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Purpose: Stroke is the leading cause of disability in adults worldwide, with hemiparesis being the most prevalent consequence. The use of video games and movement sensors could contribute to improving patients’ chances of recovery. We performed a supervised pilot study to validate the safety, feasibility, and acceptability of a new virtual rehabilitation platform in patients with chronic post-stroke upper limb hemiparesis. Methods: The participants (n = 9) participated in 40 rehabilitation sessions, twice a week, for a period of 20 weeks. Their experiences with the platform were documented using a Likert-scale survey. Changes in motor function were evaluated using the Chedoke Arm and Hand Activity Inventory (CAHAI) and the Wolf Motor Function Test (WMFT). Results and conclusions: All participants expressed that they enjoyed the experience and felt comfortable using the platform. Preliminary results showed significant motor recovery ( P = .0039) according to the WMFT scores. Patients with significant impairment showed no improvement in upper limb task-oriented motor function after therapy. The new platform is safe and well-accepted by patients. The improvement in motor function observed in some of the participants should be attributed to the therapy since spontaneous functional recovery is not expected in chronic stroke patients.

Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton’s jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.

Neuronal activity in the retina generates osmotic gradients that lead to Müller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Müller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca 2+ release from intracellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Müller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca 2+ dependency in MIO-M1 cells. Swelling-induced [ 3 H]Tau/ [ 3 H]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [ 3 H]Tau and [ 3 H]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [ 3 H]Tau efflux was mainly dependent on Ca 2+ release from intracellular stores. RVD was unaffected in a Ca 2+ -free medium, probably due to Ca 2+ -independent Tau and Glu release, but was reduced by chelating intracellular Ca 2+ . The inhibition of phosphatidylinositol-3-kinase reduced [ 3 H]Glu efflux but also the Ca 2+ -insensitive [ 3 H]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca 2+ -independent pathway. We propose that VRAC-mediated Tau and Glu release has a relevant role in RVD in Müller cells. The observed disparities in Ca 2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology. NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Müller cells are still unknown. We show that swelling-induced taurine and glutamate release mediated by the volume-regulated anion channel (VRAC) largely contributes the to the regu- latory volume decrease response in a human Müller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca 2+ -dependent and-independent regulatory mechanisms, which strongly suggests that Müller cells may express different VRAC heteromers formed by the recently discovered leu-cine-rich repeat containing 8 (LRRC8) proteins. Fil: Netti, Vanina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Pizzoni, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Peréz Domínguez, Martha. Universidad Nacional Autónoma de México; México Fil: Ford, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Pasantes Morales, Herminia. Universidad Nacional Autónoma de México; México Fil: Ramos Mandujano, Gerardo. Universidad Nacional Autónoma de México; México Fil: Capurro, Claudia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

Background The amino acid taurine (2-Aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. This study aimed to determine if the dual action of taurine on GABAC-ρ1R relates to its structure. To address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate GABAC-ρ1R. Results In Xenopus laevis oocytes, hypotaurine and homotaurine partially activate heterologously expressed GABAC-ρ1R, showing an increment in its deactivation time with no changes in channel permeability, whereas isethionic acid showed no effect. Competitive assays suggest that hypotaurine and homotaurine compete for the GABA-binding site. In addition, their effects were blocked by the ion-channel blockers picrotixin and Methyl(1,2,5,6-tetrahydropyridine-4-yl) phosphinic acid. In contrast to taurine, co-application of GABA with hypotaurine or homotaurine revealed that the dual effect is present separately for each compound: hypotaurine modulates positively the GABA current, while homotaurine shows a negative modulation, both in a dose-dependent manner. Interestingly, homotaurine diminished hypotaurine-induced currents. Thus, these results strongly suggest a competitive interaction between GABA and homotaurine or hypotaurine for the same binding site. “In silico” modeling confirms these observations, but it also shows a second binding site for homotaurine, which could explain the negative effect of this compound on the current generated by GABA or hypotaurine, during co-application protocols. Conclusions The sulfur-containing compounds structurally related to taurine are partial agonists of GABAC-ρ1R that occupy the agonist binding site. The dual effect is unique to taurine, whereas in the case of hypotaurine and homotaurine it presents separately; hypotaurine increases and homotaurine decreases the GABA current. Electronic supplementary material The online version of this article (10.1186/s12868-018-0448-6) contains supplementary material, which is available to authorized users.

Taurine content is high (mM) in mammalian brain. By its major role as an osmolyte, taurine contributes to the cell volume control, which is particularly critical in the brain. Taurine participates in osmotic adjustments required to maintain the organization and size of intracellular compartments. It counteracts volume fluctuations in unbalanced transmembrane fluxes of ions and neurotransmitters, preserving the functional synaptic contacts. Taurine has a key role in the long-term adaptation to chronic hyponatremia as well as in other pathologies leading to brain edema. Together with other osmolytes, taurine corrects cell shrinkage, preventing mysfunction of organelles and apoptosis. Swelling corrective taurine efflux occurs through a leak pathway, likely formed by LCRR8 protein isoforms. Shrinkage-activated influx comes largely by the increased activity of the Na

Taurine content is high (mM) in mammalian brain. By its major role as an osmolyte, taurine contributes to the cell volume control, which is particularly critical in the brain. Taurine participates in osmotic adjustments required to maintain the organization and size of intracellular compartments. It counteracts volume fluctuations in unbalanced transmembrane fluxes of ions and neurotransmitters, preserving the functional synaptic contacts. Taurine has a key role in the long-term adaptation to chronic hyponatremia as well as in other pathologies leading to brain edema. Together with other osmolytes, taurine corrects cell shrinkage, preventing mysfunction of organelles and apoptosis. Swelling corrective taurine efflux occurs through a leak pathway, likely formed by LCRR8 protein isoforms. Shrinkage-activated influx comes largely by the increased activity of the Na+/Cl−-dependent transporter. The brain taurine pool results from the equilibrium between (i) dietary intake and active transport into the cell, (ii) synthesis in the brain itself or import of that synthesized elsewhere, and (iii) leak and posterior excretion. The interplay between these elements preserves brain taurine homeostasis in physiological conditions and permits the proper adjustments upon deviations of normal in the internal/external environment.

Thrombin effect increasing swelling-induced glutamate efflux was examined in cultured cortical astrocytes, cerebellar granule neurons (CGN), hippocampal and cortical neurons. Hypotonic glutamate efflux (monitored by D-[(3)H]aspartate) from cortical astrocytes was increased by thrombin (5 U/mL) to reach 16% of the cell pool in 5 min. Thrombin had lower effects in CGN, and marginal effects in hippocampal and cortical neurons. These differences were related to the magnitude of thrombin-evoked cytosolic calcium rise. The protease-activated receptor 1 is expressed in astrocytes and neurons. In astrocytes exposed to chemical ischemia (sodium iodoacetate plus sodium azide) D-[(3)H]aspartate release showed a first phase (20-40 min) of initial low efflux which progressively increases; and a second phase (40-60 min) of larger efflux coincident with cell swelling. Efflux at the first phase was 52% inhibited by the glutamate transporter blocker DL-threo-β-benzyloxyaspartic-acid (TBOA) and 11% by the volume-sensitive pathway blocker phloretin. At the second phase, efflux was reduced 52 and 38% respectively, by these blockers. In CGN D-[(3)H]aspartate efflux increased sharply and then decreased. This efflux was 32% reduced by calcium omission, 46% by TBOA and 32% by 4-[(2-butyl-6,7dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1H-inden-5-yl)oxy] butanoic-acid. Thrombin enhanced this release by 32%. Ischemic treatment increased astrocyte mortality from 4% in controls to 39 and 61% in ischemia and ischemia plus thrombin, respectively. Cell death was prevented by phloretin. CGN viability was unaffected by the treatment. These results suggest that coincidence of swelling and thrombin during ischemia elevates extracellular glutamate prominently from astrocyte efflux, which may endanger neurons in vivo.

Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

Brain edema is a severe clinical complication in a number of pathologies and is a major cause of increased morbidity and death. The swelling of astrocytes caused by a disruption of water and ion homeostasis, is the primary event contributing to the cytotoxic form of brain edema. Astrocyte cytotoxic swelling ultimately leads to transcapillary fluxes of ions and water into the brain parenchyma. This review focuses on the implication of transporters and channels in cytotoxic astrocyte swelling in hyponatremia, ischemia, trauma and hepatic encephalopathy. Emphasis is put on some salient features of the astrocyte physiology, all related to cell swelling, i.e. predominance of aquaporins, control of K(+) homeostasis and ammonia accumulation during the brain ammonia-detoxifying process.

Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

With-no-lysine kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3–18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3–18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18a+22, 18b, and 18b+22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.

The decline of taurine content during brain maturation as well as the consequences of taurine deficiency disturbing brain development, suggest its involvement in basic processes of developing brain cells. If taurine participates in cell protection, differentiation or proliferation in the developing brain is as yet unclear. Extensive and solid evidence supports taurine cytoprotective actions, directly or indirectly related to an antioxidant effect. Since redox status and oxidative stress are now implicated in signalling processes regulating cell differentiation and proliferation, the question is raised of whether the taurine antioxidant activity is on the basis of its requirement during brain development.

Cerebral water control is critical to maintain neuronal excitability, and to prevent injuries derived from brain swelling or shrinkage. The influence of aquaporins (AQPs) in the balance of water distribution between intracranial compartments is getting much experimental support. The importance of AQPs in fluid clearance during vasogenic brain edema seems well established but their role in cytotoxic swelling and in brain cell shrinkage is not known in detail. The main AQPs function as water channels anticipates their influence on cell volume changes as well as on the mechanisms of volume recovery, which include notably the osmolyte translocation across the cell membrane. Osmolyte fluxes permit the reestablishment of an osmotic balance and volume recovery in anisosmotic-elicited cell volume changes, but are also causal factors per se of brain cell swelling or shrinkage in pathological situations. This review aims to inform on the so far described functional interactions between AQPs and osmolyte fluxes and their volume-sensitive pathways. It also points to the coincidence of AQPs and activation of osmolyte fluxes in physiological and pathological conditions and to the importance of finding possible functional links between these two events, thus enlarging the possibilities via AQP manipulations, to prevent the adverse consequences of cell volume changes in brain.

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl− current ( $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ ) and increased RVD rate. These effects were reduced (50–65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30–50%, respectively, the thrombin effects on hyposmotic taurine efflux, $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC–Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and $$ {\text{ICI}}^{ - }_{{{\text{swell}}}} $$ under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of (3)H-glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5-5 U/mL) elicited small (3)H-glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of (3)H-glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca(2+) (Ca(2+) (i)) concentration ([Ca(2+)](i)). Preventing Ca(2+) (i) rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%-22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca(2+)-sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC(50) 15.8 microM), DCPIB (IC(50) 4.2 microM), and tamoxifen (IC(50) 6.6 microM. These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma.

Cells respond to a condition of hypo-osmolality by rapid swelling followed by an adaptive response that tends to recover the normal cell volume despite the persistence of the hypo-osmotic condition. This is an active process accomplished by the extrusion of intracellular osmolytes, essentially K+, Cl-, and small organic molecules. This regulatory process operates through a chain of events that essentially consists of a sensor or sensing mechanism to detect changes in cell volume, a signaling cascade to amplify the sensing signal and orient it to activate pathways for osmolyte extrusion, and a memory of the original cell volume, which sets the timing for inactivation of the volume-regulatory process. This article presents a brief overview of recent progress in these different aspects of the volume-regulatory process, including (1) the mechanisms and/or candidate molecules serving the role of volume sensors, (2) the osmosignaling network and the interplay and hierarchy of the different elements in this chain, and (3) the nature and properties of the osmolyte extrusion pathways. Emphasis is placed on some of the main unsolved questions concerning different aspects of the volume-regulatory process. Recent findings regarding the effect of hypo-osmolality on synaptic function are briefly discussed in terms of the possible molecular basis for the neurologic symptoms induced by hyponatremia.

Background/Aims: Neural stem/ progenitor cells (NPCs) endure important changes in cell volume during growth, proliferation and migration. As a first approach to know about NPC response to cell volume changes, the Regulatory Volume Decrease (RVD) subsequent to hypotonic swelling was investigated. Methods: NPCs obtained from the mesencephalon and the subventricular zone of embryonic and adult mice, respectively, were grown and cultured as neurospheres. Cell volume changes were measured by large-angle light-scattering and taurine efflux by [3H]-taurine. Expression of genes encoding molecules related to RVD was analysed using a DNA microarray obtained from NPC samples. Results: Embryonic and adult NPCs exposed to osmolarity reduction (H15, H30, H40) exhibited rapid swelling followed by RVD. The magnitude, efficiency and pharmacological profile, of RVD and of [3H]-taurine osmosensitive efflux were comparable to those found in cultured brain cells, astrocytes and neurons. The relative expression of genes encoding molecules related to volume regulation, i.e. K+ and Cl- channels, cotransporters, exchangers and aquaporins were identified in NPCs. Conclusion: NPCs show the ability to respond to hypotonic-evoked volume changes by adaptative recovery processes, similar to those found in other cultured brain cells. Genes related to molecules involved in RVD were found expressed in NPCs.

Osmolarity reduction (20%) elicited 3 H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na + -dependent and La 3 + -, Gd 3 + - and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca 2 + ([Ca 2 + ]i) rise with contributions from external Ca 2 + influx and internal Ca 2 + release, probably through the mitochondrial Na + -Ca 2 + exchanger; and (iii) activation of a [Ca 2 + ]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca 2 + ]i rise by preventing depolarization with La 3 + , and by the inhibitory effects of Ca 2 + -free medium (EGTA; 50%), CGP37157 (the mitochondrial Na + -Ca 2 + exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na + omission, 75% by La 3 + , 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca 2 + release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K + -stimulated NE release, studied in parallel, was unaffected by Na + omission, or by La 3 + , Gd 3 + or ruthenium red. It was fully dependent on external Ca 2 + , insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K + -evoked depolarization and [Ca 2 + ]i rise mechanisms, are able to trigger a depolarization-dependent, Ca 2 + -dependent and TeTX-sensitive mechanism for neurotransmitter release.

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Reducing osmolarity by 35% increased (3)H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10-100 microM; EC(50) 1.5 microM) increased taurine efflux up to 10%, and decreased the set point for hyposmotically stimulated taurine release (HTR). ATP potentiation was mimicked by UTP, reduced by addition of suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and unaffected by ADP, beta,gamma-methylene-ATP (beta,gamma-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y(2) and P2Y(4) metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca(2+)] ([Ca(2+)](i)) markedly, but did not increase taurine release. HTR was independent of external Ca(2+) but was reduced (by 56-59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca(2+) stores, or phospholipase C (PLC) inhibition. Blockade of calmodulin (CaM) or calmodulin kinase II (CaMKII) reduced HTR by 54% and 76%, respectively. The ATP-mediated potentiation was prevented fully by all these treatments. HTR was reduced by 30-50% by blockers of protein tyrosine kinases (AG18), phosphoinositide 3-kinase (PI3K) (wortmannin), p21rho (toxin B), p21rho-kinase (Y27632) and the stress-activated kinase p38 (PD169316). ATP-mediated potentiation was reduced similarly by these blockers. Simultaneous inhibition of PI3K and CaMKII abolished HTR. Altogether, these results suggest a modulatory effect of ATP, probably exerted by a potentiation of the Ca(2+)-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca(2+)](i) increase, resulting from Ca(2+) released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca(2+)/ATP effect operates only when the Ca(2+)-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.

Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter's reduction when EGFR phosphorylation was blocked by 50 microM AG213 or AG112 and, conversely, its potentiation by EGF (200 ng/ml). The relationship between hyposmotically induced taurine efflux and reduced osmolarity showed saturable kinetics, following a sigmoidal function. EGF shifted the relationship to the left, implying an increase in sensitivity to hyposmolarity. EGF increased taurine efflux only marginally under isosmotic conditions. EGF and agglutinins also potentiated the hyposmotically induced release of 86Rb but, in contrast to taurine, the efflux was unaffected by EGFR inhibition. EGF and agglutinins markedly increased 86Rb release under isosmotic conditions. The EGF-evoked isosmotic 86Rb release, together with the hyposmotic efflux, accounted fully for the observed potentiation by EGF, raising the possibility of an overlapping of these two effects, rather than a true potentiation. A link between EGFR, phosphatidylinositide-3-kinase (PI3K) and hyposmotically induced taurine (but not 86Rb) release is suggested by the increase in PI3K activity elicited by hyposmolarity, which was fully prevented by EGFR inhibition, and by a marked reduction of hyposmotically induced taurine (but not 86Rb) release, by wortmannin. The present findings, together with results showing EGF activation of osmosensitive Cl- fluxes implicate EGFR as an important modulator of osmolyte efflux pathways.

External osmolarity reduction (20%) led to labelled glutamate, GABA and taurine release from rat brain cortical synaptosomes. A Cl--independent, Na+-dependent, La3+-sensitive and tetrodotoxin (TTX) reduced depolarization of synaptosomes occurred upon hyposmolarity, suggestive of Na+ entry through nonselective cation channels. This depolarization, together with cytosolic Ca2+ ([Ca2+]I) increase, resulted in exocytosis, monitored by FM1-43. The release fraction resulting from these phenomena was estimated, by its decrease, by La3+, EGTA-AM and tetanus toxin (TeTX), as 34-44% for glutamate, 21-29% for GABA and 18-22% for taurine. Protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate (PMA) increased the hyposmolarity-elicited exocytosis and this activation increased glutamate (80%), GABA (51%) and taurine (42%) hyposmotic efflux. Inhibition by chelerythrine reduced glutamate, GABA and taurine efflux by 64%, 50% and 24%, respectively. The Na+-dependence of amino acid release (glutamate 63%, GABA 46% and taurine 29%) may result from both, prevention of the depolarization-exocytosis efflux, and blockade of the carrier reversal operation. Carrier blockade by dl-threo-beta-benzyloxy aspartate (TBOA) and NO-711 resulted in 37% and 28% reduction of glutamate and GABA release, respectively. Contribution of the osmolyte leak pathway to amino acid release, estimated by the influence of Cl- (NPPB) and tyrosine kinase (AG18) blocker, was up to 55% for taurine, but only 10-18% for GABA, with apparently no contribution for glutamate. The predominant osmolyte-type mechanism of taurine release suggest its function in volume control in nerve endings, while glutamate and GABA respond to events concurrent with hyposmolarity by a neurotransmitter-like release mechanism. The hyposmolarity-induced amino acid efflux from nerve endings may have consequences for neuronal excitability during hyponatremia.

Hyposmolarity (-30%) in cultured cerebellar astrocytes raised cytosolic Ca2+ concentration ([Ca2+]i) from 160 to 400 nM and activated the osmosensitive taurine release (OTR) pathway. Although OTR is essentially [Ca2+]i-independent, further increase in [Ca2+]i by ionomycin strongly enhanced OTR, with a more robust effect at low and mild osmolarity reductions. Ionomycin did not affect isosmotic taurine efflux. OTR was decreased by tyrphostin A25 and increased by ortho-vanadate, suggesting a modulation by tyrosine kinase or phosphorylation state. Inhibition of phosphatidylinositol-3-kinase activity by wortmannin markedly decreased OTR and the ionomycin increase. Conversely, OTR and the ionomycin effect were independent of ERK1/ERK2 activation. OTR and its potentiation by ionomycin differed in their sensitivity to CaM and CaMK blockers and in the requirement of an intact cytoskeleton for the ionomycin effect, but not for normal OTR. Changes in the actin cytoskeleton organization elicited by hyposmolarity were not observed in ionomycin-treated cells, which may permit the operation of CaM/CaMK pathways involved in the OTR potentiation by [Ca2+]i rise. OTR potentiation by [Ca2+]i requires the previous or simultaneous activation/operation of the taurine release mechanism and is not modifying its set point, but rather increasing the effectiveness of the pathway, resulting in a more efficient volume regulation. This may have a beneficial effect in pathological situations with concurrent swelling and [Ca2+]i elevation in astrocytes.

Taurine efflux occurs in association with cell swelling in both hyposmotic and isosmotic conditions and during cell shrinkage in apoptotic death. Release occurs through a leak pathway, is largely Ca2+-independent and is sensitive to Cl- channel blockers. Taurine efflux elicited by hyposmolarity is reduced or suppressed by tyrosine kinase blockers and increased by tyrosine phosphatase inhibitors. The specific kinases involved are still unknown and may be different in the various cell types. Non-receptor and scr-related protein kinases have been identified in some cells as elements that directly phosphorylate the taurine efflux pathway. Possible tyrosine kinase targets are the phosphinositide kinase (PI3K), which if inhibited, prevents the osmosensitive taurine efflux in brain cells, or the small GTP-binding proteins associated with remodeling of the cytoskeleton. The similar effects of tyrosine kinase modulators on volume-activated taurine fluxes and Cl- currents are suggestive of either a shared translocation pathway or a common step in the signaling network. The effects of tyrosine kinases on taurine efflux activated in isosmotic swelling and in the release associated with apoptosis are essentially unexplored.

The purpose of this study to assess the effect of the formula taurine/diltiazem/vitamin E on the progression of visual field loss in retinitis pigmentosa. A double blind, placebo controlled study in 62 patients: visual field threshold values were obtained in a Humphrey Field Analyzer from center (30°) and periphery (30–60°), every 4 months during 3-year follow-up. Data were analyzed by univariate regression, with slopes obtained from the best fit lines. Based on slope values, three groups of patients were identified as those showing negative, positive, or zero slope: ≥-1 to ≤ + 1. In controls (32 patients), at central area, the distribution in negative, zero, or positive slope was, respectively, 16 (50%),11 (35%), and 5 (15%). In the treated group (30 patients) this distribution was 6 (20%) negative, 17 (53%) zero, and 7 (23%) positive slope. In periphery, 16 control patients were distributed as 11 (69%) negative, 4 (25%) zero, and 1 (6%) positive slope. In the treated group (17 patients), the distribution was opposite: 1 (6%) negative, 7 (41%) zero, and 9 (53%) positive slope. Nineteen patients receiving treatment up to 6 years showed similar distribution by slope values. Eight out of 9 patients switched from placebo (2 years) to treatment (2–3 years), showed improving changes in their slope values. A beneficial effect of the treatment decreasing the rate of visual field loss was observed, likely through a protective action from free radical reactions in affected photoreceptors.

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl− channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.

A 30% decrease in osmolarity stimulated 3H-taurine, 3H-GABA and glutamate (followed as 3H-D-aspartate) efflux from rat hippocampal slices. 3H-taurine efflux was activated rapidly but inactivated slowly. It was decreased markedly by 100 microM 5-nitro-(3-phenylpropylamino)benzoic acid (NPPB) and 600 microM niflumic acid and inhibited strongly by tyrphostins AG18, AG879 and AG112 (25-100 microM), suggesting a tyrosine kinase-mediated mechanism. Hyposmolarity activated the mitogen-activated protein kinases (MAPK) extracellular-signal-related kinase-1/2 (ERK1/ERK2) and p38, but blockade of this reaction did not affect 3H-taurine efflux. Hyposmosis also activated phosphatidylinositide 3-kinase (PI3K) and its prevention by wortmannin (100 nM) essentially abolished 3H-taurine efflux. 3H-taurine efflux was insensitive to the protein kinase C (PKC) blocker chelerythrine (2.5 microM) or to cytochalasin E (3 microM). The release of 3H-GABA and 3H-D-aspartate occurred by a different mechanism, characterized by rapid activation and inactivation, insensitivity to NPPB, niflumic acid, tyrphostins or wortmannin. 3H-GABA and 3H-D-aspartate efflux was not due to external [NaCl] decrease, cytosolic Ca2+ increase or depolarization, or to reverse operation of the carrier. This novel mechanism of amino acid release may be mediated by Ca2+-independent exocytosis and modulated by PKC and actin cytoskeleton disruption, as suggested by its inhibition by chelerythrine and potentiation by 100 nM phorbol-12-myristate-13 acetate (PMA) and cytochalasin E. GABA and glutamate osmosensitive efflux may explain the hyposmolarity-elicited increase in amplitude of inhibitory and excitatory postsynaptic potentials in hippocampal slices as well as the hyperexcitability associated with hyponatraemia.

Taurine was previously reported to increase the proliferation of neural precursor cells (NPCs) from subventricular zone of the mouse brain. The results of a study that aimed to understand the mechanisms of this effect are presented here. Because taurine was not found in NPC nuclei, direct interactions with nuclear elements seem unlikely. A gene expression profile analysis indicated that genes that are regulated by taurine have roles in i) proliferation, including the Shh and Wnt pathways; ii) cellular adhesion; iii) cell survival; and iv) mitochondrial functioning. Cell cycle analysis of propidium iodide and CFSE-labeled cells using flow cytometry revealed an increase in the number of cells in the S-phase and a decrease in those in the G0/G1 phase in taurine-treated cultures. No changes in the length of the cell cycle were observed. Quantification of the viable, apoptotic, and necrotic cells in cultures using flow cytometry and calcein-AM, annexin-V, and propidium iodide staining showed reductions in the number of apoptotic and necrotic cells (18% to 11% and 13% to 10%, respectively) and increases in the number of viable cells (61% to 69%) in the taurine-treated cultures. Examination of the relative mitochondrial potential values by flow cytometry and rhodamine123 or JC-1 staining showed a 44% increase in the number of cells with higher mitochondrial potential and a 38% increase in the mitochondrial membrane potential in taurine cultures compared with those of controls. Taken together, the results suggest that taurine provides more favorable conditions for cell proliferation by improving mitochondrial functioning.

K+-Cl− cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K+ diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K+ diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.

Brain cell swelling compromises neuronal function and survival by the risk of generation of ischemia episodes as compression of small vessels occurs due to the limits to expansion imposed by the rigid skull. External osmolarity reductions or intracellular accumulation of osmotically active solutes result in cell swelling which can be counteracted by extrusion of osmolytes through specific efflux pathways. Characterization of these pathways has received considerable attention, and there is now interest in the understanding of the intracellular signaling events involved in their activation and regulation. Calcium and calmodulin, phosphoinositides and cAMP may act as second messengers, carrying the information about a cell volume change into signaling enzymes. Small GTPases, protein tyrosine kinases and phospholipases, also appear to be part of the signaling cascades ultimately modulating the osmolyte efflux pathways. This review focus on i) the influence of hyposmotic and isosmotic swelling on these signaling events and molecules and ii) the effects of manipulating their function on the osmolyte fluxes, particularly K+, CI− and amino acids, and on the consequent efficiency of cell volume adjustment.

Cell shrinkage is a distinctive feature of apoptotic death, but the mechanisms leading to cell volume loss are unclear at present. Activation of pathways extruding intracellular osmolytes such as K+, Cl- and organic molecules may be part of these mechanisms. This was examined in the present work measuring the release of taurine, gamma-amino-butyric acid (GABA) and glutamate in cerebellar granule neurons cultured in conditions resulting in apoptotic death after 4-7 days in vitro (DIV). The basal release of [3H]taurine from cells started to increase (38%) after 3 DIV and reached a maximal enhancement (250%) at 5 DIV. The increase in taurine efflux closely followed the occurrence of apoptotic death markers such as caspase induction and chromatin condensation. The efflux of glutamate (traced as D-aspartate) and [3H]GABA also increased but notably less than that of taurine (90% and 75%, respectively) at 5 DIV. Taurine release associated with apoptosis was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), blockers of the diffusive pathway activated during cell volume regulation in hyposmotic conditions. Taurine efflux was increased in Cl(-)-free (replaced by gluconate) and decreased in Na+-free media. Blockers of the energy-dependent glutamate and taurine carriers, dihydrokainate and guanidinoethane sulfonate, respectively, did not affect the release associated with apoptosis. These results implicate taurine in the mechanism of cell shrinkage during apoptosis.

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments 86Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased 86Rb efflux rate from 0.015 min−1 to a maximal of 0.049 min−1 and a net release of 30%. This osmosensitive efflux was inhibited by Ba2+ (0.028 min−1), quinidine (0.024 min−1), and charybdotoxin (0.040 min−1), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased 86Rb efflux to a maximal rate constant of 0.056 min−1 and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic 86Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect 86Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor 86Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased 86Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated 86Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 μM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that 86Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin. J. Neurosci. Res. 57:350–358, 1999. © 1999 Wiley-Liss, Inc.

A decrease in intracellular ionic strength appears involved in the activation of swelling-elicited 3H-taurine efflux in cortical cultured astrocytes. Hyposmotic (50%) or isosmotic urea-induced swelling leading to a decrease of intracellular ionic strength, activated 3H-taurine efflux from a rate constant of about 0.008 min(-1) to 0.33 min(-1) (hyposmotic) and 0.59 min(-1) (urea). This efflux rate was markedly lower (maximal 0.03 min(-1)) in isosmotic swelling caused by K+ accumulation, where there is no decrease in ionic strength, or in cold (10 degrees C) hyposmotic medium (maximal 0.18 min(-1)), where swelling is reduced and consequently intracellular ionic strength is less affected. Also, astrocytes pretreated with hyperosmotic medium, which recover cell volume by ion accumulation, did not release 3H-taurine when they swelled by switching to isosmotic medium, but when volume was recovered by accumulation of urea, taurine release was restored. These results point to a key role of ionic strength in the activation of osmosensitive 3H-taurine efflux. In contrast, its inactivation was independent of the change in ionic strength but appears related to the reduction in cell volume after swelling, since despite the extent or direction of the change in ionic strength, the 3H-taurine efflux did not inactivate in isosmotic KCl-elicited swelling when cell volume did not recover nor in hyposmotic swelling when RVD was impaired by replacing NaCl in the medium by permeant osmolytes.

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl−-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl− and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]o, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels. J. Neurosci. Res. 53:626–635, 1998. © 1998 Wiley-Liss, Inc.

Cultured cerebellar astrocytes exhibit regulatory volume decrease (RVD) after anisosmotic swelling by extrusion of potassium (K), chloride (Cl), and organic osmolytes. The volume decay curve after swelling in 50% hyposmotic solutions has a first-order rate constant k min-1 of 0.18 and 70% cell volume recovery is attained after 15 min. Replacing NaCl in the hyposmotic medium by sugars and polyalcohols markedly accelerated RVD increasing k to 0.37–0.39 min-1 and full recovery in 3–5 min. NaCl removal increased (80%) K efflux (measured as 86Rb), delayed the inactivation of Cl efflux (measured as 125I) and enhanced taurine and D-aspartate release by 25%. These effects appear due to NaCl removal rather than to specific actions of the organic molecules. Replacing Na by other cations or Cl by gluconate increased k to 0.22 min-1 and 0.26 min-1, respectively, and 86Rb efflux by 4–23% and 39, respectively. It is concluded that the electrochemical gradient after NaCl removal increasing the K driving force, delaying the equilibrium of Cl and increasing amino acid efflux, accounts for the observed RVD acceleration. Other consequences of NaCl removal possibly modulating the K efflux pathway include changes in the surface charge screening, decreased ion strength, a rise in [Ca]i and a decrease in intracellular Na. J. Neurosci. Res. 53:195–202, 1998. © 1998 Wiley-Liss, Inc.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

All cells including neurons and glial cells are able to keep their volume within a very limited range. The volume regulatory mechanism involves changes in the concentration of osmolytes of which taurine appears to be of particular importance in brain cells. Swelling in brain cells may occur as a result of depolarization or small fluctuations in osmolarity. In isolated brain cells these conditions will always lead to a release of taurine, the time course of which is superimposable on that of the volume regulatory decrease which follows the initial cell swelling. The mechanism responsible for taurine release associated with swelling has not been fully elucidated but a large body of evidence seems to exclude participation of the taurme high affinity carrier. Using a number of inhibitors of anion exchangers it has been demonstrated that both volume regulation and taurine release in brain cells are inhibited by these drugs, implicating an anion channel in the process. It has be controversial issue as to whether or not taurine release is Ca++ dependent. Recent evidence appears to suggest that the release process is not associated with Ca++ or Ca++ channels. It is, however, quite possible that the swelling process may involve the Ca++ calmodulin system or other second messengers. Taurine also contributes to volume regulation after shrinkage of brain cells, in this case by increasing its intracellular concentration. This change is accomplished byan upregulation of the Na+/taurine cotransporter, together with reduced passive fluxes and increased endogenous synthesis.

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

Microwell cultures of dissociated tissue from prenatal rat hippocampus and cerebral cortex as well as from early postnatal cerebellum were used for quantification of neuronal aggregation, process extension, and fasciculation. It was shown that the cells in culture from these different brain regions developed differently with regard to both architecture and rate of differentiation. The effect of a polyclonal antibody against the neural cell adhesion molecule (NCAM), the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA), and the neurotoxin acrylamide on aggregation and fiber formation was investigated. Exposure to the NCAM antibody led to formation of fewer but larger aggregates and stimulated the morphological development of the cultures. Acrylamide affected aggregate formation, leading to smaller but more numerous aggregates, and it inhibited process extension and fasciculation. Treatment with NMDA affected process formation and led to formation of more numerous but smaller aggregates. Some of these effects were strongly tissue-dependent. Thus, large differences were seen regarding the effect of the NCAM antibody on aggregation and process extension in cultures from the different brain areas. The culture systems appear to represent convenient and reliable screening tools to study the influence of putative morphoregulatory substances on cell-cell interactions during early neuronal development.