Extracellular Cl(-) modulates shrinkage-induced activation of Na(+)/H(+) exchanger in rat mesangial cells

To examine theeffect of hyperosmolality on Na⁺/H⁺ exchanger(NHE) activity in mesangial cells (MCs), we used apH-sensitive dye,2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH_i) in a single MC from ratglomeruli. All the experiments were performed inCO₂/HCO₃-free HEPESsolutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH₂O) treated with mannitol caused cellalkalinization. The hyperosmolality-induced cell alkalinization wasinhibited by 100 µM ethylisopropylamiloride, a specific NHEinhibitor, and was dependent on extracellular Na⁺. Thehyperosmolality shifted the Na⁺-dependent acid extrusionrate vs. pH_i by 0.15-0.3 pH units in thealkaline direction. Removal of extracellular Cl byreplacement with gluconate completely abolished the rate of cellalkalinization induced by hyperosmolality and inhibited the Na⁺-dependent acid extrusion rate, whereas, under isosmoticconditions, it caused no effect on Na⁺-dependentpH_i recovery rate or Na⁺-dependent acidextrusion rate. The Cl-dependent cell alkalinizationrate under hyperosmotic conditions was partially inhibited bypretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS,and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acidextrusion rate via NHE is greater under hyperosmotic conditions thanunder isosmotic conditions at a wide range of pH_i,3) the NHE activation under hyperosmotic conditions, but notunder isosmotic conditions, requires extracellularCl, and 4) theCl-dependent NHE activation under hyperosmoticconditions partly occurs via Cl channel andmicrotubule-dependent processes.

     

Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes

Malignantgliomas exhibit alkaline intracellular pH (pH_i) and acidicextracellular pH (pH_e) compared with nontransformedastrocytes, despite increased metabolic H⁺ production. Theacidic pH_e limits the availability ofHCO₃, thereby reducing both passiveand dynamic HCO₃-dependent buffering.This implies that gliomas are dependent upon dynamic HCO₃-independent H⁺buffering pathways such as the type 1 Na⁺/H⁺exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH_i(pH_i = 7.31-7.48) than normal astrocytes(pH_i = 6.98), and increased rates of recovery fromacidification, under nominallyCO₂/HCO₃-free conditions.Inhibition of NHE1 in the absence ofCO₂/HCO₃ resulted inpronounced acidification of gliomas, whereas normal astrocytes wereunaffected. When suspended inCO₂/HCO₃ medium astrocytepH_i increased, yet glioma pH_i unexpectedlyacidified, suggesting the presence of anHCO₃-dependent acid loadingpathway. Nucleotide sequencing of NHE1 cDNA from the gliomasdemonstrated that genetic alterations were not responsible for thisaltered NHE1 function. The data suggest that NHE1 activity issignificantly elevated in gliomas and may provide a useful target forthe development of tumor-selective therapies.

     

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.

     

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats

-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa²⁺ ([Ca²⁺]_i) transient andincreased the intracellular pH (pH_i) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na⁺/H⁺exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca²⁺]_i transient andpH_i were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca²⁺]_i and pH_i responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

     

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts

The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO₃exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pH_i) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO₂-25 mMHCO₃ were nearly the same. However,replacement of external Clwith gluconate caused anH₂DIDS-inhibitable (100 µM)increase in the pH_i ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO₂/HCO₃caused the pH_i of both cell typesto increase. The pH_i remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepH_i peaked and then recoveredtoward control values. This pH_irecovery phase was completely blocked by 100 µMH₂DIDS. In the presence ofCO₂/HCO₃, there was an H₂DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO₂/HCO₃),the H₂DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H₂DIDS-sensitive, netCl efflux decreased aspH_i increased, whereas forHCMV-infected cells, efflux increased aspH_i increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO₃exchanger activity.

     

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons

To determine the role ofmembrane transporters in intracellular pH (pH_i) regulationunder conditions of low microenvironmental O₂, we monitoredpH_i in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O₂ deprivation or anoxia (PO₂ 0 Torr), a large increase in pH_i was seen in CA1neurons in HEPES buffer, but a drop in pH_i, albeit small,was observed in the presence of HCO. Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pH_i increase in HEPES buffer was totallyNa⁺ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na⁺/H⁺exchanger (NHE). Also, this pH_i increase in HEPES bufferwas significantly smaller in Na⁺/H⁺ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pH_iincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pH_iincrease in the absence of HCO. We conclude that1) O₂ deprivation activatesNa⁺/H⁺ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pH_i during low-O₂states in neurons.

     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.

     

Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle

The role of Na⁺/Ca²⁺ exchange inregulating intracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na⁺ concentration resulted in agraded rise of [Ca²⁺]_i; 50-100 µMstrophanthidin also increased [Ca²⁺]_i. Asmall outward current accompanied the rise of[Ca²⁺]_i in low-Na⁺ solutions(17.1 ± 1.8 pA in 29.4 mM Na⁺). The quantity ofCa²⁺ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca²⁺]_i,after correction was made for intracellular Ca²⁺ buffering.Ca²⁺ influx through the exchanger was able to loadintracellular Ca²⁺ stores. It is concluded that the levelof resting [Ca²⁺]_i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na⁺ concentration would besufficient to generate significant net Ca²⁺ influx.

     

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells

Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pH_i) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K⁺concentration ([K⁺])is adjusted to match the predicted intracellular[K⁺]. ForpH_i values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH₂O) is threefold larger than in unshrunken cells (~300mosmol/kgH₂O). In the nominalabsence ofCO₂/HCO₃,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH₂O) causes steady-statepH_i to increase by ~0.4. The pH_i increase is due to activationofNa⁺/H⁺exchange because, in single cells, it is blocked in the absence ofexternal Na⁺ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pH_i increase by 80%. Wecalculated the pH_i dependence oftheNa⁺/H⁺exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepH_i dependence of the totalacid-extrusion rate and 2) thepH_i dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepH_i dependence ofNa⁺/H⁺exchange by ~0.7 pH units.     

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.     

Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na⁺/H⁺ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pH_i) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pH_i recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na⁺-dependent pH_i recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 µM) completely abolished the pH_i recovery by NHE. 18-Glycyrrhetinic acid (18-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pH_i recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined. Na⁺/H⁺ exchanger type 1; intracellular pH; aqueous humor     

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.     

Hyposmolality stimulates Na(+)/H(+) exchange and HCO(3)(-) absorption in thick ascending limb via PI 3-kinase

The signal transduction mechanisms that mediateosmotic regulation of Na⁺/H⁺ exchange are notunderstood. Recently we demonstrated that hyposmolality increasesHCO₃ absorption in the renal medullary thickascending limb (MTAL) through stimulation of the apical membraneNa⁺/H⁺ exchanger NHE3. To investigate themechanism of this stimulation, MTALs from rats were isolated andperfused in vitro with 25 mM HCO₃-containingsolutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitorswortmannin (100 nM) and LY-294002 (20 µM) blocked completely thestimulation of HCO₃ absorption by hyposmolality. Intissue strips dissected from the inner stripe of the outer medulla, theregion of the kidney highly enriched in MTALs, hyposmolality increasedPI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-inducedPI 3-K activation. Further studies examined the interaction betweenhyposmolality and vasopressin, which inhibits HCO₃absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO₃ absorption, due to aneffect of cAMP to inhibit hyposmolality- induced activation of PI 3-K.In contrast to their effects to block stimulation by hyposmolality, PI3-K inhibitors and vasopressin have no effect on inhibition of apicalNa⁺/H⁺ exchange (NHE3) andHCO₃ absorption by hyperosmolality. These resultsindicate that hyposmolality increases NHE3 activity andHCO₃ absorption in the MTAL through activation of aPI 3-K-dependent pathway that is inhibited by vasopressin and cAMP.Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediatedthrough different signal transduction mechanisms.

     

Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH(2)-terminal cytoplasmic domain

The role ofintracellular pH (pH_i) in regulation of AE2 function inXenopus oocytes remains unclear. We therefore compared AE2-mediated ³⁶Cl efflux fromXenopus oocytes during imposed variation of extracellular pH(pH_o) or variation of pH_i at constantpH_o. Wild-type AE2-mediated ³⁶Clefflux displayed a steep pH_o vs. activity curve, withpH_o(50) = 6.91 ± 0.04. SequentialNH₂-terminal deletion of amino acid residues in tworegions, between amino acids 328 and 347 or between amino acids 391 and510, shifted pH_o(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocytepH_i at constant pH_o and were shown to beneither substrates nor inhibitors of AE2-mediated Cltransport. At constant pH_o, AE2 was inhibited byintracellular acidification and activated by intracellularalkalinization. Our data define structure-function relationships withinthe AE2 NH₂-terminal cytoplasmic domain, which demonstratesdistinct structural requirements for AE2 regulation by intracellularand extracellular protons.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

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.

     

Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes

In the course of studying the hypertonicity-activated iontransporters in Xenopus oocytes, we found that activation ofendogenous oocyte Na⁺/H⁺ exchange activity(xoNHE) by hypertonic shrinkage required Cl, with anEC₅₀ for bath [Cl] of ~3 mM. Thisrequirement for chloride was not supported by several nonhalide anionsand was not shared by xoNHE activated by acid loading.Hypertonicity-activated xoNHE exhibited an unusual rank order ofinhibitory potency among amiloride derivatives and was blocked byCl transport inhibitors. Chelation of intracellularCa²⁺ by injection of EGTA blocked hypertonic activation ofxoNHE, although many inhibitors of Ca²⁺-related signalingpathways were without inhibitory effect. Hypertonicity activated oocyteextracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors ofneither ERK1/2 nor p38 prevented hypertonic activation of xoNHE.However, hypertonicity also stimulated a Cl-dependentincrease in c-Jun NH₂-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE butnot activation by acid loading. We conclude that hypertonic activationof Na⁺/H⁺ exchange in Xenopusoocytes requires Cl and is mediated by activation of JNK.

     

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.

     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.