Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.

Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca(2+)-dependent K+ channel.

The Cl- secretory response of colonic cells to Ca(2+)-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca(2+)-mediated Cl- secretion on the basolateral membrane, Ca(2+)-dependent K+ channel (Kca) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected Kca in excised inside-out patches. In contrast, arachidonic acid (AA; 3 microM) potently inhibited Kca, reducing NP0, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited Kca in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on Kca was reversed. A similar effect of AA was observed on Kca during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 microM, all of these fatty acids inhibited Kca, reducing NP0 by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current (Isc) response was determined. In the presence of AACOCF3, the peak carbachol-induced Isc response was increased approximately 2.5-fold. Our results suggest that AA generation induced by Ca(2+)-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca(2+)-independent K+ channel.

We used single-channel recording techniques to identify and characterize a large-conductance, Ca(2+)-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability (Po) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel Po was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3<-->C2<-->C1<-->O. Arachidonic acid (AA) increased the Po of the channel, with an apparent stimulatory constant (Ks) of 1.39 microM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased Po also, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect Po. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 microM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl- secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

UTP inhibits Na+ absorption in wild-type and DeltaF508 CFTR-expressing human bronchial epithelia.

Ca2+-mediated agonists, including UTP, are being developed for therapeutic use in cystic fibrosis (CF) based on their ability to modulate alternative Cl- conductances. As CF is also characterized by hyperabsorption of Na+, we determined the effect of mucosal UTP on transepithelial Na+ transport in primary cultures of human bronchial epithelia (HBE). In symmetrical NaCl, UTP induced an initial increase in short-circuit current (Isc) followed by a sustained inhibition. To differentiate between effects on Na+ absorption and Cl- secretion, Isc was measured in the absence of mucosal and serosal Cl- (INa). Again, mucosal UTP induced an initial increase and then a sustained decrease that reduced amiloride-sensitive INa by 73%. The Ca2+-dependent agonists histamine, bradykinin, serosal UTP, and thapsigargin similarly induced sustained inhibition (62-84%) of INa. Mucosal UTP induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of INa in primary cultures of human CF airway homozygous for the DeltaF508 mutation. BAPTA-AM blunted UTP-dependent inhibition of INa, but inhibitors of protein kinase C (PKC) and phospholipase A2 had no effect. Indeed, direct activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit Na+ absorption. Apyrase, a tri- and diphosphatase, did not reverse inhibitory effects of UTP on INa, suggesting a long-term inhibitory effect of UTP that is independent of receptor occupancy. After establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin, mucosal UTP induced an initial increase in K+ current followed by a sustained inhibition. We conclude that increasing cellular Ca2+ induces a long-term inhibition of transepithelial Na+ transport across normal and CF HBE at least partly due to downregulation of a basolateral membrane K+ conductance. Thus UTP may have a dual therapeutic effect in CF airway: 1) stimulation of a Cl- secretory response and 2) inhibition of Na+ transport.

Intracellular pH and membrane potassium conductance in rabbit distal colon.

Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.

Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells.

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Cholinergic stimulation produces oscillations of cytosolic Ca2+ in a secretory epithelial cell line, T84.

The effects of carbamylcholine (carbachol) on intracellular Ca2+ concentration ([Ca2+]c) of T84 cells were examined using the fluorescent Ca2+ indicator fura-2 and microfluorometric techniques. In single isolated cells, carbachol (100 microM) caused a rapid increase in [Ca2+]c of 184 +/- 15 nM (SE, n = 44) from a resting value of 56 +/- 7 nM. This initial transient was followed by a series of oscillations in 68% of the cells. Atropine (10 microM) blocked this response. Removal of bath Ca2+ did not inhibit the rise in [Ca2+]c or oscillations, but the response duration was shortened in 47% of the cells. The amplitude and latency of the initial Ca2+ rise, frequency of oscillations, and number of responding cells varied with the agonist concentration. We have previously shown that carbachol induces an oscillating K+ conductance in T84 cells [D. Devor, S. Simasko, and M. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990]. Simultaneous measurement of membrane K+ current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]c and increase in K+ current. These results show that a rise in [Ca2+]c and oscillations is likely to underlie the membrane K+ current responses to carbachol in T84 cells. Responses from a single cell within a subconfluent monolayer were different from those of isolated cells. In cells of a monolayer the initial [Ca2+]c rise (111 +/- 8 nM; n = 41) was followed by a decline to a stable plateau, and oscillations were not seen. Removal of bath Ca2+ both reduced the initial transient and eliminated the plateau phase of the response. These results suggest that cell-to-cell contact or differentiation during monolayer formation influences the Ca2+ handling mechanisms of T84 cells.

Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1.

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.

Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84.

Effects of carbachol on membrane potential and current in T84 cells were determined using whole cell patch-clamp techniques. When the pipettes contained a standard KCl solution and the bath contained a standard NaCl solution, carbachol (100 microM) caused a rapid hyperpolarization to the K+ equilibrium potential (EK+), followed by potential oscillations. When membrane potential was clamped to 0 mV, carbachol induced an outwardly directed K+ current in 31 of 37 cells, with a peak value of 618 +/- 51 (SE) pA. In 77% of these cells the current oscillated and gradually declined to base line. Atropine (20 microM) blocked this response. In symmetric KCl solutions the carbachol-induced current reversed at 0 mV with no rectification. Ba2+ or Cs+ did not block the current, but tetraethylammonium ion (TEA) reduced the number of responding cells. Although a Cl- conductance was found in resting cells, carbachol did not cause an increase in Cl- current when the cells were voltage-clamped to EK+, or when voltage-clamped to +/- 60 mV while bathed in symmetric NaCl solutions. When the Ca2(+)-buffering capacity of the pipette solution was increased, 80% of the cells responded to carbachol, but only 10% oscillated; however, no K+ current was induced by carbachol when the pipette was made nominally Ca2+ free. The current was not affected by removal of Ca2+ from the bath. These results show that carbachol induces an oscillating Ca2(+)-activated K+ conductance in T84 cells, but no Cl- conductance. This K+ conductance is dependent on the mechanisms that regulate intracellular Ca2+.

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 their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and DeltaF508 (DeltaF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current (I(sc)) averaged 27.0 +/- 0.6 microA/cm(2) (n = 350). Amiloride reduced this I(sc) by 13.5 +/- 0.5 microA/cm(2) (n = 317). In DeltaF-HBE, baseline I(sc) was 33.8 +/- 1.2 microA/cm(2) (n = 200), and amiloride reduced this by 29.6 +/- 1.5 microA/cm(2) (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) (DeltaI(sc) = 8.4 +/- 0.8 microA/cm(2); n = 119). Addition of acetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4, 4'-dinitrostilben-2,2'-disulfonic acid further reduced I(sc), suggesting forskolin also stimulates HCO(3)(-) secretion. This was confirmed by ion substitution studies. The forskolin-induced I(sc) was inhibited by 293B, Ba(2+), clofilium, and quinine, whereas charybdotoxin was without effect. In DeltaF-HBE the forskolin I(sc) response was reduced to 1.2 +/- 0.3 microA/cm(2) (n = 30). In wt HBE, mucosal UTP induced a transient increase in I(sc) (Delta I(sc) = 15. 5 +/- 1.1 microA/cm(2); n = 44) followed by a sustained plateau, whereas in DeltaF-HBE the increase in I(sc) was reduced to 5.8 +/- 0. 7 microA/cm(2) (n = 13). In wt HBE, 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 microA/cm(2) (n = 17), respectively. In DeltaF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl(-) secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl(-) secretory response (2.1 +/- 0.3 microA/cm(2), n = 21). In DeltaF-HBE, genistein alone stimulated Cl(-) secretion (2.5 +/- 0.5 microA/cm(2), n = 11). After incubation of DeltaF-HBE at 26 degrees C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na(+) absorption across DeltaF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca(2+)-, but not cAMP-mediated agonists, stimulated K(+) secretion across both wt HBE and DeltaF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K(+) and apical Cl(-) conductances directly modulate Cl(-) secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Pharmacology of CFTR chloride channel activity.

Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

cAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland.

Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.

Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca(2+)-dependent K+ channel.

We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 microM. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (Isc) with an inhibitory constant (Ki) of 3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced Isc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca(2+)-dependent K+ channel (KCa) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (IK). Under these conditions, 1-EBIO induced a large increase in IK that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated 86Rb+ uptake in a CTX-sensitive manner; the Ki for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this 86Rb+ uptake was not blocked by 293B. The effects of 1-EBIO on the KCa in T84 cells was determined in excised inside-out patches. 1-EBIO (100 microM) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on KCa activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a Kca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.

Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK.

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.

Psoralens: novel modulators of Cl- secretion.

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

Benzimidazolone activators of chloride secretion: potential therapeutics for cystic fibrosis and chronic obstructive pulmonary disease.

The diseases of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by mucus-congested airways. Agents that stimulate the secretion of Cl- are anticipated to facilitate mucociliary clearance and thus be of benefit in the treatment of CF and COPD. Recently 1-EBIO (1-ethyl-2-benzimidazolinone or 1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) was shown to stimulate chloride secretion albeit at relatively high concentrations (0.6-1 mM). The studies reported here were undertaken to develop a more potent benzimidazolone. Structure activity studies with 30 benzimidazolone derivatives revealed that ethyl and hydrogen groups at the 1 and 3 nitrogen positions, respectively, were critical for the activation of hIK1 K+ channels and that other alkyl groups were not tolerated at these positions without some loss in potency. Substitutions at the 5 and 6 positions improved the potency of 1-EBIO. Compared with 1-EBIO, the most potent of these derivatives, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) was severalfold better in a 86Rb+ uptake assay, 20-fold better in short circuit current measurements on T84 monolayers, and 100-fold better in patch-clamp assays of hIK1 activity. Short circuit current studies revealed DCEBIO stimulates Cl- secretion via the activation of hIK1 K+ channels and the activation of an apical membrane Cl- conductance. The improved potency of DCEBIO strengthens the possibility that compounds in this class may be of therapeutic benefit in the treatment of CF and COPD.