Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC(swell)) in the Brain

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl⁻- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC(swell)) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC(swell) in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl⁻ conductance (I(Cl,swell)) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I(Cl,swell) with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I(Cl,swell) observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC(swell) in the brain.

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia / 亚洲男科学杂志(英文版)

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l-1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l-1 ) to a hypotonic solution (290 mOsm l-1 ), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'- isulfonic acid (DiDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DiDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC.

Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC.

Sexual hormones modulate compensatory renal growth and function / Las hormonas sexuales modulan el crecimiento renal compensador (CRC) y la función renal en la uninefrectomía (uNx)

The role played by sexual hormones and vasoactive substances in the compensatory renal growth (CRG) that follows uninephrectomy (uNx) is still controversial. Intact and gonadectomized adult Wistar rats of both sexes, with and without uNx, performed at 90 days age, were studied at age 150 days. Daily urine volume, electrolyte excretion and kallikrein activity (UKa) were determined. Afterwards, glomerular filtration rate and blood pressure were measured, the kidneys weighed and DNA, protein and RNA studied to determine nuclei content and cell size. When the remnant kidney weight at age 150 days was compared with the weight of the kidney removed at the time of uNx, male uNx rats showed the greatest CRG (50%) while growth in the other uNx groups was 25%, 15% and 19% in orchidectomized, female and ovariectomized rats, respectively. The small CRG observed in the uNx female rats was accompanied by the lowest glomerular filtration value, 0.56 ± 0.02 ml/min/g kwt compared, with the other uNx groups, p < 0.05. Cell size (protein or RNA/DNA) was similar for all the groups except for uNx orchidectomized rats. In this group the cytoplasmatic protein or RNA content was lower than in the other groups while DNA (nuclei content) was similar. Some degree of hyperplasia was determined by DNA content in the uNx groups. Male sexual hormones positively influenced CRG and its absence modulated cell size. Female sexual hormones, instead, did not appear to stimulate CRG. The kallikrein kinin system may not be involved in CRG.

La importancia que pueden tener las hormonas sexuales y sustancias vasoactivas sobre el crecimiento renal compensador (CRC) que sigue a la uninefrectomía es aún materia de debate. Se estudiaron ratas Wistar de ambos sexos, a los 150 días de vida, intactas y gonadectomizadas con y sin uNx, realizada a los 90 días de vida. Se midió volumen urinario diario y excreción de electrolitos y actividad de kalikreína urinaria. Se midió filtrado glomerular y presión arterial media extrayéndose luego los riñones que fueron pesados y preparados para estudios histológicos y determinación de ADN, ARN y proteínas para estimar contenido nuclear y tamaño celular. El CRC fue calculado comparando el peso del riñón al momento de las uNx (90 dias de vida) con aquel obtenido a los 150 días de vida. En las ratas macho uNx se observó el mayor CRC (50%) mientras que, en los otros grupos uNx solo alcanzó un 25%, 15% y 19%. El filtrado glomerular acompañó los cambios morfológicos observándose el menor filtrado en las ratas hembras uNx respecto al resto de los grupos 0.56 ± 0.02, p < 0.05. El tamaño celular (proteína o ARN/ ADN) fue similar para todos los grupos excepto para los orquidectomizados uNx, cuyo contenido citoplasmático fue menor. El contenido nuclear (ADN) fue semejante en todos los grupos. Se observó que el CRC está influenciado positivamente por las hormonas sexuales masculinas y su ausencia modula el tamaño celular. La falta de hormonas sexuales femeninas, en cambio, afecta negativamente el CRC. El sistema kalikreína kinina no parecería estar involucrado en el CRC.

Relation of the baroreflex mechanism with the photoplethysmographic volume in healthy humans during orthostatism / Relación del mecanismo barorreflejo con el volumen fotopletismográfico en humanos sanos durante ortostatismo

Objective: Our aim was to evaluate the effect of the baroreflex mechanism upon peripheral blood volume during sympathetic stimulation by orthostatism. Methods: Nineteen clinically healthy volunteers were included (12 men), 28.4 ± 6.2 years old. Blood pressure was monitored with a Finometer and blood volume with a photoplethysmo-graph during supine position and orthostatism (15 minutes each), in order to obtain systolic blood pressure (SBP), diastolic blood pressure (DBP), systolic volume (SysV), diastolic volume (DiaV), and inter beat intervals (IBI) measurements. Baroreflex sensitivity index (IBI/SBP) and baroreflex effect on blood volume (IBI/SysV) were estimated by the sequence method. The pertinence of using only systolic values was tested by linear regression analysis of systolic versus diastolic measurements. Results: More than 70% of DBP and DiaV variations can be explained by SBP and SysV, respectively (p<0.001), with coherence >0.5 in frequencies between 0.04 and 0.15 Hz. IBI/SBP and IBI/SysV were linearly correlated (R>0.4) and both decreased during orthostatism (p<0.05). Conclusion: The sequence method showed a strong baroreflex effect upon peripheral blood volume that became more apparent during sympathetic stimulation with orthostatism. This approach could be clinically useful for the evaluation of blood volume regulation for many diseases such as diabetes mellitus and heart failure, and during therapeutic interventions such as hemodialysis.

Objetivo: Evaluar el efecto del mecanismo barorreflejo sobre el volumen sanguíneo periférico durante estimulación inducida por ortostatismo. Métodos: Se incluyeron 19 voluntarios sanos (12 hombres), con edad de 28.4 ± 6.2 años. La presión arterial se midió con un Finometer y el volumen sanguíneo con un fotopletismógrafo, ambos durante posiciones supina y ortostatismo activo (15 minutos cada una), para obtener los valores de presión arterial sistólica (PAS), presión arterial diastólica (PAD), volumen sistólico (VS), volumen diastólico (VD) e intervalo inter pulso (IIP). Se estimó la sensibilidad barorrefleja (IIP/PAS) y el efecto barorreflejo sobre el volumen sanguíneo (IIP/VS) mediante el método de secuencias. La pertinencia de usar sólo variables sistólicas, se evaluó mediante análisis de regresión lineal de las mediciones sistólicas versus las diastólicas. Resultados: Más de 70% de las variaciones de presión arterial diastólica y volumen diastólico pueden ser explicadas mediante presión arterial sistólica y volumen sistólico, respectivamente (p<0.001), con coherencia >0.5 en frecuencias entre 0.04 y 0.15 Hz. IIP/PAS y IIP/VS tuvieron correlación positiva (R>0.4) y ambos disminuyeron durante ortostatismo (p<0.05). Conclusiones: El método de secuencias demostró un importante efecto barorreflejo sobre el volumen sanguíneo periférico que se hizo más notable durante estimulación simpática con ortostatismo. Este enfoque podría ser clínicamente útil en la evaluación de la regulación del volumen sanguíneo en distintas enfermedades como diabetes mellitus o falla cardiaca, y durante intervenciones terapéuticas como la hemodiálisis.

Hypernatemia:Successful Treatment

Hypernatremia reflects a net water loss or a hypertonic sodium gain, with inevitable hyperosmolality. Severe symptoms are usually evident only with acute and large increases in plasma sodium concentrations to above 158-160 mmol/l. Importantly, the sensation of intense thirst that protects against severe hypernatremia in health may be absent or reduced in patients with altered mental status or with hypothalamic lesions affecting their sense of thirst and in infants and elderly people. Non-specific symptoms such as anorexia, muscle weakness, restlessness, nausea, and vomiting tend to occur early. More serious signs follow, with altered mental status, lethargy, irritability, stupor, and coma. Acute brain shrinkage can induce vascular rupture, with cerebral bleeding and subarachnoid hemorrhage. However, in the vast majority of cases, the onset of hypertonicity is low enough to allow the brain to adapt and thereby to minimize cerebral dehydration. Organic osmolytes accumulated during the adaptation to hypernatremia are slow to leave the cell during rehydration. Therefore, if the hypernatremia is corrected too rapidly, cerebral edema results as the relatively more hypertonic ICF accumulates water. To be safe, the rate of correction should not exceed 12 mEq/liter/day.

Expression and localization of aquaporin 8 in human WISH cells / 重庆医科大学学报

Objective:To determine the expression of aquaporin 8 and its localization in human WISH cells and to explore the molecular and cellular mechanisms for water absorption across the amniotic membranes.Methods:Human amnion-derive WISH cells were cultured.Western analysis was used to quantify AQP8 expression level.Reverse transcripton-polymerase chain reaction(RT-PCR)was used to quantify AQP8 mRNA expression levels.Immunofluorescence was used to determine localization of AQP8 in WISH cells.Results:AQP8 mRNA protein was found in human WISH cells.By western-blot,the AQP8 proteim was detected at 34ku in human WISH cells.AQP8 labeling was observed in intracellular vesicular structures throughout the cytosol and plasma membrane.Conclusion:The study demonstrates the expression of aquaporin 8 in human WISH cells.These results suggest that aquaporin 8 may be a channel that mediates amniotic fluid resorption by way of intramembranous pathway.

Expression of volume-activated anion channels in exocrine acinar cells

No abstract available.

Volume Changes under Isotonic Condition Containing Thiopental in Brain Astrocytoma Cells / 대한구급학회지

BACKGOUND: Cell volume regulation is especially important in the brain because the brain is confined within a non-compliant vault and cannot tolerate significant perturbations in cell size. Cerebral cell volume regulation mechanisms are activated by sustained disturbances in plasma osmolality. The constancy of cell volume under physiological conditions is generally thought to reflex a balance between influx and efflux of solute and is therefore critically dependent on the properties of the plasma membrane. Cell volume regulation have not been described under isoosmotic solution. The object of the study was to know the effects of thiopental on cell volume change in isoosmotic condition. METHODS: We made isoosmotic solution without thiopental (Group 1) and isoosmotic solution with 22.9 mM (Group 2), 16.8 mM (Group 3), 13.3 mM (Group 4) thiopental, separately, in order to study changes in cell volume under isoosmotic solution. We put cultured human brain astrocytoma cells into isoosmotic solution for each group and calculated cell volume using Coulter Counter after 30 minutes. RESULTS: Cell volume was shown to be 5084+/-8580 (micrometer3)in Group 1, 501+/-854 (micrometer3) in Group 2, 1183+/-3839 (micrometer3) in Group 3, and 624+/-1100 (micrometer3) in Group 4. We discovered that cells in Group 2,3,4 were shrunk relative to cells in Group 1 (p<0.01). And there were significant differences in cell volume among thiopental groups. CONCLUSIONS: Thiopental may has an effect on cell membrane properties and decrease cell volume under isoosmotic solution in brain astrocytoma cell.

Regulation of Astroglial Volume by Ketamine in Glutamate Induced Cellular Volume Changes / 대한마취과학회지

BACKGROUND: Relative changes of astroglial volume constitute the major part of brain edema, which is related to delayed neuronal damage. Several factors including glutamate may contribute to astroglial swelling. Intravenous anesthetic, ketamine was known to restore neuronal damage by inhibiting NMDA receptor activity. Therefore, we decided to investigate the effect of ketamine on the astrocyte swelling by glutamate in the present study. METHODS: To analyze cell swelling in vitro, glial cell line, U1242MG was used. The effects of glutamate (1, 2, 3 mM), and glutamate with ketamine (1 mM) on the regulation of astrocyte volume were achieved by flow cytometry system. To eliminate the dead cells from experimental cell suspension and to assess cell viability, fluorescent dye propidium iodide was used. RESULTS: Glutamate addition (1, 2, 3mM) caused astroglial swelling both in calcium present and calcium absent buffer. The difference of cellular swelling dependent on glutamate concentration was only seen in calcium free buffer (p<0.05). Ketamine per se did not affect astroglial volume. However, when it was added to glutamate perfusion, 1 mM ketamine diminished cellular swelling by glutamate during first 10 minutes (p<0.05), and cellular shrinkage by glutamate after 1 hour incubation (p<0.05). CONCLUSIONS: Ketamine (1 mM) is effective in the regulation of astroglial volume alterations induced by glutamate in both short time and long time perfusion.