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.

     

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.

     

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.

     

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.

     

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.

     

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].

     

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-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

Transport of fluid by lens epithelium

We report for the first time that cultured lens epithelial celllayers and rabbit lenses in vitro transport fluid. Layers of the TN4mouse cell line and bovine cell cultures were grown to confluence onpermeable membrane inserts. Fluid movement across cultured layers andexcised rabbit lenses was determined by volume clamp (37°C).Cultured layers transported fluid from their basal to their apicalsides against a pressure head of 3 cmH₂O. Rates were (inµl · h¹ · cm²)3.3 ± 0.3 for TN4 cells (n = 27) and 4.7 ± 1.0 for bovine layers (n = 6). Quinidine, a blocker ofK⁺ channels, andp-chloromercuribenzenesulfonate andHgCl₂, inhibitors of aquaporins,inhibited fluid transport. Rabbit lenses transported fluid from theiranterior to their posterior sides against a2.5-cmH₂O pressure head at 10.3 ± 0.62 µl · h¹ · lens¹(n = 5) and along the same pressurehead at 12.5 ± 1.1 µl · h¹ · lens¹(n = 6). We calculate that this flowcould wash the lens extracellular space by convection about once every2 h and therefore might contribute to lens homeostasis and transparency.     

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells

Adenosinestimulates Cl channels ofthe nonpigmented (NPE) cells of the ciliary epithelium. We sought toidentify the specific adenosine receptors mediating this action.Cl channel activity inimmortalized human (HCE) NPE cells was determined by monitoring cellvolume in isotonic suspensions with the cationic ionophore gramicidinpresent. The A₃-selective agonistN⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide(IB-MECA) triggered shrinkage (apparentK_d = 55 ± 10 nM). A₃-selective antagonists blocked IB-MECA-triggered shrinkage, andA₃-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 µMadenosine when all four known receptor subtypes are occupied. TheA₁-selective agonistN⁶-cyclopentyladenosineexerted a small effect at 100 nM but not at higher or lowerconcentrations. The A_2A agonistCGS-21680 triggered shrinkage only at high concentration (3 µM), aneffect blocked by MRS-1191. IB-MECA increased intracellularCa²⁺ in HCE cells and alsostimulated short-circuit current across rabbit ciliary epithelium.A₃ message was detected in bothHCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possessA₃ receptors and that adenosinecan activate Cl channels inNPE cells by stimulating these A₃ receptors.     

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.

     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

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

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

     

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia

Fluid transport across cultures of bovine tracheal epitheliumwas measured with a capacitance probe technique. Baseline fluid absorption (J_v)across bovine cells of 3.2 µl · cm² · h¹was inhibited by ~78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with aconcomitant decrease in transepithelial potential (TEP) and increase intransepithelial resistance(R_t). Effectsof P. aeruginosa were blocked byamiloride, which decreased J_v by 112% frombaseline of 2.35 ± 1.25 µl · cm² · h¹,increased R_t by101% from baseline of 610 ± 257  · cm², anddecreased TEP by 91% from baseline of 55 ± 18.5 mV.Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption weredue in part to a block of basolateral membrane K channels. In thepresence of Cl transport inhibitors[5-nitro-2-(3-phenylpropylamino)-benzoic acid,H₂-DIDS, and bumetanide],P. aeruginosa induced a fluid secretion of ~2.5 ± 0.4 µl · cm² · h¹and decreased R_twithout changing TEP. However, these changes were abolished when thetransport inhibitors were used in a medium in which Cl was replaced byan impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect onJ_v, TEP, orR_t. Mutantslacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encodinga  factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial saltand water transport induced by P. aeruginosa may alter viscosity and ionic composition ofairway secretions so as to foster further bacterial colonization.

     

Desensitization of P2Y2 receptor-activated transepithelial anion secretion

Desensitization ofP2Y₂ receptor-activated anionsecretion may limit the usefulness of extracellular nucleotides insecretagogue therapy of epithelial diseases, e.g., cystic fibrosis(CF). To investigate the desensitization process for endogenousP2Y₂ receptors, freshly excised orcultured murine gallbladder epithelia (MGEP) were mounted in Ussingchambers to measure short-circuit current (I_sc), an indexof electrogenic anion secretion. Luminal treatment with nucleotidereceptor agonists increased theI_sc with apotency profile of ATP = UTP > 2-methylthioATP >>,-methylene-ATP. RT-PCR revealed the expression ofP2Y₂ receptor mRNA in the MGEPcells. The desensitization of anion secretion required a 10-minpreincubation with the P2Y₂receptor agonist UTP and increased in aconcentration-dependent manner(IC₅₀  10⁶ M). Approximately 40%of the anion secretory response was unaffected by maximal desensitizingconcentrations of UTP. Recovery from UTP-induced desensitization wasrapid (<10 min) at preincubation concentrations less than theEC₅₀ (1.9 × 10⁶ M) but requiredprogressively longer time periods at greater concentrations.UTP-induced total inositol phosphate production and intracellularCa²⁺ mobilization desensitizedwith a concentration dependence similar to that of anion secretion. Incontrast, maximal anion secretion induced byCa²⁺ ionophore ionomycin wasunaffected by preincubation with a desensitizing concentration of UTP.It was concluded that 1)desensitization of transepithelial anion secretion stimulated by theP2Y₂ receptor agonist UTP is timeand concentration dependent; 2)recovery from desensitization is prolonged (>90 min) at UTPconcentrations >10⁵ M;and 3) UTP-induced desensitizationoccurs before the operation of the anion secretory mechanism.     

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.

     

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle

Freshly dispersed sheep mesenteric lymphaticsmooth muscle cells were studied at 37°C using the perforatedpatch-clamp technique with Cs⁺- and K⁺-filledpipettes. Depolarizing steps evoked currents that consisted ofL-type Ca²⁺ [I_Ca(L)]current and a slowly developing current. The slow current reversed at1 ± 1.5 mV with symmetrical Cl concentrationscompared with 23.2 ± 1.2 mV (n = 5) and34.3 ± 3.5 mV (n = 4) when externalCl was substituted with either glutamate (86 mM) orI (125 mM). Nifedipine (1 µM) blocked and BAY K 8644 enhanced I_Ca(L), the slow-developing sustainedcurrent, and the tail current. The Cl channel blockeranthracene-9-carboxylic acid (9-AC) reduced only the slowly developinginward and tail currents. Application of caffeine (10 mM) tovoltage-clamped cells evoked currents that reversed close to theCl equilibrium potential and were sensitive to 9-AC.Small spontaneous transient depolarizations and larger actionpotentials were observed in current clamp, and these were blocked by9-AC. Evoked action potentials were triphasic and had a prominentplateau phase that was selectively blocked by 9-AC. Similarly, fluidoutput was reduced by 9-AC in doubly cannulated segments ofspontaneously pumping sheep lymphatics, suggesting that theCa²⁺-activated Cl current plays an importantrole in the electrical activity underlying spontaneous activity in this tissue.

     

ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb

Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa⁺-K⁺(NH⁺₄)-2Clcotransport. ANG II at 10¹⁶to 10¹² M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the10¹² M ANG II levelcotransport activity; eventually,10⁶ M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 10¹²M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 10¹² M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 10⁶ M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa²⁺ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus10¹² M ANG II inhibitsvia 20-HETE, whereas 5 × 10¹¹ M ANG II stimulatesvia PKCNa⁺-K⁺(NH⁺₄)-2Clcotransport; all ANG II effects involveAT₁ receptors and PLC activation.

     

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.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(_ex) 350 nm, emission wavelength (_em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (_ex 380 nm,_em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M¹)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.