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.

     

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.

     

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells

Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA₃-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (I_IB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (V_rev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO₃, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(I_Swell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothI_IB-MECA and I_Swell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A₃-subtype adenosine agonists and swellingactivate a common population of Cl channels.

     

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.     

Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions

Depolarization elicited outwardK⁺ currents from canine loweresophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitiveCa²⁺-dependentK⁺ channels. Current magnitudesvaried with pipette Ca²⁺concentration (EC₅₀ = 108.5 nM).N^G-nitro-L-arginine(L-NNA,10⁴ M), IbTX(10⁸ M), or bufferingintracellular Ca²⁺ to 8 nMdecreased outward currents >80%. Sodium nitroprusside (NaNP,10⁴ M) restoredL-NNA-inhibited or lowintracellular Ca²⁺ concentration(not IbTX)-inhibited currents.L-NNA or IbTXapplication depolarized LES cells from 43 to 35 mV. NaNPrestored the membrane potential to 46 mV afterL-NNA but not after IbTXapplication. Nifedipine (30 µM) reduced outward currents andabolished or reduced L-NNA orNaNP effects, respectively. Immunocytochemistry revealed the presenceof both argininosuccinate synthetase and argininosuccinate lyase in LESmuscle cells. L-Citrulline, likeL-arginine, reversed L-NNA inhibition of outwardcurrents; only L-argininereversed inhibition of outward currents by an antibody toargininosuccinate synthetase. Therefore, endogenous nitric oxideproduction, activated by Ca²⁺entrance involving L-typeCa²⁺ channels, may continuouslyenhance outward currents to modulate LES muscle cell membrane potentialand excitability.

     

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.

     

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

The free radicals nitric oxide(·NO) and superoxide (O₂·) react to formperoxynitrite (ONOO), a highly toxic oxidant species. Inthis study we investigated the respective effects of NO andONOO in monocytes from healthy human donors. Purifiedmonocytes were incubated for 6 or 16 h with a pure NO donor(S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an ·NO/ONOO donor(3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and withoutsuperoxide dismutase (200 IU/ml), or pure ONOO. Weprovide evidence that 3-morpholinosydnonimine chlorhydrate alonerepresents a strong stress to human monocytes leading to adose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis andnecrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO, but not ·NO, was responsible forthe observed effects. This observation was further strengthened by theabsence of a stress response in cells exposed toS-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO alone also inducedmitochondrial membrane depolarization and cell death by apoptosis andnecrosis. Thus ONOO formation may well explain the toxiceffect generally attributed to ·NO.

     

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.     

Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra

Freshly dispersedinterstitial cells from the rabbit urethra were studied by using theperforated-patch technique. When cells were voltage clamped at 60 mVand exposed to 10 µM norepinephrine (NE) at 80-s intervals, eitherlarge single inward currents or a series of oscillatory inward currentsof diminishing amplitude were evoked. These currents were blocked byeither phentolamine (1 µM) or prazosin (1 µM), suggesting that theeffects of NE were mediated via ₁-adrenoceptors.NE-evoked currents were depressed by the blockers ofCa²⁺-activated Cl currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversalpotential of the above currents changed in a predictable manner whenthe Cl equilibrium potential was altered, againsuggesting that they were due to activation of a Clconductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-releasedCa²⁺. Inhibition of NE-evoked currents by the phospholipaseC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate(100 µM) suggested that NE releases Ca²⁺ via an inositol1,4,5-trisphosphate (IP₃)-dependent mechanism. Theseresults support the idea that stimulation of₁-adrenoceptors releases Ca²⁺ from anIP₃-sensitive store, which in turn activatesCa²⁺-activated Cl current in freshlydispersed interstitial cells of the rabbit urethra. This elevates slowwave frequency in these cells and may underlie the mechanismresponsible for increased urethral tone during nerve stimulation.

     

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.

     

Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber

String-shaped reconstitutedsmooth muscle (SM) fibers were prepared in rectangular wells by thermalgelation of a mixed solution of collagen and cultured SM cells derivedfrom guinea pig stomach. The cells in the fiber exhibited an elongatedspindle shape and were aligned along the long axis. The fibercontracted in response to KCl (140 mM), norepinephrine (NE;10⁷ M), epinephrine (10⁷ M), phenylephrine(10⁶ M), serotonin (10⁶ M), and histamine(10⁵ M), but not acetylcholine (10⁵ M).Phentolamine (10⁷ M) produced a parallel rightward shiftof the NE dose-response curve. Moreover, NE-induced contractionwas partially inhibited by nifedipine and completely abolished by theintracellular Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, theRho kinase inhibitor Y-27632, and papaverine. A[³H]quinuclidinyl benzilate binding study revealed thatthe loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. Theseresults suggest that, although the fiber is not a model for fullydifferentiated SM, contractile mechanisms are maintained.

     

Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells

Corneal endothelial function is dependent onHCO₃ transport. However, the relativeHCO₃ permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO₂-HCO₃ (at constant pH) or removing HCO₃alone (at constant CO₂) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO₃ permeabilities and their dependencies on Na⁺ andCl. Removal ofCO₂-HCO₃from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO₃ at constantCO₂ indicated that the basolateralHCO₃ fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa⁺ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO₃ flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO₃ fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO₂ conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO₂-HCO₃apically suggests that CO₂diffusion may contribute to apicalHCO₃ flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO₂ fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa⁺-dependent basolateralHCO₃ permeability is consistent withNa⁺-nHCO₃cotransport. Changes inHCO₃ flux in the absence ofCl are most likely due toNa⁺-nHCO₃cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO₃ permeabilityis relatively low, but may be augmented byCO₂ diffusion in conjunction witha CAIV.

     

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.

     

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3

Although its primary function is monovalent anion exchange, theband 3 protein also cotransports divalent anions together with protonsat low pH. The putative proton binding site, Glu-681 in humanerythrocyte band 3, is conserved throughout the anion exchanger family(AE family). To determine whether or not the monovalent anion bindingsite is located near Glu-681, we modified this residue with Woodward'sreagent K(N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements ofCl binding by³⁵Cl-NMR show that externalCl binds to band 3 evenwhen Cl transport isinhibited ~95% by WRK modification of Glu-681. This indicates thatthe external Cl bindingsite is not located near Glu-681 and thus presumably is distant fromthe proton binding site. DIDS inhibitsCl binding even when WRK isbound to Glu-681, indicating that the DIDS binding site is also distantfrom Glu-681. Our data suggest that the DIDS site and probably also theexternally facing Cltransport site are located nearer to the external surface of themembrane than Glu-681.

     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

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.

     

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.

     

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.     

Inhibition of phosphatidylinositol 3-kinase does not alter forskolin-stimulated Cl(-) secretion by T84 cells

Wortmannin is a potent inhibitor ofphosphatidylinositol 3-kinase (PI3K) and membrane trafficking in manycells. To test the hypothesis that cystic fibrosis transmembraneconductance regulator (CFTR) traffics into and out of the plasmamembrane during cAMP-stimulated epithelial Clsecretion, we have studied the effects of wortmannin onforskolin-stimulated Cl secretion by the humancolonic cell line T84. At the PI3K inhibitory concentration of 100 nM,wortmannin did not affect significantly forskolin-stimulatedCl secretion measured as short-circuit current(I_SC). However, 500 nM wortmannin significantlyinhibited forskolin-stimulated I_SC. cAMP activationof apical membrane CFTR Cl channels in-toxin-permeabilized monolayers was not reduced by 500 nMwortmannin, suggesting that inhibition of other transporters accountsfor the observed reduction in T84 Cl secretion.Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP),but wortmannin did not alter forskolin inhibition of apical HRPendocytosis. In the absence of forskolin, wortmannin stimulated HRPendocytosis significantly. We conclude that, in T84 cells, apical fluidphase endocytosis is not dependent on PI3K activity and that CFTR doesnot recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

     

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.