminae terminalis (OVLT). Hypertonicity excites ONs in part through the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1). However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC) that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ.

Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force.

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Differential effects of isotonic and hypotonic 4% albumin solution on intracranial pressure and renal perfusion and function.

OBJECTIVES: Albumin is used to resuscitate trauma patients but may increase intracranial pressure (ICP). Its effects on renal blood flow and function are unknown. Our aim was to examine the effects of hypertonic albumin on ICP and renal function, and if any effects are due to the hypotonicity of the solution containing albumin or to albumin itself. DESIGN, SETTING AND SUBJECTS: Cross-over, randomised controlled experimental study of six adult Merino ewes in the animal facility of a research institute. METHOD: Sheep were implanted with flow probes around the pulmonary and renal arteries and an ICP monitoring catheter in a lateral cerebral ventricle. Conscious sheep received normal saline, commercially available hypotonic 4% albumin solution (4% Albumex [278 mOsm/kg]) or a novel isotonic 4% albumin solution (288 mOsm/kg), with at least 48 hours between each intervention. RESULTS: Commercial hypotonic albumin solution increased ICP (by 8.5 mmHg [SEM, 2.1 mmHg]; P < 0.01), but neither isotonic albumin solution nor saline significantly changed ICP. The increase in ICP with hypotonic albumin solution was associated with an increase in central venous pressure (CVP) (by 5.4 mmHg [SEM, 0.6 mmHg]; P < 0.001), but no significant changes in cardiac output or stroke volume. None of the infusions changed renal blood flow, plasma creatinine level, creatinine clearance or plasma or urinary electrolyte levels. CONCLUSION: Compared with saline or isotonic albumin solution, hypotonic albumin solution increased ICP and CVP, but did not alter arterial pressure, cardiac output renal blood flow or renal function. Our findings support the view that the tonicity of the albumin solution, rather than the albumin itself, is responsible for increasing ICP.

The effect of substrate stiffness on cancer cell volume homeostasis.

Existing studies on the mechanism of cell volume regulation are mainly relevant to ion channels and osmosis in extracellular fluid. Recently, accumulating evidence has shown that cellular mechanical microenvironment also influences the cell volume. Herein, we investigated the regulation of substrate stiffness on the cell volume homeostasis of MCF-7 cells and their following migration behaviors. We found that cell volume increases with increasing substrate stiffness, which could be affected by blocking the cell membrane anion permeability and dopamine receptor. In addition, the cell migration is significantly inhibited by decreasing the cell volume using tamoxifen and such inhibition effect on migration is enhanced by increasing substrate stiffness. The cell membrane anion permeability might be the linker between cellular mechanical microenvironment and cellular volume homeostasis regulation. This work revealed the regulation of substrate stiffness on cell volume homeostasis for the first time, which would provide a new perspective into the understanding of cancer metastasis and a promising anti-cancer therapy through regulation of cell volume homeostasis.