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.

     

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.

     

Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump

A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa⁺-K⁺ pump current (I_p)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured I_p of myocytes voltageclamped with wide-tipped patch pipettes. I_p ofmyocytes from captopril-treated rabbits was larger thanI_p of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedI_p to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on I_p that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa⁺-K⁺ pump.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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].

     

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms

The cardiacNa⁺/Ca²⁺ exchanger (NCX), an importantregulator of cytosolic Ca²⁺ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (I_NCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheI_NCX for both isoforms exhibitedNa⁺-dependent inactivation and Ca²⁺-dependentpositive regulation. The I_NCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ₁₀ values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the I_NCX ofNCX-TR1.0 was relatively temperature insensitive with Q₁₀values of 1.2 and 1.1 for peak and steady-state currents, respectively.I_NCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in I_NCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in I_NCX temperature dependence between the twoisoforms are not due to potential disparities in either theI_NCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.

     

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.

     

Chloride secretion by semicircular canal duct epithelium is stimulated via beta 2-adrenergic receptors

The ductalepithelium of the semicircular canal forms much of the boundary betweenthe K⁺-rich luminal fluid and the Na⁺-richabluminal fluid. We sought to determine whether the net ion fluxproducing the apical-to-basal short-circuit current(I_sc) in primary cultures was due to anionsecretion and/or cation absorption and under control of receptoragonists. Net fluxes of ²²Na, ⁸⁶Rb, and³⁶Cl demonstrated a basal-to-apical Clsecretion that was stimulated by isoproterenol. Isoproterenol andnorepinephrine increased I_sc with anEC₅₀ of 3 and 15 nM, respectively, and isoproterenolincreased tissue cAMP of native canals with an EC₅₀ of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had noeffect on I_sc. Isoproterenol stimulation ofI_sc and cAMP was inhibited by ICI-118551(IC₅₀ = 6 µM for I_sc) but notby CGP-20712A (1 µM) in primary cultures, and similar results werefound in native epithelium. I_sc was partially inhibited by basolateral Ba²⁺ (IC₅₀ = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, andDIDS did not fully fit the profile for CFTR. Our findings show that thecanal epithelium contributes to endolymph homeostasis by secretion ofCl under ₂-adrenergic control with cAMP assecond messenger, a process that parallels the adrenergic control ofK⁺ secretion by vestibular dark cells. The current workpoints to one possible etiology of endolymphatic hydrops in Meniere'sdisease and may provide a basis for intervention.

     

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.

     

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.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

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.

     

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.

     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

Serotonin-elicited inhibition of Cl(-) secretion in the rabbit conjunctival epithelium

The effects of serotonin[5-hydroxytryptamine (5-HT)] on the transepithelial electricalproperties of the short-circuited rabbit conjunctiva were examined.With this epithelium, the short-circuit current(I_sc) measures Cl secretion plusan amiloride-resistant Na⁺ absorptive process. Apicaladdition of 5-HT (10 µM) elicited a prompt I_screduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm² and increased transepithelial resistance from0.89 ± 0.05 to 1.03 ± 0.06 k · cm²(means ± SE, n = 21, P < 0.05).Similar changes were obtained with conjunctivae bathed withoutNa⁺ in the apical bath, as well as with conjunctivaepreexposed to bumetanide with the Cl-dependentI_sc sustained by the parallel activities ofbasolateral Na⁺/H⁺ andCl/HCO exchangers. In contrast, the5-HT-evoked effects were attenuated by the absence of Cl(I_sc = 0.5 ± 0.2, n = 5), suggesting that reduced Clconductance(s) is an effect of 5-HT exposure. In amphotericin B-treatedconjunctiva and in the presence of a transepithelial K⁺gradient, 5-HT addition reduced K⁺ diffusion across thepreparation by 13% and increased transepithelial resistance by 4%(n = 6, P < 0.05), indicating that aninhibition in K⁺ conductance(s) was also detectable.Significant electrical responses also occurred under physiologicalconditions when 5-HT was introduced to epithelia pretreated withadrenergic agonists or protein kinase C, phospholipase C,phosphodiesterase, or adenylyl cyclase inhibitors or after perturbationof Ca²⁺ homeostasis. Briefly, the conjunctiva harbors theonly known Cl-secreting epithelium in which 5-HT evokesCl transport inhibition; receptor subtype and signaltransduction mechanism were not determined.

     

Pharmacological modulation of ion transport across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia

Forskolin,UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen(Methoxsalen; 8-MOP), and genistein were evaluated for theireffects on ion transport across primary cultures of human bronchialepithelium (HBE) expressing wild-type (wt HBE) and F508(F-HBE) cystic fibrosis transmembrane conductance regulator. In wtHBE, the baseline short-circuit current (I_sc)averaged 27.0 ± 0.6 µA/cm² (n = 350). Amiloride reduced this I_sc by 13.5 ± 0.5 µA/cm² (n = 317). In F-HBE,baseline I_sc was 33.8 ± 1.2 µA/cm² (n = 200), and amiloride reducedthis by 29.6 ± 1.5 µA/cm² (n = 116), demonstrating the characteristic hyperabsorption of Na⁺ associated with cystic fibrosis (CF). In wt HBE,subsequent to amiloride, forskolin induced a sustained,bumetanide-sensitive I_sc(I_sc = 8.4 ± 0.8 µA/cm²; n = 119). Addition ofacetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced I_sc, suggesting forskolin also stimulatesHCO₃ secretion. This was confirmed by ionsubstitution studies. The forskolin-induced I_scwas inhibited by 293B, Ba²⁺, clofilium, and quinine,whereas charybdotoxin was without effect. In F-HBE the forskolinI_sc response was reduced to 1.2 ± 0.3 µA/cm² (n = 30). In wt HBE, mucosal UTPinduced a transient increase in I_sc ( I_sc = 15.5 ± 1.1 µA/cm²;n = 44) followed by a sustained plateau, whereas inF-HBE the increase in I_sc was reduced to5.8 ± 0.7 µA/cm² (n = 13). In wtHBE, 1-EBIO, NS004, 8-MOP, and genistein increased I_sc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm²(n = 17), respectively. In F-HBE, 1-EBIO, NS004, and8-MOP failed to stimulate Cl secretion. However, additionof NS004 subsequent to forskolin induced a sustained Clsecretory response (2.1 ± 0.3 µA/cm²,n = 21). In F-HBE, genistein alone stimulatedCl secretion (2.5 ± 0.5 µA/cm²,n = 11). After incubation of F-HBE at 26°C for24 h, the responses to 1-EBIO, NS004, and genistein were allpotentiated. 1-EBIO and genistein increased Na⁺ absorptionacross F-HBE, whereas NS004 and 8-MOP had no effect. Finally,Ca²⁺-, but not cAMP-mediated agonists, stimulatedK⁺ secretion across both wt HBE and F-HBE in aglibenclamide-dependent fashion. Our results demonstrate thatpharmacological agents directed at both basolateral K⁺ andapical Cl conductances directly modulate Clsecretion across HBE, indicating they may be useful in ameliorating theion transport defect associated with CF.