ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

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.

     

Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Cl{-} and fluid secretion

Rabbit conjunctival epithelium exhibits UTP-dependentCl secretion into the tears. We investigated whetherfluid secretion also takes place. Short-circuit current(I_sc) was 14.9 ± 1.4 µA/cm²(n = 16). Four P2Y₂ purinergic receptoragonists [UTP and the novel compounds INS365, INS306, and INS440(Inspire Pharmaceuticals)] added apically (10 µM) resulted intemporary (~30 min) I_sc increases (88%, 66%,57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of6.5 ± 0.7 µl · h¹ · cm² (range2.1-15.3, n = 20). Fluid transport was stimulatedby mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h¹ · cm²,n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h¹ · cm²,n = 5. Fluid transport was abolished by 1 mMouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h¹ · cm²,n = 4). We conclude that this epithelium secretes fluidactively and that P2Y₂ agonists stimulate bothCl and fluid secretions.

     

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.

     

Functional and molecular identification of ERG channels in murine portal vein myocytes

Ion channels encoded byether-à-go-go-related genes (ERG) have been implicatedin repolarization of the cardiac action potential and also ascomponents of the resting membrane conductance in various cells. Theaim of the present study was to determine whether ERG channels wereexpressed in smooth muscle cells isolated from portal vein. RT-PCRdemonstrated the expression of murine ERG (mERG), and real-timequantitative PCR showed that the mERG1b isoform predominated over themERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portalvein displayed membrane staining with an ERG1-specific antibody. Wholecell voltage-clamp experiments were performed to determine whetherportal vein myocytes expressed functional ERG channels. Large inwardcurrents with distinctive kinetics were elicited that were inhibitedrapidly by E-4031 (mean amplitude of the E-4031-sensitive current at120 mV was 205 ± 24 pA; n = 14). Deactivationof the E-4031-sensitive current was voltage dependent (mean timeconstants at 80 and 120 mV were 103 ± 9 and 33 ± 2 ms,respectively; n = 13). Because of the rapid kinetics ofmERG currents at more negative potentials, there was a substantialnoninactivating "window" current that reached a maximum of66 ± 10 pA at 70 mV. Complete portal veins exhibitedspontaneous contractile activity in isometric tension experiments, andthis activity was modified significantly by E-4031. These data showthat ERG channels are expressed in murine portal vein myocytes that maycontribute to the resting membrane conductance.

     

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity

Human endothelial cells wereexposed to 5 mM glucose (control), 25 mM (high) glucose, or osmoticcontrol for 72 h. TGF-1 production, cell growth, death, andcell cycle progression, and the effects of TGF-1 and TGF-neutralization on these parameters were studied. High glucose andhyperosmolarity increased endothelial TGF-1 secretion(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducingendothelial cell number (P < 0.001) and increasingcellular protein content (P < 0.001) than the osmoticcontrol. TGF- antibody only reversed the antiproliferative andhypertrophic effects of high glucose. High glucose altered cell cycleprogression and cyclin-dependent kinase inhibitor expressionindependently of hyperosmolarity. High glucose increased endothelialcell apoptosis (P < 0.01), whereashyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF- antibody did not reverse the apoptotic effectsobserved with high glucose. Exogenous TGF-1 mimicked the increased Sphase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cellcycle progression. These growth effects occurred principally via aTGF-1 autocrine pathway. In contrast, apoptosis andendoreduplication occurred independently of this cytokine and hyperosmolarity.

     

Fast-twitch skeletal muscles of dystrophic mouse pups are resistant to injury from acute mechanical stress

Loss of the dystrophin-glycoproteincomplex from muscle sarcolemma in Duchenne's muscular dystrophy (DMD)renders the membrane susceptible to mechanical injury, leaky toCa²⁺, and disrupts signaling, but the precise mechanism(s)leading to the onset of DMD remain unclear. To assess the role ofmechanical injury in the onset of DMD, extensor digitorum longus (EDL)muscles from C57 (control), mdx, andmdx-utrophin-deficient [mdx:utrn(/); dystrophic] pups aged 9-12 days were subjected to an acutestretch-injury or no-stretch protocol in vitro. Before the stretches,isometric stress was attenuated for mdx:utrn(/) comparedwith control muscles at all stimulation frequencies (P < 0.05). During the stretches, EDL muscles for each genotypedemonstrated similar mean stiffness values. After the stretches,isometric stress during a tetanus was decreased significantly for bothmdx and mdx:utrn(/) muscles compared withcontrol muscles (P < 0.05). Membrane injury assessedby uptake of procion orange dye was greater for dystrophic comparedwith control EDL (P < 0.05), but, within eachgenotype, the percentage of total cells taking up dye was not different for the no-stretch vs. stretch condition. These data suggest that thesarcolemma of maturing dystrophic EDL muscles are resistant to acutemechanical injury.

     

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

The phorbol ester phorbol12-myristate 13-acetate (PMA) inhibits Cl secretion(short-circuit current, I_sc) and decreasesbarrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes inthis response, we compared PMA with two non-phorbol activators of PKC(bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozymeselectivity profiles. PMA sequentially inhibited cAMP-stimulatedI_sc and decreased TER, as measured byvoltage-current clamp. By subcellular fractionation and Western blot,PMA (100 nM) induced sequential membrane translocation of the novelPKC followed by the conventional PKC and activated both isozymesby in vitro kinase assay. PKC was activated by PMA but did nottranslocate. By immunofluorescence, PKC redistributed to thebasolateral domain in response to PMA, whereas PKC moved apically.Inhibition of I_sc by PMA was prevented by theconventional and novel PKC inhibitor Gö-6850 (5 µM) but not theconventional isoform inhibitor Gö-6976 (5 µM) or the PKCinhibitor rottlerin (10 µM), implicating PKC in inhibition ofCl secretion. In contrast, both Gö-6976 andGö-6850 prevented the decline of TER, suggesting involvement ofPKC. Bryostatin-1 (100 nM) translocated PKC and PKC andinhibited cAMP-elicited I_sc. However, unlikePMA, bryostatin-1 downregulated PKC protein, and the decrease in TERwas only transient. Carbachol (100 µM) translocated only PKC andinhibited I_sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1and carbachol inhibition of I_sc. We concludethat basolateral translocation of PKC inhibits Clsecretion, while apical translocation of PKC decreases TER. Thesedata suggest that epithelial transport and barrier function can bemodulated by distinct PKC isoforms.

     

p38 MAPK mediates renal tubular cell TNF-{alpha} production and TNF-{alpha}-dependent apoptosis during simulated ischemia

Ischemia causes renal tubular cellloss through apoptosis; however, the mechanisms of this processremain unclear. Using the renal tubular epithelial cell lineLLC-PK₁, we developed a model of simulated ischemia(SI) to investigate the role of p38 MAPK (mitogen-activated proteinkinase) in renal cell tumor necrosis factor- (TNF-) mRNAproduction, protein bioactivity, and apoptosis. Resultsdemonstrate that 60 min of SI induced maximal TNF- mRNA productionand bioactivity. Furthermore, 60 min of ischemia induced renaltubular cell apoptosis at all substrate replacement time pointsexamined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF- mRNA production andTNF- bioactivity, and both p38 MAPK inhibition and TNF- neutralization (anti-porcine TNF- antibody) preventedapoptosis after 60 min of SI. These results constitute theinitial demonstration that 1) renal tubular cells produceTNF- mRNA and biologically active TNF- and undergoapoptosis in response to SI, and 2) p38 MAPKmediates renal tubular cell TNF- production and TNF--dependent apoptosis after SI.

     

Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor

The nonsense codonsuppression technique was used to incorporate o-nitrobenzylcysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) intothe 9' position of the M2 transmembrane segment of the -subunit ofthe muscle nicotinic ACh receptor expressed in Xenopusoocytes. The caged amino acids replaced an endogenous Leu residue thathas been implicated in channel gating. ACh-induced current increasedsubstantially after ultraviolet (UV) irradiation to remove the caginggroup. This represents the first successful incorporation of caged Cysinto a protein in vivo and the first incorporation of caged amino acidswithin a transmembrane segment of a membrane protein. The bulkynitrobenzyl group does not prevent the synthesis, assembly, ortrafficking of the ACh receptor. When side chains were decaged using1-ms UV light flashes, the channels with caged Cys or caged Tyrresponded with strikingly different kinetics. The increase in currentupon photolysis of caged Cys was too rapid for resolution by thevoltage-clamp circuit [time constant () <10 ms], whereas theincrease in current upon photolysis of caged Tyr was dominated by aphase with  ~500 ms. Apparently, the presence of a bulkyo-nitrobenzyl Tyr residue distorts the receptor into anabnormal conformation. Upon release of the caging group, the receptorrelaxes, with  ~500 ms, into a conformation that allows thechannel to open. Tyr at the 9' position of the -subunit greatlyincreases the ability of ACh to block the channel by binding within thechannel pore. This is manifested in two ways. 1) A"rebound," or increase in current, occurs upon removal of ACh fromthe bathing medium; and 2) at ACh concentrations >400 µM,inward currents are decreased through the mutated channel. The abilityto incorporate caged amino acids into proteins should have widespread utility.

     

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.

     

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.

     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.

     

Measurement of stretch-induced loss of alveolar epithelial barrier integrity with a novel in vitro method

Mechanical ventilation with hightidal volumes has been shown to contribute to the formation orworsening of interstitial and alveolar edema. Previously we showed thatapplication of large biaxial deformations in vitro perturbs theconcentration and distribution of functional tight junction proteins inalveolar epithelial cells. Using a novel method, we determined thatapplied epithelial strain increases paracellular permeability in adose- and rate-dependent manner. Primary rat alveolar epithelial cellswere subjected to 12%, 25%, or 37% change in surface area (SA)cyclic equibiaxial stretch for 1 h. Cells were also stretchednoncyclically at 25% SA for 1 h. During the experimentalperiod, a fluorescently tagged ouabain derivative was added to theapical fluid. Evidence of binding indicated functional failure of theparacellular transport barrier. The percentage of field area stainedwas quantified from microscopic images. There was no significantevidence of basolateral fluorescent staining at 12% SA or at 25%SA applied cyclically or statically. However, cyclic stretch at 37%SA resulted in significantly more staining than in unstretched cells(P < 0.0001) or those stretched at either 12%(P < 0.0001) or 25% cyclic (P < 0.0005) or static (P < 0.05) SA. These resultssuggest that large cyclic tidal volumes may increase paracellularpermeability, potentially resulting in alveolar flooding.

     

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes

To investigate theeffects of reactive oxygen species (ROS) on NHpermeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (V_m) and intracellular pH(pH_i) induced by a 20 mM NH₄Cl-containingsolution. Under control conditions, NH₄Cl exposure induceda large membrane depolarization (to V_m = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pH_i(pH_i) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpH_i/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H₂O₂ or -amyloid protein had nomarked effect on the NH₄Cl-induced pH_i. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pH_i anddpH_i/dt. These increases in pH_iand dpH_i/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies ¹gO₂ and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (G_m). ThisRB-induced G_m increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na⁺concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway.

     

Immunocompetence of macrophages in rats exposed to Candida albicans infection and stress

The integration of innate andadaptive immune responses is required for efficient control ofCandida albicans. The present work aimed to assess, at thelocal site of the infection, the immunocompetence of macrophages inrats infected intraperitoneally with C. albicans and exposedsimultaneously to stress during 3 days (CaS group). We studied the1) ability to remove and kill C. albicans,2) tumor necrosis factor- (TNF-) release,3) balance of the inducible enzymes NO synthase (iNOS) andarginase, and 4) expression of interleukin (IL)-1 andIL-1 receptor antagonist (ra) mRNA. Compared with only infected animals(Ca group), the number of colony-forming units was significantly higherin CaS rats (P < 0.01), and the macrophagecandidicidal activity was ~2.5-fold lower (P < 0.01). Release of TNF- was diminished in both unstimulated andheat-killed C. albicans restimulated macrophages of the CaSgroup (Ca vs. CaS, P < 0.03 and P < 0.05, respectively). In Ca- and CaS-group rats, the rates for both thearginase activity and the NO synthesis were significantly enhanced.However, the stress exposure downregulated the activity of both enzymes(CaS vs. Ca, P < 0.05). After in vitro restimulation,the IL-1ra/IL-1 ratio was significantly diminished in CaS-group rats(P < 0.05). Our results indicate that a correlationexists between early impairment of macrophage function and stress exposure.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor

Growthfactors affect a variety of epithelial functions. We examined theability of TGF- to modulate epithelial ion transport andpermeability. Filter-grown monolayers of human colonic epithelia, T84and HT-29 cells, were treated with TGF- (0.1-100 ng/ml,15 min-72 h) or infected with an adenoviral vector encodingTGF- (Ad-TGF) for 144 h. Ion transport (i.e., short-circuitcurrent, I_sc) and transepithelial resistance(TER) were assessed in Ussing chambers. Neither recombinant TGF- norAd-TGF infection affected baseline I_sc;however, exposure to 1 ng/ml TGF- led to a significant (30-50%) reduction in the I_sc responses toforskolin, vasoactive intestinal peptide, and cholera toxin (agentsthat evoke Cl secretion via cAMP mobilization) and to thecell-permeant dibutyryl cAMP. Pharmacological analysis of signalingpathways revealed that the inhibition of cAMP-driven epithelialCl secretion by TGF- was blocked by pretreatment withSB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors ofJNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF- enhanced the barrier function of the treated monolayers by up to threefold asassessed by TER; however, this event was temporally displaced from thealtered I_sc response, being statisticallysignificant only at 72 h posttreatment. Thus, in addition toTGF- promotion of epithelial barrier function, we show that thisgrowth factor also reduces responsiveness to cAMP-dependentsecretagogues in a chronic manner and speculate that this serves as abraking mechanism to limit secretory enteropathies.

     

Direct estimate of 1:1 stoichiometry of K(+)-Cl(-) cotransport in rabbit erythrocytes

This work was undertaken toobtain a direct measure of the stoichiometry ofNa⁺-independent K⁺-Cl cotransport(KCC), with rabbit red blood cells as a model system. To determinewhether ⁸⁶Rb⁺ can be used quantitatively as atracer for KCC, ⁸⁶Rb⁺ and K⁺effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulatedK⁺ efflux into isosmotic NaCl was smaller than that for⁸⁶Rb⁺ by a factor of 0.68 ± 0.11 (SD,n = 5). This correction factor was used in all otherexperiments to calculate the K⁺ efflux from the measured⁸⁶Rb⁺ efflux. To minimize interference from theanion exchanger, extracellular Cl was replaced withSO, and4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clampednear 0 mV with the protonophore 2,4-dinitrophenol. The Clefflux at 25°C under these conditions is ~100,000-fold smaller thanthe uninhibited Cl/Cl exchange flux and isstimulated ~2-fold by NEM. The NEM-stimulated ³⁶Cl flux is inhibited by okadaic acid andcalyculin A, as expected for KCC. The ratio of the NEM-stimulatedK⁺ to Cl efflux is 1.12 ± 0.26 (SD,n = 5). We conclude thatK⁺-Cl cotransport in rabbit red blood cellshas a stoichiometry of 1:1.