Extracellular Hypotonicity Research Articles

Change in Metabolomic Profile Associated with an Average Increase in Plain Water Intake of >+ 1 L/Day, Sustained Over 4 Weeks, in Healthy Young Men with Initial Total Water Intake Below 2 L/Day.

Cells adapt to chronic extracellular hypotonicity by altering metabolism. Corresponding effects of sustained hypotonic exposure at the whole-person level remain to be confirmed and characterized in clinical and population-based studies. This analysis aimed to 1) describe changes in urine and serum metabolomic profiles associated with four weeks of sustained > +1 L/d drinking water in healthy, normal weight, young men, 2) identify metabolic pathways potentially impacted by chronic hypotonicity, and 3) explore if effects of chronic hypotonicity differ by type of specimen and/or acute hydration condition. Untargeted metabolomic assays were completed for specimen stored from Week 1 and Week 6 of the Adapt Study for four men (20-25 years) who changed hydration classification during that period. Each week, first-morning urine was collected after overnight food and water restriction, and urine (t+60 min) and serum (t+90 min) were collected after a 750 mL bolus of drinking water. Metaboanalyst 5.0 was used to compare metabolomic profiles. In association with four weeks of > + 1 L/d drinking water, urine osmolality decreased below 800 mOsm/kg H2O and saliva osmolality decreased below 100 mOsm/kg H2O. Between Week 1 and Week 6, 325 of 562 metabolic features in serum changed by 2-fold or more relative to creatinine. Based on hypergeometric test p-value <0.05 or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact factor >0.2, the sustained > + 1 L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a metabolomic pattern of carbohydrate oxidation via the tricarboxylic acid (TCA) cycle, instead of glycolysis to lactate, and a reduction of chronic disease risk factors in Week 6. Similar metabolic pathways appeared potentially impacted in urine, but the directions of impact differed by specimen type. In healthy, normal weight, young men with initial total water intake below 2 L/d, sustained > + 1 L/d drinking water was associated with profound changes in serum and urine metabolomic profile, which suggested normalization of an aestivation-like metabolic pattern and a switch away from a Warburg-like pattern. Further research is warranted to pursue whole-body effects of chronic hypotonicity that reflect cell-level effects and potential beneficial effects of drinking water on chronic disease risk.

Change in Metabolomic Profile Associated with an Average Increase in Plain Water Intake of &gt;+1 L/day, Sustained over 4 Weeks, in Healthy Young Men with Initial Total Water Intake below 2 L/day

Cells adapt to chronic extracellular hypotonicity by altering metabolism, such that cell‐level chronic disease risk factors are reduced. Corresponding effects of sustained whole‐body hydration are hypothesized but remain to be confirmed and translated into population‐level chronic disease prevention.This analysis aimed to 1) describe change in urine and serum metabolomic profiles associated with change in hydration, induced by four weeks of sustained higher intake of drinking water, 2) check if features recognized as key osmolytes in specific tissues changed in urine and/or serum, and 3) identify which metabolic pathways might potentially be impacted by sustained hypotonic hydration.Adapt Study [Stookey JD et al., Physiol Rep 2013;1(5)] data from for 4 healthy, normal weight men (20‐25 years) who transitioned from not meeting to meeting 3 osmolality criteria (urine:&lt;800mmol/kg, serum:&lt;295mmol/kg and saliva:&lt;100mmol/kg) over 4 weeks of &gt;+1L/d intake of drinking water were used for this analysis. Untargeted metabolomic assays were performed by the UC Davis West Coast Metabolomic Center on first‐morning urine collected after overnight food and water restriction and urine (t+60min) and serum (t+90min) collected after a 750 ml bolus of drinking water. Metaboanalyst 5.0 was used to compare Week 1 vs Week 6 metabolomic profiles, using heatmap, OPLS‐DA, fold‐change, and pathway analyses.In heatmap and OPLS‐DA plots, metabolomic profiles appeared more similar in Week 6.Over 350 metabolic features decreased by 2‐fold or more relative to creatinine for at least 3 out of 4 participants in first‐morning and post‐bolus urine. Over 200 features increased by 2‐fold or more relative to creatinine in serum. A wide variety of known compounds changed by 2‐fold or more in at least one specimen type, including alcohols, monosaccharides, disaccharides, sugar acids, polyols, azoline, lactone, nucleosides, phenols, purines, pyridines, pyrimidines, fatty acids, cholesterol, glycerolipids, amino acids, carboxylic acids, phosphate, and urea. Based on hypergeometric test p‐value &lt;0.05 or pathway impact factor &gt;0.2, sustained +1L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a ‘reverse’ Warburg‐like pattern favoring carbohydrate oxidation via the TCA cycle in Week 6.The results warrant further work to leverage cell hydration effects for chronic disease prevention.

P2X-GCaMPs as Versatile Tools for Imaging Extracellular ATP Signaling.

ATP is an extracellular signaling molecule involved in numerous physiological and pathologic processes. However, in situ characterization of the spatiotemporal dynamic of extracellular ATP is still challenging because of the lack of sensor with appropriate specificity, sensitivity, and kinetics. Here, we report the development of biosensors based on the fusion of cation permeable ATP receptors (P2X) to genetically encoded calcium sensors [genetically encoded calcium indicator (GECI)]. By combining the features of P2X receptors with the high signal-to-noise ratio of GECIs, we generated ultrasensitive green and red fluorescent sniffers that detect nanomolar ATP concentrations in situ and also enable the tracking of P2X receptor activity. We provide the proof of concept that these sensors can dynamically track ATP release evoked by depolarization in mouse neurons or by extracellular hypotonicity. Targeting these P2X-based biosensors to diverse cell types should advance our knowledge of extracellular ATP dynamics in vivo.

TRPC3 is a critical contributor to the osmosensitivity and renal water handling in the mouse collecting duct

Kidney plays a central role in control of systemic water balance depending on hydration status. Arginine vasopressin (AVP) augments water permeability of the collecting duct (CD) by facilitating aquaporin 2 (AQP2) translocation to the apical plasma membrane. Inability of CD to respond to AVP signal causes Nephrogenic Diabetes Insipidus (NDI) leading to polyuria, dehydration and thirst. AVP stimulates CD to reabsorb water, but this can only occur in presence of positive osmotic difference between the cytosol and luminal fluid. Extracellular hypotonicity increases [Ca2+]i and causes cell swelling due to AQP2‐driven water influx. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remains unknown. TRPC3 is a Ca2+ permeable mechano‐activated channel abundantly expressed in the CD. Using ratiometric Fura 2‐based [Ca2+]i imaging, we simultaneously measured [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2‐dependent water reabsorption in freshly isolated split‐opened CDs of wild type and TRPC3 −/− mice and combined it with immunofluorescent detection of AVP‐induced AQP2 trafficking and assessment of systemic water balance. We found that TRPC3 inhibition or deletion precluded [Ca2+]i elevations induced hypotonicity and remarkably slowed the rate of cell swelling indicative of diminished water transport in the CD. TRPC3 −/− mice had comparable with WT serum and urine osmolarity in the presence of ad libitum water intake, but exhibited a significantly greater bodyweight loss, and larger urinary volume after 24 h water deprivation despite higher AVP levels when compared to WT. Furthermore, osmosensitive [Ca2+]i elevations were greatly increased in CDs from WT but not in TRPC3 −/− animals after 24 h water deprivation. The rate of cell swelling was greatly accelerated in WT, while was only modestly increased in TRPC3 −/− mice under this condition. Using immunofluorescent microscopy, we found that AQP2 translocated to the apical plasma membrane in WT, while remained largely cytosolic in TRPC3 −/− after 24 h water deprivation. Consistently, TRPC3 inhibition interferes with AVP‐induced AQP2 trafficking in cultured mpkCCDc14 cells. In summary, we demonstrate a significant role of TRPC3 in osmosensitivity and regulation of AVP‐dependent AQP2 trafficking in the CD. TRPC3 deletion compromises systemic water balance producing an NDI‐like phenotype.Support or Funding InformationAmerican Heart Association Career Development Award for Viktor Tomilin (19CDA34660148)NIH RO1 awards for Oleh Pochynyuk (DK117865 and DK119170)

Interaction between cAMP, volume-regulated anion channels and the Na+-HCO3−-cotransporter, NBCe1, in the regulation of nutrient- and hypotonicity-induced insulin release from isolated rat pancreatic islets and tumoral insulin-producing BRIN-BD11 cells

Soluble adenylyl cyclase (sAC) has been hypothesized to play a role in insulin secretion. The present study aimed to investigate the interaction between adenosine 3',5'‑cyclic monophosphate (cAMP), volume‑regulated anion channels (VRACs) and the electrogenic sodium bicarbonate (Na+‑HCO3‑) cotransporter, NBCe1, in the regulation of nutrient‑ and hypotonicity‑induced insulin release from rat pancreatic islets and tumoral insulin‑producing BRIN‑BD11 cells. In the islets, 5‑nitro‑2‑(3‑phenylpropylamino)benzoic acid (NPPB) and 5‑chloro‑2‑hydroxy‑3‑(thiophene‑2‑carbonyl)indole‑1‑carboxamide (tenidap) reduced glucose‑stimulated insulin release, however, only NPPB suppressed the enhancing action of cAMP analogs upon such a release. Insulin output from the BRIN‑BD11 cells was stimulated by 2‑ketoisocaproate (KIC) or extracellular hypoosmolarity. cAMP analogs and 3‑isobutyl‑1‑methylxanthine increased the insulin output recorded in the isotonic medium to a greater relative extent than that in the hypotonic medium. The secretory response to KIC or hypotonicity was inhibited by NPPB or tenidap, which both also opposed the enhancing action of cAMP analogs. Inhibitors of mitogen‑activated protein (MAP) kinase decreased insulin output in isotonic and hypotonic media. The inhibitor of sAC, 2‑hydroxyestriol, caused only a modest inhibition of insulin release, whether in the isotonic or hypotonic medium, even when tested at a concentration of 100µM. The omission of NaHCO3 markedly decreased the secretory response to KIC or extracellular hypotonicity. The omission of Na+ suppressed the secretory response to extracellular hypotonicity. The observations of the present study do not support the hypothesis of a major role for sAC in the regulation of insulin release.

Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in β-cells?

NAD(P)H oxidase (NOX)-derived H(2)O(2) 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 H(2)O(2) (100 to 200μM) at basal glucose concentration (1.1 to 2.8mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) 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 H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity.

The TRPV4 channel is a novel regulator of intracellular Ca2+ in human esophageal epithelial cells

The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca(2+) in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 μM 4α-PDD, whereas higher concentrations (10 μM and 30 μM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1β-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca(2+)-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

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+. The application of a P2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+]i increase. The [Ca2+]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+, indicating that a Ca2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+ quenching was significantly inhibited by nicardipine.These results strongly suggested that a nicardipine-sensitive Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+]i elevation of principal cells in rat CCD.

Regulation of neuronal cell volume: from activation to inhibition to degeneration

Regulation of neuronal cell volume: from activation to inhibition to degeneration

Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2(-/ -) and AQP1(-/ -) mice, hypotonicity increased neither C(m) nor outward current, but 0.1-muM PGE(2) increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C(m) and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA(2)/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.

Extracellular hypotonicity induces disturbance of sodium currents in rat ventricular myocytes

Hypotonic solution alters ion channel activity, but little attention has been paid to voltage-dependent sodium channels. The aim of this study was to investigate the effects of hypotonic solution on transient sodium currents (I(NaT)) and persistent sodium currents (I(NaP)). We also explored whether the intracellular signal transduction systems participated in the hypotonic modifications of sodium currents. I(NaT) and I(NaP) were recorded by means of whole-cell patch-clamp technique in isolated rat ventricular myocytes. Our results revealed that hypotonic solution reduced I(NaT) and simultaneously augmented I(NaP) with the occurrence of interconversion between I(NaT) and I(NaP). Hypotonic solution shifted steady-state inactivation to a more negative potential, prolonged the time of recovery from inactivation, and enhanced intermediate inactivation (I(IM)). Ruthenium red (RR, inhibitor of TRPV4), bisindolylmaleimide VI (BIM, inhibitor of PKC), Kn-93 (inhibitor of Ca/CaMKII) and BAPTA (Ca(2+)-chelator) inhibited the effects of hypotonic solution on I(NaT) and I(NaP). Therefore we conclude that hypotonic solution inhibits I(NaT), enhances I(NaP) and I(IM) with the effects being reversible. TRPV4 and intracellular Ca(2+), PKC and Ca/CaMKII participate in the hypotonic modifications of sodium currents.

Resveratrol Inhibits Electrical Activity and Insulin Release from Insulinoma Cells by Block of Voltage-Gated Ca2+ Channels and Swelling-Dependent Cl- Currents

The phytostilbene resveratrol (RV) improves the metabolic state in animal models by increasing the insulin responsiveness of tissues and there is evidence that RV affects insulin secretion from native β-cells and insulinoma cells. In whole cell patch clamp experiments on clonal rat INS-1E cells we used high extracellular glucose (20 mM), extracellular hypotonicity (30%) or tolbutamide (100 μM) to elicit membrane depolarizations and electrical activity. Application of RV (50 μM) repolarized the cells, terminated electrical activity and prevented the hypotonicity-induced depolarization. These effects were fully reversible and intermittent application of RV restored tolbutamide-induced electrical activity after desensitization. Glucose-induced depolarization was counteracted by RV in presence of iberiotoxin (50 nM), showing that the RV effect does not depend on BK_Ca channel activation. RV dose-dependently inhibited K_ATP currents, L- and T-type Ca²⁺ currents and swelling-dependent Cl^- currents evoked by either hypotonicity or high extracellular glucose - ion conductances crucially involved in regulating the electrical activity of insulin secreting cells. We further show that RV blunts glucose-induced, but not basal insulin release. Our results indicate that RV counteracts/prevents stimulus-induced cell membrane depolarization and electrical activity by blocking voltage-gated Ca²⁺- and swelling-dependent Cl^- currents despite the inhibition of K_ATP currents.

Stimulus-secretion coupling of hypotonicity-induced insulin release in BRIN-BD11 cells

The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 mM decrease in extracellular NaCl 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 a maximal 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 Ca2+ 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 Ca2+, 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 Ca2+-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca2+ channels.

TRPV4‐mediated regulation of epithelial permeability

TRPV4 is a calcium-permeable channel activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. We show that TRPV4 is localized in the basolateral membrane of the mouse mammary cell line HC11. Activation of TRPV4 caused an increase in the intracellular Ca(2+) concentration through influx of extracellular Ca(2+), triggering two independent chains of events: 1) a rapid increase in transcellular conductance through the activation of apical large conductance calcium-activated (BK) potassium channels that were blockable by paxilline; 2) a slow increase in paracellular permeability for small solutes. The latter effect was accompanied by a down-regulation of the tight junctional proteins claudin -1, -3, -4, -5, -7, and -8 and by dramatic changes in tight junction morphology, including frequent large breaks in the tight junction strands. This dual modulation of epithelial permeability after TRPV4 activation may be involved in regulating the tonicity across mammary gland epithelia. TRPV4 activation may also be responsible for exudation and edema formation during inflammation processes.-Reiter, B., Kraft, R., Günzel, D., Zeissig, S., Schulzke, J-D., Fromm, M., Harteneck, C. TRPV4-mediated regulation of epithelial permeability.

Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions

Aquaporin-5 (AQP5) is expressed in epithelia of lung, cornea, and various secretory glands, sites where extracellular osmolality is known to fluctuate. Hypertonic aquaporin (AQP) induction has been described, but little is known about the effects of a hypotonic environment on AQP abundance. We report that, when mouse lung epithelial cells were exposed to hypotonic medium, a dose-responsive decrease in AQP5 abundance was observed. Hypotonic reduction of AQP5 was blocked by ruthenium red, methanandamide, and miconazole, agents that inhibit the cation channel transient receptor potential vanilloid (TRPV) 4 present in lung epithelial cells. Several observations indicate that TRPV4 participates in hypotonic reduction of AQP5, including a requirement for extracellular calcium to achieve AQP5 reduction; an increase in intracellular calcium in mouse lung epithelial (MLE) cells after hypotonic stimulation; and reduction of AQP5 abundance after addition of the TRPV4 agonist 4alpha-Phorbol-12,13-didecanoate (4alpha-PDD). Similarly, addition of hypotonic PBS to mouse trachea in vivo decreased AQP5 within 1 h, an effect blocked by ruthenium red. To confirm a functional interaction, AQP5 was expressed in control or TRPV4-expressing human embryonic kidney (HEK) cells. Hypotonic reduction of AQP5 was observed only in the presence of TRPV4 and was blocked by ruthenium red. Combined with earlier studies, these observations indicate that AQP5 abundance is tightly regulated along a range of osmolalities and that AQP5 reduction by extracellular hypotonicity can be mediated by TRPV4. These findings have direct relevance to regulation of membrane water permeability and water homeostasis in epithelia of the lung and other organs.

Slow volume transients in amphibian skeletal muscle fibres studied in hypotonic solutions.

The influence of extracellular hypotonicity on the relationship between cell volume (V(c)) and resting membrane potential (E(m)) was investigated in Rana temporaria skeletal muscle. V(c) was measured by confocal microscope imaging of fibres through their transverse (xz) planes, and E(m) was determined using standard microelectrode techniques. Hypotonic solutions first elicited a rapid increase in fibre volume, DeltaV(R+) that fulfilled expectations of simple osmotic behaviour described in earlier reports. However, this was consistently followed by a slow increase in V(c) (DeltaV(S+)) to 10-15% above osmotic predictions. Longer (>1 h) exposures to hypotonic solutions permitted a subsequent slow decrease in V(c) (DeltaV(S-)), the eventual magnitude of which exceeded that of the preceding DeltaV(S+). Restoration of isotonic conditions elicited a prompt recovery in V(c) that matched simple osmotic predictions and thus left a net change in V(c). Such alterations in V(c) attributable to DeltaV(S+) then gradually reversed, while those due to DeltaV(S-) persisted. Both DeltaV(S+) and DeltaV(S-) persisted under conditions of Cl- deprivation. The depolarization of E(m) that accompanied DeltaV(R+) was consistent with dilution of intracellular [K(+)]. E(m) did not significantly alter during the subsequent DeltaV(S) transients. These empirical features of DeltaV(S+) and DeltaV(S-) were analysed using the quantitative charge-difference model of Fraser and Huang, published in 2004. This attributed the DeltaV(S+) to an electroneutral increase in the effective osmotic activity of normally membrane-impermeant intracellular anions. In contrast, the DeltaV(S-) could only be explained by an efflux of such anions and was accordingly comparable to organic anion-dependent regulatory volume decreases reported in other cell types.

Identification of swelling-activated Cl(-) current in rabbit cardiac Purkinje cells.

The presence and functional role of the swelling-activated Cl(-) current (I(Cl(swell))) in rabbit cardiac Purkinje cells was examined using patch-clamp methodology. Extracellular hypotonicity (210 or 135 mOsm) activated an outwardly rectifying, time-independent current with a reversal potential close to the calculated Cl(-) equilibrium potential (E(Cl)). The magnitude of this current was related to tonicity of the superfusate. The current was blocked by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These features are comparable to those of I(Cl(swell)) found in sinoatrial nodal, atrial, and ventricular myocytes. I(Cl(swell)) activation at 210 and 135 mOsm depolarized the resting membrane potential with 6 and 10 mV and shortened the action potential by approximately 18 and approximately 33%, respectively. DIDS partially reversed I(Cl(swell))-induced action potential changes. We conclude that I(Cl(swell)) is present in Purkinje cells and its activation leads to action potential shortening and resting membrane potential depolarization, both of which can promote the development of reentrant arrhythmias.

生体膜輸送の新たなる展開 上皮膜輸送を介する浸透圧感受機構

To sense an environmental change such as hypotonic shock is a crucial function for cells to adjust to a new environment. Na+ reabsorption in the kidney is finely regulated to maintain body Na+ content, blood pressure, extracellular fluid volume (ECF) and is stimulated by aldosterone, vasopression and extracellular hypotonicity to maintain normal plasma osmolality. On the other hand, hypotonic shock causes drastic cell volume changes; initial cell swelling followed by regulatory volume decrease (RVD). The mechanism of RVD is well understood as K+/Cl-release through volume-sensitive Cl-and K+ channels and a decrease in cytosolic Cl-concentration is associated with the RVD. In this review, we focus our study on a possible role of cytosolic Cl-for sensing extracellular hypotonicity by modulating enzyme activities to regulate tyrosine phosphorylation and suggest the possibility that ion channels and ion transporters function as regulators of signal transduction for extracellular stimuli in addition to the contribution to transepithelial ion transporter.

Calcium modulates osmosensitive taurine efflux in HeLa cells.

The role of Ca2+ in the signaling transduction pathway involved in osmosensitive taurine efflux in HeLa cells was studied using radiotracer efflux techniques. Taurine efflux induced by extracellular hypotonicity was decreased by 85% by removal of extracellular Ca2+ and simultaneous depletion of intracellular Ca2+ stores with thapsigargin. Extracellular Ca2+ removal, thapsigargin treatment, or addition of Gd3+ all decreased taurine efflux by approximately 50%. To explore the putative signal transduction pathways involved in swelling-induced taurine efflux, HeLa cells were exposed to PP1, an inhibitor of the Src family of tyrosine kinases, the phospholipase C inhibitor U73122, the IP3 receptor antagonist 2-APB, and the generic protein kinase C inhibitor chelerythrine. All of these treatments caused approximately 50% inhibition of taurine release in Ca2+-rich extracellular medium and approximately 85%-90% in Ca2+-free conditions. The inhibitors of the conventional protein kinase C isoforms BIM-1 and Gö6976 reduced taurine efflux to a lesser extent. Acute (10-min) exposure to the phorbol ester tetradecanoyl phorbol acetate (TPA) increased taurine efflux in 25%, whilst overnight exposure had an inhibitory effect decreasing efflux by 22%. A working model for activation of osmosensitive taurine efflux in HeLa cells involving different Ca2+ signaling pathways is presented.

Brain edema – a new classification

Brain edema is a reaction to any brain injury and can be the first stage in the beginning of intracranial hypertension. This paper puts forth a modern classification of brain edema types, based on a etiopathogenic interpretation. The hydroelectrolitic and/or proteinic buildup can occur within cells and/or in the extracellular space and differentiates three types of brain edema: cellular brain edema; extracellular brain edema and combined brain edema. Cellular brain edema (cytotoxic brain edema) occurs through intracellular hyperosmolarity or extracellular hypotonicity. Extracellular brain edema (interstitial) appears as a result of the buildup of edema fluid in the extracellular space of the brain parenchyma and can be: hydrostatic extracellular brain edema (through ultrafiltration), oncotical extracellular brain edema (vasogen brain edema) and hydrocephalic extracellular brain edema. Combined brain edema includes in variable ratios both types of brain edema, cellular and extracellular; they can be present together from the beginning or can appear successively.