Hypotonic Challenge Research Articles

WNK1 is required for proliferation induced by hypotonic challenge in rat vascular smooth muscle cells.

Hypotonic challenge evoked vascular cell proliferation through activation of volume-regulated Cl- channel (VRCC), leading to a decrease in the intracellular Cl- concentration ([Cl-]i). We hypothesize that the decrease in [Cl-]i may activate one or several Cl--sensitive kinases, resulting in a subsequent signaling cascade. In this study we demonstrated that WNK1, a Cl--sensitive kinase, was involved in VRCC-induced proliferative signaling pathway in A10 vascular smooth muscle cells in vitro. A10 cells were exposed to a hypotonic challenge (225 mosmol·kg-1·H20), which caused significantly increase in WNK1 phosphorylation without altering WNK1 protein expression. WNK1 overexpression significantly increased hypotonic-induced A10 cell proliferation, whereas silencing of WNK1 caused an opposite action. WNK1 mutation did not affect hypotonic-induced WNK1 phosphorylation and cell proliferation. Silencing of WNK1 caused cell cycle arrest at G0/G1 phase and prevented transition from G1 to S phase, whereas the WNK1 overexpression accelerated cell cycle transition from G1 to S phase. Silencing of WNK1 significantly inhibited cyclin D1/cyclin E1 expression and increased p27kip/p21cip expression. WNK1 overexpression significantly increased cyclin D1/cyclin E1 expression and reduced p27KIP/p21CIP expression. In addition, WNK1 knockdown or overexpression significantly attenuated or increased the hypotonic-induced phosphorylation of Akt and PI3K respectively.In conclusion, the reduction in [Cl-]i caused by hypotonic challenge-induced VRCC opening evokes WNK1 phosphorylation in A10 VSMCs, which mediates cell cycle transition from G0/G1 to S phase and proliferation through the PI3K-Akt signaling pathway.

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance.

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

Hypotonicity activates a voltage-dependent membrane conductance in N2a neuroblastoma cells

To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (Id) is insensitive to NPPB, a nonspecific blocker of Cl− channels, and intracellular Mg2+, which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na+ with NMDG+ reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells.

Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50% hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20min and followed, during the next 20min, by RVD of 60±7% of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82% of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole.

Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism.

ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production.

Osmotic water permeability diversification in primary trophoblast cultures from aquaporin 1-deficient pregnant mice.

Aquaporins (AQP) are water channel proteins, and some play an important role in maternal-fetal fluid exchange. The present study aimed to measure the osmotic water permeability in primary cultures of trophoblast cells from AQP1-deficient (AQP1(-/-) ) pregnant mice and to define the quantitative role of AQP1 in water transport across the trophoblast plasma membrane. Trophoblast cells were obtained from placental tissue cell culture of AQP1(-/-) pregnant mice and were characterized by cytokeratin 7 immunostaining. The expression of the AQP1 gene in trophoblast cells of wild-type (AQP1(+/+) ) mice was confirmed by immunofluorescence. The osmotic water permeability of trophoblast plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients. A primary cell culture system for trophoblasts was successfully established. Immunofluorescence showed the expression of AQP1 in the trophoblast cell membrane of AQP1(+/+) mice. The osmotic water permeability of AQP1(-/-) trophoblast cells was significantly lower than that in AQP1(+/+) trophoblast cells, in response to both hypotonic and hypertonic challenges. The results suggest an important role of AQP1-mediated plasma membrane water permeability in maternal-fetal fluid balance and also provide a potential direction for the identification of therapeutic targets for the treatment of abnormalities in amniotic fluid volume.

Characterisation Of Whole‐Cell Properties In Chondrocytes From An In Vitro Model Of Arthritis

Chondrocytes express a rich complement of ion channels with a range of functions including regulation of membrane potential (Vm) and cell volume. Transcript studies show changes in ion channel expression with osteoarthritis (OA) and in vitro studies show that pro‐inflammatory cytokines can induce cell apoptosis. Using an in vitro model of OA (DMEM + 10ng/ml TNFα and Il1β) we examined changes in cellular properties with patch‐clamp and intracellular Ca2+ measurements. Healthy chondrocytes have depolarised Vm (~‐10mV) so, using a whole cell tail current voltage protocol, we investigated if Vm was affected in our model of OA. Cells received a 1s hyperpolarising step with 10mV increments for 10 cycles. Cells from the model had a whole cell reversal potential (Vrev) 16±2mV more positive than healthy cells (n=5, p<0.05). Healthy cells had a greater whole‐cell conductance than cytokine treated cells (1.8±0.6nS vs 0.9±0.1nS; n=4 and 5, respectively; p<0.05). Given the positive shift in Vrev, we investigated Ca2+ handling of these cells using hypotonic challenge to increase intracellular Ca2+ entry. Healthy cells responded to the challenge with a peak Ca2+ increase of 115±15nM (n=16). When applied to cytokine treated cells the same challenge caused a greater Ca2+ increase (328±45nM; n=11; p蠄0.01). This Ca2+ increase was inhibited by a TRPC channel antagonist, PYR3 (n=69). Intracellular Ca2+ increases greater than 300nM have been shown to lead to cell apoptosis; the changes we observe here could implicate important pathological changes in cytokine‐stimulated chondrocytes. Further work is necessary to identify the mechanism by which this Ca2+ increase occurs. Funded by DBOARD (EU FP7/2007‐2013).

TRPV4 and K(Ca) ion channels functionally couple as osmosensors in the paraventricular nucleus.

Background and PurposeTransient receptor potential vanilloid type 4 (TRPV4) and calcium-activated potassium channels (KCa) mediate osmosensing in many tissues. Both TRPV4 and KCa channels are found in the paraventricular nucleus (PVN) of the hypothalamus, an area critical for sympathetic control of cardiovascular and renal function. Here, we have investigated whether TRPV4 channels functionally couple to KCa channels to mediate osmosensing in PVN parvocellular neurones and have characterized, pharmacologically, the subtype of KCa channel involved.Experimental ApproachWe investigated osmosensing roles for TRPV4 and KCa channels in parvocellular PVN neurones using cell-attached and whole-cell electrophysiology in mouse brain slices and rat isolated PVN neurons. Intracellular Ca2+ was recorded using Fura-2AM. The system was modelled in the NEURON simulation environment.Key ResultsHypotonic saline reduced action current frequency in hypothalamic slices; a response mimicked by TRPV4 channel agonists 4αPDD (1 μM) and GSK1016790A (100 nM), and blocked by inhibitors of either TRPV4 channels (RN1734 (5 μM) and HC067047 (300 nM) or the low-conductance calcium-activated potassium (SK) channel (UCL-1684 30 nM); iberiotoxin and TRAM-34 had no effect. Our model was compatible with coupling between TRPV4 and KCa channels, predicting the presence of positive and negative feedback loops. These predictions were verified using isolated PVN neurons. Both hypotonic challenge and 4αPDD increased intracellular Ca2+ and UCL-1684 reduced the action of hypotonic challenge.Conclusions and ImplicationsThere was functional coupling between TRPV4 and SK channels in parvocellular neurones. This mechanism contributes to osmosensing in the PVN and may provide a novel pharmacological target for the cardiovascular or renal systems.

Functional expression of chloride channels and their roles in the cell cycle and cell proliferation in highly differentiated nasopharyngeal carcinoma cells.

We previously demonstrated that the growth of the poorly differentiated nasopharyngeal carcinoma cells (CNE‐2Z) was more dependent on the activities of volume‐activated chloride channels than that of the normal nasopharyngeal epithelial cells (NP69‐SV40T). However, the activities and roles of such volume‐activated chloride channels in highly differentiated nasopharyngeal carcinoma cells (CNE‐1) are not clarified. In this study, it was found that a volume‐activated chloride current and a regulatory volume decrease (RVD) were induced by 47% hypotonic challenges. The current density and the capacity of RVD in the highly differentiated CNE‐1 cells were lower than those in the poorly differentiated CNE‐2Z cells, and higher than those in the normal cells (NP69‐SV40T). The chloride channel blockers, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) and tamoxifen inhibited the current and RVD. Depletion of intracellular Cl− abolished the RVD. The chloride channel blockers reversibly inhibited cell proliferation in a concentration‐ and time‐dependent manner, and arrested cells at the G0/G1 phases, but did not change cell viability. The sensitivity of the three cell lines to the chloride channel blockers was different, with the highest in poorly differentiated cells (CNE‐2Z) and the lowest in the normal cells (NP69‐SV40T). ClC‐3 proteins were expressed in the three cells and distributed inside the cells as well as on the cell membrane. In conclusion, the highly differentiated nasopharyngeal carcinoma CNE‐1 cells functionally expressed the volume‐activated chloride channels, which may play important roles in controlling cell proliferation through modulating the cell cycle, and may be associated with cell differentiation. Chloride channels may be a potential target of anticancer therapy.

Effects of airway surface liquid height on the kinetics of extracellular nucleotides in airway epithelia

Experimental techniques aimed at measuring the concentration of signaling molecules in the airway surface liquid (ASL) often require an unrealistically large ASL volume to facilitate sampling. This experimental limitation, prompted by the difficulty of pipetting liquid from a very shallow layer (~15μm), leads to dilution and the under-prediction of physiologic concentrations of signaling molecules that are vital to the regulation of mucociliary clearance. Here, we use a computational model to describe the effect of liquid height on the kinetics of extracellular nucleotides in the airway surface liquid coating respiratory epithelia. The model consists of a reaction–diffusion equation with boundary conditions that represent the enzymatic reactions occurring on the epithelial surface. The simulations reproduce successfully the kinetics of extracellular ATP following hypotonic challenge for ASL volumes ranging from 25μl to 500μl in a 12-mm diameter cell culture. The model reveals that [ATP] and [ADO] reach 1200nM and 2200nM at the epithelial surface, respectively, while their volumetric averages remain less than 200nM at all times in experiments with a large ASL volume (500μl). These findings imply that activation of P2Y2 and A2B receptors is robust after hypotonic challenge, in contrast to what could be concluded based on experimental measurements of volumetric concentrations in large ASL volumes. Finally, given the central role that ATP and ADO play in regulating mucociliary clearance, we investigated which enzymes, when inhibited, provide the greatest increase in ATP and ADO concentrations. Our findings suggest that inhibition of NTPDase1/highTNAP would cause the greatest increase in [ATP] after hypotonic challenge, while inhibition of the transporter CNT3 would provide the greatest increase in [ADO].

The Aquaporin 1 C-Terminal Tail Is Required for Migration and Growth of Pulmonary Arterial Myocytes

Pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin (AQP)1, a member of the water channel family of proteins, was expressed in PASMCs and was necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or the EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 small interference RNA (siRNA) or nontargeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail, or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability, and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal-deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.

Ion channel modulation of chondrocyte cell volume regulation (1182.1)

Healthy chondrocytes exist with depolarised resting membrane potentials (Vm); critical for volume regulation. Chondrocytes express several cell volume regulation channels, including Transient Receptor Potential (TRP) V4, V5 and V6 and epithelial sodium channels. We investigated mechanisms of cell volume regulation in healthy chondrocytes. Patch clamp electrophysiology was used to measure canine chondrocyte Vm (Vm=‐12±3mV; n=21). Hypotonic challenge caused a significant Vm hyperpolarisation of 11±1mV (n=9; p<0.05). We hypothesised this was due to Ca2+ influx activating a Ca2+‐activated potassium channel (KCa). Studies propose TRPV4 as a possible component of osmotically‐activated Ca2+ influx so we investigated this channel. However, the TRPV4 inhibitor, RN1734, failed to prevent hypotonicity‐induced hyperpolarisation (ΔVm=‐19±4mV, n=4; p<0.05). Application of a TRPV4 agonist, 4αPDD, caused no significant change in Vm (n=12), whole cell current (n=7) or cell volume (n=8). Furthermore, application of iberiotoxin or 2‐APB completely abolished the hypotonicity‐induced hyperpolarisation. This leads us to conclude that Ca2+ increase in response to hypotonicity is likely to result from an, as yet, unidentified range of ion channels and/or Ca2+ stores and the KCa channel responsible for the hyperpolarisation is a large‐KCa channel.Grant Funding Source: Supported by European Union Seventh Framework Programme(FP7/2007‐2013) under grant agreement 305815

Aquaglyceroporin HC‐3 mediates hypotonicity‐induced cell volume and shape changes in cultured erythrocytes from Cope’s gray treefrog, Hyla chrysoscelis (1099.4)

Freeze tolerant Cope’s gray treefrog, Hyla chrysoscelis, accumulates glycerol during a process of cold‐acclimation in anticipation of freezing. Glycerol acts as a cryoprotectant to control osmotic gradients formed by extracellular ice crystals during freezing. The aquaglyceroporin, HC‐3, is abundantly expressed in the membrane of erythrocytes from H. chrysoscelis, where it facilitates osmotically driven transmembrane water flux and glycerol diffusion, both important in freeze tolerance. We hypothesize that HC‐3 functions to moderate the dynamic cell shape changes that occur during the freeze/thaw process as a result of hypotonicity induced cell volume expansion. Cultured erythrocytes subjected to moderate hypotonic stress (70 mOsM) in the absence of 0.3 mM HgCl2, underwent a series of shape changes: they swelled, initially elongated, then became swollen and round, before returning to an elongated state. Erythrocytes treated with an HC‐3 morpholino to knockdown HC‐3 expression become spherical and remained spherical in response to hypotonic challenge. Within 600 seconds of the initiation of the hypotonic challenge, 80% of control cells succumbed to hypotonicity‐induced cell lysis, whereas 70% of the morpholino‐treated cells remained intact. These data indicate that HC‐3 functions in regulating erythrocyte cell volume in response to hypotonic challenge, implicating a role for HC‐3 in cellular freeze tolerance.Grant Funding Source: This research was supported by NSF Research Grant IOS‐1121457.

Environmental pollutants and osteoarthritis: Effects of non-dioxin-like polychlorinated biphenyls on cultured chondrocytes

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S173 used in experiments up to the 2nd passage. Two experimental protocols were used: a membrane water permeability assay and measurement of intracellular calcium using Fura-2AM. Results: Using an established permeability assay, whereby a hypotonic solution is applied to the cells and cell volume recorded, aquaporin permeability was measured. In healthy cells permeability was found to be 31 3x104cm.s (n 1⁄4 5). Stimulated cells did not show a significant change in aquaporin permeability (n 1⁄4 12). Calcium measurements showed that healthy cells responded to the same hypotonic challenge with an intracellular calcium increase of 115 15nM (n 1⁄4 16). This calcium increase was inhibited by the TRP channel antagonist, PYR3 (n 1⁄4 69). When applied to cells from the in vitro model of arthritis the same osmotic challenge caused a significantly greater calcium increase of 328 45nM (n 1⁄4 11; p 0.01). Conclusions: We have investigated changes to two ion channels in healthy chondrocytes and those from an in vitro model of arthritis. Aquaporin function appeared unchanged in our cytokine arthritis model, possibly suggesting that this change in gene expression occurs as a result of OA, rather than contributing to OA development. Previous work has shown that intracellular calcium increases greater than :300nM can lead to cell apoptosis and therefore the changes we observe here could implicate important pathological changes in cytokinestimulated chondrocytes. Further work is necessary to identify the exact mechanism by which this calcium increase occurs. 291 ENVIRONMENTAL POLLUTANTS AND OSTEOARTHRITIS: EFFECTS OF NON-DIOXIN-LIKE POLYCHLORINATED BIPHENYLS ON CULTURED CHONDROCYTES V. Abella yz, M. Scotece y, J. Conde y, V. Lopez y, V. Lazzaro yx, J. Pino y, J.J. Gomez-Reino y, R. Meli k, O. Gualillo y. y SERGAS-IDIS Santiago Univ. Clinical Hosp., Santiago de Compostela, SPAIN; zUniv. of A Coru~ na, A Coru~ na, Spain; xUniversit a della Magna Grecia, Catanzaro, Italy; kUniv. of Naples Federico II, Napoli, Italy a. Purpose: Non-dioxin-like polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in fatty tissues causing immune suppression and endocrine disruption. Several studies suggest that PCBs may be involved in pathogenesis of osteoarthritis (OA). Alterations in the mechanisms of programmed cell death (apoptosis) are strongly related to the degradation of extracellular matrix (ECM) in the cartilage of OA subjects. Identification of apoptosis inducers is of paramount relevance to understand the pathogenesis and/or progression of OA. Thus, the aim of the present study was to assess the effect of several PCBs on chondrocytes viability and apoptosis induction. b. Methods: The murine chondrogenic cell line ATDC5 and human juvenile costal chondrocyte cell line T/C-28a2 were treated with several doses of PCB 101, 153 and 180, alone and in combination. Cell viability was examined using a colorimetric assay based on the MTT labeling reagent. Apoptosis was evaluated by Annexin V flow cytometric assay and by the involvement of apoptotic related proteins, such as caspase-3 and Bcl-2/Bax ratio, using western blot analysis. Finally, to evaluate whether PCBs exert necrotic effect, apart from apoptosis pathways, we have also assessed lactate dehydrogenase (LDH) levels in culture supernatants. c. Results: ATDC5 and T/C-28a2 cell lines treated with PCBs, alone and in combination, showed a significant reduction of cell viability rate in a concentration-dependent manner. Neither synergisms nor additive effects were observed on cell viability with the combined treatment. Data from annexin V assays suggested that PCBs clearly induced apoptotic pathways, as well as, a certain rate of necrosis. Actually, this effect was confirmed by evaluating LDH levels that were strongly increased in supernatants of PCBs-treated cells, suggesting that necrotic mechanisms are at play too. PCBs also induced caspase-3 activation by increasing its proteolytic cleavage in a concentration-dependent manner. Finally, the Bcl2/Bax ratio was also altered. d. Conclusions: The viability of murine and human chondrocytes was reduced in presence of PCBs. The activity of PCBs on cell viability is likely to be mediated by alterations in the mechanisms of regulation of apoptosis and necrosis. Overall, this work highlights a novel role of environmental pollutants in the pathophysiology of chondrocytes. 292 IL-1b MEDIATES MMP SECRETION AND IL-1B NEOSYNTHESIS VIA UPREGULATION OF P22PHOX AND NOX4 ACTIVITY IN HUMAN ARTICULAR CHONDROCYTES F. Rousset y, F. Hazane Puch z, L. Grange yz, M. Nguyen y, C. Pinosa y, C. Combaz Lair z, B. Burroni z, B. Rubens-Duval z, F. Morel y, B. Lardy yz. yUniversit e Joseph Fourier, Grenoble, France; zCHU Grenoble, Grenoble,

Na+-K+-2Cl−cotransporter-mediated fluid secretion increases under hypotonic osmolarity in the mouse submandibular salivary gland

Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca(2+) measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mosmol/kgH2O), although the Ca(2+) response was unchanged. The lowering of the external Cl(-) by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring the NKCC1 bumetanide-dependent NH4 (+) transport; Na(+)-K(+)-2Cl(-) transport was upregulated >40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed, to a large extent, to the specific activation of the NKCC1.

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

Electrophysiological Effects of Tamoxifen

Reperfusion arrhythmias are currently attributed to ionic imbalance and oxidative stress. Tamoxifen is a potent antioxidant that also modulates some ionic transport pathways. In this work, we tried to correlate the electrophysiological effects of 1, 2, and 5 µM of tamoxifen with the incidence and severity of arrhythmias appearing on reperfusion after 10 minutes of coronary occlusion in isolated hearts from female rats. All tamoxifen concentrations inhibited the action potential shortening observed in the control hearts during late ischemia (6-10 minutes), whereas 2 and 5 µM also reduced the resting membrane potential depolarization. The incidence of sustained ventricular tachycardia and/or ventricular fibrillation on reperfusion decreased from 10 of 12 (control group) to 5 of 10 (1 µM, P = 0.1718), 4 of 12 (2 µM, P = 0.0361), and 2 of 10 (5 µM, P = 0.0083). The possible role of chloride currents activated by cell swelling in these effects was explored in hearts submitted to a 10-minute hypotonic challenge, where tamoxifen (5 µM) blocked the action potential shortening and the late resting membrane potential depolarization produced by hypotonicity, mimicking its action in late ischemia. Tamoxifen produced a similar increase of the total antioxidant capacity of myocardial samples at all the concentration tested. In conclusion, our data strongly suggest that the antiarrhythmic action of this agent is mediated by its electrophysiological effect derived from modulation of chloride currents activated by cell swelling.

Involvement of Volume-Activated Chloride Channels in H2O2 Preconditioning Against Oxidant-Induced Injury Through Modulating Cell Volume Regulation Mechanisms and Membrane Permeability in PC12 Cells

The functions of chloride channels in preconditioning-induced cell protection remain unclear. In this report, we show that the volume-activated chloride channels play a key role in hydrogen peroxide (H2O2) preconditioning-induced cell protection in pheochromocytoma PC12 cells. The preconditioning with 100 μM H2O2 for 90 min protected the cells from injury induced by long period exposure to 300 μM H2O2. The protective effect was attenuated by pretreatment with the chloride channel blockers, 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) and tamoxifen. H2O2 preconditioning directly activated a chloride current, which was moderately outward-rectified and sensitive to the chloride channel blockers and hypertonicity-induced cell shrinkage. H2O2 preconditioning functionally up-regulated the activities of volume-activated chloride channels and enhanced the regulatory volume decrease when exposure to extracellular hypotonic challenges. In addition, acute application of H2O2 showed distinctive actions on cell volume and membrane permeability in H2O2 preconditioned cells. In H2O2 preconditioned cells, acute application of 300 μM H2O2 first promptly induced a decrease of cell volume and enhancement of cell membrane permeability, and then, cell volume was maintained at a relatively stable level and the facilitation of membrane permeability was reduced. Conversely, in control cells, 300 μM H2O2 induced a slow but persistent apoptotic volume decrease (AVD) and facilitation of membrane permeability. H2O2 preconditioning also significantly up-regulated the expression of ClC-3 protein, the molecular candidate of the volume-activated chloride channel. These results suggest that H2O2 preconditioning can enhance the expression and functional activities of volume-activated chloride channels, thereby modulate cell volume and cell membrane permeability, which may contribute to neuroprotection against oxidant-induced injury.

The C‐terminal tail of Aquaporin 1(AQP1) is Required for Migration and Proliferation of Pulmonary Arterial Smooth Muscle Cells (PASMCs)

Both PASMC proliferation and migration are important contributors to the vascular remodeling that occurs during the development of pulmonary hypertension. AQP1 is a member of the water channel family of proteins. We previously demonstrated that AQP1 is expressed in and mediates migration and proliferation of PASMCs; however, the mechanism by which AQP1 controls these responses is unclear. The C‐terminal tail of AQP1 contains putative calcium and protein binding sites. Thus, we wanted to explore whether the C‐terminal tail of AQP1 was required for migration and proliferation. Rat PASMCs were harvested from distal pulmonary arteries enzymatically and plated onto culture dishes. Proliferation and migration were measured using BrdU incorporation and transwell filters. PASMCs were treated with 50 ifu/cell for 4 h with an adenoviral construct expressing green fluorescent protein (AdGFP), the control for infection, wild‐type AQP1 (AdAQP1) or AQP1 lacking the entire cytoplasmic C‐terminal tail (AdAQP1CT). Compared to AdGFP cells, infection with AdAQP1 or AdAQP1CT increased total and surface AQP1 protein levels and the rate of change in cell volume in response to hypotonic challenge to a similar level. Infection with AdAQP1, but not AdAQP1CT, increased migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs via a mechanism that includes the C‐terminal tail but is independent of water transport.