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

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

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

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

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Isotonic intravenous maintenance fluid reduces hospital acquired hyponatremia in young children with central nervous system infections.

OBJECTIVE: To find the appropriate type of intravenous fluid (isotonic vs. hypotonic saline in 5 % dextrose) for empiric maintenance fluid therapy in children with central nervous system (CNS) infections that reduces the incidence of hospital acquired hyponatremia. METHODS: This blinded randomized controlled trial included hospitalized children aged 3 mo to 5 y with suspected CNS infections requiring intravenous maintenance fluid for at least 24 h. The subjects were randomized to receive 0.9 % saline (Group-A), 0.45 % saline (Group-B) and 0.18 % saline (Group-C) at standard maintenance rate. The outcome measures were proportion of patients developing hyponatremia (serum sodium < 135 mmol/L) after 24 h and serum sodium values at 6, 12, 18, 24 h of receiving maintenance fluids. RESULTS: Of the 92 patients enrolled, 31, 30 and 31 patients were randomized to Group A, B and C, respectively. Majority (60.7 %) of the patients in Group-C developed hyponatremia compared with 7.1 % of the children in Group-A and 46.1 % in Group-B. During first 24 h of fluid administration successive fall in the serum sodium values was observed in patients receiving hypotonic fluids. The risk of developing hyponatremia was nearly 6½ (95 % confidence interval (CI) 1.6-26) to 8.5 (95 % CI 2.16-33.39) times more in patients who received hypotonic saline compared to those who received isotonic saline. CONCLUSIONS: Administration of 0.9 % saline in 5 % dextrose as intravenous maintenance fluid in children with CNS infection leads to significantly less incidence of hyponatremia when compared to that with hypotonic fluids.

Erythrocyte swelling and membrane hole formation in hypotonic media as studied by conductometry.

Hypoosmotic swelling of erythrocytes and the formation of membrane holes were studied by measuring the dc conductance (G). In accordance with the theoretical predictions, these processes are manifested by a decrease in G followed by its increase. Thus, unlike the conventional osmotic fragility test, the proposed methodological approach allows investigations of both the kinetics of swelling and the erythrocyte fragility. It is shown that the initial rate of swelling and the equilibrium size of the cells are affected by the tonicity of a hypotonic solution and the membrane rheological properties. Because the rupture of biological membranes is a stochastic process, a time-dependent increase in the conductance follows an integral distribution function of the membrane lifetime. The main conclusion which stems from reported results is that information about rheological properties of red blood cell (RBC) membranes and the resistivity of RBCs to a certain osmotic shock may be extracted from conductance signals.

Membrane tension homeostasis of epithelial cells through surface area regulation in response to osmotic stress.

Osmotic stress poses one of the most fundamental challenges to living cells. Particularly, the largely inextensible plasma membrane of eukaryotic cells easily ruptures under in-plane tension calling for sophisticated strategies to readily respond to osmotic stress. We describe how epithelial cells react and adapt mechanically to the exposure to hypotonic and hypertonic solutions in the context of a confluent monolayer. Site-specific indentation experiments in conjunction with tether pulling on individual cells have been carried out with an atomic force microscope to reveal spatio-temporal changes in membrane tension and surface area. We found that cells compensate for an increase in lateral tension due to hypoosmotic stress by sacrificing excess of membrane area stored in protrusions and invaginations such as microvilli and caveolae. At mild hypotonic conditions lateral tension increases partly compensated by surface are regulation, i.e. the cell sacrifices some of its membrane reservoirs. A loss of membrane-actin contacts occurs upon exposure to stronger hypotonic solutions giving rise to a drop in lateral tension. Tension release recovers on longer time scales by an increasing endocytosis, which efficiently removes excess membrane from the apical side to restore the initial pre-stress. Hypertonic solutions lead to shrinkage of cells and collapse of the apical membrane onto the cortex. Exposure to distilled water leads to stiffening of cells due to removal of excess surface area and tension increase due to elevated osmotic pressure across the plasma membrane.

Control of ion transport by mitochondrion-rich chloride cells of eurythermic teleost fish: Cold shock vs. cold acclimation.

Seawater-acclimated eurythermic mummichogs (Fundulus heteroclitus L.) were acclimated to cold and warm conditions (5 and 20 °C, 4 weeks). Opercular epithelia (OE) from 20 °C-acclimated animals, containing numerous mitochondrion-rich chloride cells were mounted in Ussing-style membrane chambers, cooled to 16, 13, 10, 5 and 2.5 °C, then subjected to hypotonic shock that normally inhibits Cl(-) secretion (as short-circuit current, I(sc)). Cold exposure to 10 °C slowed Cl(-) secretion (Q(10)=1.62 ± 0.204 95% CI) and OEs responded rapidly and reversibly to hypotonic shock, but below 8.0 °C a sharp decrease (Q(10)=5.63 ± 0.736) occurred and the tissue was unresponsive to hypotonicity. By immunocytochemistry, Focal Adhesion Kinase (FAK) phosphorylated at tyrosine-407 (pY(407)) colocalized with CFTR in apical membrane and dephosphorylated with hypotonic shock at 20 °C but failed to dephosphorylate at 5 °C, while opercular epithelia from cold-acclimated fish at 5 and 20 °C responded normally to hypotonic shock. Cold-shock of warm-acclimated OEs also stimulated covering over of mitochondrion- rich cell apical crypts, detected by SEM. Cold-acclimation increased C18:1 and decreased C18:0 fatty acids in liver, indicating homeoviscous adaptation. Eurythermic fish acclimate osmoregulatory systems to cold by maintaining membrane fluidity and preserving complex transport regulation pathways.

Involvements of the ABC protein ABCF2 and α-actinin-4 in regulation of cell volume and anion channels in human epithelial cells.

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin-binding proteins are involved in the RVD process. In the present study, an involvement of an actin-binding protein, α-actinin-4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA-mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 × g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4-ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH(2) -terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume-sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl(-) efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole-cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA-mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling-induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells.

Neurites outgrowth and amino acids levels in goldfish retina under hypo-osmotic or hyper-osmotic conditions.

Amino acids are known to play relevant roles as osmolytes in various tissues, including the retina. Taurine is one of these active molecules. In addition, taurine stimulates outgrowth from the goldfish retina by mechanisms that include extracellular matrix, calcium fluxes and protein phosphorylation. The present report aims to explore the effect of medium osmolarity on goldfish retinal outgrowth and the possible modifications produced by changing eye osmolarity on amino acid levels in the retina. Goldfish retinal explants were obtained 10 days after crush of the optic nerve and cultured under iso-, hypo- or hyper-osmotic conditions. Hypo-osmotic medium was prepared by diluting the solutions 10% twice, preserving fetal calf serum concentration. Hyper-osmotic medium was done by adding 50 or 100 mM urea or mannitol. Evaluation of length and density of neurites was performed 5 days after plating. Outgrowth was reduced in hypo- and in hyper-osmotic conditions. Taurine, 4 mM, increased length and density of neurites in iso-osmotic, and produced stimulatory effects under both hyper-osmotic conditions. The in vivo modification of osmolarity by intraocular injection of water or 100 mM urea modified levels of free amino acids in the retina. Taurine and aspartate retinal levels increased in a time-dependent manner after hypo- and hyper-osmotic solution injections. Serine, threonine, arginine, γ-aminobutyric acid, alanine and tyrosine were elevated in hyper-osmotic conditions. Outgrowth in vitro, after in vivo osmolarity changes, was higher in the absence of taurine, but did not increase in the presence of the amino acid. The fact that certain outgrowth took place in these conditions support that the impairment was not due to tissue damage. Rather, the effects might be related to the cascade of kinase events described during osmolarity variations. The time course under these conditions produced adjustments in ganglion cells probably related to taurine transporter, and phosphorylation of specific proteins.

OmpR may regulate the putative YehU/YehT two-component system in Salmonella enterica serovar Typhi under hypotonic growth condition.

Decreased expression (twofold) of a putative yehUTS operon of which yehUT encodes a putative YehU/YehT two-component system in the ompR mutant from Salmonella enterica serovar Typhi (S. Typhi) GIFU10007 under hypotonic growth condition was observed by qRT-PCR. Purified recombinant protein OmpR(His6) of GIFU10007 was shown to bind the upstream region of the yehU gene by the gel-shift assay. In addition, the yehT deletion mutant (ΔyehT) displayed differential expression (twofold or higher) of 26 genes under the condition by the DNA microarray analysis. Altogether, OmpR might regulate the YehUT system in S. Typhi under hypotonic growth condition.

Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension.

Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.

Shear mechanical force induces an increase of intracellular Ca2+ in cultured Merkel cells prepared from rat vibrissal hair follicles.

Merkel cells have been proposed to play a role in mechanical transduction of light touch in mammals. In the present study, Merkel cells were prepared from upper segments of rat vibrissal hair follicles and maintained in culture. Reponses of these cells to shear mechanical forces were examined by Ca(2+) imaging technique. Shear forces of ≥ 0.8 dyn/cm(2) that were delivered to the cells by the application of normal bath solution significantly increased intracellular Ca(2+) levels ([Ca(2+)](i)) in some of these cells, and up to 30% cells responded to 1.6 dyn/cm(2) shear force applied for 20 s. Gd(3+) (100 µM), a compound widely used to inhibit mechanically activated channels, abolished shear force-induced increases of [Ca(2+)](i) in these cells. Reduction of extracellular Ca(2+) concentration from 2 mM to 0.2 mM also abolished shear force-induced increases of [Ca(2+)](i) in these cells. In addition to shear force, we found that many shear force-responding cells also responded to hypotonic solution. However, the response to hypotonic solution was not abolished by Gd(3+) (100 µM). We also found that all shear force-responding cells responded to ATP (100 µM) with large increases of [Ca(2+)](i). The responses to ATP remained in the presence of Gd(3+). Taken together, our results suggest that Merkel cells in culture are sensitive to shear force stress, osmotic, and chemical stimuli and that shear force-induced increases of [Ca(2+)](i) may be mediated by the activation of mechanically activated channels.

LSm4 associates with the plasma membrane and acts as a co-factor in cell volume regulation.

ICln is a ubiquitous, multifunctional protein with functions in cell volume regulation and RNA processing, and is thus part of an intricate protein network critically involved in the homoeostasis of cells. To better understand this vital protein network in health and disease it is fundamental to characterize the interactions between the physiological pathways in which ICln is involved, as well as the spatio-temporal regulation of these interactions. In this study, we focused on the interaction between the two best studied pathways in which ICln is involved--regulatory volume decrease and RNA processing--and asked, whether or not the RNA processing factor and ICln interaction partner LSm4 may also have a function in cell volume regulation in NIH3T3 fibroblasts or HEK293 Phoenix cells. To address this question, we studied in isotonic and hypotonic conditions by FRET, biochemistry and electrophysiology, the intracellular distribution of the RNA processing factor LSm4, its interaction with ICln, as well as the involvement of LSm4 in the activation of the swelling dependent anion and osmolyte channel IClswell. In isotonic conditions, LSm4 associates with ICln, and the plasma membrane. Hypotonic cell swelling leads to the dissociation of LSm4 from the plasma membrane, and from ICln. Over-expression of LSm4 affects the translocation of ICln to the cell membrane and markedly inhibits the activation kinetics and current density of IClswell. These findings indicate that LSm4 not only acts in RNA processing, but also as a co-factor in cell volume regulation.

Dynamic effects of Hg2+-induced changes in cell volume.

Using a microfluidic volume sensor, we studied the dynamic effects of Hg2+ on hypotonic stress-induced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 muM HgCl2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na+ with impermeant NMDG+ in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg2+-induced excess swelling. The volume decrease in the presence of Hg2+ was inhibited by tetraethylammonium and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, blockers of K+ and Cl(-) channels, respectively, suggesting that part of the Hg2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steady-state volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.

Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells.

Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell types, impaired in the presence of BEL and DPI and following restoration of the cell volume. Thus, potentiation of the volume-sensitive taurine efflux pathway following inhibition of tyrosine phosphatase activity reflects increased arachidonic acid mobilization and ROS production for downstream signaling. Vanadate delays the inactivation of volume-sensitive taurine efflux in NIH3T3 cells, and this delay is impaired in the presence of DPI. Vanadate has no effect on the inactivation of swelling-induced taurine efflux in Ehrlich Lettre cells. It is suggested that increased tyrosine phosphorylation of regulatory components of NADPH oxidase leads to increased ROS production and a subsequent delay in inactivation of the volume-sensitive taurine efflux pathway and that NADPH oxidase or antioxidative capacity differ between NIH3T3 and Ehrlich Lettre cells.

Hypotonic swelling of salicylate-treated cochlear outer hair cells.

The outer hair cell (OHC) is a hydrostat with a low hydraulic conductivity of Pf=3x10(-4) cm/s across the plasma membrane (PM) and subsurface cisterna that make up the OHC's lateral wall. The SSC is structurally and functionally a transport barrier in normal cells that is known to be disrupted by salicylate. The effect of sodium salicylate on Pf is determined from osmotic experiments in which isolated, control and salicylate-treated OHCs were exposed to hypotonic solutions in a constant flow chamber. The value of Pf=3.5+/-0.5x10(-4) cm/s (mean+/-s.e.m., n=34) for salicylate-treated OHCs was not significantly different from Pf=2.4+/-0.3x10(-4) cm/s (mean+/-s.e.m., n=31) for untreated OHCs (p=.3302). Thus Pf is determined by the PM and is unaffected by salicylate treatment. The ratio of longitudinal strain to radial strain epsilonz/epsilonc=-0.76 for salicylate-treated OHCs was significantly smaller (p=.0143) from -0.72 for untreated OHCs, and is also independent of the magnitude of the applied osmotic challenge. Salicylate-treated OHCs took longer to attain a steady-state volume which is larger than that for untreated OHCs and increased in volume by 8-15% prior to hypotonic perfusion unlike sodium alpha-ketoglutarate-treated OHCs. It is suggested that depolymerization of cytoskeletal proteins and/or glycogen may be responsible for the large volume increase in salicylate-treated OHCs as well as the different responses to different modes of application of the hypotonic solution.

Stress-induced changes in neuronal Aquaporin-9 (AQP9) in a retinal ganglion cell-line.

The water channel, Aquaporin-9 (AQP9) is enriched in selected neuronal populations and is unique its ability to act as a lactate-glycerol channel supplying neurons with alternative fuel under ischaemic conditions. AQP9 was detected in RGC-5 cells, a retinal ganglion cell-line, primary RGCs, and retina by Western blotting, real-time PCR (RT-PCR) and immunohistochemistry. RGC-5 cells subjected to a hypotonic stress increased their cell volume that was blocked by known inhibitor of AQP9 (phloretin (40 microM)). RGC-5 cells subjected to hypoxia, showed an up-regulation in AQP9 expression as judged by Western blotting and RT-PCR. Similarly, hypotonic shock (50%) increased AQP9 expression as determined by RT-PCR. AQP9 is involved in energy balance as a glycerol-lactate channel and also appears to regulate cell volume in retinal ganglion neurons. This water channel may play a key role in retinal ganglion pathology.

Substantia nigra osmoregulation: taurine and ATP involvement.

An extracellular nonsynaptic taurine pool of glial origin was recently reported in the substantia nigra (SN). There is previous evidence showing taurine as an inhibitory neurotransmitter in the SN, but the physiological role of this nonsynaptic pool of taurine has not been explored. By using microdialysis methods, we studied the action of local osmolarity on the nonsynaptic taurine pool in the SN of the rat. Hypoosmolar pulses (285-80 mosM) administered in the SN by the microdialysis probe increased extrasynaptic taurine in a dose-dependent way, a response that was counteracted by compensating osmolarity with choline. The opposite effect (taurine decrease) was observed when osmolarity was increased. Under basal conditions, the blockade of either the AMPA-kainate glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dionine disodium or the purinergic receptors with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid modified the taurine concentration, suggesting that both receptors modulate the extrasynaptic pool of taurine. In addition, these drugs decreased the taurine response to hypoosmolar pulses, suggesting roles for glutamatergic and purinergic receptors in the taurine response to osmolarity. The participation of purinergic receptors was also supported by the fact that ATP (which, under basal conditions, increased the extrasynaptic taurine in a dose-dependent way) administered in doses saturating purinergic receptors also decreased the taurine response to hypoosmolarity. Taken together, present data suggest osmoregulation as a role of the nonsynaptic taurine pool of the SN, a function that also involves glutamate and ATP and that could influence the nigral cell vulnerability in Parkinson's disease.

Hyposmotic stimulation-induced nitric oxide production in outer hair cells of the guinea pig cochlea.

Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca(2+)-free solution. L-N(G)-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca(2+) response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca(2+)](i) increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca(2+) response via the NO-cGMP-PKG pathway by a feedback mechanism.

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.