Francisco J. Alvarez-Leefmans

Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.

1. Intracellular Cl‐ activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double‐barrelled Cl‐ ‐selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was ‐57.7 +/‐ 1.0 mV and aiCl was 23.6 +/‐ 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl‐ led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min‐1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl‐ occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl‐ was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl‐ was dependent on the extracellular Cl‐ activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half‐maximal activation of about 50 mM‐external Cl‐. 4. The steady‐state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl‐ after intracellular Cl‐ depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl‐ was inhibited by furosemide (0.5‐1 x 10(‐3) M) or bumetanide (10(‐5) M). The decrease in aiCl observed in Cl‐ ‐free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl‐ ‐free solutions to about 75% their initial volume, at an initial rate of 6% min‐1. 7. The present results provide direct evidence for the active accumulation of Cl‐ in DRG neurones. The mechanism of Cl‐ transport is electrically silent, dependent on the simultaneous presence of external Cl‐, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl‐ co‐transport system mediates the active transport of Cl‐ across the cell membrane of DRG neurones.

Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus

Summary:  Intracellular chloride concentration, [Cl−]i, determines the polarity of GABAA‐induced neuronal Cl− currents. In neurons, [Cl−]i is set by the activity of Na+, K+, 2Cl− cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl− in the cell, and Cl− extruding K+, Cl− cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex.

Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe

A non-invasive microspectrofluorimetric technique was used to investigate experimentally induced changes in cell water volume in single N1E-115 murine neuroblastoma cells, using calcein, a derivative of fluorescein, as a marker of the intracellular water compartment. The osmotic behavior of N1E-115 cells exposed to media of various osmolalities was studied. Exposure to hyperosmotic (up to +28%) or hyposmotic (up to -17%) solutions produced reversible decreases and increases in cell water volume, respectively, which agreed with near-osmometric behavior. Increases in [Ca2+]i produced by exposing the cells to the ionophore ionomycin (1 microM) in isosmotic medium, resulted in a gradual decrease in cell water volume. Cells shrank to 40 +/- 7% (n = 7) below their initial water volume at an initial rate of -1.2 +/- 0.2%/min. It is concluded that N1E-115 cells are endowed with Ca2+-sensitive mechanisms for volume control, which can produce cell shrinkage when activated under isosmotic conditions. Because the technique used for measuring cell water volume changes is new, we describe it in detail. It is based on the principle that relative cell water volume in single cells can be measured by introducing an impermeant probe into cells and measuring its changes in concentration. If the intracellular content of the probe is constant, changes in its concentration reflect changes in cell water volume. Calcein was used as the probe because its fluorescence intensity is directly proportional to its concentration and independent of changes in the concentration of native intracellular ions within the physiological range. Because calcein is two to three times more fluorescent that other fluorophores such as 2,7,-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein or Fura-2, and it is used at its peak excitation and emission wavelengths, it has a better signal to noise ratio and baseline stability than the other dyes. Calcein can also be esterified allowing for cell loading and because of the possibility of reducing the intensity of the excitation light, measurements can be performed producing minimal photodynamic damage. The technique allows for measurements of cell water volume changes of < 5% and it can be applied to single cells which can be grown or affixed to a rigid substratum, e.g., a coverslip.

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down‐regulated by the activation of anion channels. The role of cell swelling‐activated chloride channels in cell volume regulation has been studied using the patch‐clamp technique and a non‐invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P‐glycoprotein (P‐gp), increased the rate of channel activation and the rate of RVD. In addition, P‐gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P‐gp. The data show that, in these cell types, swelling‐activated chloride channels have a central role in RVD. Moreover, they clarify the role of P‐gp in channel activation and provide direct evidence that P‐gp, through its effect on chloride channel activation, enhances the ability of cells to down‐regulate their volume.

Chloride channels and carriers in nerve, muscle, and glial cells

1. Methods for Measuring Chloride Transport across Nerve, Muscle, and Glial Cells.- 2. Principles of Cell Volume Regulation: Ion Flux Pathways and the Roles of Anions.- 3. Chloride Transport in the Squid Giant Axon.- 4. Intracellular Cl? Regulation and Synaptic Inhibition in Vertebrate and Invertebrate Neurons.- 5. Chloride Transport across Glial Membranes.- 6. Chloride Channels and Carriers in Cultured Glial Cells.- 7. Chloride Transport across the Sarcolemma of Vertebrate Smooth and Skeletal Muscle.- 8. Biophysical Aspects of GABA- and Glycine-Gated Cl? Channels in Mouse Cultured Spinal Neurons.- 9. GABA-Gated Cl? Currents and Their Regulation by Intracellular Free Ca2+.- 10. Pharmacology and Physiology of Cl? Conductances Activated by GABA in Cultured Mammalian Central Neurons.- 11. Acetylcholine-Activated Cl? Channels in Molluscan Nerve Cells.- 12. GABA-Activated Bicarbonate Conductance: Influence on EGABA and on Postsynaptic pH Regulation.- 13. Calcium-Dependent Chloride Currents in Vertebrate Central Neurons.- 14. Hyperpolarization-Activated Chloride Channels in Aplysia Neurons.- 15. The Voltage-Dependent Chloride Channel of Torpedo Electroplax: Intimations of Molecular Structure from Quirks of Single-Channel Function.- 16. Chloride Channels in Skeletal Muscle.

Peripheral and central antinociceptive action of Na+–K+–2Cl− cotransporter blockers on formalin-induced nociception in rats

&NA; The possible local peripheral and spinal (intrathecal) antinociceptive effect of Na+–K+–2Cl− cotransporter (NKCC) inhibitors was investigated in the rat formalin test. Nociceptive flinching behavior induced by formalin (1%) injection in the hind paw was assessed following administration of cotransporter inhibitors. Local peripheral pretreatment in the ipsilateral paw with bumetanide (ED30, 27.1±12.7 μg/paw), piretanide (ED30, 109.2±21.6 μg/paw) or furosemide (ED30, 34.3±5.0 μg/paw), but not vehicle (DMSO 100%), produced dose‐dependent antinociception in phase 2 of the test. Local bumetanide had the greatest effect (∼70% antinociception). Bumetanide also inhibited formalin‐induced flinching behavior during phase 1 (ED30, 105.6±99.1 μg/paw). Spinal intrathecal pretreatment with bumetanide (ED30, 194.6±97.9 μg), piretanide (ED30, 254.4±104.9 μg) or furosemide (ED30, 32.0±6.9 μg), but not vehicle (DMSO 100%), also produced antinociception in phase 2. In this case, only intrathecal furosemide reduced flinching behavior during phase 1 (ED30, 99.4±51.4 μg) and had the maximal antinociceptive effect in phase 2 (∼65% antinociception). The opioid receptor‐antagonist naloxone (2 mg/kg, s.c.) did not reverse antinociception induced by either peripheral or spinal administration of NKCC blockers. Our data suggest that the Na+–K+–2Cl− cotransporter localized in sensory neurons at intraspinal and peripheral sites is involved in formalin‐induced nociception.

Immunolocalization of the Na + –K + –2Cl - Cotransporter in Peripheral Nervous Tissue of Vertebrates

Efflux of Cl(-) through GABA(A)-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Cl(-) inside sensory neurons occurs through an Na(+)-K(+)-2Cl(-) cotransport system that generates and maintains the electrochemical gradient for this outward Cl(-) current. We studied the immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at approximately 160kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to approximately 135kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwann cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and beta-mercaptoethanol also uncovered intense cotransporter immunoreactivity in Schmidt-Lanterman incisures at the light microscopic level. The localization of the Na(+)-K(+)-2Cl(-) cotransporter protein is consistent with its function as a Cl(-)-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K(+) uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons.

Na + ,K + ,2Cl − Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects

Adult primary afferent neurons are depolarized by GABA throughout their entire surface, including their somata located in dorsal root ganglia (DRG). Primary afferent depolarization (PAD) mediated by GABA released from spinal interneurons determines presynaptic inhibition, a key mechanism in somatosensory processing. The depolarization is due to Cl(-) efflux through GABA(A) channels; the outward Cl(-) gradient is generated by a Na+,K+,2Cl(-) cotransporter (NKCC) as first established in amphibians. Using fluorescence imaging microscopy we measured [Cl(-)]i and cell water volume (CWV) in dissociated rat DRG cells (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and calcein, respectively. Basal [Cl(-)]i was 44.2 +/- 1.2 mM (mean +/- SE), Cl(-) equilibrium potential (E Cl) was -27.0 +/- 0.7 mV (n = 75). This [Cl(-)]i is about four times higher than electrochemical equilibrium. On isosmotic removal of external Cl(-), cells lost Cl(-) and shrank. On returning to control solution, cells reaccumulated Cl(-) and recovered CWV. Cl(-) reaccumulation had Na+-dependent (SDC) and Na+-independent (SIC) components. The SIC stabilized at [Cl(-)]i = 13.2 +/- 1.2 mM, suggesting that it was passive (E(Cl) = -60.5 +/- 3 mV). Bumetanide blocked CWV recovery and most (65%) of the SDC (IC50 = 5.7 microM), indicating that both were mediated by NKCC. Active Cl(-) uptake fell with increasing [Cl(-)]i and became negligible when [Cl(-)]i reached basal levels. The kinetics of active Cl(-) uptake suggests a negative feedback system in which intracellular Cl(-)regulates its own influx thereby keeping [Cl(-)]i constant, above electrochemical equilibrium but below the value that would attain if NKCC reached thermodynamic equilibrium.

Cotransport of water by the Na+ −K+ −2Cl− cotransporter NKCC1 in mammalian epithelial cells

Water transport by the Na+–K+–2Cl− cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl− fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self‐quenching of the fluorescent dye calcein. Isosmotic removal of external Cl− or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na+ and Cl−. The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl−] saturated in a sigmoidal fashion with a Km of 60 mm, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells.

Water permeability of Na+–K+–2Cl− cotransporters in mammalian epithelial cells

Water transport properties of the Na+–K+–2Cl− cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide‐sensitive component of 86Rb+ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (Lp) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide‐sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l−1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi‐osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide‐sensitive component of the Lp was 21 kcal mol−1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na+, K+ and Cl−. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide‐sensitive 86Rb+, the NKCCs of this tissue did not contribute any bumetanide‐sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.