Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl- channels in human osteoblasts

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl^– channels expressed in these cells as described previously. The reversal potential of the induced Cl^– current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl^–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl^– current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl^– channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling. potasium-chloride cotransporter; KCC1–KCC4; chloride channels; extracellular potassium concentration buffering     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

We isolated and characterized a novelK-Cl cotransporter, KCC3, from human placenta. The deduced proteincontains 1,150 amino acids. KCC3 shares 75-76% identity at theamino acid level with human, pig, rat, and rabbit KCC1 and 67%identity with rat KCC2. KCC3 is 40 and 33% identical to twoCaenorhabditis elegans K-Cl cotransporters and ~20%identical to other members of the cation-chloride cotransporter family(CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Clcotransporter (NCC). Hydropathy analysis indicates a typical KCCtopology with 12 transmembrane domains, a large extracellular loopbetween transmembrane domains 5 and 6 (unique to KCCs), and largeNH₂ and COOH termini. KCC3 is predominantly expressed inkidney, heart, and brain, and is also expressed in skeletal muscle,placenta, lung, liver, and pancreas. KCC3 was localized to chromosome15. KCC3 transiently expressed in human embryonic kidney (HEK)-293cells fulfilled three criteria for increased expression of K-Clcotransport: stimulation of cotransport by swelling, treatment withN-ethylmaleimide, or treatment with staurosporine.

     

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role inmodulating intraocular pressure by regulating aqueous humor outflow,although the mechanisms are largely unknown. We and others have shownpreviously that aqueous humor outflow facility is modulated byconditions that alter TM cell volume. We have also shown that theNa-K-Cl cotransport system is a primary regulator of TM cell volume andthat its activity appears to be coordinated with net efflux pathways tomaintain steady-state volume. However, the cellular mechanisms thatregulate cotransport activity and cell volume in TM cells have yet tobe elucidated. The present study was conducted to investigate thehypothesis that intracellular Cl concentration([Cl]_i) acts toregulate TM cell Na-K-Cl cotransport activity, as has been shownpreviously for some other cell types. We demonstrate here that thehuman TM cell Na-K-Cl cotransporter is highly sensitive to changes in[Cl]_i. Our findingsreveal a marked stimulation of Na-K-Cl cotransport activity, assessedas ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means ofreducing [Cl]_i. Incontrast, preincubation of cells with media containing elevated Kconcentrations as a means of increasing [Cl]_i results ininhibition of Na-K-Cl cotransport activity. The effects of reducing[Cl]_i, as well aselevating [Cl]_i, onNa-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced[Cl]_i is additive withcell-shrinkage-induced stimulation of the cotransporter. Our studiesalso show that TM cell Na-K-Cl cotransport activity is altered by avariety of Cl channel modulators, presumably through changes in[Cl]_i. These findingssupport the hypothesis that regulation of Na-K-Cl cotransport activity,and thus cell volume, by[Cl]_i may participatein modulating outflow facility across the TM.

     

Arginine vasopressin stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransporter activity is V1 receptor and [Ca] dependent

Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally located BBB Na-K-Cl cotransporter is stimulated during cerebral ischemia to increase transport of Na and Cl into the brain. The main focus of the present study was to evaluate the effects of arginine vasopressin (AVP), previously shown to be increased in the brain during ischemia and to promote edema formation, on activity of the BBB cotransporter. Cerebral microvascular endothelial cell (CMEC) monolayers were cultured in astroglial cell conditioned medium, and Na-K-Cl cotransporter activity was assessed as bumetanide-sensitive ⁸⁶Rb influx. In both human and bovine CMECs, as well as in freshly isolated microvessels, AVP stimulated cotransport activity. This stimulatory effect was mimicked by V₁ but not V₂ vasopressin agonists and was blocked by V₁ but not V₂ vasopressin antagonists. Consistent with a V₁ vasopressin receptor mechanism of action, AVP caused an increase in CMEC intracellular [Ca] that was blocked by a V₁ antagonist. Exposing the cells to [Ca]-free media and/or reducing intracellular [Ca] by BAPTA also blocked AVP stimulation of CMEC cotransporter activity, as did the phospholipase C inhibitor U-73122. Finally, we found that while stimulation of CMEC cotransporter activity by AVP occurred within minutes, it was also sustained for hours in the continued presence of AVP. These findings support the hypothesis that AVP, through a V₁ receptor- and [Ca]-dependent mechanism, stimulates the BBB Na-K-Cl cotransporter to participate in ischemia-induced edema formation. blood-brain barrier; stroke; cerebral ischemia; brain edema     

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

K-Cl cotransporters (KCC) playfundamental roles in ionic and osmotic homeostasis. To date, fourmammalian KCC genes have been identified. KCC2 is expressed exclusivelyin neurons. Injection of Xenopus oocytes with KCC2cRNA induced a 20-fold increase in Cl-dependent,furosemide-sensitive K⁺ uptake. Oocyte swelling increasedKCC2 activity 2-3 fold. A canonical tyrosine phosphorylationsite is located in the carboxy termini of KCC2 (R1081-Y1087) andKCC4, but not in other KCC isoforms. Pharmacological studies, however,revealed no regulatory role for phosphorylation of KCC2 tyrosineresidues. Replacement of Y1087 with aspartate or arginine dramaticallyreduced K⁺ uptake under isotonic and hypotonic conditions.Normal or near-normal cotransporter activity was observed when Y1087was mutated to phenylalanine, alanine, or isoleucine. A tyrosineresidue equivalent to Y1087 is conserved in all identified KCCs fromnematodes to humans. Mutation of the Y1087 congener in KCC1 toaspartate also dramatically inhibited cotransporter activity. Takentogether, these results suggest that replacement of Y1087 and itscongeners with charged residues disrupts the conformational state ofthe carboxy terminus. We postulate that the carboxy terminus plays anessential role in maintaining the functional conformation of KCCcotransporters and/or is involved in essential regulatory protein-protein interactions.

     

K-Cl cotransporter expression in the human kidney

The K-Cl cotransporter protein KCC1 is a membrane transportprotein that mediates the coupled, electroneutral transport of K and Clacross plasma membranes. The precise cell type(s) in the kidney thatexpress the K-Cl cotransporter have remained unknown. The aim of thepresent investigation was to define the distribution of KCC1 mRNA inthe human kidney. We used in situ hybridization with a nonradioactivedigoxigenin-labeled riboprobe. We identified abundant KCC1 mRNAexpression in the epithelial cells throughout the distal and proximalrenal tubular epithelium. The transporter was also expressed inglomerular mesangial cells and endothelial cells of the renal vessels.These findings suggest that the K-Cl cotransporter may have animportant role in transepithelial K and Cl reabsorption.

     

Inducible expression of erythrocyte band 3protein

A permanent cell line with inducible expression of the humananion exchanger protein 1 (hAE1) was constructed in a derivative ofhuman embryonic kidney cells (HEK-293). In the absence of the inducer,muristerone A, the new cell line had no detectable hAE1 protein byWestern analysis or additional³⁶Cl flux. Increasing dose andincubation time with muristerone A increased the amount of protein(both unglycosylated and glycosylated). The4,4'-dinitrostilbene-2,2'-disulfonate(DNDS)-inhibitable rapid Cl exchange flux was increased up to40-fold in induced cells compared with noninduced cells. There was noDNDS-inhibitable rapid flux component in noninduced cells. This resultdemonstrates inducible expression of a new rapid Cl transport pathwaythat is DNDS sensitive. The additional transport of³⁶Cl and³⁵SO₄had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 µM DNDS,2) activation of³⁶Cl efflux by external Cl with aconcentration producing half-maximal effect of 4.8 mM,3) activation of³⁶Cl efflux by external anionsthat was selective in the orderNO₃ = Cl > Br > I, and4) activation of³⁵SO₄influx by external protons. Under the assumption that the turnovernumbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies ofglycosylated hAE1 and the induced tracer fluxes. This is the firstexpression of hAE1 in a mammalian system to track the kineticcharacteristics of the native protein.

     

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA

Human NBC3 is an electroneutral Na⁺/HCO₃^– cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO₃^– metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO₂ + H₂O HCO₃^– + H⁺ in many tissues. We investigated whether NBC3, like some Cl^–/HCO₃^– exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K_d = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH_i) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH_i recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [-³²P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^– transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon     

The Na-K-Cl Cotransporters

The Na-K-Cl cotransporters are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. The transport process mediated by Na-K-Cl cotransporters is characterized by electroneutrality (almost always with stoichiometry of 1Na:1K:2Cl) and inhibition by the loop diuretics bumetanide, benzmetanide, and furosemide. Presently, two distinct Na-K-Cl cotransporter isoforms have been identified by cDNA cloning and expression; genes encoding these two isoforms are located on different chromosomes and their gene products share approximately 60% amino acid sequence identity. The NKCC1 (CCC1, BSC2) isoform is present in a wide variety of tissues; most epithelia containing NKCC1 are secretory epithelia with the Na-K-Cl cotransporter localized to the basolateral membrane. By contrast, NKCC2 (CCC2, BSC1) is found only in the kidney, localized to the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop and of the macula densa. Mutations in the NKCC2 gene result in Bartter's syndrome, an inherited disease characterized by hypokalemic metabolic alkalosis, hypercalciuria, salt wasting, and volume depletion. The two Na-K-Cl cotransporter isoforms are also part of a superfamily of cation-chloride cotransporters, which includes electroneutral K-Cl and Na-Cl cotransporters. Na-K-Cl cotransporter activity is affected by a large variety of hormonal stimuli as well as by changes in cell volume; in many tissues this regulation (particularly of the NKCCl isoform) occurs through direct phosphorylation/dephosphorylation of the cotransport protein itself though the specific protein kinases involved remain unknown. An important regulator of cotransporter activity in secretory epithelia and other cells as well is intracellular [Cl] ([Cl]_i), with a reduction in [Cl]_i being the apparent means by which basolateral Na-K-Cl cotransport activity is increased and thus coordinated with that of stimulated apical Cl channels in actively secreting epithelia.     

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis.

     

Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d 33.4 g/dl) has only about 5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d 24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d_50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d_50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r² = 0.995) and sickle erythrocytes (r² = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity. phosphorylation; protein phosphatase; urea; cell size; density     

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes

Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K⁺ currents(I_ir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca²⁺]_i) produced by Ca²⁺ storedepletion with thapsigargin (TG) and readdition of external CaCl₂ in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca²⁺]_i in monocytesbound to glass for 5 h in response to TG and CaCl₂ addition, adherence to VCAM-1 produced a 5-fold increase in[Ca²⁺]_i. Depolarization of monocytes adherentto VCAM-1 by I_ir blockade or exposure to high[K⁺] abolished the enhancement of the peak[Ca²⁺]_i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK⁺ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca²⁺]_i. Adherence of monocytes to E-selectinproduced a similar peak [Ca²⁺]_i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa²⁺ entry and may impact Ca²⁺-dependentmonocyte function.

     

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells

Thiol reagents activateK-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independentouabain-resistant K flux, in red blood cells (RBCs) of several species,upon depletion of cellular glutathione (GSH). K-Cl COT isphysiologically active in high potassium (HK), high GSH (HG) dog RBCs.In this unique model, we studied whether the same inverse relationshipexists between GSH levels and K-Cl COT activity found in other species.The effects of GSH depletion by three different chemical reactions[nitrite (NO₂)-mediated oxidation, diazene dicarboxylicacid bis-N,N-dimethylamide (diamide)-induceddithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD).After 85% GSH depletion, all three interventions stimulated K-Cl COThalf-maximally with the following order of potency: diamide > NO₂ > CDNB. Repletion of GSH reversed K-Cl COTstimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activationby NO₂ and diamide. K-Cl COT activation at concentrationratios of oxidant/GSH greater than unity was irreversible, suggestingeither nitrosothiolation, heterodithiol formation, or GST-mediateddinitrophenylation of protein thiols. The data support the hypothesisthat an intact redox system, rather than the absolute GSH levels,protects K-Cl COT activity and cell volume regulation from thiol modification.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species