Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

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.

     

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.

     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.

     

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.

     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.

     

Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

The hypothesisthat amiloride-sensitive Na⁺channels (ENaC) are involved in cell volume regulation was tested.Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used tosuperfuse Xenopus oocytes expressing-rat ENaC (-rENaC). Whole cell currents werereversibly dependent on external osmolarity. Under conditions ofswelling (70 mosM) or shrinkage (450 mosM), current amplitude decreasedand increased, respectively. In contrast, there was no change incurrent amplitude of H₂O-injectedoocytes to the above osmotic insults. Currents recorded from-rENaC-injected oocytes were not sensitive to externalCl concentration or to theK⁺ channel inhibitorBaCl₂. They were sensitive toamiloride. The concentration of amiloride necessary to inhibit one-halfof the maximal rENaC current expressed in oocytes(K_i; apparentdissociation constant) decreased in swollen cells and increased inshrunken oocytes. The osmotic pressure-inducedNa⁺ currents showed propertiessimilar to those of stretch-activated channels, including inhibition byGd³⁺ andLa³⁺, and decreased selectivityfor Na⁺.-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the firstdemonstration that -rENaC heterologously expressed inXenopus oocytes may contribute tooocyte volume regulation following shrinkage.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

We evaluated theeffects of acute hyperoxic exposure on alveolar epithelial cell (AEC)active ion transport and on expression ofNa⁺ pump(Na⁺-K⁺-ATPase)and rat epithelial Na⁺ channelsubunits. Rat AEC were cultivated in minimal defined serum-free medium(MDSF) on polycarbonate filters. Beginning on day5, confluent monolayers were exposedto either 95% air-5% CO₂(normoxia) or 95% O₂-5%CO₂ (hyperoxia) for 48 h.Transepithelial resistance(R_t) andshort-circuit current(I_sc) weredetermined before and after exposure.Na⁺ channel -, -, and-subunit andNa⁺-K⁺-ATPase₁- and₁-subunit mRNA levels werequantified by Northern analysis.Na⁺ pump₁- and₁-subunit protein abundance wasquantified by Western blotting. After hyperoxic exposure,I_sc across AECmonolayers decreased by ~60% at 48 h relative to monolayersmaintained under normoxic conditions.Na⁺ channel -subunit mRNAexpression was reduced by hyperoxia, whereas - and -subunit mRNAexpression was unchanged. Na⁺ pump₁-subunit mRNA was unchanged,whereas ₁-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in₁-subunit protein. Becausekeratinocyte growth factor (KGF) has recently been shown to upregulateAEC active ion transport and expression ofNa⁺-K⁺-ATPaseunder normoxic conditions, we assessed the ability of KGF to preventhyperoxia-induced changes in active ion transport by supplementingmedium with KGF (10 ng/ml) from day2. The presence of KGF prevented theeffects of hyperoxia on ion transport (as measured byI_sc) relativeto normoxic controls. Levels of₁ mRNA and protein wererelatively preserved in monolayers maintained in MDSF and KGF comparedwith those cultivated in MDSF alone. These results indicate that AECnet active ion transport is decreased after 48 h of hyperoxia, likelyas a result of a decrease in the number of functionalNa⁺ pumps per cell. KGF largelyprevents this decrease in active ion transport, at least in part, bypreserving Na⁺ pump expression.

     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

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.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

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.

     

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels

Alveolar epithelial cells were isolated from adultSprague-Dawley rats and grown to confluence on membrane filters. Mostof the basal short-circuit current(I_sc; 60%) wasinhibited by amiloride (IC₅₀ 0.96 µM) or benzamil (IC₅₀ 0.5 µM).Basolateral addition of terbutaline (2 µM) produced a rapid decreasein I_sc, followed by a slow recovery back to its initial amplitude. WhenCl was replaced withmethanesulfonic acid, the basalI_sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of28 mV. Increasing Cl concentration in thebasolateral solution shifted the reversal potential to more depolarizedvoltages. These results were consistent with the existence of aterbutaline-activated Cl conductance in the apicalmembrane. Terbutaline did not increase the amiloride-sensitiveNa⁺ conductance. We conclude that -adrenergicstimulation of adult alveolar epithelial cells results in an increasein apical Cl permeability and thatamiloride-sensitive Na⁺ channels are not directly affectedby this stimulation.

     

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.

     

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC)

Our group recentlycloned the electrogenicNa⁺-HCO₃cotransporter (NBC) from salamander kidney and later from mammaliankidney. Here we report cloning an NBC isoform (hhNBC) from a humanheart cDNA library. hhNBC is identical to human renal NBC (hkNBC),except for the amino terminus, where the first 85 amino acids in hhNBCreplace the first 41 amino acids of hkNBC. About 50% of the amino acidresidues in this unique amino terminus are charged, compared with~22% for the corresponding 41 residues in hkNBC. Northern blotanalysis, with the use of the unique 5' fragment of hhNBC as aprobe, shows strong expression in pancreas and expression in heart andbrain, although at much lower levels. InXenopus oocytes expressing hhNBC,adding 1.5% CO₂/10 mMHCO₃ hyperpolarizes the membrane andcauses a rapid fall in intracellular pH(pH_i), followed by apH_i recovery. Subsequent removalof Na⁺ causes a depolarization anda reduced rate of pH_i recovery.Removal of Cl from the bathdoes not affect the pH_i recovery.The stilbene derivative DIDS (200 µM) greatly reduces thehyperpolarization caused by addingCO₂/HCO₃.In oocytes expressing hkNBC, the effects of addingCO₂/HCO₃and then removing Na⁺ were similarto those observed in oocytes expressing hhNBC. We conclude that hhNBCis an electrogenicNa⁺-HCO₃cotransporter and that hkNBC is also electrogenic.     

An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

We screened rat brain cDNA libraries and used 5'rapid amplification of cDNA ends to clone two electrogenicNa⁺-HCO₃ cotransporter(NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acidlevel, one clone (rb1NBC) is 96% identical to human pancreas NBC. Theother clone (rb2NBC) is identical to rb1NBC except for 61 uniqueCOOH-terminal amino acids, the result of a 97-bp deletion near the3' end of the open-reading frame. Using RT-PCR, we confirmed thatmRNA from rat brain contains this 97-bp deletion. Furthermore, wegenerated rabbit polyclonal antibodies that distinguish between theunique COOH-termini of rb1NBC (rb1NBC) and rb2NBC (rb2NBC).rb1NBC labels an ~130-kDa protein predominantly from kidney, andrb2NBC labels an ~130-kDa protein predominantly from brain.rb2NBC labels a protein that is more highly expressed in corticalneurons than astrocytes cultured from rat brain; rb1NBC exhibits theopposite pattern. In expression studies, applying 1.5%CO₂/10 mM HCO₃ toXenopus oocytes injected with rb2NBC cRNA causes 1)pH_i to recover from the initial CO₂-inducedacidification and 2) the cell to hyperpolarize. Subsequently,removing external Na⁺ reverses the pH_i increaseand elicits a rapid depolarization. In the presence of 450 µM DIDS,removing external Na⁺ has no effect on pH_i andelicits a small hyperpolarization. The rate of the pH_idecrease elicited by removing Na⁺ is insensitive toremoving external Cl. Thus rb2NBC is aDIDS-sensitive, electrogenic NBC that is predominantly expressed inbrain of at least rat.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.