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.

     

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].

     

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.

     

Secretory modulation of basolateral membrane inwardly rectified K(+) channel in guinea pig distal colonic crypts

Cell-attached recordings revealedK⁺ channel activity in basolateral membranes ofguinea pig distal colonic crypts. Inwardly rectified currents wereapparent with a pipette solution containing 140 mM K⁺.Single-channel conductance () was 9 pS at the resting membrane potential. Another inward rectifier with  of 19 pS was observed occasionally. At a holding potential of 80 mV,  was 21 and 41 pS,respectively. Identity as K⁺ channels was confirmed afterpatch excision by changing the bath ion composition. From reversalpotentials, relative permeability of Na⁺ overK⁺ (P_Na/P_K)was 0.02 ± 0.02, withP_Rb/P_K = 1.1 andP_Cl/P_K < 0.03. Spontaneous open probability (P_o) of the 9-pSinward rectifier (^gpK_ir) was voltageindependent in cell-attached patches. Both a low(P_o = 0.09 ± 0.01) and a moderate(P_o = 0.41 ± 0.01) activity mode wereobserved. Excision moved ^gpK_ir to the mediumactivity mode; P_o of^gpK_ir was independent of bath Ca²⁺activity and bath acidification. Addition of Cl andK⁺ secretagogues altered P_o of^gpK_ir. Forskolin or carbachol (10 µM)activated the small-conductance ^gpK_ir inquiescent patches and increased P_o inlow-activity patches. K⁺ secretagogues, either epinephrine(5 µM) or prostaglandin E₂ (100 nM), decreasedP_o of ^gpK_ir in activepatches. This ^gpK_ir may be involved inelectrogenic secretion of Cl and K⁺ acrossthe colonic epithelium, which requires a large basolateral membraneK⁺ conductance during maximal Cl secretionand, presumably, a lower K⁺ conductance during primaryelectrogenic K⁺ secretion.

     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

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.

     

Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

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.

     

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.

     

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.

     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

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.

     

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.

     

Characterization of L-glutamine transport by a human neuroblastoma cell line

This study characterized theNa⁺-dependent transport of L-glutamine by ahuman neuroblastoma cell line, SK-N-SH. The Na⁺-dependentcomponent represented >95% of the total glutamine uptake. Kineticstudies showed a single saturable high-affinity carrier with aMichaelis constant (K_m) of 163 ± 23 µMand a maximum transport velocity (V_max) of13,713 ± 803 pmol · mgprotein¹ · min¹. Glutamine uptakewas markedly inhibited in the presence of L-alanine, L-asparagine, and L-serine. Li⁺ didnot substitute for Na⁺. These data show thatL-glutamine is predominantly taken up through systemASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreasedV_max without affectingK_m. The expression of the system ASC subtypeASCT2 decreased in the glutamine-deprived group, whereas glutaminedeprivation did not induce changes in system ASC subtype ASCT1 mRNAexpression. Adaptive increases in Na⁺-dependent glutamate,Na⁺-dependent 2-(methylamino)isobutyric acid, andNa⁺-independent leucine transport were observed underglutamine-deprived conditions, which were completely blocked byactinomycin D and cycloheximide. These mechanisms may allow cells tosurvive and even grow under nutrient-deprived conditions.

     

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes

We investigated the regulation ofATP-sensitive K⁺ (K_ATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(10⁵ M) activated inward currents in the presence of highexternal K⁺ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (10⁵ M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 10⁷ M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of K_ATP channels wasincreased by pinacidil, and PDBu suppressed openings ofK_ATP channels. When cells were pretreated withchelerythrine (10⁶ M) or calphostin C (10⁷M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (10⁶ M).Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(10⁷ M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on K_ATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh onK_ATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates K_ATP in murine colonic myocytes.

     

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (I_sc) averaged 20.8 ± 0.7 µA/cm² (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, I_sc averaged 14.4 ± 1.1 µA/cm² (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (K_i) of 0.68 µM. The remainingamiloride-insensitive I_sc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO₃. It was further stimulated bybasolateral application of forskolin (10⁵ M), which increasedI_sc by 3.1 ± 0.3 µA/cm² (n=65) in epididymis and 0.9 ± 0.1 µA/cm² (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na⁺ viaamiloride-sensitive epithelial Na⁺ channels (ENaC) andsecrete Cl and HCO₃via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA.

     

Intrahepatic bile ducts transport water in response to absorbed glucose

The physiological relevance of theabsorption of glucose from bile by cholangiocytes remains unclear. Theaim of this study was to test the hypothesis that absorbed glucosedrives aquaporin (AQP)-mediated water transport by biliary epitheliaand is thus involved in ductal bile formation. Glucose absorption andwater transport by biliary epithelia were studied in vitro bymicroperfusing intrahepatic bile duct units (IBDUs) isolated from ratliver. In a separate set of in vivo experiments, bile flow andabsorption of biliary glucose were measured after intraportal infusionof D-glucose or phlorizin. IBDUs absorbedD-glucose in a dose- and phlorizin-dependent manner with anabsorption maximum of 92.8 ± 6.2 pmol · min¹ · mm¹.Absorption of D-glucose by microperfused IBDUs resulted inan increase of water absorption (J_v = 310nl · min¹ · mm¹,P_f = 40 × 10³ cm/sec).Glucose-driven water absorption by IBDUs was inhibited byHgCl₂, suggesting that water passively followsabsorbed D-glucose mainly transcellularly viamercury-sensitive AQPs. In vivo studies showed that as the amount ofabsorbed biliary glucose increased after intraportal infusion ofD-glucose, bile flow decreased. In contrast, as theabsorption of biliary glucose decreased after phlorizin, bile flowincreased. Results support the hypothesis that the physiologicalsignificance of the absorption of biliary glucose by cholangiocytes islikely related to regulation of ductal bile formation.

     

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

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

     

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.

     

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

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