Changes In Intracellular Cl Research Articles

The chloride anion as a signalling effector.

The specific role of the chloride anion (Cl- ) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl- concentration affect diverse cellular functions such as gene and protein expression and activities, post-translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl- also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl- signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl- transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl- as a signalling effector.

Protein‐Protein Interaction Prediction Yields WNK‐SPAK/OSR1 Pathway as Regulator of Kir2.1 and Kir2.3 K+ Channels

The WNK‐SPAK/OSR1 pathway is a key regulator of cellular ion transport and homeostasis, and has a role in physiological processes such as neurotransmission and blood pressure regulation. WNK kinases respond to changes in intracellular Cl−, osmolarity, and cell volume by activating the closely related kinases, SPAK and OSR1. The pathway mediates ion flux through Na+, K+, and Cl‐− cotransporters and ion channels via SPAK and OSR1‐dependent and independent mechanisms. SPAK and OSR1 use a protein interaction domain to bind linear R‐F‐x‐V/I sequence motifs in WNKs, cotransporters, and other proteins. Our goal was to identify novel SPAK and OSR1 interacting proteins by further defining the specificity determinants of the interaction, and predicting new interactions using the refined specificity profile. We used peptide arrays and biophysical measurements to define specificity, and used this information to search and rank motifs in the Uniprot database. All motifs not conserved in mice, not solvent exposed, and not present in the cytosol or nucleus were excluded. Nearly all of the previously validated interactions clustered within the top ten percent of our rankings and none were excluded, thus providing strong evidence that our ranking methodology was successful. Members of the inward rectifier K+ channel family were predicted to have a high probability of interacting with SPAK and OSR1 based on our rankings. We determined that OSR1 kinase activity activates two of the channels, Kir2.1 and Kir2.3, and activation is dependent on the presence of the binding motif. SiRNA knockdown of OSR1 and chemical inhibition of WNK kinases disrupted NaCl‐induced plasma membrane localization of Kir2.3. Our results suggest that activation of the WNK‐SPAK/OSR1 pathway enhances Kir2.1 and Kir2.3 channel activity by increasing plasma membrane localization of the channels. Our interaction predictions imply that additional inward rectifier K+ channel family members may be regulated in a similar manner. On a broader scale, our interaction predictions may help identify additional novel regulatory functions of the WNK‐SPAK/OSR1 pathway.Support or Funding InformationThis work was supported by National Institutes of Health GM53032 and Welch Foundation I‐1243 for M.H.C. Additional funding support to C.L.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid regulation of tonic GABA currents in cultured rat hippocampal neurons

Subacute and chronic changes in tonic GABAergic inhibition occur in human and experimental epilepsy. Less is known about how tonic inhibition is modulated over shorter time frames (seconds). We measured endogenous tonic GABA currents from cultured rat hippocampal neurons to evaluate how they are affected by 1) transient increases in extracellular GABA concentration ([GABA]), 2) transient postsynaptic depolarization, and 3) depolarization of presynaptic cells. Transient increases in [GABA] (1 μM) reduced tonic currents; this reduction resulted from GABA-induced shifts in the reversal potential for GABA currents (E(GABA)). Transient depolarization of postsynaptic neurons reversed the effects of exogenous GABA and potentiated tonic currents. The voltage-dependent potentiation of tonic GABA currents was independent of E(GABA) shifts and represented postdepolarization potentiation (PDP), an intrinsic GABA(A) receptor property (Ransom CB, Wu Y, Richerson GB. J Neurosci 30: 7672-7684, 2010). Inhibition of vesicular GABA release with concanamycin A (ConA) did not affect tonic currents. In ConA-treated cells, transient application of 12 mM K(+) to depolarize presynaptic neurons and glia produced a persistent increase in tonic current amplitude. The K(+)-induced increase in tonic current was reversibly inhibited by SKF89976a (40 μM), indicating that this was caused by nonvesicular GABA release from GABA transporter type 1 (GAT1). Nonvesicular GABA release due to GAT1 reversal also occurred in acute hippocampal brain slices. Our results indicate that tonic GABA currents are rapidly regulated by GABA-induced changes in intracellular Cl(-) concentration, PDP of extrasynaptic GABA(A) receptors, and nonvesicular GABA release. These mechanisms may influence tonic inhibition during seizures when neurons are robustly depolarized and extracellular GABA and K(+) concentrations are elevated.

Coupling Molecular Motions of Prestin to Cellular Motility: Effects of Voltage, Chloride and Cytoskeleton

Mammalian outer hair cell (OHC) electro-motility results from voltage-dependent conformational changes of the membrane protein prestin, with the cell's nonlinear capacitance (NLC) being considered a surrogate measure of electromotility. A number of physiological factors shift prestin's voltage dependence, including changes in intracellular Cl and membrane holding potential (pre-pulse effect), each presumably affecting electromotility and NLC equivalently.Here we study the consequences of combined Cl and pre-pulse manipulations on electromotility with simultaneous measurements of NLC and video imaging. We found, as expected from our previous work, that OHCs patched with 140 mM intracellular Cl show robust depolarizing shifts of NLC upon negative pre-pulse, with electromotility displaying a similar shift. However, lower, more physiological levels of intracellular chloride (1 and 10 mM), induce an uncoupling of mechanical responses from NLC. That is, voltage dependence of motility is shifted in the depolarizing direction compared to that of NLC. The uncoupling shows hysteresis, being greater with depolarizing voltage polarity. We have found disparities as large as 50 mV between charge movement and electromotility. Interestingly, we find that diamide can reduce the disparity, suggesting that chloride modulates a cytoskeletal coupling between prestin's conformational state and whole cell mechanics. Finally, the uncoupling we find imposes an additional challenge for the voltage-sensitive protein prestin to drive the cochlear partition during acoustic stimulation, since the whole cell mechanical response is variably uncoupled from voltage depending on phase of receptor potential.(Supported by NIH/NIDCD DC 000273 to JSS)

Cl− concentration changes and desensitization of GABAA and glycine receptors

Desensitization of ligand-gated ion channels plays a critical role for the information transfer between neurons. The current view on γ-aminobutyric acid (GABA)A and glycine receptors includes significant rapid components of desensitization as well as cross-desensitization between the two receptor types. Here, we analyze the mechanism of apparent cross-desensitization between native GABAA and glycine receptors in rat central neurons and quantify to what extent the current decay in the presence of ligand is a result of desensitization versus changes in intracellular Cl− concentration ([Cl−]i). We show that apparent cross-desensitization of currents evoked by GABA and by glycine is caused by changes in [Cl−]i. We also show that changes in [Cl−]i are critical for the decay of current in the presence of either GABA or glycine, whereas changes in conductance often play a minor role only. Thus, the currents decayed significantly quicker than the conductances, which decayed with time constants of several seconds and in some cells did not decay below the value at peak current during 20-s agonist application. By taking the cytosolic volume into account and numerically computing the membrane currents and expected changes in [Cl−]i, we provide a theoretical framework for the observed effects. Modeling diffusional exchange of Cl− between cytosol and patch pipettes, we also show that considerable changes in [Cl−]i may be expected and cause rapidly decaying current components in conventional whole cell or outside-out patch recordings. The findings imply that a reevaluation of the desensitization properties of GABAA and glycine receptors is needed.

Phosphoregulation of K+‐Cl− cotransporter 4 during changes in intracellular Cl− and cell volume

It has long been stated that the K(+)-Cl(-) cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N-ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C(VOL)) and intracellular [Cl(-)] ([Cl(-)](i)) on the activity and phosphorylation levels (P(LEV)) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)-sensitive effectors. We found that (1) low [Cl(-)](i) or low C(VOL) leads to decreased activity but increased P(LEV), (2) high C(VOL) leads to increased activity but no decrease in P(LEV) and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P(LEV). Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl(-)](i) or C(VOL) modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A-sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787-796, 2009. (c) 2009 Wiley-Liss, Inc.

Ca 2+ -Induced Cl− Efflux at Rat Distal Colonic Epithelium

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl(-) concentration were measured to characterize the role of Ca(2+)-dependent Cl(-) channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl(-) exit (i.e., mediated by opening of Ca(2+)-dependent K(+) channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K(+) concentrations. The Ca(2+)-dependent secretagogue carbachol induced a stilbene-sensitive Cl(-) efflux, which was mimicked by the Ca(2+) ionophore ionomycin. Surprisingly, the activation of Ca(2+)-dependent Cl(-) efflux was resistant against blockers of classical Ca(2+) signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl(- )efflux. Vice versa, the NO synthase inhibitor N-omega-monomethyl-arginine (L: -NMMA) reduced the carbachol-induced Cl(- )efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca(2+)-dependent Cl(-) channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl(-) efflux. Consequently, the activation of apical Cl(-) channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.

The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.

Changes in intracellular chloride after oxygen-glucose deprivation of the adult hippocampal slice: effect of diazepam.

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca2+ is well established, but there are few studies of changes in intracellular Cl- ([Cl-]i) after ischemia. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to examine changes in [Cl-]i and GABA(A) receptor-mediated responses in hippocampal slices from adult rats. Changes in [Cl-]i were measured in area CA1 pyramidal neurons using optical imaging of 6-methoxy-N-ethylquinolinium chloride, a Cl--sensitive fluorescent indicator. Oxygen-glucose deprivation induced an immediate rise in [Cl-]i, which recovered within 20 min. A second and more prolonged rise in [Cl-]i occurred within the next hour, during which postsynaptic field potentials failed to recover. The sustained increase in [Cl-]i was not blocked by GABA(A) receptor antagonists. However, oxygen-glucose deprivation caused a progressive downregulation of the K+-Cl- cotransporter (KCC2), which may have contributed to the Cl- accumulation. The rise in [Cl-]i was accompanied by an inability of the GABA(A) agonist muscimol to cause Cl- influx. In vivo, diazepam is neuroprotective when given early after ischemia, although the mechanism by which this occurs is not well understood. Here, we added diazepam early after oxygen-glucose deprivation and prevented the downregulation of KCC2 and the accumulation of [Cl-]i. Consequently, both GABA(A) responses and synaptic transmission within the hippocampus were restored. Thus, after oxygen-glucose deprivation, diazepam may decrease neuronal excitability, thereby reducing the energy demands of the neuron. This may prevent the activation of downstream cell death mechanisms and restore Cl- homeostasis and neuronal function

Experience-dependent changes in intracellular Cl − regulation in developing auditory neurons

A developmental change in GABA and glycine responses, from a depolarization to a hyperpolarization, have been reported for a range of CNS neurons, and has been demonstrated to be due to a developmental decrease in the intracellular Cl − concentration ([Cl −] i). We examined [Cl −] i in isolated rat lateral superior olive (LSO) neurons using patch-clamp recordings of glycine gated Cl − currents and by measuring intracellular Cl −-fluorescence. In neurons from 14–16-day-old rats (P14–P16), which had previously received unilateral or bilateral cochlear ablations before the onset of hearing, there was no developmental decrease in [Cl −] i. No significant differences in [Cl −] i were observed amongst rats with either ipsi- and contralateral ablations. Implanted strychnine pellets also prevented the decrease in [Cl −] i in most neurons. In some of these neurons in which [Cl −] i remained high, there was a lack of expression of the K +–Cl − cotransporter 2 (KCC2) mRNA. These results demonstrate that the developmental decrease in [Cl −] i in LSO neurons is dependent on neuronal activity and that both GABAergic/glycinergic and glutamatergic afferent activity contribute to this maturation of the Cl − regulatory mechanisms.

Modulation of intracellular Cl − homeostasis by lectin-stimulation in Jurkat T lymphocytes

We investigated changes in intracellular Cl − concentration ([Cl −] i) during lectin-induced activation and proliferation in human Jurkat T lymphocytes. [Cl −] i was measured using Cl − fluorescence dye ( N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE) methods. Lectins, phytohemagglutinin and concanavalin A, dose-dependently increased [Cl −] i and triggered intracellular Cl − oscillation in human Jurkat T lymphocytes. However, some mitochondria metabolism inhibitors, such as m-chlorocarbonylcyanide phenylhydrazone (CCP) and 2,4-dinitrophenol, increased [Cl −] i without triggering any Cl − oscillation. Furthermore, both lectins and metabolism inhibitors-induced elevation in [Cl −] i were blocked by removal of extracellular Cl − from perfusion solution or by application of anthracene-9-carboxylate, a blocker of Cl − channels. Since an extracellular Cl −-free condition and application of 9-AC also inhibited PHA-induced proliferation, we suggested that elevation of [Cl −] i via activation of Cl − channels and increase in incidence of Cl − oscillation would play an important role in modulation of Jurkat T cell activation and proliferation.

Diazepam prevents changes in intracellular Cl− and Ca2+ and restores neuronal activity after ischemia in vitro

We have shown that diazepam (DZ) protects hippocampal neurons when administered early after transient cerebral ischemia. To examine mechanisms of DZ action, in the absence of systemic effects, we used an in vitro model of ischemia [oxygen‐glucose deprivation (OGD)] in the adult hippocampal slice. We are interested in determining if DZ can prevent upstream events such as loss of ionic homeostasis and its consequences on neuronal transmission. We measured the effect of OGD on changes in intracellular Cl− and Ca2+. Application of DZ after OGD prevented the increase in intracellular Cl− in area CA1 pyramidal neurons, measured by optical imaging with the Cl− sensitive fluorescent probe, MEQ. The increase in intracellular Cl− after OGD led to a reduction in GABA responses 2 h later, measured in the presence of the GABA agonist, muscimol. When DZ was added for 1 h after OGD, muscimol responses were restored completely. To measure changes in intracellular Ca2+ after OGD, we bath‐loaded adult hippocampal slices with calcium green‐1 AM. OGD increased intracellular Ca2+ in CA1 pyramidal neurons. The addition of DZ prior to OGD reduced the increase in intracellular Ca2+ by approximately 50%. To determine the effect of DZ on neuronal transmission within the hippocampus, we measured field responses in area CA1 pyramidal neurons. Area CA1 population spikes were suppressed by OGD and they did not recover 1 h later. When DZ was included in the reoxygenation buffer for only 30 min, it restored the population spike amplitude to within 85% of that in normal slices. Thus, after OGD, DZ restores ionic homeostasis, preserves GABA responses and restores neuronal activity within the hippocampus.Acknowledgements: Supported by NIH grant #NS28791.

Pacemaker Channels Produce an Instantaneous Current

Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I(h)), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I(inst(HCN2))) in addition to the well characterized, slowly activating I(h). I(inst(HCN2)) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I(h). The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl(-) and cAMP. A mutation in the S4 domain of HCN2 (S306Q) decreased I(h) but did not alter I(inst(HCN2)), and instantaneous currents in cells expressing either wild type HCN2 or mutant S306Q channels were insensitive to block by Cs(+). Co-expression of HCN2 with the accessory subunit, MiRP1, decreased I(h) and increased I(inst(HCN2)), suggesting a mechanism for modulation of both currents in vivo. These data suggest that expression of HCN channels may be accompanied by a background conductance in native tissues and are consistent with at least two open states of HCN channels: I(inst(HCN2)) is produced by a Cs(+)-open state; hyperpolarization produces an additional Cs(+)-sensitive open state, which results in I(h).

GABA-Induced Cl- current in cultured embryonic human dorsal root ganglion neurons.

gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.

Fluorescence Imaging of Changes in Intracellular Chloride in Living Brain Slices

In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl− tracer methods, Cl−-selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noinvasive technique that uses the fluorescent Cl− ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl− by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl−-sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl− concentrations can be calibrated from the Stern–Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl−, Cl− flux through the γ-aminobutyric acid (GABA)-gated Cl− channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl− quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl− can also be studied under various conditions such as oxygen/glucose deprivation (“in vitro ischemia”) and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl− flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl− and an increase in cell volume.

26] Using confocal microscopy and the fluorescent indicator, 6-methoxy-N-ethylquinolinium iodide, to measure changes in intracellular chloride

The chapter presents approaches of studying dynamic changes in intracellular Cl – in living neurons with the laser scanning confocal microscope (LSCM) and a fluorescent Cl – indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ). The chapter discusses the brain slice preparation. Brain slices of the hippocampus (containing somatosensory cortex) or cerebellum are prepared from rats between 8 and 21 days old. All laser scanning confocal microscopes have an improved signal-to-noise ratio and the ability to penetrate into tissues, although slit variations. The Cl – channels in the areas studied, include the recently discovered ClC-2 family as well as GABA A (γ-aminobutyric acid) receptors; activation of the latter in adult animals leads to neuronal inhibition via a stimulated Cl – conductance. Applying MEQ and confocal microscopy to the brain slice preparation, an appropriate GABA A receptor pharmacology is demonstrated. In addition, a comparison of simulated ischemia versus activation of excitatory amino acid receptor subtypes identified different mechanisms for Cl – influx and reduced GABAergic function. The presence of cell swelling can be investigated with fluorescence microscopy or microfluorimetry with the volume-sensitive fluorescent dye, calcein-AM.

Simultaneous optical imaging of intracellular Cl − in neurons in different layers of rat neocortical slices: advantages and limitations

Simultaneous recording of changes in intracellular Cl − concentration ([Cl −] i) in individual neurons situated in different layers (e.g. II/III–VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy- N -ethylquinolinium iodide (MEQ). γ-Aminobutyric acid (GABA) caused a measurable increase in [Cl −] i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl − pump and cation–Cl − co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl − and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at>30°C. Estimation of [Cl −] i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl − concentrations ([Cl −]) in the presence of tributyltin, a Cl −–OH − antiporter, nigericin, a K +–H +antiporter, and KSCN. This enables comparison of [Cl −] i between neurons in different slices. Thus, optical imaging of [Cl −] i in brain slices can provide valuable spatial information about [Cl −] i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.

Biphasic changes in intracellular Cl − induced by oxygen-glucose deprivation in rat neocortical slices: A study by optical imaging

Biphasic changes in intracellular Cl − induced by oxygen-glucose deprivation in rat neocortical slices: A study by optical imaging

Elevated Cytoplasmic Sodium and Chloride Is Associated With Purinergic Receptor Stimulation of Rat Ventricular Myocytes: Implications For Myocardial Ischemia

During myocardial ischemia, several factors have been identified which contribute to the development of cardiac arrhythmias. These include membrane depolarization, increased cytosolic Na+ and decreased intracellular pH. This increase in intracellular Na+ has been shown to be associated with the development of ventricular arrhythmias. Changes in intracellular Cl- may also be involved in development of cardiac arrhythmias since decreasing extracellular Cl- concentration in the perfusion medium can prevent development of arrhythmias during myocardial ischemia in vitro. Extracellular ATP is known to increase in the heart during myocardial ischemia. ATP activation of p2 purinergic receptors on cardiac myocytes has been proposed to contribute to the initiation of the arrhythmias and ventricular fibrillations which characterize myocardial ischemia. Activation of p2 purinergic receptors results in membrane depolarization and decreased intracellular pH, thus reproducing some of the changes that occur during ischemia. We recently showed that activation of p2 purinergic receptors in both quiescent and electrically stimulated ventricular myocytes triggers spontaneous oscillatory contractions and Ca2+ transients.

Chloride and fluid secretion by cultured human polycystic kidney cells

Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3',5'-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended within a polymerized collagen matrix and stimulated to form cysts. Individual cultured cysts were placed in a chamber on the stage of an inverted microscope equipped with epifluorescent and video analysis attachments. The rate of fluid secretion, cell volume and changes in intracellular Cl- were measured. In the absence of secretagogues, fluid was absorbed from the cyst cavity (-2.36 +/- 0.64 nl/min/cm2 inner surface area). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-1-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 +/- 1.28 nl/min/cm2). Bumetanide reversibly reduced fluid secretion to 0.95 +/- 0.60 nl/min/cm2. Cell volume rapidly decreased by 7.5 +/- 0.9% with the initiation of secretion and bumetanide caused an additional loss (4.2 +/- 1.0%). Furosemide had a similar effect on forskolin-induced fluid secretion. Cellular chloride concentration was monitored with the use of the indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ). Removal of Cl- from the bath reduced intracellular [Cl-] (MEQ fluorescence increased by 11.4 +/- 2.3%). In cysts pretreated with furosemide to prevent Cl- entry, the application of forskolin caused a decrease in Cl- concentration (MEQ fluorescence increased by 9.3 +/- 2.6%). Using monolayers of cultured ADPKD cells, grown on permeant supports, we compared the changes in short circuit current (ISC) induced by forskolin in the presence and absence of external Cl-. Forskolin increased ISC (from 8.9 +/- 2.7 to 10.6 +/- 2.7 microA/cm2) in the presence of Cl-, but did not significantly affect ISC in its absence. These data indicate that cultured ADPKD cells can direct fluid transport in either the absorptive or the secretory direction, and that cAMP stimulates secretion and this secretion is accompanied by a net loss of cell solute. Inhibition of secretion by bumetanide or furosemide caused an additional loss of cell solute, including Cl-. The ionic transepithelial current induced by forskolin is dependent on the presence of Cl-. These data support the thesis that chloride secretion drives fluid secretion by cultured ADPKD cells.