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Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypotonic swelling should be reduced chloride concentration, we tested if chloride depletion can act as a signal for vacuolation. Indeed, four different low-chloride buffers resulted in the development of similar vacuoles. Moreover, vacuolation was suppressed in WNK1/WNK3 double knockouts or by the inhibition of WNK kinase, which is activated by low chloride; in hypotonic media, the WNK inhibitor had a similar effect. However, exposing cells to a low-sodium, high-potassium medium also resulted in vacuoles, which were insensitive to WNK. We conclude that vacuole development can be triggered either by the loss of chloride or by the loss of sodium. [ABSTRACT FROM AUTHOR]

Abstract Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypotonic swelling should be reduced chloride concentration, we tested if chloride depletion can act as a signal for vacuolation. Indeed, four different low-chloride buffers resulted in the development of similar vacuoles. Moreover, vacuolation was suppressed in WNK1/WNK3 double knockouts or by the inhibition of WNK kinase, which is activated by low chloride; in hypotonic media, the WNK inhibitor had a similar effect. However, exposing cells to a low-sodium, high-potassium medium also resulted in vacuoles, which were insensitive to WNK. We conclude that vacuole development can be triggered either by the loss of chloride or by the loss of sodium.

The first and foremost step in in vitro drug designing was the screening of phytochemical constituents of the traditional plants. Literature survey on traditional plant indicated that, Prosopis chilensis was a frequently used plant for its high medicinal properties. The plant can be further explored for the study of traditionally claimed unexplored activities, as well as isolation and identification of active constituents may lead to new findings. In the present study, the flowers of Prosopis chilensis were collected, dried and extracted with various solvents according to solubility index, and finally five extracts were studied. The isolated compounds were identified by chromatography, UV and NMR studies. In the present study, phytochemical investigation and hypotonicity induced membrane stabilization studies of Prosopis chilensis was carried out. [ABSTRACT FROM AUTHOR]

The current research is to study anti-inflammatory and mechanisms of anti-inflammatory activities of thymoquinone (t), which is used in 50 mg/kg to investigate the anti-inflammatory and its activities. Fresh egg albumin model, leukocyte migration and heat induced hemolysis of human red blood cells (RBCs) were used to study the anti-inflammatory activities. Also, Hypotonicity-induced hemolysis was applied to evaluated the mechanisms of anti-inflammatory activities compared to dexamethasone and Piroxicam. As a result, t has made obvious reduction (P>0.05) of inflammation and suppressed paw edema (PE) in rats induced by egg albumin. t was efficient for heat inhibition, in total leukocyte migration inhibition and in hypotonicity induced hemolysis of human red blood cells (RBCs) showing better anti-inflammatory profile that might be phytochemical constituents especially flavonoid. [ABSTRACT FROM AUTHOR]

Introduction: Pompe disease (PD), glycogen storage disease Type II (GSD II), is an autosomal recessive inherited lysosomal storage disease caused by pathogenic variants in the GAA gene that encodes lysosomal acid α-glucosidadase (GAA) enzyme. The incidence of the disease varies from country to country. PD is mainly presents as two groups of phenotypes as infantile-onset Pompe disease (IOPD) and late-onset Pompe disease. Objective: The aim of this study is to discuss the molecular and clinical characteristics of infantile-onset Pompe disease (IOPD) and late-onset pompe disease (LOPD) followed-up in our center. Method: A total of 10 patients diagnosed with IOPD and 4 patients diagnosed with LOPD in Izmir Dr. Behcet Uz Pediatric Health and Diseases and Surgery Training and Research Hospital Pediatric Metabolism Unit between 06.01.2015 and 06.01. 2019 were included in the study. The patients' demographic characteristics, clinical findings at the time of diagnosis and during the folllow-up period, biochemical findings, muscle biopsy data, results of enzymatic analyses and moleculargenetic characteristics were recorded retrospectively. Results: A total of 10 patients were included in the study. 7 patients were diagnosed with IOPD and 3 patients with LOPD. The median follow-up period of all patients was 26 months (range: 6-42 months). The c.896 C> T (8/32, 25%) is detected as the most common variant. 1237G>T (p.Asp413Tyr), c.2019 C>A (p.Asn673Lys), c.418A>T (p.Asn140Tyr) variants were detected for the first time. Conclusion: Pompe disease is one of the most important congenital metabolic diseases in which early diagnosis and treatment are of great importance. Despite the significant improvement in disease prognosis with the introduction of enzyme replacement therapy, there are still patients with poor prognosis despite early diagnosis. Phenotype-genotype studies are crucial in this respect. [ABSTRACT FROM AUTHOR]

We report a morphological manipulation of cell division which was achieved by changing the environment from isotonic to highly hypotonic. Cells at telophase were observed to undergo a morphological reversal to anaphase, with the contractile ring being reopened and the cell shape reversing from dumb-bell back to spherical. Once restored to isosmotic environment, the reversed cells would either continue to divide or instead to form binuclear cells that further proliferated in runaway fashions. The immunofluorescent staining of tubulins and myosin II indicated that the hypotonic stress affected the accumulation of tubulins and myosin II at the contractile ring. Distinct from previous studies using specific chemical reagents, the present study provides a simple method to manipulate cell division. The morphological reversal is the adaption of dividing cells to the environmental change. The observation opens a new window to understand cell division mechanisms and runaways.

The closure of gaps within epithelia is an essential part of many physiological and pathological processes, such as embryonic development, organ remodeling and wound healing. Emerging evidence proved that the physical microenvironment plays important roles in cell behaviors. However, the effect of osmolarity of extracellular medium on gap closure is least understood. Using a gap closure model of epithelial cells, we found that hypotonic condition significantly facilitated the process of gap closure. Moreover, instead of actomyosin ring, enhanced migration leading by lamellipodia primarily contributed to the rapid gap closure in hypotonic condition. These findings provide insights for understanding the physiology of epithelial gap closure. [ABSTRACT FROM AUTHOR]

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation.

This review contends that “healthy” water in terms of electrolyte balance is as important as “pure” water in promoting public health. It considers the growing use of desalination (demineralization) technologies in drinking water treatment which often results in tap water with very low concentrations of sodium, potassium, magnesium and calcium. Ingestion of such water can lead to electrolyte abnormalities marked by hyponatremia, hypokalemia, hypomagnesemia and hypocalcemia which are among the most common and recognizable features in cancer patients. The causal relationships between exposure to demineralized water and malignancies are poorly understood. This review highlights some of the epidemiological and in vivo evidence that link dysregulated electrolyte metabolism with carcinogenesis and the development of cancer hallmarks. It discusses how ingestion of demineralized water can have a procarcinogenic effect through mediating some of the critical pathways and processes in the cancer microenvironment such as angiogenesis, genomic instability, resistance to programmed cell death, sustained proliferative signaling, cell immortalization and tumorigenic inflammation. Evidence that hypoosmotic stress-response processes can upregulate a number of potential oncogenes is well supported by a number studies. In view of the rising production and consumption of demineralized water in most parts of the world, there is a strong need for further research on the biological importance and protean roles of electrolyte abnormalities in promoting, antagonizing or otherwise enabling the development of cancer. The countries of the Gulf Cooperative Council (GCC) where most people consume desalinated water would be a logical place to start this research. [ABSTRACT FROM AUTHOR]

Background We evaluated the neurodevelopmental (ND) outcome at one year of age for congenital diaphragmatic hernia (CDH) children who have not undergone extracorporeal membrane oxygenation (ECMO) treatment during the neonatal period. Material and methods Between 01/2005 and 06/2012, 63 consecutive CDH patients underwent ND assessment using the BSID-III at a median age of 12 months. ND delay was defined by a score of ≤ 85 in any of the composite scales. Severe impairment was defined as a score of ≤ 69 in at least one domain. Results Mean ± SD cognitive, language, and motor functions were 94 ± 14, 86 ± 14, 90 ± 15, respectively (normal 100 ± 15, P < 0.01 for each). Forty-three-percent scored within the average range for all scales. Forty-four-percent had mild, and 13% had severe delays in at least one domain. Prolonged NICU stay, intubation and O 2 requirement, fundoplication, abnormal BAERs, and tracheostomy were associated with lower scores in all domains. Right-sided CDH, male gender, lower 5 min APGAR, pulmonary hypertension, and delayed start of enteral feeding were predictive of lower cognitive and/or language scores. Conclusion At one year of age, a high percentage of CDH children whose illness did not necessitate ECMO have below normal ND scores. Modifiable and non-modifiable factors are significant determinants of adverse outcomes. [ABSTRACT FROM AUTHOR]

Hyponatremia has complex pathophysiology, is frequent and has potentially severe clinical manifestations, and its treatment is associated with high risks. Hyponatremia can be hypertonic, isotonic or hypotonic. Hypotonic hyponatremia has multiple etiologies, but only two general mechanisms of development, defective water excretion, usually because of elevated serum vasopressin levels, or excessive fluid intake. The acute treatment of symptomatic hypotonic hyponatremia requires understanding of its targets and risks and requires continuous monitoring of the patient's clinical status and relevant serum biochemical values. The principles of fluid restriction, which is the mainstay of management of all types of hypotonic hyponatremia, should be clearly understood and followed. Treatment methods specific to various categories of hyponatremia are available. The indications and risks of these treatments should also be well understood. Rapid correction of chronic hypotonic hyponatremia may lead to osmotic demyelination syndrome, which has severe clinical manifestations, and may lead to permanent neurological disability or death. Prevention of this syndrome should be a prime concern of the treatment of hypotonic hyponatremia. [ABSTRACT FROM AUTHOR]

The aim of the study was to explore the effect of distilled water on killing tumour cells attached to the surgery instruments during operation. Tumour cells were collected from the suspected tumour cell-contaminated surgery instruments and then cultured. Then the tumour cells were treated by distilled water at different gradient temperature for different time periods. The morphology of the tumour cells was observed by inverted microscope after hematoxylin-eosin staining. The results showed that positive tumour cell culture rate was 34.3%. After soaked in distilled water for 60 s at 55°C, the tumour cells were inactive, and the death rate was 100%. We also found that no active cells were seen to grow adherently after recultured. In conclusion, tumour cells can be killed by distilled water for 60 s at 55°C, which provides a new fast and low-cost tumour-free technique to inactivate tumour cells attached to surgery instruments. [ABSTRACT FROM AUTHOR]

Aim and methods Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 ( TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. Results Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (f EPSP) was enhanced by hypotonicity and TRPV4 agonist 4α- PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in f EPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( AMPA) receptor antagonist, but not N-methyl- d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current ( ICa) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current ( IAMPA) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α- PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA, whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. Conclusion TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function. [ABSTRACT FROM AUTHOR]

Kidneys are complex highly organized paired organs of nearly one million nephrons each. They rigorously process about 180 l of plasma daily to keep whole body homeostasis. To effectively perform such a titanic work, kidneys rely on mechanisms able to sense dynamic changes in composition and flow rates of protourine along the renal tubule. It is envisioned that Ca-permeable transient receptor potential (TRP) channels, and specifically mechanosensitive TRPV4, can serve to interpret these external mechanical cues in the form of elevated intracellular Ca concentration. This, in turn, initiates multiple cellular responses and adaptation mechanisms. The current review summarizes up-to-date knowledge about the sites of TRPV4 expression in renal tissue as well as discusses the functional role of the channel in cellular responses to hypotonicity and tubular flow. We will also provide insights as to how TRPV4 fits into classical polycystin mechanosensory complex in cilia and will speculate about previously underappreciated clinical implication of pharmacological TRPV4 targeting in treatment of polycystic kidney disease. [ABSTRACT FROM AUTHOR]

NAD(P)H oxidase (NOX)-derived HO was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous HO (100 to 200 μM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous HO. In patch clamp experiments, exogenous HO was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by HO. Taken together, these data suggest that NOX-derived HO plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity. [ABSTRACT FROM AUTHOR]

Hypotonicity following water intoxication and/or salt loss leads to mainly astrocytic brain swelling. Astrocytic swelling also occurs following brain trauma or ischemia, together with an increase in extracellular K ([K]), stimulating a bumetanide/furosemide/ethacrynic acid-inhibitable cotransporter, NKCC1, that accumulates Na and K together with 2 Cl and osmotically obliged water. Either type of swelling may become fatal and is associated with phosphorylation of extracellular regulated kinases 1 and 2 (ERK). Only the swelling associated with elevated [K], leads to an increase in astrocytic proliferation and in expression of the astrocytic marker, glial fibrillary acidic protein. These differences prompted us to investigate key aspects of the molecular pathways between hypotonicity-induced and high-K-mediated swelling in primary cultures of mouse astrocytes. In the latter Ca-mediated, AG1478-inhibitable transactivation of the epidermal growth factor (EGF) receptor leads, via bumetanide-inhibitable activation of the mitogen activated protein (MAP) kinase pathway to ERK phosphorylation and to NKCC1-mediated swelling. In the former, inhibition of the MAP kinase pathway, but not of EGF receptor activation, abolishes ERK phosphorylation, but has no effect on swelling, indicating that activation of ERK is a result, not a cause, of the swelling. [ABSTRACT FROM AUTHOR]

It is well established that hypotonicity generates a marked and unexpected increase in active Na+ efflux in frog muscle fibers as well as in other cells like cardiac myocytes, astrocytes, brain synaptosomes and renal cells. The effect of hypotonicity on the electrical activity of skeletal muscle related to Na+ and K+ voltage-gated channels, however, has not been specifically addressed. The results of the present investigation show that the changes in resting and action potentials produced by hypotonicity can be fully explained by the reduction of intracellular [Na+] and [K+] due to the increase in cellular water content. [ABSTRACT FROM AUTHOR]

Abstract: We examined the mechanisms involved in the [Ca2+] i response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195mosmol/kgH2O (hypotonic) evoked transient increase in [Ca2+] i of principal cells of rat CCDs. The [Ca2+] i increase was markedly attenuated by the removal of extracellular Ca2+ . purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+] i increase. The [Ca2+ increase in response to extracellular hypotonicity was not influenced by application of Gd i increase in response to extracellular hypotonicity was not influenced by application of Gd3+ and ruthenium red. On the other hand, a voltage-gated Ca2+ channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca2+] i increase in the principal cells. In order to assess Ca2+ entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn2+. The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+ ] i elevation of principal cells in rat CCD. [Copyright &y& Elsevier]

Hypotonic fluids are commonly used for treating hospitalized children. However, an excess of arginine vasopressin (AVP) with impaired free water excretion is thought to contribute to the development of hyponatremia in febrile children. The aim of this two-part study was to define the clinical relationship between hyponatremia and excess AVP. In a retrospective study carried out between 2001 and 2005, we found that approximately 17% of the hospitalized patients had hyponatremia [serum sodium (Na) < 135 mEq/l] upon admission and that the ratio of patients with hyponatremia was significantly higher among febrile patients than among afebrile patients. In a subsequent prospective study, we examined 73 hospitalized patients who presented with acute febrile diseases accompanied by hyponatremia (serum Na <134 mEq/l). Almost all of these patients demonstrated excess AVP, defined as high plasma AVP levels (>1 pg/ml). There were no significant relationships between the levels of AVP and other laboratory variables, including serum sodium, serum osmolality, atrial natriuretic peptide, and brain natriuretic peptide. About 30% (22/73) of the patients fulfilled the criteria of the syndrome of inappropriate secretion of antidiuretic hormone. These findings suggest that fever and other nonosmotic stimuli lead directly to excess AVP and hyponatremia. We therefore recommend that isotonic fluids should be used for patients with prolonged fever and hyponatremia. [ABSTRACT FROM AUTHOR]

Abstract: Voltage-gated potassium channels (VGPCs) play an important role in many physiological functions by controlling the electrical properties and excitability of cells. Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Here, using whole cell patch clamp techniques, we explore how hypo- and hypertonicity modulate VGPCs in cultured rat and mouse trigeminal ganglion (TG) neurons. We found that hypo- and hypertonicity had different effects on slow-inactivating K+ current (I K) and fast-inactivating K+ current (I A): hypotonicity increased I K but had no effect on I A while hypertonicity depressed both I K and I A. The increase of I K by hypotonicity was mimicked by transient receptor potential vanilloid 4 (TRPV4) receptor activator 4α-phorbol-12,13-didecanoate (4α-PDD) but hypotonicity did not exhibit increase in TRPV4−/− mice TG neurons, suggesting that TRPV4 receptor was involved in hypotonicity-induced response. We also found that inactivation of PKC selectively reversed the increase of I K by hypotonicity, whereas antagonism of G-protein selectively rescued the inhibitions of I K and I A by hypertonicity, indicating that different intracellular signaling pathways were required for the modulation by hypo- and hypertonicity. In summary, changes in osmolality have various effects on I K and I A and different receptors and second messenger systems are selective for the modulation of VGPCs induced by hypo- versus hypertonicity. [Copyright &y& Elsevier]

Abstract: Acid-sensing ion channels (ASICs) are strong candidates for mammalian mechanoreceptors. We investigated whether mouse acid-sensing ion channel-1b (ASIC1b) is sensitive to mechanical stimuli using oocyte electrophysiology, because ASIC1b is located in the mechanosensory stereocilia of cochlear hair cells. Hypotonic stimuli that induced membrane stretch of oocytes evoked no significant current in ASIC1b-expressing oocytes at pH 7.5. However, acid (pH 4.0 or 5.0)-evoked currents in the oocytes were substantially enhanced by the hypotonicity, showing mechanosensitivity of ASIC1b and possible mechanogating of the channel in the presence of other components. Interestingly, the ASIC1b channel was permeable to K+ (a principal charge carrier for cochlear sensory transduction) and the affinity of the channel for amiloride (IC50 (inhibition constant)=approximately 48.3μM) was quite similar to that described for the mouse hair cell mechanotransducer current. Taken together, these data raise the possibility that ASIC1b participates in cochlear mechanoelectrical transduction. [Copyright &y& Elsevier]

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-l cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4α-PDD and hypotonic medium, induced strong calcium influx in M-l cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD. [ABSTRACT FROM AUTHOR]

The roles of various inorganic ions and taurine, an organic osmolyte, in cell volume regulation were investigated in the perfused liver of a freshwater air-breathing catfish Clarias batrachus under aniso-osmotic conditions. There was a transient increase and decrease of liver cell volume following hypotonic (-80 mOsmol/l) and hypertonic (+80 mOsmol/l) exposures, respectively, which gradually decreased/increased near to the control level due to release/ uptake of water within a period of 25-30 min. Liver volume decrease was accompanied by enhanced efflux of K+ (9.45 ± 0.54 µmol/g liver) due to activation of Ba2+- and quinidine-sensitive K+ channel, and to a lesser extent due to enhanced efflux of Cl- (4.35 ± 0.25 µmol/g liver) and Na+ (3.68 ± 0.37 µmol/g liver). Conversely, upon hypertonic exposure, there was amiloride- and ouabain-sensitive uptake of K+ (9.78 ± 0.65 µmol/g liver), and also Cl- (3.72 ± 0.25 µmol/g liver). The alkalization/acidification of the liver effluents under hypo-/hypertonicity was mainly due to movement of various ions during volume regulatory processes. Taurine, an important organic osmolyte, appears also to play a very important role in hepatocyte cell volume regulation in the walking catfish as evidenced by the fact that hypo- and hyper-osmolarity caused transient efflux (5.68 ± 0.38 µmol/g liver) and uptake (6.38 ± 0.45 µmol/g liver) of taurine, respectively. The taurine efflux was sensitive to 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS, an anion channel blocker), but the uptake was insensitive to DIDS, thus indicating that the release and uptake of taurine during volume regulatory processes are unidirectional. Although the liver of walking catfish possesses the RVD and RVI mechanisms, it is to be noted that liver cells remain partly swollen and shrunken during anisotonic exposures, thereby possibly causing various volume-sensitive metabolic changes in the liver as reported earlier. [ABSTRACT FROM AUTHOR]

The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 m M decrease in extracellular NaCI caused a twofold increase in insulin release. The release of insulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellular hypotonicity displayed a threshold value close to 20 mOsmol/L and amaximal response at about 70 mOsmol/L. Hypotonicity also caused a rapid increase in cell volume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca concentration. 5-Nitro-2-(3-phenylpropylamino) benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca, thapsigargin, furosemide, tributyltin, and diazoxide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K concentration, while augmenting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca channels. [ABSTRACT FROM AUTHOR]

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane. [ABSTRACT FROM AUTHOR]

TRPV4, a renally expressed nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Kinases of the WNK family influence expression and function of the thiazide-sensitive Na+-Cl- cotransporter, and monogenic human hypertension has been linked to mutations in the gene coding for WNK4. Along with TRPV4, WNK isoforms are highly expressed in the distal nephron. We show here that coexpression of WNK4 downregulates TRPV4 function in human embryonic kidney (HEK-293) cells and that this effect is mediated via decreased cell surface expression of TRPV4; total abundance of TRPV4 in whole cell lysates is unaffected. The effect of the related kinase WNKI on TRPV4 function and surface expression was similar to that of WNK4. Disease-causing point mutations in WNK4 abrogate, but do not eliminate, the inhibitory effect on TRPV4 function. In contrast to wild-type WNK4, a kinase-dead WNK4 point mutant failed to influence TRPV4 trafficking; however, deletion of the entire WNK4 kinase domain did not blunt the effect of WNK4 on localization of TRPV4. Deletion of the extreme COOH-terminal putative coiled-coil domain of WNK4 abolished its effect. In immunoprecipitation experiments, we were unable to detect direct interaction between TRPV4 and either WNK kinase. In aggregate, these data indicate that TRPV4 is functionally regulated by WNK family kinases at the level of cell surface expression. Because TRPV4 and WNK kinases are coexpressed in the distal nephron in vivo and because there is a tendency toward hypercalcemia in TRPV4-/- mice, we speculate that this pathway may impact systemic Ca2+ balance. In addition, because WNK kinases and TRPV4 are activated by anisotonicity, they may comprise elements of an osmosensing or osmotically responsive signal transduction cascade in the distal nephron. [ABSTRACT FROM AUTHOR]

We identified a consensus N-linked glycosylation motif within the pore-forming loop between the fifth and sixth transmembrane segments of the osmoresponsive transient receptor potential (TRP) channel TRPV4. Mutation of this residue from Asn to Gin (i.e., TRPV4N651Q) resulted in loss of a slower migrating band on anti-TRPV4 immunoblots and a marked reduction in lectin-precipitable TRPV4 immunoreactivity. HEK293 cells transiently transfected with the mutant TRPV4N651Q exhibited increased calcium entry in response to hypotonic stress relative to wild-type TRPV4 transfectants. This increase in hypotonicity responsiveness was associated with an increase in plasma membrane targeting of TRPV4N651Q relative to wild-type TRPV4 in both HEK293 and COS-7 cells but had no effect on overall channel abundance in whole cell lysates. Residue N651 of TRPV4 is immediately adjacent to the pore-forming loop. Although glycosylation in this vicinity has not been reported for a TRP channel, the structurally related hexahelical hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, and the voltage-gated potassium channel, human ether-a-go-go-related (HERG), share a nearly identically situated and experimentally confirmed N-linked glycosylation site which promotes rather than limits channel insertion into the plasma membrane. These data point to a potentially conserved structural and functional feature influencing membrane trafficking across diverse members of the voltage-gated-like ion channel superfamily. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroper-oxyeicosatetrenoic acid (5-HPETE), induced Ca2+ entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca2+ entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca2+-per-meant nonselective cation channels (NSCC5) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

SRC family kinases are a group of nine cytoplasmic protein tyrosine kinases essential for many cell functions. Some appear to be ubiquitously expressed, whereas others are highly tissue specific. The ability of members of the SRC family to influence ion transport has been recognized for several years. Mounting evidence suggests a broad role for SRC family kinases in the cell response to both hypertonic and hypotonic stress, and in the ensuing regulatory volume increase or decrease. In addition, members of this tyrosine kinase family participate in the mechanotransduction that accompanies cell membrane deformation. Finally, at least one SRC family member operates in concert with the p38 MAPK to regulate tonicity-dependent gene transcription. [ABSTRACT FROM AUTHOR]

1: We have investigated the inhibitory effects of blockers of volume-activated (Clvol) and calcium-activated (ClCa) chloride channels on hypotonic solution (HS)-induced contractions of rat trachea, comparing their effects with those of the voltage-dependent calcium channel (VDCC) blocker nifedpine. 2: HS elicited large, stable contractions that were partially dependent on the cellular chloride gradient; a reduction to 41.45±7.71% of the control response was obtained when extracellular chloride was removed. In addition, HS-induced responses were reduced to 26.8±5.6% of the control by 1?µM nifedipine, and abolished under calcium-free conditions, indicating a substantial requirement for extracellular calcium entry, principally via VDCCs. 3: The established Clvol blockers tamoxifen (?10?µM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (1-100?µM), at concentrations previously reported to inhibit Clvol in smooth muscle, did not significantly inhibit HS-induced contractions. 4: In contrast, the recognized ClCa blocker niflumic acid (NFA; 1-100?µM) produced a reversible, concentration-dependent inhibition of HS responses, with a reduction to 36.6±6.4% of control contractions at the highest concentration. The mixed Clvol and ClCa blocker, 5-nitro 2-(3-phenylpropylamine) benzoic acid (NPPB; 10-100?µM) also elicited concentration-related inhibition of HS-induced contractions, producing a decrease to 35.9±11.3% of the control at 100?µM. 5: Our results show that HS induces reversible, chloride-dependent contractions of rat isolated trachea that were inhibited by NFA and NPPB, while exhibiting little sensitivity to recognized blockers of Clvol. The data support the possibility that opening of calcium-activated chloride channels under hypotonic conditions in respiratory smooth muscle may ultimately lead to VDCC-mediated calcium entry and contraction.British Journal of Pharmacology (2004) 141, 367-373. doi:10.1038/sj.bjp.0705615 [ABSTRACT FROM AUTHOR]

TonEBP (NFAT5) is a newly identified member of the Rel family of transcriptional activators that include NF-κB and NFAT1 to NFAT4. Activated in response to hypertonicity, TonEBP stimulates transcription of transporters of organic osmolytes, certain cytokines, and a molecular chaperone. We provide biochemical data demonstrating that full-length TonEBP dimerizes via the C-terminus of the Rel-homology domain (CRHD). The two polyglutamine motifs were not involved. The dimerization was not affected by nucleocytoplasmic shifts in TonEBP in response to changes in ambient tonicity. Preventing the dimer formation by deleting the CRHD did not affect the nucleocytoplasmic shifts. On the other hand, deletion of the CRHD prevented DNA binding and eliminated the dominant negative activity of a C-terminal truncated TonEBP. Furthermore, phosphorylation was dramatically reduced especially in hypertonic conditions by deletion of the CRHD. We conclude that dimerization is required for proper phosphorylation of TonEBP as well as DNA binding. [Copyright &y& Elsevier]

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (CT) by 57.3±2.3% after 16 min. The transepithelial Na+ current (INa) increased concomitantly from 4.2±0.7 to 26.1±2.6 µA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (GT) increased synchronously with INa. Analysis of blocker-induced noise in INa, using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (NT) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced CT increase to 14.3±0.5%, whereas the INa increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of CT. At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased INa, leaving CT unaffected, whereas 10 mM Ca2+ stimulated both INa and CT. In the presence of 1 mM Mg2+, however, the Ca2+-induced CT increase was abolished. The failure of CT to increase during stimulation of INa by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and INa, reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg2+-sensitive increase in CT, not correlated with increases in GT and INa, seems to be unrelated to Na+ channel recruitment. [ABSTRACT FROM AUTHOR]

A6 epithelia, a cell line originating from the distal tubular part of the kidney of Xenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness ( T), short-circuit current ( I) and transepithelial conductance ( G) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease of T described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na uptake by replacing apical Na by N-methyl- d-glucamine (NMDG) did not affect cell volume under isotonic conditions. An increase of T by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na uptake or Na pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K channels by 5 mM Ba elicited a significant increase in T of 16.3% above control. Quinine, a potent blocker of volume-activated K channels, did not change T significantly. Basolateral hypotonicity elicited a rapid rise in T followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na uptake as well as after stimulating apical Na uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K efflux at the basolateral side with Ba or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K, whereas the effects of Ba and quinine are most likely due to the blockage of the basolateral K pathway. [ABSTRACT FROM AUTHOR]

This paper describes a Ca-blockable, poorly selective cation pathway in the apical membrane of A6 epithelia. This pathway has properties that resemble the cation-selective channels in the toad urinary bladder and frog skin. Transepithelial short circuit currents ( I) and power density spectra (PDS) of the fluctuations in current were recorded. The basolateral surface of the tissues was exposed to Cl or SOsolutions with Na as the major cation. Ca-blockable inward oriented currents and Lorentzian noise were recorded with isotonic (215 mosmol/kg) mucosal Cl and hypotonic (144 mos-mol/kg) serosal SOsolution with Na, K, Rb or Cs as the major mucosal cation. Experiments with mucosal K demonstrated that the cation-selective channel was markedly activated by serosal hypotonicity. Effects of an increased electrical driving force were excluded on the basis of the results obtained with microelectrode experiments and transepithelial voltage clamping. Cell volume expansion induced by isotonic replacements of serosal sucrose by glycerol or urea also activated the cation-selective pathway. Furthermore, the presence of Cl in the mucosal solution was a prerequisite for a sustained response to hypotonicity or replacements of the organic compounds. Moreover, we found that the cationselective channels are mainly expressed in the cells during the early period of epithelial growth. [ABSTRACT FROM AUTHOR]

The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in ( i) active K influx, ( ii) passive Na and K fluxes, and ( iii) the number ofH-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (π) to one-half of its normal value (π=0.5) produced the following effects: ( i) an increase in active K influx on the order of 160%, ( ii) a 20% reduction in Na influx and K permeability ( P), and ( iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na pump is not caused by an increased leak of either Na (inward) or K (outward). It is unlikely that the stimulation of active Na extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance π=2 to π=1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na and K fluxes were not significantly altered by hypertonicity (π=2). [ABSTRACT FROM AUTHOR]

The ability of early proximal tubule cells of the Necturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes. Necturus proximal tubule cells regulate volume in both hyper- and hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO3- dependent and is associated with a decrease in the relative K+ conductance of the basolateral cell membrane and a decrease in the resistance ratio, Ra/Rbl. Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO3- but is also inhibited by 2 mM Ba2+ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K+ conductance of the basolateral cell membrane and an increase in Ra/Rbl. Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO3- removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO3(-)-dependent transport systems and the baso-lateral K+ conductance. [ABSTRACT FROM AUTHOR]

The isolated and perfused cortical collecting tubule of the rabbit was examined by differential interference contrast microscopy in order to characterize the morphologic response of this nephron segment to peritubular hypotonicity. Computer-assisted, morphometric procedures were developed to obtain measurements of cell volume and lateral intercellular space geometry from interference contrast images of perfused nephron segments. Following dilution of the bath from 290 to 190 mOsm in the absence of antidiuretic hormone (T = 25 degrees C), the cells swelled rapidly to a new steady-state volume which was maintained for at least 20 to 30 min and which was about 90% of that predicted for ideal osmometric behavior. The increase in cell volume was accomplished entirely by bulging of the cells into the lumen; lateral space width and outside tubule diameter were unaffected by peritubular hypotonicity. In addition, the swelling of the cells was associated with an apparent swelling of intracellular organelles, e.g., nuclei and mitochondria. Our results indicate that cells of the mammalian collecting tubule swell without the capacity for significant volume regulation at 25 degrees C and without the cytoplasmic vacuolation and dilation of the lateral intercellular spaces observed following the onset of antidiuretic hormone-dependent volume reabsorption (E. Ganote , J. Grantham , H. Moses, M. Burg and J. Orloff , J. Cell Biol. 36:355, 1968). [ABSTRACT FROM AUTHOR]

Abstract Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability. We previously reported that the excitability of nociceptor was increased by hypotonic stimulation. The present study tested the effect of hypotonicity on tetrodotoxin-sensitive sodium current (TTX-S current) in cultured trigeminal ganglion (TG) neurons. Our data show that after hypotonic treatment, TTX-S current was increased. In the presence of hypotonicity, voltage-dependent activation curve shifted to the hyperpolarizing direction, while the voltage-dependent inactivation curve was not affected. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers. We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response. We conclude that hypotonic stimulation enhances TTX-S current, which contributes to hypotonicity-induced nociception. TRPV4 receptor and PKC intracellular pathway are involved in the increase of TTX-S current by hypotonicity.