Contractile regulation of the Na+-K+-2Cl{-} cotransporter in vascular smooth muscle

Vasoconstrictors activate theNa⁺-K⁺-2Cl cotransporter NKCC1 inrat aortic smooth muscle, but the mechanism is unknown. Efflux of⁸⁶Rb⁺ from rat aorta in response tophenylephrine (PE) was measured in the absence and presence ofbumetanide, a specific inhibitor of NKCC1. Removal of extracellularCa²⁺ completely abolished the activation of NKCC1 by PE.This was not due to inhibition of Ca²⁺-dependentK⁺ channels since blocking these channels withBa²⁺ in Ca²⁺-replete solution did not preventactivation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited70% by 75 µM ML-9, 97% by 2 µM wortmannin, and 70% by 2 mM2,3-butanedione monoxime, each of which inhibited isometric forcegeneration in aortic rings. Bumetanide-insensitive Rb⁺efflux, an indication of Ca²⁺-dependent K⁺channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to120% of normal almost completely blocked the stimulation of NKCC1 byPE without inhibiting the stimulation by hypertonic shrinkage. Weconclude that activation of theNa⁺-K⁺-2Cl cotransporter by PE isthe direct result of smooth muscle contraction throughCa²⁺-dependent activation of myosin light chain kinase.This indicates that theNa⁺-K⁺-2Cl cotransporter isregulated by the contractile state of vascular smooth muscle.

     

Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmalNa⁺ transport, we measuredouabain-sensitive electrogenicNa⁺-K⁺pump current(I_p) involtage-clamped ventricular myocytes and intracellularNa⁺ activity(aⁱ_Na) in right ventricularpapillary muscles. Aldosterone (10 nM) induced an increase in bothI_p and the rateof rise of aⁱ_Na duringNa⁺-K⁺pump blockade with the fast-acting cardiac steroid dihydroouabain. Thealdosterone-induced increase inI_p and rate ofrise of aⁱ_Na was eliminated bybumetanide, suggesting that aldosterone activates Na⁺ influx through theNa⁺-K⁺-2Clcotransporter. To obtain independent support for this, theNa⁺,K⁺, andCl concentrations in thesuperfusate and solution of pipettes used to voltage clamp myocyteswere set at levels designed to abolish the inward electrochemicaldriving force for theNa⁺-K⁺-2Clcotransporter. This eliminated the aldosterone-induced increase inI_p. We concludethat in vitro exposure of cardiac myocytes to aldosterone activates theNa⁺-K⁺-2Clcotransporter to enhance Na⁺influx and stimulate theNa⁺-K⁺pump.

     

CFTR upregulates the expression of the basolateral Na+-K+-2Cl- cotransporter in cultured pancreatic duct cells

The purpose ofthe current experiments was 1) toassess basolateralNa⁺-K⁺-2Clcotransporter (NKCC1) expression and2) to ascertain the role of cysticfibrosis transmembrane conductance regulator (CFTR) in the regulationof this transporter in a prototypical pancreatic duct epithelial cellline. Previously validated human pancreatic duct celllines (CFPAC-1), which exhibit physiological features prototypical ofcystic fibrosis, and normal pancreatic duct epithelia (stablerecombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown toconfluence before molecular and functional studies. High-stringencyNorthern blot hybridization, utilizing specific cDNA probes, confirmedthat NKCC1 was expressed in both cell lines and its mRNA levels weretwofold higher in CFPAC-WT cells than in CFPAC-1 cells(P < 0.01, n = 3).Na⁺-K⁺-2Clcotransporter activity, assayed as the bumetanide-sensitive, Na⁺- andCl-dependentNH⁺₄ entry into the cell (withNH⁺₄ acting as a substitute forK⁺), increased by ~115% inCFPAC-WT cells compared with CFPAC-1 cells(P < 0.01, n = 6). Reducing the intracellularCl by incubating the cellsin a Cl-free mediumincreasedNa⁺-K⁺-2Clcotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cellsand mediates the entry ofCl. NKCC1 activity isenhanced in the presence of an inwardCl gradient. The resultsfurther indicate that the presence of functional CFTR enhances theexpression of NKCC1. We speculate that CFTR regulates this process in aCl-dependent manner.

     

Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate

Na⁺-K⁺-2Clcotransporters are important in renal salt reabsorption and in saltsecretion by epithelia. They are also essential in maintenance andregulation of ion gradients and cell volume in both epithelial andnonepithelial cells. Expression ofNa⁺-K⁺-2Clcotransporters in brain tissues is high; however, little is known abouttheir function and regulation in neurons. In this study, we examinedregulation of theNa⁺-K⁺-2Clcotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitiveK⁺ influx, and protein expressionwas evaluated by Western blot analysis. Glutamate was found to induce adose- and time-dependent stimulation ofNa⁺-K⁺-2Clcotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonistN-methyl-D-asparticacid (NMDA) and the metabotropic receptor agonist(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantlystimulated the cotransport activity in these cells.NMDA-mediated stimulation of theNa⁺-K⁺-2Clcotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate.trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)--methyl-(4-carboxyphenyl)glycine. The results demonstrate thatNa⁺-K⁺-2Clcotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

     

Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

To identify protein kinases (PK) and phosphatases (PP) involvedin regulation of theNa⁺-K⁺-2Clcotransporter in Ehrlich cells, the effect of various PK and PPinhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculinA (Cal-A) was a potent activator ofNa⁺-K⁺-2Clcotransport (EC₅₀ = 35 nM).Activation by Cal-A was rapid (<1 min) but transient. Inactivation isprobably due to a 10% cell swelling and/or the concurrentincrease in intracellularCl concentration. Cellshrinkage also activates theNa⁺-K⁺-2Clcotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation withCal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH andCa²⁺/calmodulin dependent.Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA byH-89 and KT-5720 inhibited cotransport activity induced by Cal-A and bycell shrinkage, with IC₅₀ values similar to reported inhibition constants of the respective kinases invitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of theNa⁺-K⁺-2Clcotransport activity during regulatory volume increase.

     

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells

The function of the apicalNa⁺-K⁺-2Clcotransporter in mammalian choroid plexus (CP) is uncertain andcontroversial. To investigate cotransporter function, we developed anovel dissociated rat CP cell preparation in which single, isolatedcells maintain normal polarized morphology. Immunofluorescencedemonstrated that in isolated cells theNa⁺-K⁺-ATPase,Na⁺-K⁺-2Clcotransporter, and aquaporin 1 water channel remained localized to thebrush border, whereas theCl/HCO₃(anion) exchanger type 2 was confined to the basolateral membrane. Weutilized video-enhanced microscopy and cell volume measurementtechniques to investigate cotransporter function. Application of 100 µM bumetanide caused CP cells to shrink rapidly. Elevation ofextracellular K⁺ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling wasblocked completely by Na⁺ removalor by addition of 100 µM bumetanide. Exposure of CP cells to 5 mMBaCl₂ induced rapid swelling thatwas inhibited by 100 µM bumetanide. We conclude that the CPcotransporter is constitutively active and propose that it functions inseries with Ba²⁺-sensitiveK⁺ channels to reabsorbK⁺ from cerebrospinal fluid to blood.

     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

We examined the effects of human cytomegalovirus (HCMV)infection on theNa⁺-K⁺-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]_i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO₃-free media) was reduced inthe absence of external Na⁺ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]_iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa⁺ sensitivities of[Cl]_iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

     

Myosin regulation of NKCC1: effects on cAMP-mediated Cl- secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstratedpreviously to regulate Clsecretion from intestinal epithelia via its effects on theNa⁺-K⁺-2Clcotransporter (NKCC1). In nontransporting epithelia, inhibition ofmyosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the roleof myosin in the regulation of secretagogue-stimulated Cl secretion in intestinalepithelia. The human intestinal epithelial cell line T84 was used forthese studies. Prevention of myosin light chain phosphorylation withthe MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase withbutanedione monoxime (BDM) attenuated cAMP but notCa²⁺-mediatedCl secretion. Both ML-9 andBDM diminished cAMP activation of NKCC1. Neither apicalCl channel activity,basolateral K⁺ channel activity,norNa⁺-K⁺-ATPasewere affected by these agents. Cytochalasin D prevented suchattenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin lightchain and subsequent contraction of basal actin-myosin bundles arecrucial to the cAMP-driven activation of NKCC1 and subsequent apicalCl efflux.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.

     

Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtainedduring the last two decades show that spontaneously hypertensive rats,an acceptable experimental model of primary human hypertension, possessincreased activity of both ubiquitous and renal cell-specific isoformsof theNa⁺/H⁺exchanger (NHE) andNa⁺-K⁺-2Clcotransporter. Abnormalities of these ion transporters have been foundin patients suffering from essential hypertension. Recent geneticstudies demonstrate that genes encoding the - and -subunits ofENaC, a renal cell-specific isoform of theNa⁺-K⁺-2Clcotransporter, and 3-, 1-, and 2-subunits of theNa⁺-K⁺pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis ofthe homology of genome maps, several other genes encoding these transporters, as well as theNa⁺/H⁺exchanger andNa⁺-K⁺-2Clcotransporter, can be predicted in QTL related to the pathogenesis ofhypertension. However, despite their location within QTL, analysis ofcDNA structure did not reveal any mutation in the coding region of theabove-listed transporters in primary hypertension, with the exceptionof G276L substitution in the1-Na⁺-K⁺pump from Dahl salt-sensitive rats and a higher occurrence of T594Mmutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities ofsystems involved in the regulation of their expression and/or function.Further analysis of QTL in F₂hybrids of normotensive and hypertensive rats and in affected siblingpairs will allow mapping of genes causing abnormalities ofthese regulatory pathways.     

Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport: role of Cl- and F-actin

Previous data indicate that adenosine 3',5'-cyclicmonophosphate activates the epithelial basolateralNa⁺-K⁺-Clcotransporter in microfilament-dependent fashion in part by direct action but also in response to apicalCl loss (due to cellshrinkage or decreased intracellularCl). To further addressthe actin dependence ofNa⁺-K⁺-Clcotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed.Na⁺-K⁺-Clcotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellularCl concentration and alsoto activation of K⁺ andCl efflux pathways.Stabilization of actin with phalloidin blunted cotransporter activationby hypotonicity and abolished hypotonic activation ofK⁺ andCl efflux. However,phalloidin did not prevent activation of cotransport by hypertonicityor isosmotic reduction of extracellularCl. Conversely, hypertonicbut not hypotonic activation was attenuated by the microfilamentdisassembler cytochalasin D. The results emphasize the complexinterrelationship among intracellularCl activity, cell volume,and the actin cytoskeleton in the regulation of epithelialCl transport.

     

Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o)

In this study, we examined theNa⁺-K⁺-Cl cotransporter activityand expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05).Expression of an ~161-kDa cotransporter protein was increased by 91%in the DBcAMP-treated astrocytes. Moreover, the specific[³H]bumetanide binding was increased by 67% in theDBcAMP-treated astrocytes. Inhibition of protein synthesis bycyclohexamide (2-3 µg/ml) significantly attenuated theDBcAMP-mediated upregulation of the cotransporter activity andexpression. The Na⁺-K⁺-Clcotransporter in astrocytes has been suggested to play a role inK⁺ uptake. In 75 mM extracellular K⁺concentration, the cotransporter-mediated K⁺ influx wasstimulated by 147% in nontreated cells and 79% in DBcAMP-treatedcells (P < 0.05). To study whether this highK⁺-induced stimulation of the cotransporter is attributedto membrane depolarization and Ca²⁺ influx, the role of theL-type voltage-dependent Ca²⁺ channel was investigated. Thehigh-K⁺-mediated stimulation of the cotransporter activitywas abolished in the presence of either 0.5 or 1.0 µM of the L-typechannel blocker nifedipine or Ca²⁺-free HEPES buffer. Arise in intracellular free Ca²⁺ in astrocytes was observedin high K⁺. These results provide the first evidence thatthe Na⁺-K⁺-Cl cotransporterprotein expression can be regulated selectively when intracellular cAMPis elevated. The study also demonstrates that the cotransporter inastrocytes is stimulated by high K⁺ in aCa²⁺-dependent manner.

     

Duality of G protein-coupled mechanisms for beta-adrenergic activation of NKCC activity in skeletal muscle

Skeletal muscleNa⁺-K⁺-2Cl cotransporter (NKCC)activity provides a potential mechanism for regulated K⁺uptake. -Adrenergic receptor (-AR) activation stimulatesskeletal muscle NKCC activity in a MAPK pathway-dependent manner. Weexamined potential G protein-coupled pathways for -AR-stimulatedNKCC activity. Inhibition of G_s-coupled PKA blockedisoproterenol-stimulated NKCC activity in both the slow-twitch soleusmuscle and the fast-twitch plantaris muscle. However, thePKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP(8-BrcAMP) were not sufficient to activate NKCC in the plantaris andpartially stimulated NKCC activity in the soleus.Isoproterenol-stimulated NKCC activity in the soleus was abolished bypretreatment with pertussis toxin (PTX), indicating aG_i-coupled mechanism. PTX did not affect the8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded theisoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus,consistent with NKCC's MAPK dependency. Inhibition ofisoproterenol-stimulated ERK activity by PTX treatment was associatedwith an increase in Akt activation and phosphorylation of Raf-1 on theinhibitory residue Ser²⁵⁹. These results demonstrate anovel, muscle phenotype-dependent mechanism for -AR-mediated NKCCactivation that involves both G_s and G_iprotein-coupled mechanisms.

     

ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb

Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa⁺-K⁺(NH⁺₄)-2Clcotransport. ANG II at 10¹⁶to 10¹² M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the10¹² M ANG II levelcotransport activity; eventually,10⁶ M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 10¹²M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 10¹² M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 10⁶ M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa²⁺ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus10¹² M ANG II inhibitsvia 20-HETE, whereas 5 × 10¹¹ M ANG II stimulatesvia PKCNa⁺-K⁺(NH⁺₄)-2Clcotransport; all ANG II effects involveAT₁ receptors and PLC activation.

     

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

AlthoughNa⁺-K⁺-2Cl cotransport has beendemonstrated in cultured bovine corneal endothelial cells, its presenceand role in the native tissue have been disputed. Using RT-PCR we havenow identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99%identical to that of the bovine isoform (bNKCC1).[³H]bumetanide binding shows that the cotransporter sitesare located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium.Immunocytochemistry confirms the basolateral location of thecotransporter. We calculate the turnover rate of the cotransporter tobe 83 s¹. Transendothelial fluid transport, determinedfrom deepithelialized rabbit corneal thickness measurements, ispartially inhibited (30%) by bumetanide in a dose-dependent manner.Our results demonstrate thatNa⁺-K⁺-2Cl cotransporters arepresent in the basolateral domain of freshly dissected bovine cornealendothelial cells and contribute to fluid transport across cornealendothelial preparations.

     

Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl--dependent mechanism

The Na-K-2Clcotransporter NKCC1 is an important volume-regulatory transporter thatis regulated by cell volume and intracellular Cl. Thisregulation appears to be mediated by phosphorylation of NKCC1, althoughthere is evidence for additional, cytoskeletal regulation via myosinlight chain (MLC) kinase. NKCC1 is also activated by growth factors andmay contribute to cell hypertrophy, but the mechanism is unknown. Inaortic endothelial cells, NKCC1 (measured as bumetanide-sensitive⁸⁶Rb⁺ influx) was rapidly stimulated by serum,lysophosphatidic acid, and fibroblast growth factor, with the greateststimulation by serum. Serum increased bumetanide-sensitive influxsignificantly more than bumetanide-sensitive efflux (131% vs. 44%),indicating asymmetric stimulation of NKCC1, and produced a 17%increase in cell volume and a 25% increase in Cl contentover 15 min. Stimulation by serum and hypertonic shrinkage wereadditive, and serum did not increase phosphorylation of NKCC1 or MLC,and did not decrease cellular Cl content. When cellularCl was replaced with methanesulfonate, influx via NKCC1increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we proposea novel mechanism whereby serum activates NKCC1 by reducing itssensitivity to inhibition by intracellular Cl. Thisresetting of the Cl set point of the transporter enablesthe cotransporter to produce a hypertrophic volume increase.

     

Evidence for an Na+-K+-Cl- cotransporter in mammalian type I vestibular hair cells

In amniotes,there are two types of hair cells, designated I and II, that differ intheir morphology, innervation pattern, and ionic membrane properties.Type I cells are unique among hair cells in that their basolateralsurfaces are almost completely enclosed by an afferent calyceal nerveterminal. Recently, several lines of evidence have ascribed a motilefunction to type I hair cells. To investigate this, elevated externalK⁺, which had been used previouslyto induce hair cell shortening, was used to induce shape changes indissociated mammalian type I vestibular hair cells. Morphologicallyidentified type I cells shortened and widened when the externalK⁺ concentration was raisedisotonically from 2 to 125 mM. The shortening did not require externalCa²⁺ but was abolished whenexternal Cl was replacedwith gluconate or sulfate and when externalNa⁺ was replaced withN-methyl-D-glucamine.Bumetanide (10-100 µM), a specific blocker of theNa⁺-K⁺-Cl cotransporter,significantly reduced K⁺-inducedshortening. Hyposmotic solution resulted in type I cell shape changessimilar to those seen with highK⁺, i.e., shortening and widening.Type I cells became more spherical in hyposmotic solution, presumablyas a result of a volume increase due to water influx. In hypertonicsolution, cells became narrower and increased in length. These resultssuggest that shape changes in type I hair cells induced by highK⁺ are due, at least in part, toion and solute entry via anNa⁺-K⁺-Cl cotransporter, whichresults in cell swelling. A scheme is proposed whereby the type I haircell depolarizes and K⁺ leaves thecell via voltage-dependent K⁺channels and accumulates in the synaptic space between the type I haircell and calyx. Excess K⁺ couldthen be removed from the intercellular space by uptake via thecotransporter.

     

Human trabecular meshwork cell volume regulation

The volume ofcertain subpopulations of trabecular meshwork (TM) cells may modifyoutflow resistance of aqueous humor, thereby altering intraocularpressure. This study examines the contribution thatNa⁺/H⁺, Cl/HCOexchange, and K⁺-Cl efflux mechanisms have onthe volume of TM cells. Volume, Cl currents, andintracellular Ca²⁺ activity of cultured human TM cells werestudied with calcein fluorescence, whole cell patch clamping, and fura2 fluorescence, respectively. At physiological bicarbonateconcentration, the selective Na⁺/H⁺ antiportinhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicitytriggered a regulatory volume decrease (RVD), which could be inhibitedby the Cl channel blocker5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K⁺channel blockers Ba²⁺ and tetraethylammonium, and theK⁺-Cl symport blocker[(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism inisotonic conditions was dependent on bicarbonate; at physiologicallevels, the Na⁺/H⁺ exchange inhibitordimethylamiloride reduced cell volume, whereas at low levels theNa⁺-K⁺-2Cl symport inhibitorbumetanide had the predominant effect. Patch-clamp measurements showedthat hypotonicity activated an outwardly rectifying, NPPB-sensitiveCl channel displaying the permeability rankingCl > methylsulfonate > aspartate.2,3-Butanedione 2-monoxime antagonized actomyosin activity and bothincreased baseline [Ca²⁺] and abolishedswelling-activated increase in [Ca²⁺], but it did notaffect RVD. Results indicate that human TM cells display aCa²⁺-independent RVD and that volume is regulated byswelling-activated K⁺ and Cl channels,Na⁺/H⁺ antiports, and possiblyK⁺-Cl symports in addition toNa⁺-K⁺-2Cl symports.

     

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Cl- cotransport

The role ofNa⁺-K⁺-2Clcotransport in ion and fluid transport of the corneal endothelium wasexamined by measuring changes in corneal hydration and uptake of⁸⁶Rb by the endothelial celllayer. Isolated, intact rabbit corneas maintain normal hydration whenthey are superfused at the endothelial surface with bicarbonate()-Ringer solutions as aresult of equilibrium between active ion and fluid transport out of thestromal tissue and leak of fluid into stromal tissue from the aqueoushumor. Furosemide and bumetanide did not alter this equilibrium whenthey were added to the superfusion medium. Uptake of⁸⁶Rb by the endothelium of theincubated cornea was increased 25% by bumetanide, but uptake in thepresence of ouabain (70% less than that of controls) was not changedby bumetanide. In Na⁺-free medium,uptake of ⁸⁶Rb was reduced by58%, but it was unchanged inCl-free medium. CalyculinA, a protein phosphatase inhibitor and activator ofNa⁺-K⁺-Clcotransport, was without effect on⁸⁶Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes wassignificantly different when bumetanide was present in the media. It isconcluded thatNa⁺-K⁺-2Clcotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport ofthis cell layer. Its presence in cultured endothelial cells may reflectthe reported importance of this protein in growth, proliferation, anddifferentiation.